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Name_______________________________ TISSUE STRUCTURE LECTURE AND LAB MANUAL 2011/2012 JS (3rd yr) Physiology Students (8 Lectures & 8 Labs)
SF (2nd yr) Human Health and Disease Students (8 Lectures & 1 Lab) JF (1st yr) Medical Students (8 Lectures & 1 Lab) JF (1st yr) Physiotherapy Students (8 Lectures) Professor Kumlesh K. Dev Department of Physiology Acknowledgements: We are grateful to Dr Alan Tuffery (Department of Physiology, Medical School, Trinity College Dublin) who developed the original version of this course manual and Lesley Penny (Department of Physiology, Medical School, Trinity College Dublin) for helping with the annotations of Digital Slidebox Slides. This Manual is produced in conformity with College’s policy on accessibility and may be requested in other formats (e.g. electronic, large print) Note. In the event of any conflict or inconsistency between the general regulations published in the University Calendar and information contained in this Handbook, the provisions of the General Regulations will prevail. CONTENTS Lessons and Lectures Introduction to Histology Lecture Classes : All Students Lecture 1 — Epithelial Tissues (KKD) Lecture 2 — Excitable Tissues (KKD) Lecture 3 — Connective Tissues (KKD) Lecture 4 — Bone & Bone Formation (KKD) Lecture 5 — Body Fluids (KKD) Lecture 6 — Blood & Blood Pathophysiology (TC) Lecture 7 — Immune System (TC) Lecture 8 — Thermoregulation (TC) Practical Classes: JS (3rd yr) Physiology Lab 1 — Digital SlideBox Lab 1 (KKD) Lab 2 — Digital SlideBox Lab 2 (KKD) Lab 3 — Digital SlideBox Lab 3 (KKD) Lab 4 — Immunocytochem (LAB COATS) (KKD) Lab 5 — Confocal Imaging (KKD) Lab 6 — Haematology (TC) Lab 7 — Digital SlideBox Revision Lab Lab 8 — In course Assessment Practical Classes: JF (1st yr ) Medicine / SF (2nd yr) Human Health & Disease Group A/B — Digital SlideBox Lab (KKD) Group C/D — Digital SlideBox Lab (KKD)
Lecturers & Staff KKD ‐ Professor Kumlesh K. Dev TC ‐ Professor Thomas Connor NB ‐ Dr. Noreen Boyle LESSONS AND LECTURES This course will provide knowledge of tissue structure for the understanding of Physiology. At the same time a historical perspective will be introduced as well as some key concepts in Physiology. 1. Course Structure and Lectures Please note that some parts of this Manual are of specific relevance to Physiology, Medical or Physiotherapy students, and may not apply to you. JS (3rd yr) Physiology Students There are 8 lectures and 8 x 3hr Labs. There will be lab‐based assessments at the end of each lab. A lab‐based examination will be held at the end of the Michaelmas Term. More details of this assessment will be given during the course. JF (1st yr) Medical Students There are 8 Lectures and 1 x 4hr Lab. Take note of the group you are in for the lab. There will be a lab‐based assessment at the end of the lab. These lectures and lab form Part of Human Form & Function Module. These lectures deal and lab deal with Basic Tissues, Body Fluids and Blood (including basic immunology etc) and thermoregulation. SF (2nd yr) Human Health and Disease Students There are 8 Lectures and 1 x 4hr Lab. Take note of the group you are in for the lab. There will be a lab‐based assessment at the end of the lab. These lectures and lab form Part of Human Form & Function Module. These lectures deal and lab deal with Basic Tissues, Body Fluids and Blood (including basic immunology etc) and thermoregulation. JF (1st yr) Physiotherapy Students There are 8 Lectures. There are NO associated labs. These lectures deal with Basic Tissues, Body Fluids and Blood (including basic immunology etc) and thermoregulation. 2. Lessons Synopses This course manual is designed to allow you to get the most out of this course, and includes learning outcomes for each lecture. You are encouraged to read the Lessons Synopses before the corresponding lecture to leave you free focus on the key concepts. The material is arranged by topic and corresponds mostly to each lecture. Note each Lesson does not directly correspond to a Lecture and that one or more Lesson may apply to each Lecture. Each topic/lecture and practical class has learning outcomes and associated test questions. The Lecture Synopses defines the specialist terminology. The histological terminology used in this course is agreed by international convention, as set out in Nomina Histologica (1975), and may differ from textbooks. The most important difference is the deletion of all eponymous terms (i.e. those bearing individuals' names). The modern terminology tends to be usefully descriptive (e.g. ‘intestinal crypts’ for ‘crypts of Lieberkühn’). I
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3. Approach to Study It is important to keep up during the year so that you understand the material. Concepts are repeated and built on so you will get useful reinforcement. Look for the underlying themes and organisation of information and focus on the basic physiological mechanism (e.g. compartments, the negative feedback loop). Preparation for lectures is important. Review the lecture synopses before each lecture and if possible read the related sections of the textbook indicated in lecture outlines. The Lectures will define the content, especially the level of detail required. Make your own notes and don’t fully rely on the lecture slides. These are not a substitute for you notes and note taking is an important skill to develop. The learning outcomes should be reviewed and used as the basis for your private study. They are in many respects versions of exam questions. Past papers are also a crucial part of revision, but do not rely on question‐spotting! 4. Assumed background knowledge Students who have not previously studied Biology should get a basic grounding from the JF Biology text. The key areas are cell structure and function and the basic functions of the organs of the body. The JF Biology text is Biology by Campbell, NA & Reece, JB. There are multiple copies of the 8th edition (2008) in the university library. The following topics should also be revised: (i)
Basic cell structure and function, including electron microscopy and cell organelles. (ii) Methods of tissue preparation: fixing, sectioning and staining. These topics can be covered by reading the first one or two chapters of almost any Histology textbook (see recommended textbooks). It is important to know how the method of preparation affects the tissue. In particular, the effects of making a virtually two‐dimensional section of a three‐
dimensional structure must always be taken into account. The functional state of the tissue should also be taken into account: a histological preparation is a static representation of a dynamic process. 5. Recommended Textbooks The recommended textbook is: – Young, B & Heath, JW (2006). Wheater’s Functional Histology — A Text and Colour Atlas 5th edn. London: Churchill Livingstone. [Hamilton 599 L93*4,] – Sherwood (2009). Human Physiology from Cells to Systems. Thomson Other books in the University Library: – McCann, S et al. (2009) Clinical Cases Uncovered: Haematology. Wiley‐
Blackwell: Oxford I
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6. Learning Outcomes Learning outcomes specify what students should be able to do as result of studying the material from each lecture or lab class, as well as some more general outcomes which should result from the course as a whole. General learning outcomes – interpret two‐dimensional images as three‐dimensional structures – relate structure to function – give examples of changes in tissue structure in physiological, pathophysiological and developmental states – list the Basic Tissues and their general functions – explain basis of classification of tissues according to different criteria 7. Further Support Students who need additional supports because of a disability should arrange to discuss with the Head of Department, Dr. Aine Kelly, Contact Person for Disability Support in the Department of Physiology. This manual and all lecture slides are available in electronic (PDF) format from http://www.medicine.tcd.ie/physiology/student/. LAB ASSESSMENTS Please note that there will be assessments associated with each of your laboratory classes. These assessments will be given at the end of each lab. I
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INTRODUCTION TO HISTOLOGY 1. Objectives To be able to: a) explain methods of sectioning, staining and preparation artefacts b) explain appearance of cell components in terms of common stains c) list classification of four basic tissues and general properties: epithelium, connective tissue, muscle and nerve 2. What is Histology? Histology is the study of tissue structure, from the individual cell, to organs, to systems. Histology relates to Cell Biology (Cytology) and Anatomy. It also forms the structural basis for understanding Function (Physiology) and abnormal structure and function (Pathology). Pathophysiological (clinical) examples will be used to illustrate aspects of functions and their significance. Tissues may be regarded as aggregations of cells of one or several types, which serve a particular function or set of functions. The concepts of structure and function are essential to the study of Histology. Particular skills which will be emphasised are: (i)
Classification of tissues. This requires close attention to the way tissues are described. (ii) Recognition of specific features. This requires the application of classification criteria, observational and reasoning skills. (iii) Relationship between structure and function. This is most essential and requires synthetic and deductive skills. It requires bringing together knowledge from different fields of Anatomy, Biochemistry and Physiology. Your ability to combine information from these fields is critical for your understanding. 3. Cells and Tissue An essential requirement in Histology is to be able to describe cells and tissues unambiguously. The following list gives the criteria which can be used at light microscope level. Sometimes you will be asked to provide a full written description of a cell or tissue, on other occasions you may be asked to select from this list, for example: ‘Describe the shape and arrangement of...’ Some criteria are especially important in certain cases. (i)
Relative size. Compared to other cells in the tissue. (ii) Shape. Such as columnar, cuboidal, flattened, polyhedral. (iii) Cytoplasmic reaction. Usually refers to pH of the cytoplasm as demonstrated by staining: acidophilia (usually eosinophilia, i.e. affinity for eosin, a red, acidic dye) or basophilia (usually affinity for haematoxylin, a blue, basic dye). Special stains may be used for specific substances (e.g. fat or glycogen)(see below). (iv) Cytoplasmic inclusions. Such as granules, vacuoles. (v) Nuclear characteristics. Such as shape, position within the cell, size, staining pattern, presence or absence of nucleoli. I
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(vi) Surface specialisations. Such as cilia. (vii) Arrangement. Cells are arranged to form tissues, e.g. in single or multiple layers, cords or clumps; with variable amounts of intercellular matrix which may be solid, fluid or fibrous. 4. Heamatoxylin & Eosin Staining Haematoxylin and eosin (usually abbreviated H&E) are two very commonly used histological stains. It is most important to be clear about their properties because the interpretation of cell function depends upon knowledge of the reaction (pH) of organelles. Haematoxylin is a base and therefore tends to bind to acidic structures. It stains blue. The most distinctive acid in cells is nuclear DNA, consequently nuclei appear blue. Structures which are acidic are said to be basophilic, i.e. they attract basic stains. Protein‐producing cells have lots of rough endoplasmic reticulum (RER) containing RNA and hence are basophilic and appear blue. Eosin is acidic and therefore stains basic structures. It stains red. The cytoplasm of most cells is slightly basic and therefore stains pink and is said to be acidophilic. Note that knowledge of these properties allows you to interpret a preparation made with an unknown stain. The nucleus is always acidic and therefore defines the basic stain. 5. Basic Tissues Classically, the Basic Tissues are: Epithelia, Connective Tissue and the Excitable Tissues (Nerve and Muscle). This is a core concept of this course I
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LESSON 1: EPITHELIAL (LINING) TISSUES 1. Objectives To be able to: a) distinguish structure and function of lining versus glandular epithelia b) state the general function of lining epithelia. c) classify lining epithelia according to morphological criteria. d) relate structure and function in lining epithelia (permeability/transport) e) give examples of named epithelia: structure, location, function f) describe cell surface specialisation and functions g) describe proliferation/differentiation of epithelia and relate it to function h) give examples of pathophysiological changes in a lining epithelium. 2. Introduction Definition of Epithelium is: a single tissue; plural, epithelia; adjective, epithelial Epithelium is a Basic Tissue, characterised by closely‐packed cells with minimal intercellular material and an absence of nerves and blood vessels. Nutrition is by diffusion from the highly vascular connective tissue known as the lamina propria underlying all epithelia. There are two functional types of epithelium: lining epithelia and glandular epithelium. Most epithelia are lining tissues that cover free surfaces of the body and its cavities, e.g. epidermis (skin), lining of the gastrointestinal tract and ducts. Other epithelia are secretory and are the glandular epithelia (see Secretion). The position of epithelium in contact with the environment gives them great importance in regulating the composition of the body by controlling the movement of materials in and out. The structure of epithelia can be correlated with their function. Thus the epithelium of larger ducts is thicker than in smaller ducts; and in sites exposed to desiccation or friction the epithelium may have a surface coat of keratin, a tough protein, and is said to be keratinised. For specific examples of the adaptation of epithelia to particular function see the organ systems. Many epithelia have a high rate of renewal of their constituent cells. For example the entire epithelium of the gut is replaced every 6‐7 days, an equivalent to a daily loss of 1.38 x 109 cells from the small intestine. 3. Structural Characteristics of Epithelia The structural characteristics of epithelia are defined by three factors listed below: a) absence of nerves (except for a few axons in the deeper layers) b) absence of blood vessels — nutrition is by diffusion from the highly vascular connective tissue (known as the lamina propria) underlying all epithelia c) close packing of the constituent cells with minimal intercellular substance I
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4. Morphological Classification of Epithelia The morphological characterisation of epithelia is more commonly used and depends on the following 3 factors: a) number of layers of cells: an epithelium with only one layer is described as simple; with more layers — stratified. (Note that cells are not described as ‘simple’ or ‘stratified’, only the layer.) N.B. Pseudostratified epithelium appears to have more than one layer since the nuclei lie at different heights, but in fact all cells are in contact with the basement membrane (see Figure 5g). b)
shape of cells at free surface: e.g. squamous (flattened), cuboidal, columnar. c)
surface specialisation (if any) e.g. keratinised, ciliated. 5. Types of Lining Epithelium The types of Lining Epithelium are listed in the figure and text below: a) Simple squamous epithelium: a single layer of flattened cells Function. Thinness provides minimal barrier to the movement of materials. Examples. Alveolar lining of the lung, renal corpuscle. Figure Diagram of types of lining epithelium. b) Stratified squamous epithelium: many layers; those at surface are flattened Function. To withstand mechanical wear and tear; resist desiccation Example. Lining of mouth, vagina and rectum. Stratified squamous epithelium (keratinised): surface cells are dead and filled with an inert protein, keratin, forming flakes or squames. Function. As above, but more so; particularly adapted to a drier site. Example. Skin. c) Simple cuboidal epithelium: single layer of box‐shaped (cuboidal) cells. I
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Function. Usually have a role in active transport or synthesis. Examples. Lining of ducts, thyroid follicles. d) Transitional epithelium: many layers of cells; those at surface are irregularly polyhedral (like squashed bubbles) and it is called transitional. Function. To allow large changes in the volume of the lumen. Examples. Bladder, ureter. e) Simple columnar epithelium: single layer of tall cells Function. Metabolically active cells — absorption, synthesis. Example. Lining of intestine. f) Stratified columnar epithelium: many layers; cells at surface are columnar. Function. Largely support (possibly some modification of secretion) Example. Very large ducts g) Pseudostratified columnar ciliated epithelium: although nuclei lie at different levels, all cells are attached to basement membrane and so there is only one layer. Cells that reach free surface are tall (columnar) and bear cilia. Function. Complex — several different functions. Example. Lining of trachea. 6. Self Test Questions 1. List the three criteria used to classify lining epithelia. 2. Give a brief description of the terms used to describe the shape of the surface cells in lining epithelia. 3. List two types of surface specialisation found in lining epithelia. I
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LESSON 2: SECRETION (GLANDULAR EPITHELIA) 1. Objectives To be able to: a) distinguish endocrine and exocrine glands b) apply functional classification by secretion: mucous, serous, steroid etc c) give examples of the different cellular mechanisms of secretion d) describe physiological/transport functional significance of ducts e) relate ultrastructural, LM, EM and functional properties of secretory cells 2. Introduction Glands are composed of secretory epithelial cells, together with supporting connective tissue containing nerves and blood vessels. They are specialised in production of secretions. This process normally involves production of macromolecules which are stored as membrane‐bound vesicles within secretory cell and liberated via apical membrane. The secretion may be inorganic (e.g. gastric cells, pancreatic duct cells), protein (e.g. pancreatic islet cells), lipid (e.g. sebaceous glands), steroid (e.g. adrenal) or carbohydrate (e.g. mucous cells). Glandular tissue may be divided into two basic types. a)
Exocrine glands which retain their connection with the surface from which they originated, by means of ducts, and liberate their fluid secretions onto the surface. b)
Endocrine glands which are ductless and liberate their secretion directly into the blood. 3. Classification of Exocrine Glands This is a good example of different systems of classification evolving as knowledge accumulated and also having usefulness for different purposes. Morphological classification This is a classical but not very useful classification. According to morphological classification, glands may be simple (with one duct) or compound (with a branching duct system), and with a variety of forms of secretory unit: tubular, rounded acini, oval alveoli. Functional classification This classification is based on type of secretion, which may be mucous or serous (containing enzymes) or mixed (secretion contains both mucus and enzymes). Note
different spellings: mucus is substance; mucous is adjective, meaning
containing mucus. Histological characteristics of some principal types in wax‐
embedded sections stained with H&E are summarised below. a) Mucous cells. Typically pale (vacuolated), due to unstained mucus, with squashed, basal nuclei. b) Serous (enzyme‐containing) cells. With basophilic cytoplasm (with or without stained secretory granules), due to large amounts of RNA (in rough ER) for protein synthesis. Nuclei are rounded and near base of the cell. c) Pancreatic acinar cell. This cell is not evenly basophilic. Enzyme synthesis takes place in rough ER in the basal portion of cell so only that area is I
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basophilic. Enzyme precursors are stored as granules in apical part of cell and may be unstained, giving a pale, vacuolated appearance. d) Duodenal [Brunner’s] glands. Cells are unusual in that they share features of mucous and serous cells: pale, vacuolated cytoplasm (‘mucous’) with rounded nuclei near the base of the cell (‘serous’). They produce a watery mucus containing some enzymes. 4. Mechanism of Liberation of Secretion The 3 types of secretion is as follows: a) Merocrine exocytosis of secretory material only, without loss of cell cytoplasm, e.g. pancreatic acinar cells, salivary glands. b) Holocrine secretory material liberated as a result of cell breakdown and death, e.g. sebaceous glands. c) Apocrine secretion liberated together with loss of some cytoplasm, but not cell death, e.g. mammary gland. The process of secretion is controlled by the autonomic nervous system and hormones. Myoepithelial (basket) cells possess long contractile extensions which embrace secretory acini and aid expression of the secretory product. 5. Role of Ducts Ducts are not merely passive conduits for secretions. They may influence composition of secretion. For example, ducts of salivary glands remove Na+, and add K+. The extent of change in composition is inversely proportional to rate of secretion. Na+ HCO3‐ Cl‐ K+ 50 200 50 300 In general, the type of epithelium varies with the calibre of the duct. Smaller ducts usually have simple cuboidal epithelium; larger ones have columnar or occasionally, stratified cuboidal epithelium; the largest ducts have stratified columnar epithelium. Table Relative composition of saliva. Numbers are percentage of concentration in plasma (Parotid gland; flow ~2.5 ml/min) [Berne & Levy Fig. 38‐3] 6. Self Test Questions 1.
2.
3.
What is an exocrine gland? Describe the appearance of a pancreatic acinar cell as seen in an H&E‐stained, wax‐embedded section. Define the three mechanisms of liberation of secretion in exocrine glands and name a cell type for each. I
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LESSON 3: SKIN 1. Objectives To be able to: a) describe general structure and function of dermis and epidermis b) describe accessory cell types and functions c) describe differentiation of epidermis and keratinocytes. d) relate structure to functions of skin: sense organs, thermoregulation 2. Introduction Skin covers the whole of the body. It is arguably the largest organ of the body (1.5‐
2.0 m2). As an organ, it has components from all of the Basic Tissues (see Connective Tissue). The epithelial cells of the skin are constantly being renewed by proliferation of stem cells in the basal layer. 3. Functions of Skin a)
b)
c)
d)
e)
protection against mechanical, chemical and thermal stresses and micro‐
organisms. Sensation via receptors for touch, pressure, pain, temperature etc. thermoregulation by (i) insulation by adipose tissue and hair, and (ii) regulation of blood flow through superficial capillaries evaporation of sweat resists dehydration by having a waterproof external layer of horny protein (keratin). 4. Structure of Skin Essentially the skin consists of an epithelium, referred to as the epidermis, and an underlying layer of loose (or areolar) connective tissue, called the dermis. a) Epidermis The principal cell type is the keratinocyte, which forms keratin. The epidermis consists of up to five layers, here listed from the base up, i.e. in the order of increasing age of cells. 1. Basal layer of cuboidal cells: Cell division occurs here to allow renewal and repair 2. Prickle cell layer: Consisting of several layers of cells, each of which is tightly joined to its neighbouring cells by desmosomes. This layer gets its name from the spiny appearance of the cells in some preparations in which the cells are shrunken so placing tension on the desmosomes. 3. Granular layer: The cells contain granules of keratohyalin, a keratin precursor. 4. Stratum lucidum: A clear layer (often absent). 5. Keratin: Dead cells, which are dehydrated and lacking almost all cellular characteristics, being reduced to flakes (or squames) of keratin which are lost from skin surface. I
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Two other cell types are commonly found in the epidermis are melanocytes and ‘Langerhans cells’ (nonpigmented granular dendrocytes). 1. Melanocytes: These cells are dendritic (branching) cells among the basal cells. Since they are only 10‐20% of basal cells they are difficult to detect at LM level. They produce the pigment, melanin, which is taken up by adjacent keratinocytes. It is the persistence of the melanin in the keratinocytes that causes different skin colours. 2. Langerhans cells: ‘Langerhans cells’ are also dendritic and show properties similar to cells of the lymphoid organs and have a role in immunological reactions of skin, e.g. contact allergic dermatitis. They are regarded as related to macrophages. b) Dermis The dermis onsists of loose CT, containing nerves, blood vessels and various appendages. The dermal‐epidermal junction is not always smooth, but may form a series of ridges, the dermal papillae. The height of the ridges is associated with the proliferation of the tissue and is sometimes used as an index of tissue damage, e.g. oesophageal epithelium in gastric reflux. Blood Circulation in Skin flows via arteries in dermis branch to form the following: 1. A plexus deep in the dermis: is associated with adipose tissue, hair follicles and sweat glands 2. A plexus near the dermal papillae, and a capillary loop in each dermal papilla: This nourishes the epidermis and brings the blood vessels into closer contact with the skin surface aiding heat loss. Blood flow in a capillary loop is controlled by changes in the degree of contraction of the smooth muscle of the supplying arteriole. 5. Appendages of Skin a) Hairs Hairs are keratinised structures produced by a hair follicle. The follicle is a down‐growth of surface epithelium surrounded by connective tissue. Hair formation takes place at the base of the follicle in the hair bulb. Associated with the follicle, in the dermis, are groups of smooth muscle fibres, called the arrector pili muscles which connect the hair follicle to the dermal papillae. They are under autonomic nervous control, and contract in response to cold and fear. b) Sweat Glands These consist of coiled tubular glands deep in the dermis which liberate a watery secretion onto the skin surface. These glands are surrounded by many capillaries and are involved in thermoregulation. Myoepithelial cells assist secretion. Eccrine (= merocrine) sweat glands open directly onto the skin surface and are solely concerned with heat loss. Apocrine sweat glands are associated with hair follicles (see Figure) in restricted areas of the body. They store their secretions in their lumina. The surfaces of the secretory cells bear protrusions which suggested an apocrine (‘pinching off’) mechanism of secretion, but, in fact their mechanism of secretion is probably merocrine (see Secretion). The function of apocrine sweat glands may be to produce pheromones. I
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Figure Diagram of the appendages of skin. [From Weiss, 1983] c) Sebaceous Glands These are small branched alveolar glands associated with the hair follicle which liberate an oily secretion (sebum) by holocrine secretion (see Secretion), to waterproof the hair and surrounding skin. d) Receptors A variety of receptor on skin cells allow for sensation of touch, pressure, heat, cold, etc. These include: a) Meissner's Corpuscles — in dermal papillae; respond to light touch. b) Pacinian Corpuscles — deep in dermis; respond to deep pressure and vibration. c) Merkel’s Receptor — close to basal cells; touch. d) Free nerve endings of small unmyelinated sensory axons; respond to pain and temperature as well as other modalities of sensation. 6. Self Test Questions 1.
2.
List the three epidermal cell types and their principal functions. Briefly describe the development of a basal keratinocyte as it migrates upwards. I
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LESSON 4: THERMOREGULATION 1. Objectives To be able to: a) describe normal and pathophysiological range of core temperature b) describe compensatory mechanism of temperature regulation c) outline mechanism of heat production and loss; thermoneutral zone d) explain measures of metabolism: in different activities; BMR e) outline role of skin thermoreceptors as a feed‐forward homeostatic mechanism f) explain thermoregulatory responses and treatments in extreme environments, fever, hypothermia, hyperthermia g) give pathophysiological examples of thermoregulation: fever, malaria, malignant hyperthermia 2. Introduction Thermoregulation is maintenance of body’s temperature ~37.8 °C. Temperature usually refers to core temperature (tc), essentially temperature of deeper parts of body, especially CNS. It usually measured via external auditory meatus (team). Temperature is regulated within a narrow range (± 2 °C). The feedback mechanism involves thermoreceptors, especially central thermoreceptors in hypothalamus which monitor temperature of arterial blood; and peripheral thermoreceptors (mostly in skin). Inputs are integrated in hypothalamus to maintain tc at set point and there are 3 effectors: sweat glands, skin blood vessels, and skeletal muscles. The following concepts are important in understanding temperature regulation: a) Heat Movement Heat passes into and out of a physical object by: (i) radiation (heat loss over the skin) (ii) conduction (contact with objects) (iii) convection (transfer by air) (iv) evaporation (sweating) b) Negative Feedback The regulated variable tc deviates from normal range. Deviation is detected by receptors whose inputs are integrated and effectors adjust heat production and loss to restore regulated variable tc to its normal value. c) Compartment Material flows through spaces and flow is regulated. For example heat may be transferred from core (organs) to shell (skin) where it can be lost. 3. Heat Production and Loss Temperature (tc) can be regulated by increasing or decreasing production and loss to maintain homeostasis. Heat is produced from 2 main sources, metabolism and brown fat: a) Metabolism Heat is generated in the body by metabolism which is about 50% efficient. This means that for every unit of work, one unit of heat is generated. I
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However, muscles are only 25% efficient (i.e. work:heat ratio of 1:3) and this is an important mechanism for heat generation in the cold (increased muscle tone or shivering). b) Brown Fat Brown fat (BAT) produces heat by uncoupling mitochondrial metabolism and is regulated by the thyroid hormones and the sympathetic nervous system. BAT is important in humans in the newborn (which cannot shiver) and in some forms of obesity. Heat is lost from 3 main sources, metabolism and brown fat: a) Skin and blood flow The regulation of arteriolar tone to control skin blood flow is significant in control of heat loss. Note that skin thermoreceptors monitor ambient temperature and cause a change in skin blood flow before there is a change in core temperature. This is a ‘feed‐forward’ response, ‘anticipating the change in tc. The Thermoneutral Zone is the range of core temperatures (c. 20‐30 °C) at which body temperature can be maintained solely by regulation of skin blood flow. That is, neither shivering nor sweating is required. b) Sweating Sweating is stimulated by sympathetic nervous system and heat loss occurs by evaporation. c) Metabolic Rate Metabolic rate is defined as total energy used at a given time. Basal Metabolic Rate (BMR) is a physiological measurement made under very specific, controlled conditions and is about 80 kcal/h (330 kJ/h). In sleep, metabolic rate is about 65 kcal/h. Exercise increases metabolic rate. Climbing stairs requires 110 kcal/h, walking 250 kcal/hr and biking or swimming 500 kcal/hr. 4. Pathophysiology Many of the processes of temperature control in pathophysiological conditions are complex responses, which you will revisit in other parts of the course, especially in Cardiovascular System. a) Cold Environment Heat production is increased by increasing muscle tone and/or shivering. Voluntary exercise is increased and metabolism may be increased. (Longer term adjustments are made by thyroid hormones.) Heat loss is decreased by skin vasoconstriction, huddling, and adding clothing. (Note that some of these responses are behavioural.) b) Hot Environment Heat production is decreased: muscles relax and movement is reduced. Heat loss is increased by increased vasoconstriction and sweating. Cool clothing is preferred. c) Heat exhaustion Occurs when excessive sweating reduces blood volume so that blood pressure cannot be maintained. d) Heat stroke Is an uncompensated increase in tc and will lead to CNS malfunction; the temperature‐regulating centre fails and sweating stops. I
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e) Fever This is caused by a rising of the set point by chemical mediators (endogenous pyrogens or from pathogens) which release prostaglandins which act on hypothalamic neurones. 5. Self Test Questions 1. Why is the metabolic rate in sleep lower than BMR? 2. Explain sweating when fever breaks. I
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LESSON 5: EXCITABLE NERVE TISSUES 1. Objectives To be able to: a) describe structure of processes, fibres, synapses of nerve cells b) outline stages of communication between nerve cells (neurotransmission) c) describe myelinated and nonmyelinated nerve axons 2. Introduction Muscle & Nerve: together the Excitable Tissues, characterised by response to external stimuli; part of response is electrical. Nerve cells are adapted to conduct electrical impulses (action potentials). Transmission between one nerve terminal and another or an end‐organ is by chemical transmission across the synaptic gap. It is important to distinguish nerve cell bodies, nerve processes (axons, dendrites), bundles of nerve fibres and nerve fibres. The nerve cell body and its processes comprise the neuron. The function of nervous tissue is to integrate stimuli, generate and transmit action potentials. Nerve cells (neurones) are very variable in size and shape, but essentially are relatively large cells with processes. The main process is the axon, smaller processes are dendrites. Axons contact an end‐organ (effector) or another neuron. Dendrites contact other neurons. Motoneurons have up to 1000 dendritic connections each. Neurons typically have relatively large, pale nuclei, each with a prominent nucleolus. Often only the ‘cell body’ is seen in section. The cytoplasm contains many granules which are clusters of free ribosomes (the high RNA content makes them basophilic). The prominent nucleolus and abundant free ribosomes together indicate a high level of synthesis. Nuclei are long‐lived and often have long axons, so they may have a large mass of cytoplasm to maintain; hence the need to have a capacity for synthesis of cellular components. A typical motor neurone receives about 1000 connections, reflecting the general function of neurones of integrating inputs and giving a single output, the action potential. A neurone’s connections may be relatively short and non‐myelinated (‘dendrites’) or longer (axons). Glial cells provide structural and physiological support for neurones and lie between blood vessels and neurone. About 90% of CNS cells are glial. 3. Nerve Fibres The vast majority of nerve fibres in the body are very thin (<2µm) and invisible with the light microscope. These extensions of the cell body may be very long as in the case of those from motor neurones to the lower limb. Thicker axons become myelinated, that is, they acquire a wrapping of Schwann cell cytoplasm giving an insulating layer. (The Schwann cell is a special type of glial cell.) Thinner axons (the vast majority) remain nonmyelinated. Myelin is an effective insulator and assists the rapid conduction of the action potential from node to node. The larger the axon, the thicker the myelin and the faster the speed of conduction. I
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All neurons are have associated glial cells which have a very close metabolic relationship and are ‘supporting’ (trophic) cells. The glial cells cover all the surface of the nerve cell body. Glial cells have important immunological functions. 4. Synapses The junction between a neuron and an effector or other neuron is a synapse which has a gap across which the stimulus is passed by a chemical transmitter. This means that there is a synaptic delay and the synapse acts as a filter or gate for the stimulus. This is an important part of the cleaning up of the signal. I
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LESSON 6: NERVE CELLS 1. Introduction There are three types of neurons. Motor neuron, which runs from CNS to effector such as muscle. Sensory neuron, which runs from a receptor to the CNS. Interneuron, which link other neuron within the CNS (they are often short but can be very complicated especially in the brain). The basic characteristics of neurons are listed below. A neuron contains 1. Cell body (soma) 2. Dendrites (dendritic tree) that carry signals to soma 3. Axon hillock 4. Axon that may be more than 1m long and carry signals away from soma, except in sensory fibres where it is reverse 5. Colaterals 6. Axonal branches and terminals Neurons contain the usual organelles 1. Nucleus with nucleolus 2. Mitochondria, which are also found in the axon terminals. 3. Endoplasmic reticulum with ribosomes (rough ER) 4. Golgi apparatus 5. Neurofibrils that add support 6. microtubules which are important for axonal transport 2. Supporting cells Only about 10% of brain cells are neurons. A large numbers of supporting cells known as glial (or neuroglia) are present. There are 4 types of these cells, these include astrocytes, microglia, oligodendrocytes and ependymal cells.. Astrocytes: take up and recycle certain neurotransmitters and growth factors, and their perivascular feet surround blood vessels & help maintain blood‐brain barrier. Oligodendrocytes (in CNS) and Schwann cells (in periphery) that myelinate neurons. Microglia are of the macrophage lineage (they are fixed macrophages) and act as phagocytic scavengers for defence. Ependymal cells line cavities in brain and spinal cord. 3. Nerve Conduction 1. Nerve Coding The nervous system only has one unit of code ‐ the Action Potential (AP). Nervous information must convey information about the nature of the signal (eg light, temperature, pressure etc) and the strength of the stimulus (eg how bright the light, how high the temp). Nature of the signal (modality) depends on anatomy. Nerve impulses from eye are interpreted as light (even if caused by some other method ‐ punch in eye). Nerve impulses to muscle contract muscle even if elicited electrically. Strength is encoded as frequency of AP ‐ the faster the frequency the stronger the signal and the number of fibres used (recruitment). I
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2. Action potential A change in electrical potential in a neuron can stimulate voltage gated Na+ channels to open, which cause an influx of Na+ into the neuron causing a further potential change across the membrane opening more channels. Eventually with a strong enough stimulus (supra‐threshold) there is positive feedback and a large number of Na+ channels open (Na+ Gate). In this state the Na+ permeability is some 20 times greater than potassium permeability. Since Na+ carries a positive charge into the cell the inside of the membrane becomes positive. The potential rises to almost Na+ equlibrium potential (+60mV) to about +40mV. This sudden depolarisation of the membrane opens more K+ channels (also voltage gated). K+ permeability increases (K+ gate) and at the same time Na+ permeability returns to below normal (to inactivated state). Thus cell membrane repolarises. In fact the potential actually drops slightly below the resting membrane potential, for a few milliseconds ‐ hyperpolarisation. This whole process of events is known as the Action Potential. It lasts about 1 ms. in nerve (longer in muscle). It is important to note that very few ions actually move so many APs can occur in rapid sucession up to 200/sec for many sec before any serious depletion occurs. The action potential is propergated along the axon. This action is an ALL OR NONE event. 3. Refractory periods During and shortly after an AP the membrane cannot be excited again. This is the Absolute Refractory Period . The membrane then gradually returns to normal Relative Refractory Period. Early in the RRP the membrane can be excited but it requires a much higher voltage (stimulus). 4. Myelin sheath Many neurones (but NOT all) are insulated with a layer of Myelin, which is created by Oligodendrocytes in the CNS and by Schwann cells in peripheral nerves. The glial cell wraps itself around the axon and the fatty Myelin is secreted in the folds. Oligodendrocytes add Myelin sheaths to several axons. Schwann cells to only one. There are gaps in the myelin sheath known as Nodes of Ranvier 5. Un‐myelinated fibres Currents flow around the depolarised area. These flow through the next bit of the membrane and alter its permeability to Na+ ions. The next bit of membrane depolarises. Every tiny increment of the membrane must be depolarised, and depolarisation is a relatively slow process (0.5ms). So conduction is slow. Speed depends on Diameter of fibre. Conduction In both directions but refractory period prevents change of direction. Unmyelinated fibres is only about 1 m/s. Clearly this is too slow for large animals (2 sec from brain to foot!). Speed can be increased by making the fibres larger. Some invertebrate giant fibres conduct at > 10 m/s. Squid giant fibre reaches 20 m/s but it is almost 1mm in diameter. Can’t have many of these as nerves become very large. 6. Myelinated fibres Currents flow around the depolarised area. But current cannot flow through the myelin sheath. Therefore current flows to the next Node of Ranvier (the inter‐node section acting as an electric cable). So only a few depolarisations take place ‐ so much faster conduction. Speed depends on: Diameter of I
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fibre and Internode distance. Thus myelinated fibres conduct over 100 times faster than the same size unmyelinated fibre. A fibre only 10µm in diameter can reach a speed of almost 100 m/s. The advantages of myelination are: faster conduction, reduced energy expenditure (less ion movement across membrane so less ions have to be pumped back). I
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LESSON 7: NEUROMUSCULAR JUNCTION Neurons communicate across cell‐cell junctions known as synapses. The junction between a nerve and a muscle fibre is known as a Neuromuscular junction (NMJ), and is a special kind of synapse. 1. Muscle Innervation 1. Motor unit Each motor nerve fibre divides into many terminal branches (unmyelinated), each of which forms one NMJ with ONE muscle fibre. Each muscle fibre only has one NMJ. The group of muscle fibres innervated by one motor nerve fibre, together with that fibre, is called a MOTOR UNIT. Large motor units may have 1000 muscle fibres. Small fine control muscles (eg orbital muscles) may have as few as 10 fibres per motor unit. 2. Mechanism of action The end of a motor nerve fibre forms a swelling (Terminal Knob) which is imbedded in the surface of the muscle fibre. The muscle membrane is folded to increase its surface area (Motor end plate). The neuron and muscle membranes are separated by a narrow cleft. The action potential in the neuron cannot cross this gap. The neuronal terminal knob is well supplied with mitochondria and with small vesicles that contain neurotransmitters. The depolarisation of the neuron terminal knob causes the opening of voltage gated Ca2+ channels. Ca2+ enters the neuron (since it is in low conc inside & high outside). The Ca2+ ions trigger reactions which cause the vesicles containing neurotransmitters to migrate towards the junction membrane. Some vesicles fuse with the neuronal surface membrane and burst releasing their neurotransmitter content into the cleft. The neurotransmitter in this case is Acetylcholine (ACh). The ACh diffuses rapidly across the narrow cleft and attaches to receptors on the muscle membrane. These receptor operated channels (ROCs) are chemical‐gated and thus differ from voltage gated channels (VOCs). These receptors are connected to cation channels for example Na+ and K+ which allow these ions to pass across the muscle membrane (esp. Na+, which is driven by both concentration and electrical gradients) thus depolarising the muscle membrane. This depolarisation is known as the end plate potential (EPP). If the NMJ is partially blocked with curare no action potential is seen but these small local EPP are recorded. The EPP is always supra‐threshold so always initiates an AP in the muscle. The AP is initiated in the region on either side of the end plate. The AP passes along the muscle fibre (in both directions) as in unmyelinated nerve fibre. Importantly, the ACh only stays bound to its receptor molecules for a very short time, then it is released to either bind to another receptor or finds its way to the bottom of the folds and bind to the enzyme acetylcholineesterase which breaks it down. 3. Quantal Release of Ach Even in the absence of stimulation, odd vesicles of ACh are released giving tiny depolarisations of the muscle membrane (≈400µV) known as miniature end plate potentials (MEPPs). If the NMJ is almost completely blocked, so that each AP only releases a very few vesicles, EPPs can be seen to occur in steps of 400µV, indicating the quantal release of Ach from each vesicle . I
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2. Toxins and Disease The neuromuscular junction is very susceptible to toxins, drugs & diseases. Curare binds to the ACh receptor preventing ACh attaching. Botulinum toxin (food poisoning) blocks release of Ach. In both these cases the muscles stay relaxed. Black widow venom causes excess release of ACh leading to prolonged depolarisation and contraction. Organophosphates (in pesticides and nerve gas) irreversibly bind to the ACh esterase thus preventing breakdown of Ach In these cases the muscles stay contracted. These toxins are mainly dangerous because of their effect on the respiratory muscle which must contract & relax rhymically to maintain oxygen supply & sustain life. Myasthenia gravis is an autoimmune condition which depletes ACh receptors leading to muscle weakness. Neostigmine is an anticholinesterase drug that counteracts above condition. I
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LESSON 8: SYNAPTIC TRANSMISSION 1. Mechanism of transmission The mechanism of neurotransmission at nerve‐nerve synapses (also known as a general synapse) are similar to NMJ. The following events occur: 1. Pre‐synaptic AP depolarises the terminal knob 2. Opens voltage‐gated Ca2+ channels 3. Vesicles of transmitter migrate to pre‐synaptic membrane and burst. 4. Transmitter crosses cleft and attaches to receptors on post‐synaptic membrane 5. These receptors are bound to chemical‐gated channels 6. Ions cross post‐synaptic membrane and change its trans‐membrane potential There are some key differences between nerve‐nerve synapses and NMJ: 1. Nerve‐nerve synapses are much smaller, so potential change/AP is far less 2. No folding of neuronal post‐synaptic membrane ‐ smaller surface area 3. Synapses introduce a delay (≈ 0.5 ‐1.0 mS) which is significant in complex circuits 4. In Nerve‐nerve synapses transmitter substance is not always ACh. There are many known transmitters 5. One AP hardly ever raises trans‐membrane potential to threshold. It usually takes many presynaptic APs to reach threshold and produce a post‐synaptic AP. Potential is therefore lower, usually sub‐threshold in nerve‐nerve synapses and requires summation 2. Summation The cell bodies of the postsynaptic neuron receive many synaptic endings from many per‐synaptic fibres (up to 100,000!). If 1 presynaptic AP is not sufficient to cause a post‐synaptic AP there must be summation to raise the Membrane potential to threshold. Each depolarisation of a terminal knob produces a small local (graded) excitatory postsynaptic potential (EPSP). Since the resistance of the soma is very low the EPSP spreads throughout the soma. 1. Spatial summation Most presynaptic neurones have more than one ending, so contribute more to the membrane potential (EPSP). Usually many presynaptic fibres have to fire together to raise the membrane potential to threshold. 2. Temporal Summation Alternatively one (or a few) fibres may fire repeatedly at high frequency. The individual EPSPs will summate and may raise the membrane potential (EPSP) to threshold. 3. Inhibition Not all presynaptic fibres are the same. Some have a different transmitter, which opens either K+ or Cl‐ channels, and hyper‐polerises the membrane giving rise to an inhibitory postsynaptic potential (IPSP). IPSPs summate by spatial & temporal summation but have opposite effect (cancelling out EPSPs). I
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3. Neurotransmitters There are a number of known neurotransmitters and many more suspected ones. Some are always excitatory, some always inhibitory. Some may be excitatory or inhibitory depending on the synapse. However each ending only has 1 neurotransmitter and is either excitatory or inhibitory. Neurotransmitters do not survive for long in the synaptic cleft. They are either broken down by enzymes or taken back into the presynaptic terminal to be recycled. Many neuropeptides are also released by presynaptic neurones. They are produced in the soma, passed down to the terminal and released along with the transmitter. Their effect is slow and long‐term, usually enhancing or supressing the effects of tramsmitters by altering availability of transmitter or receptors. 4. Receptors It is necessary to get information INTO the system (from the environment or body) and OUT OF the system (as muscular work). The body is well supplied with sensory receptors of many types: − Chemo receptors Taste/smell, O2/CO2/pH − Photo receptors Vision − Mechano receptors touch, pressure, balance, motion, blood pressure, hear − Thermo receptors skin − Osmo receptors blood osmolarity − Nociceptors Non‐specific nerve endings ‐ pain The general mechanism is − The stimulus brings about a change in permeability of membrane to small ions. Na+ in particular moves into the cell (as its concentration and electrical gradients are strongest) − Na+ movement brings about a depolarisation − These potentials are graded potentials akin to EPSPs. If stimulus is strong enough they will exceed threshold and initiate an action potential. The Frequency of Action potentials depends on the size of the potential. Hence frequency signals stimulus strength (Frequency code). Stimulus strength may also be reflected in Number of endings which reach threshold, for example a light touch fires one ending and a heavy prod excites several (Population code). The modality of the stimulus is signaled by the anatomical arrangement of the nerves (where they come from & where they go to) (Place Code) eg. blow to the eye gives ‘stars’. Receptor desensitisation/adaption: Many sensory receptors do not keep firing at the same rate if stimulus is maintained. They adapt. Tonic receptors show little or no adaptation (eg joint position receptors or temperature receptors. Phasic receptors adapt rapidly to a constant stimulus (eg skin touch receptors). 5. Summary The EPSP of the post‐synaptic soma does not remain at resting membrane potential for long. It fluctuates up and down depending on the nett input from all the presynaptic fibres acting together by spatial & temporal summation of EPSPs & IPSPs. In this way synaptic connections are far more than just junctions between nerve fibres. Furthermore, the amount of transmitter released by one ending may be altered by presynaptic inhibition/facilitation from a second neuron. These processes give the CNS its powers of calculation and computation. I
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LESSO
ON 9: EXCITABLE
X
E MUSCL
LE TISSUES 1. Obje
ectives To be ab
ble to: a) deescribe the sttructure, inneervation and function of the t different types of musscle (sm
mooth, cardiaac and skeletaal) b) coomment on thheir appearancce in section c) deescribe the fibbre‐types of skkeletal musclee d) deefine a motorr endplate an
nd motor unit e) exxplain fibre‐ttype groupin
ng 2. Intro
oduction Muscle is adapted for contracction. There are three types t
of muscle: smoo
oth, cardiac a
and skeletal.. They can be considered
d as a series of increasing
g specialisattion for conttraction. Exccitable Tissu
ues respond to approprriate stimuli by generatting electricaal responses. In the casee of muscle, the electricaal response is translated
d to contracttion. There are three types of muscle m
whicch can be seen s
as beeing increasin
ngly specialised for contraction. Figu
ure Diagrram of Muscle Types. a) Skkeletal Muscle; b
b) Cardiac Muscle; c) Smooth m
muscle. The right‐‐hand pictures r
represent transverse sections o
of cells/ffibres. [Passmore & Robsoon (1968). Fig.155.1] 3. Smo
ooth Muscle Smooth muscle con
nsists of small spindle‐sshaped slen
nder cells with w a relativvely undevelo
oped contraactile apparaatus. The ceells are not striated. The cells tend
d to aggregaate into bund
dles or sheeets (e.g. gut)). Each cell has h a single nucleus in tthe slightly w
wider centree. Note that because thee cells are much longer t
than the nucclei, long, maany profiles o
of transversee sections will not contaiin a nucleus. ‐
In
nnervation Du
ual innervattion with bo
oth excitatory and inhib
bitory inputss. The balan
nce beetween them
m gives rise to a partial state of con
ntraction or t
tone. In sing
gle‐
un
nit muscle (e.g. gut, uteerus) not all cells are directly innerrvated; insteead th
hey are coup
pled more or less indirecttly, so that e
excitation sp
preads from t
the directly‐innervvated cells to t those thaat do not recceive innervaation. In mu
ulti‐
un
nit muscle (ee.g. airways, large arteriees) each cell is innervated
d. I
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‐
Function Relatively weak slow, spreading contraction (obviously related to the above properties. The contraction of each cell is graded (cf. ‘all‐or none’ of cardiac /skeletal muscle). 4. Cardiac Muscle Cardiac muscle consists of large cells or fibres which are closely attached to one another end‐to‐end at intercalated discs (modified Z discs). The fibres are striated indicating an elaborate well developed contractile apparatus. Fibres may branch and have electrical continuity; may be binucleate. The nuclei are centrally placed in the large cells, so that nuclei are not visible in all profiles. The electrical junctions between cells are indicated by modified Z discs; the intercalated discs. Contraction is faster than in smooth muscle and all‐or‐none. Not all cells are directly innervated and electrical excitation spreads through the tissue via electrical continuity and also by specialised cardiac muscle cells, the Purkinje fibres. The latter are pale because they are rich in glycogen which is unstained in conventional (H&E) preparations. ‐
Innervation Within the heart pacemaker cells control the rate of contraction and excitation spreads via the left and right branch bundles of Purkinje fibres: modified cardiac muscle cells containing glycogen. ‐
Function These muscles are non‐fatiguing, powerful, co‐ordinated contraction; rich in mitochondria; rich blood supply. 5. Skeletal Muscle Skeletal muscle consists of large fibres, each of which is formed by the fusion of primitive cells; thus multinucleate with relatively prominent cross‐striations. The contractile apparatus almost completely fills the fibres and is very regularly arranged to give the characteristic striations. The many nuclei are at the very edges of the fibres (i.e. peripheral). ‐
Innervation Each muscle fibre receives a specialised nerve terminal i.e. a single axon of an alpha motor neurone forming a motor endplate. The area of the fibre immediately below the endplate is modified; a specialised form of neuromuscular junction, characterised by much folding of the post‐synaptic (muscle) surface to give a large surface area for transmission. One spinal motor neurone (motoneuron) can innervate hundreds or even thousands of muscle fibres in a given muscle. These fibres comprise the motor unit. All the fibres of a motor unit contract at the same time and are all of the same type (below). Contractions are powerful due to the density of the contractile apparatus and are all‐or‐none. There are many motor units in a muscle; force is increased by increasing ‘recruitment’. ‐
Function These muscles are fast, powerful, ‘all or nothing’ (‘twitch’) contraction. I
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6. Muscle‐Fibre Types Skeletal muscle has essentially two types of fibre according to enzyme profile and physiological characteristics, such as velocity of contraction and resistance to fatigue. The principal enzymes used are succinic dehydrogenase (SDH), a mitochondrial oxidative enzyme, and myofibrillar ATPase (mATPase). The Table below summarises the enzyme and physiological differences between Type I and Type II fibres. Further subdivisions of these two types are recognised. The fibre‐
type is determined largely by the neuron innervating it. The constituent fibres of a motor unit are scattered throughout a muscle (‘mosaic’ not ‘random’). The properties of an anatomical muscle depend upon the proportions of I and II fibres.
Enzyme activity mATPase activity (anaerobic) Oxidative capacity (SDH, aerobic) Speed of contraction Type I low high slow Type II high low fast Table Properties of Types I and II Muscle Fibres a) Type I fibres have a relatively high activity of mitochondrial oxidative enzymes (e.g. succinic dehydrogenase, SDH) and lower activity of phosphorylytic enzymes (especially myofibrillar ATPases). The velocity of contraction is relatively slow (for skeletal muscle); the resistance to fatigue is high. b) Type II fibres in contrast have a relatively high activity of ATPases and lower activities of oxidative enzymes. The velocity of contraction is very high and resistance to fatigue is relatively poor. 7. Muscle‐Fibre Type Grouping Nerve damage may lead to loss of innervation to individual muscle fibres which will be re‐innervated by adjacent nerve fibres (‘collateral re‐innervation’). This will lead to an increase in motor‐unit size resulting in loss of fine control. Conversion of the histochemical fibre‐types will lead to fibre‐type grouping. 8. Self Test Questions 1.
2.
3.
Explain the following histological observation made in wax‐embedded, H&E‐stained sections. a. In smooth muscle cut in transverse section, not all profiles contain nuclei. b. White adipose tissue shows no sign of fat in a wax‐embedded section What do the blue granules in conventional sections of neurones represent? Define a motor unit. I
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LESSON 10: CONNECTIVE TISSUES 1. Objectives To be able to: a) describe morphological/functional classification of types of connective tissue b) list components of CT (cell types, fibres, matrix) and their functions c) outline the role of the matrix in conferring differing properties of CT d) recognise the inter‐relatedness of all CT cells (incl. Blood) e) define types and appearance of adipose tissue in sections (BAT, WAT) f) define 3 types of cartilage and mechanisms of cartilage growth g) explain effect of vitamin C deficiency and lupus in terms of CT lesions. [Note — Wheater prefers the term ‘supporting tissues’ to reflect the wide range of important functions, but this is not generally accepted.] 2. Introduction Connective Tissue (CT) occurs everywhere in the body in a variety of forms. Its principal function is support, both structural and physiological. It is both a skeletal framework for tissues and also the route through which blood vessels and nerves run. CT binds organs as in fascia and capsules of organs, and supports the other components of organs e.g. interlobular CT of glands and the lamina propria of epithelia. CT has an important function in modulating the differentiation and division of the overlying cells. Histologically, CT is characterised by having cells scattered within varying amounts of extracellular material, which consists of fibres and ground substance (matrix). 3. Components of Connective Tissues a)
Fibres collagenous, reticular or elastic, all chemically similar and produced by fibroblasts. b)
Cells of many types, including white blood cells which have left the blood vessels (i.e. are extravasated). Macrophages and fibroblasts are the commonest cell types. The cells are derived from a common precursor (ancestral) cell which is closely related to the precursor of blood cells (see Figure). c)
Ground substance typically amorphous, may be sol/gel and is mainly composed of hyaluronic acid and glycoproteins (especially chondroitin sulphate). To a very large extent, it is the properties of the different glycoproteins which determine the different properties of connective tissues. The types and arrangement of the glycoproteins varies within connective tissues and according to age 4. Classification of Connective Tissue Proper a)
Proportion of fibres Loose (Low) or Dense (High). In practice, there is a graded series of density. b)
Arrangement of fibres Regular (in parallel bundles) or Irregular (in a coarse feltwork). I
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. Figure Relationship of connective tissue cell type and blood cells. The upper half of the figure shows the various types of connective tissue cells and emphasises their inter‐
relatedness. They all arise from one single precursor cell. The lower part of the figure shows the blood cells. They arise from a single precursor cell which is closely related to the precursor cell of the other connective tissue cells. The lineage of the developing blood cells is shown in abbreviated form. [From Leeson & Leeson 3rd edn]. 5. Types of Connective Tissue Proper Loose Connective Tissues Characterised by having a relatively low proportion of fibres. a)
Mesenchyme Foetal, unspecialised from which other tissues are derived (see below). b)
Loose (Areolar) Connective Tissue Develops from mesenchyme. It occurs as packing and support of most structures (e.g. the lamina propria underlying epithelia). Has all types of fibre with collagen the most conspicuous. Reticular fibres may be abundant at the edges of other structures. The unstained ground‐substance occurs in patches (areolae). Is well‐supplied with nerves and blood vessels which supply the overlying epithelium. Macrophages and fibroblasts are the predominant cell types. Forms the lamina propria underlying epithelia. I
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c) Adipose Tissue Fat cells are the main cell type and are surrounded by reticular fibres. Because fat is dissolved out in by the alcohols used in histological preparation, the cells normally appear empty with a thin ring of cytoplasm. Note that Brown Adipose Tissue (BAT) has many small lipid droplets (multilocular), in contrast to White Adipose Tissue (WAT) which has the single droplet (unilocular). Adipose tissue is highly vascular reflecting the dynamic state of rapid metabolism and turnover of lipid. d) Reticular tissue 'Primitive', composed of probably pluripotent cells and reticular fibres. Found only in lymphoid tissues. Other principal cell types: lymphoid cells, eosinophils and mast cells. Dense Connective Tissues Characterised by having a relatively high proportion of fibres. a) Dense Irregular Connective Tissue This is a coarse feltwork of mainly collagenous fibres forming sheets. The type of connective tissue withstands multidirectional stress. b) Dense Regular Connective Tissue Parallel fibres, which withstand unidirectional stress. Fibroblasts are the predominant cell type. The predominant fibre type is collagen, except in special elastic ligaments. Examples are Tendons and ligaments, where the collagen fibres are arranged into bundles or fascicles. c) Fibrocartilage Occurs at the insertion of tendon on the surface of bone, Dense Regular CT blends into the cartilage. Thus, at this zone of transition there is a gradation from dense fibrous tissue of the tendon through calcified fibrocartilage to bone. The proportion of fibrocartilage may vary in specific sites according to the differing stresses. For example, the lateral menisci of the knee‐joint have more fibrocartilage than the medial because there is more movement in the former. 6. Specialised Connective Tissues In addition to Connective Tissue Proper, there are the Specialised Connective Tissues: Blood, Cartilage, Bone and the Lymphoid organs (see Lessons below). Since cartilage precedes bone in development, the process of Ossification will be outlined. All three of the specialised CTs differ from CT Proper in that they have a greater proportion of extracellular matrix; in cartilage and bone the matrix is solid. 7. Cartilage Develops from mesenchyme. Provides good support while retaining flexibility. Uniquely among connective tissues, it lacks blood vessels and nerves. There are three types of cartilage. a)
Hyaline (glassy) Cartilage This is the commonest type of cartilage. Cartilage cells (chondrocytes) occupy spaces (lacunae; singular, lacuna) in the matrix which is slightly basophilic due to high content of glycoproteins. The chondrocytes shrink in preparation and are vacuolated (due to the presence of unstained glycogen I
33
and lipid). Collagen fibres form a fine feltwork but are not visible with normal staining methods. The matrix is densest at the edges of the lacunae. Located in, for example, larynx, trachea, bronchi, articular surfaces of joints. b)
Elastic Cartilage contains a large proportion of elastic fibres (demonstrable by special staining methods) for greater flexibility. Located in, for example, external ear. c)
Fibrocartilage Located in, for example, tendinous insertions, menisci, vertebral disc. (See above). 7. Self Test Questions 1. What are the components of Connective Tissues? 2. What criteria are used to classify Connective Tissue Proper? 3. White Adipose Tissue is pale in a conventional, wax‐embedded preparation because [Choose one answer]: a) fat is dissolved out in fixing b) fat is dissolved out in dehydration c) fat is dissolved out in staining d) fat stains only very lightly e) fat doesn’t stain 4. How can fat be preserved in a slide? I
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LESSON 11: BONE AND BONE FORMATION 1. Objectives To be able to: a) describe structure of bone (gross, histology, components) b) describe bone formation cellular process (intracartilaginous ossification) c) describe bone growth, destruction of cartilage, ossification, remodelling d) describe cells involved in bone dynamics (osteoblasts and osteoclasts) e) outline role of bone as a Ca2+ reservoir and outline its regulation 2. Introduction Bone is one of the three Specialised Connective Tissues. It is rigid for weight‐
bearing. Bone also provides a specialised environment for the production of blood cells. The matrix is largely composed of calcium salts (60% by weight). Collagen fibres are present. The cells are called osteocytes, and lie in lacunae (holes) which interconnect via narrow tunnels (canaliculi). Processes of the osteocytes extend throughout the canaliculi, which are continuous with the longitudinal, central canals and the radial, perforating canals, which carry blood vessels and nerves. The concentric layers or lamellae of matrix around a central canal form osteons, the structural and functional units of bone. The inside and outside free surfaces of bone are covered by a layer of dense connective tissue, (endosteum and periosteum, respectively), which contains collagen fibres and undifferentiated, pluripotent mesenchymal cells. Bone is not fixed, but is continuously being remodelled in response to physical stresses by organised processes of breakdown and formation of new osteons. The cells responsible for the resorption of bone are osteoclasts, which are giant cells with 200‐300 nuclei, 15‐20 of which may be visible in a single section. Osteoclasts are invariably found lying on the surface of the bone. Their cytoplasm is acidophilic (red with H&E) and highly vacuolated ('foamy') and they contain hydrolytic enzymes. Bone may be classified as spongy or dense. Spongy (also known as ‘cancellous’) bone consists of a trabecular (lattice‐like) structure, with many spaces between the strands. The strands of bone are covered by a thin layer of CT — endosteum. Dense (or ‘compact’) bone has no spaces within it. Compact bone is almost entirely composed of osteons, cylinders of layers of compact bone. Spongy bone may contain osteons; if it does not, it is known as ‘woven bone’. 3. Bone as a Calcium Reserve Almost 99% of the calcium in the body is in the skeleton — which therefore acts as a Ca2+ reservoir. Ca2+ may be mobilised by transfer of Ca2+ from newly‐formed regions of bone to the interstitial fluid. Two hormones, calcitonin and parathyroid hormone (PTH) act on the osteoclasts to regulate the release of Ca2+ from bone. PTH is the principal regulator of plasma Ca2+ concentration and stimulates osteoclasts to increase bone breakdown and hence increase the release of calcium from bone. Calcitonin only acts when plasma Ca2+ is extremely high and inhibits the osteoclasts and reduces the mobilisation of calcium. [These hormones also act on renal handling of Ca2+.] Lack of Ca2+ can lead to malformation or decalcification I
35
of the bones. Both Vitamin D and Vitamin A are required for the effective assimilation of Ca2+. 4. Bone Formation (Ossification) Bone is always formed by the conversion of an already existing tissue. Woven bone is formed first and then converted to lamellar (compact) bone. The foetal skeleton is made of cartilage which forms the template for bone formation by intracartilaginous ossification. This is best seen in the epiphysis of a growing bone, where there are two processes occurring simultaneously: a) growth in length; b) ossification. It is these two processes that cause the characteristic zonation of growing bone. This zonation is the production of a dynamic process — the image we have is static. [It is not necessary to remember the names of the zones but you should know what processes are occurring.] Sequence proceeds from 1‐10. Cartilage is represented by pale blue (eroding cartilage, dark blue), bone by pink; blood vessels by red. Process is as follows: Figure Bone formation (ossification) 1) 2) 3) 4) cartilaginous model of bone forms bony periosteal collar forms erosion of oldest cartilage erosion forms primary marrow cavity. Blood vessels and CT enter primary marrow cavity 5) cavity enlarges rapidly. Erosion of cartilage forms trabeculae. 6‐9) secondary ossification centre is formed, resulting in restriction of bone formation to epiphyseal discs (as well as the sub‐periosteum). 10) eventually epiphyseal disc is obliterated and no further growth can occur. (Remodelling continues throughout life.) 5. Summary of Bone Formation a) Growth in length occurs by addition of new cartilage at neck of bone (epiphysis). At extremity of epiphysis the chondrocytes are small (Quiescent I
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Zone), but towards shaft (diaphysis) they are mitotic, forming columns of small cells (Proliferative Zone), which then increase in size (Maturation Zone). b) Ossification. Cells in the connective tissue sheath around the cartilage (perichondrium) differentiate into osteoblasts and form bone on the surface of the cartilage (the periosteal collar — pink in Fig. 3.2). This process continues, adding to the growth of the bone. c) At the same time, the chondrocytes in the middle of the shaft hypertrophy, the lacunae enlarge and amount of matrix is correspondingly reduced and calcified (becoming basophilic). In favourable circumstances it may be possible to see a ‘tide‐mark’ of calcification where matrix becomes basophilic, blue with H&E. The chondrocytes then die and the matrix is dissolved leaving only the thicker plates (trabeculae) like stalactites hanging down into marrow cavity of the bone. d) Blood vessels and cells grow into the spaces that are enlarged to form primary marrow cavity. Some cells become osteoblasts and form bone on remaining matrix — the whole constituting the primary ossification centre. This process extends outwards from the centre. The periosteal collar thickens to support the eroded cartilage. e) Finally, bone is resorbed in centre so that thickness of wall remains approximately constant while overall diameter increases. Primary marrow cavity becomes filled with small precursor cells of blood and is therefore densely basophilic because their nuclei are so close together. g) Thus, ossification forms four more zones (from end to middle): Calcification, Retrogression (death of chondroblasts and dissolution of matrix), Ossification, Resorption. h) A secondary ossification centre develops at epiphysis and expands, leaving cartilage only on articular surface and as a thin epiphyseal plate or disc. It is from diaphyseal edge of this plate that further cartilage formation and growth occurs in childhood. i) Growth in length of bone ceases when proliferation of chondrocytes is not sufficient to keep pace with rate of ossification and epiphyseal disc becomes completely ossified. 6. Self Test Questions 1. 2. 3. Briefly describe the functions of the following cells: ‐ chondrocytes ‐ osteoclasts ‐ osteoblasts Name the principal processes involved in bone formation. What is the probable origin of the cells involved in fracture repair? I
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LESSON 12: BODY FLUIDS 1. Objectives To be able to: a) describe different fluid compartments of body and outline principles of their measurement b) state approximate volumes of major compartments of body water c) describe movement of water and other molecules between compartments d) list various forms of water input and output and state which are subject to physiological regulation e) explain role of plasma proteins in movement of fluid across capillaries f) describe differences in composition of different compartments g) explain flow of water/solutes (bulk flow) between plasma and interstitial fluid h) explain pathophysiological changes in body fluids (oedema) i) explain difference between osmolarity, osmolality and tonicity j) describe regulation and significance of composition of Plasma 2. Introduction a) Both composition (ions and proteins) and volume must be kept within narrow limits to maintain normal body function. b) ‘Normal’ input is by ingestion and output via gut, urine, respiration and sweat. Of outputs, only urine can be regulated in relation to water balance. c) In ‘Abnormal’ or clinical and pathological situations there may be inputs via injection or infusion. Abnormal outputs are vomiting, diarrhoea, burns, haemorrhage. (consider whether water, salts, proteins or cells are lost or gained in each case.) d) Physiological responses to perturbations of fluid composition or volume are complex. Consider general mechanisms of response to fluid loss. e) Volume Regulation is by increasing intake of water (thirst centre etc.) and reducing output of urine. Osmolarity of blood is monitored in kidneys and concentration of the urine adjusted accordingly. f)
Regulation of Composition. Na+ monitored in kidneys; erythrocyte levels monitored (see Haemopoiesis). In both cases appropriate physiological adjustments are made to counteract deviation from normal values. g) Key Concept. The concept of compartments occurs frequently in Physiology. The movement of material between compartments in Physiology of body fluids is an excellent example. Other ‘compartment’ systems might be the digestive system, circulation and blood glucose concentration. Body fluids are regulated in compartments by specific forces and flows. All cells are bathed in fluid and therefore depend for their functional integrity on the maintenance of the volume and composition of that fluid. h) If there is a large and rapid interchange, then if one compartment (Plasma) is regulated (volume and composition), the other (interstitial fluid) is also regulated. I
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3. Definition of Compartments The fluid compartments and their volumes in a healthy 70 kg male is shown in the table below: Compartment Volume (litres) Total Body Fluids (H2O) is 40‐80% of body weight 42 Intracellular Fluid (ICF) in cells 28 Extracellular Fluid (ECF) 14 ECF sub‐compartments Plasma (Pl) in blood 2.8 Interstitial Fluid (IF) bathes cells 11.2 Total body water accounts for 40‐80% of body weight. Most of variation between individuals is accounted for by variations in amount of body fat. Fat is only about 10% water. Table Fluid compartments and their volumes. Sherwood, Fig. 12.2 Intracellular Fluid (ICF) is water contained in cells. Composition of each cell is separately regulated by intracellular mechanisms (ion pumps etc). Thus, different cells can have different compositions according to their functions. ICF is separated from Interstitial Fluid by cell membranes. Interstitial Fluid (IF) bathes all cells and is internal medium. Its maintenance is critical to function of cells and hence body as a whole. Interstitial Fluid is one component of the Extracellular Fluid (ECF) and is separated from the Plasma by the walls of the blood vessels. There is a continuous passive interchange (filtration, osmosis, diffusion) between the Plasma and the Interstitial Fluid, so their compositions are very similar with notable exception of Plasma proteins which cannot leave blood vessels. There are also Lymph and Transcellular compartments, which are so small that they need not be considered in calculating volumes of compartments. Lymph compartment comprises fluid passing to plasma via lymph nodes. Transcellular compartment comprises fluid secreted by cells e.g. CSF, bile, urine etc. 4. Comparison of ICF and ECF There are important differences in composition because of the selective permeability of cell membranes (active and passive mechanisms) and the non‐
diffusibility of the intracellular proteins. The principal differences are as follows. a) Specific non‐diffusible proteins on both sides of the cell membrane. b) Na+/K+/ATPase pump moves Na+ out and K+ into cells. Consequently, Na+ is predominate cation outside the cells and K+ predominant cation inside cells. Cl‐ follows Na+ (so does HCO3‐). c) Inside cells, principal anions are PO43‐ and Proteins. 5. Osmotic Function of Plasma Proteins Plasma proteins are too large to leave capillaries and provide an osmotic pressure tending to draw water into capillaries from interstitial fluid. This ‘colloid osmotic pressure’ (oncotic pressure) of about 25 mmHg is important in understanding process of ultrafiltration and reabsorption of fluid in capillaries, which is essential mechanism of interchange between Plasma and Interstitial Fluid. I
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6. Lymph Vessels Since net inward pressure is less than net outward pressure there is an imbalance and not all fluid that leaves capillaries re‐enters them. This 3 litres per day passes into lymph vessels and via lymph nodes eventually returns to circulation close to right atrium. Lymph vessels are contractile and have valves. ————————————————————————— Arteriole Capillary BP Colloid OP Capillary BP Venule 37 mmHg 25 mmHg 17 mmHg ————————————————————————— NET OUTWARD PRESSURE NET INWARD PRESSURE You should consider the differences in forces. Osmotic pressure due to Plasma Proteins (Colloid OP, oncotic pressure) acts to draw fluid into capillary. Blood pressure inside capillary acts to force fluid out of capillary. Since blood pressure falls over length of capillary, there is net outward flow at arteriolar end and net inward flow at venular end. [Add your own arrows to show the directions of these principal forces.]
Figure Starling’s Law of the Capillary. Forces involved in Bulk Flow across wall of a systemic capillary outside pulmonary system. 7. Physiological Significance of Bulk Flow The capillary wall is very permeable so that water and solutes (but not large molecules, proteins and cells) pass freely across in both directions. Since there is a large and rapid interchange, between plasma and interstitial fluid (IF), and volume and composition of plasma are regulated, thus the volume and composition of IF compartment are also regulated. The composition (and volume) of plasma is carefully regulated (kidney); hence IF composition is carefully regulated. 8. Oedema [U.S. edema] Oedema is swelling of tissues following accumulation of interstitial fluid. In the following summary of the causes of oedema, the above diagram should be used to work out the exact cause and effects. Oedema is important because it results in a reduced exchange between blood and cells and therefore nutrition of cells may be impaired. Note particularly key role of Plasma Proteins. a) Reduced Plasma Protein Concentration Results in reduced oncotic pressure and hence reduced absorption of interstitial fluid. Reduced plasma protein concentration can be caused by increased loss of proteins (e.g. renal disease or extensive burns); reduced protein synthesis (e.g. in liver disease); or dietary deficiency. b) Increased Capillary Permeability For example, after histamine release, leads to leakage of plasma proteins in Interstitial Fluid, with a reduction of oncotic pressure and also a tendency to retain fluid in the interstitium. c) Increased Venous Pressure Causes increased capillary pressure tending to increase outward flow of fluid from capillaries. Occurs in congestive heart failure and in pregnancy when uterus presses on abdominal veins. d) Blockage of Lymph Vessels Impairs normal removal of excess Interstitial Fluid. Occurs after removal of lymph nodes or parasitic infestation of lymph vessels (elephantiasis). I
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9. Self Test Questions 1. 2. 3. What is the volume of Total Body Water in a normal, healthy 70 kg male? What are the principal differences between intracellular water (ICF and extracellular water (ECF)? Explain these differences. What is oedema and why does it occur in liver disease? I
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LESSON 13: BLOOD 1. Objectives To be able to: a) classify by morphology and function the circulating blood cells b) list relative frequencies of blood cells and factors which cause increases c) outline the stem cell processes by which blood cells are formed (haemopoiesis; erythropoiesis) d) outline the basis clotting mechanism and the role of platelets e) outline the fundamental mechanisms of anaemias f) describe function of Plasma proteins 2. Introduction Blood is a connective tissue. Its cells are dispersed in a liquid matrix (plasma); the fibrous component is 'latent', being produced only in the course of clotting. The cells occupy about 45% of the blood by volume (haematocrit). It is important to be able to recognise all the circulating blood cells, because many of them migrate into the tissues and the different types present in pathological conditions are a fundamental diagnostic aid. Special stains (e.g. Wright's, Giemsa) are used to identify blood cells in smear preparations, because they show more detail than conventional histological stains, such as H&E. The pH reaction of these stains is the same as H&E; that is, acidophilic structures are stained red and basophilic structures are blue. However, it is possible to identify many of the cells fairly reliably with conventional stains in sections by using the simple criteria of i) size and especially ii) nuclear form. 3. Erythrocytes (Red Blood Cells) a)
Shape and Lifespan Red blood cells are small (mean diameter ~8 µm), have no nucleus, with pale centres due to a biconcave shape. The cytoplasmic reaction is slightly acidophilic which means they appear pale red with the commonly‐used stains. Erythrocytes are enormously common (5 x 106 /mm3 i.e. 5 x 109/ml). The lifespan of erythrocytes can be estimated by labelling with radio‐active iron during formation (‘cohort labelling’) and monitoring the disappearance of the label from the blood. The proportion of radio‐labelled erythrocytes remains constant for several months then declines. The average life‐span is about 120 days. Clinically, the half‐life is measured with chromium (‘random labelling’) as 30 days. This is an underestimate due to loss of Cr, when corrected the half‐life is the expected 60 days. b)
Functions of Erythrocytes The principal function is carriage of O2 and CO2, bound to haemoglobin (Hb). The biconcave shape of the cells aids diffusion by giving a large area and minimising the diffusion distance. The cells are filled with Hb and have no organelles for synthesis or repair. Consequently their lifespan is limited. The cells are very flexible and can therefore pass through the smallest capillaries of around 3 µm diameter, As they age, they become less flexible and cannot pass through the small capillaries and are usually removed by phagocytic I
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cells in the spleen. This is the final common pathway of erythrocyte removal. (This is the fate of damaged cells in haemolytic anaemias.) 4. Leucocytes (White Blood Cells) These are sub‐divided into granulocytes and agranulocytes according to the presence or absence of granules in the cytoplasm. Note that the granules are visible only with special stains. Leucocytes are rare (5‐9 x 103/mm3) compared to erythrocytes. A more detailed consideration of the functions of leucocytes is in Immune System Lesson. 5. Granulocytes (i.e. containing granules) Granulocytes are distinguished primarily by the staining reaction of their granules. It is important to assess the 'colour balance' of a particular preparation to assist in determining the staining reaction. Red blood cells are slightly acidophilic, and their staining reaction can be used as an inbuilt colour comparator, to determine the reaction of the granules of granulocytes. Diagnosis of a particular cell type should be confirmed by the secondary criteria of relative frequency and nuclear form. a)
Neutrophils Neutrophils make up 70% of all leucocytes. The granules have a neutral reaction and are therefore paler than the slightly acidophilic red blood cells (rbc). In fact, they may often be quite difficult to distinguish. There is a characteristic prominent, many‐lobed nucleus (hence the alternative names of polymorphonucleocyte or 'polymorph'. Motile and phagocytic. Lifespan approximately 60 hours. A high level of circulating neutrophils is associated with bacterial infection. They are the characteristic cells of early (‘acute’) inflammation. b) Eosinophils Eosinophils make up 2‐4% of leucocytes. Granules redder (acidophilic) than the rbc. Nucleus bilobed. Lifespan 8‐12 days. A high level of eosinophils is associated with parasitic infection or allergic reactions. c) Basophils A high level of basophils are rare, less than 1% of leucocytes. The granules are large, coarse and almost always obscure the slightly‐indented nucleus. The granules are basophilic (blue) and contain heparin and histamine. 6. Agranulocytes (Lymphocytic cells) In both Lymphocytes and Monocytes the nucleus fills most of the cell, leaving a thin rim of slightly basophilic cytoplasm. The lymphocytes and monocytes are associated with immune responses (see Immune Lesson). a)
Lymphocytes 30% of leucocytes. Nucleus round or only slightly indented. Their size is variable but smaller than monocytes. Involved in immune responses. b) Monocytes 5% of leucocytes. Their nuclei are indented or kidney‐shaped. Becomes a macrophage after migrating into the tissues. They are the largest circulating blood cells. Involved in immune responses. I
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7. Platelets Platelets (thrombocytes) are not cells but are fragments of the cytoplasm of large cells (megakaryocytes) in the bone marrow. Their frequency in blood is 25 x 103/mm3. Platelets are concerned in the process of clotting. Lifespan 8‐12 days. 8. Clotting Clotting is an extremely powerful and complex homeostatic mechanism which is essential to deal with the many small leakages of blood that occur, as well as more significant haemorrhages. The first step is formation of the platelet plug to stop blood flow. That is followed by coagulation of blood involving the formation of fibrin. The basic pathway is illustrated in the Figure. a) Formation of platelet plug Platelets are activated by damaged endothelium, become sticky, release ADP and aggregate to form the platelet plug (positive feedback). Platelets also release factors that activate the coagulation cascade at various points. b) Coagulation This is the formation of fibrin whose function is to reinforce the initial platelet plug. This is a powerful cascade where the activation of one factor activates further downstream factors. Almost all of the factors are inactive forms of enzymes circulated in the blood and produced in the liver. Intrinsic Pathway
Extrinsic Pathway
(damaged vessels/foreign surface)
(tissue damage)
Factor XII
Thromboplastin
(Factor VII)
Common Pathway
Factor X
Thrombin
Fibrin
Haemostasis
Figure Outline of the Clotting Pathways. c) Pathways Damage of vessels or foreign surface activates Intrinsic pathways that involves FactorXII. Tissue damage activates Extrinsic pathways that involves Thromboplastin (Factor VII). The intrinsic pathway requires many upstream factors and is therefore slower than the extrinsic pathway. Ca2+ is required at several points in the cascade, hence ‘chelating agents’ can be used to remove Ca2+ from blood in order to prevent clotting, for example during operations. The last step is the activation of Factor X [ten] which cleaves prothrombin to form thrombin which in turn cleaves fibrinogen to form fibrin. I
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d) Inhibition of clotting Clotting must be limited or all the blood would clot every time there is damage to a blood vessel. The following processes prevent excessive clotting: a) intact endothelium releases NO and prostacyclin which both inhibit platelet adhesion. b) circulating plasmin dissolves clots and is activated by many clotting factors (incl. XII, the key initiating step of clotting! Consider the implications of this?). c) tissue Plasminogen Activator (tPA) circulates to deals with small clots. d) thrombomodulin binds thrombin, so fibrinogen is not converted to fibrin. Thrombomodulin also activates Protein C, an anticoagulant. 7. Haemopoiesis The continuous formation of cells and platelets of the blood is necessary to keep their numbers relatively constant. In the human adult, blood cells are formed in certain bones (e.g. skull, ribs, sternum, ends of long bones). Before maturity, other sites are also involved (long bones, spleen etc.). The rate of production of blood cells is regulated by a complex hormone system. It now seems most probable that all blood cells are derived from a single haemopoietic ‘stem cell’, the haemocytoblast, found only in the bone marrow. This stem cell has two possible fates: its offspring may remain as stem cells, thus maintaining the pool of stem cells; or they may differentiate into one of the five specialised ‘progenitor cells’. Each progenitor gives rise to only one of the following cell types: erythrocytes, granulocytes, lymphocytes, monocytes or platelets (from megakaryocytes) (See Fig in Connective Tissue Lesson). Each of these progenitor cells divides repeatedly (‘amplification’) to give a large number of descendants. This is an example of the progressive restriction of a cell's potential; from a cell which can potentially form any one of the circulating cell, to a more restricted range of possibilities. Thus, after one division of a haemocytoblast, the daughter cells may be able to form any one of the three types of granulocyte (but none of the other cell types). After further division the daughters acquire granules specific to one cell type (e.g. a neutrophil) and that cell line is then restricted to that type. 8. Erythropoiesis The formation of the red blood cells (rbcs), or erythrocytes, is taken as the example of haemopoiesis. The general pattern is the same for all cell types. The haemocytoblast is the putative pluripotent stem cell capable of giving rise to all circulating blood cells. There are only about 1‐2 haemocytoblasts for every 1000 nucleated marrow cells. The haemocytoblast is large, spherical with basophilic cytoplasm and a round nucleus (i.e. relatively undifferentiated). In contrast, the erythrocyte is small (~8 µm diameter), common, without a nucleus, biconcave and slightly acidophilic due to the large concentration of haemoglobin (Hb). The first recognisable stage in erythropoiesis is the pro‐normoblast, which is a moderately large cell with a round nucleus filling most of the cell. The chromatin is dispersed; there are nucleoli, and the cytoplasm is basophilic. The nucleoli disappear and the cell is now a normoblast. Several divisions (3‐5) forming 8‐32 cells over 5‐7 days result in much smaller cells. Nuclei become shrunken and dense I
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(pyknotic) and are extruded. The Hb accumulates and makes the cytoplasm more acidophilic (pink with conventional stains). Reticulocytes are formed when the division has ended; they are still larger than rbc and with less Hb. They have a residual apparatus for synthesis of Hb. The reticulocytes continue making Hb and shrinking. They usually spend 1‐2 days in the marrow before entering the circulation. Reticulocytes may be recognised after they have been released into circulation by staining for RNA. Erythropoietin (EPO), is released from the kidney in response to hypoxia. It increases the production of rbc by stimulation proliferation of progenitor cells in the rbc lineage. a) Concept of Cell Lineage Apply similar logic to all other cell types. The key points are the gradual restriction of potential and the gradual acquisition of the features of the mature, circulating cell. This is a prime example explaining the concept of Cell Lineage b) Experimental Evidence for Kinetics The spleen colony‐forming assay involves supra‐lethal radiation to kill all stem cells and injection of known numbers/types of marrow cells from a syngeneic donor. After a few days, clones can be seen in the spleen (by the naked eye). It can be shown that they are clones and hence the number of stem cells can be estimated as, for example, granulocyte colony‐forming units (G‐CFUs). Serial passaging shows that the clones are stable and self‐
replicating. This type of experiment gives a functional definition of the stem and progenitor cells according their ability to form cells of different types. Haemoglobin Haematocrit MCHC Red Cell Count Mean Corpuscular Volume Mean Corpuscular Haemoglobin Reticulocyte Count Male Female 13.5 ‐ 18.0 11.5 ‐ 16.4 43 ‐ 49 36 ‐ 45 30.8 ‐ 34.6 4.6 ‐ 5.7 4.0 ‐ 5.2 83 ‐ 89 26.7 ‐ 32.5 14.0 ‐ 100 Units g/DL % g/dL x1012/L fL pg x109/L Table Normal haematological values. Note particularly the calculation of mean corpuscular Hb. You will have an opportunity to derive these values in labs (from McCann et al., 2009). dL = decilitre (100 ml), fL = femtolitre (10‐15 litre), pg = picogram (10‐12 g) 9. Self Test Questions 1. 2. 3. 4. 5. For each cell type listed below give a clinical condition which would result in an increased numbers of those cells in the blood. a) neutrophils b) eosinophils Give the function of the following cells: neutrophils, lymphocytes. Name the cell type which is the precursor (ancestor) of all circulating blood cells. In a conventional, wax‐embedded section, which criterion for classifying circulating blood cells would be the most useful? Given the concentration of erythrocytes in blood (Table 3) and that the total blood volume is (approximately) 5 litres, derive the formula for calculating the rate of rbc formation (per second) I
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LESSON 14: IMMUNE SYSTEM 1. Objectives To be able to: a) describe differences between Innate and Adaptive immune responses: component and Immunological responses b) explain immunological memory, diversity specificity and self‐tolerance c) describe origin and roles of macrophages and lymphocytes in immune function d) describe general features of inflammatory response and phagocytosis. e) give examples of immune disorders and their basic mechanisms. 2. Introduction Immune cells have the ability to differentiate between the cells of the body (‘self’) and other organisms (‘non‐self’). This is the key element in defending the body against pathogens such as parasites, viruses, bacteria and fungi. Damaged or mutant cells are also ‘non‐self’ and are detected and removed. Immune responses are very powerful and have harmful effects: allergies and autoimmune diseases (below). Hence immune responses contain self‐limiting mechanisms (beyond the scope of this lecture, but see Suppressor T cells below). 3. Components of the Immune System The immune system consists of the white blood cells, called leucocytes (see above) and the lymphoid tissues. All the white blood cells are produced in the bone marrow but the lymphocytes divide and differentiate in the lymphoid tissue, specialised tissue scattered throughout the body especially at the ‘portals’, such as the tonsils in the throat. All leucocytes are involved in immune responses but the most important are monocytes and lymphocytes. Recall that all leucocytes are essentially transitory in the blood and have their principal functions in tissues. a) Monocytes (neutrophils and macrophages) (innate immunity) In tissues they are called macrophages. They have three main functions. ‐ Phagocytosis, engulfing and destroying pathogens, cell débris etc ‐ Secreting signalling molecules (cytokines) ‐ Processing antigens and presenting them to lymphocytes. b) Lymphocytes (B cells and T cells) (adaptive immunity) The two main functional types are B cells and T cells. B cells are derived from the bone marrow and clones in gut‐associated lymphoid tissue (GALT) and produce antibodies to specific pathogens. T cells are produced in the bone marrow and migrate to the thymus when they proliferate further and are selected to recognise ‘self’ molecules and destroy ‘non‐self’ cells. Recognition of ‘Self’ depends upon major histocompatibilty complexes (MHC) which are carried on each nucleated cell and are peculiar to each individual (shared by identical twins). (MHC is not present on rbc.) I
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4. Innate Immunity (monocytes) There are two types of immunity: Innate and Adaptive. Innate is the body’s natural defence system, irrespective of exposure to any pathogens. Adaptive mechanisms are developed in response to exposure to specific agents (pathogens, complex molecules etc). Innate Immunity is non‐specific, in other words it responds to the first exposure to any ‘non‐self’ cell. This provides an immediate response while the slower, more specialised adaptive response is mobilised. As well as the barriers to the entry of pathogens (skin and mucous membranes), the innate responses include (i) cellular phagocytosis, (ii) the inflammatory response, (iii) specialised chemical responses, (iv) natural killer cells, and (v) the complement system, discussed below: a) Cellular Phagocytosis Both neutrophils and macrophages are phagocytic. ‘Non‐self’ material is attached to the surface of the cell, taken into the cell (internalised) in a membrane‐bound vesicle (phagosome). Lysosomes containing hydrolytic enzymes fuse with the phagosome and break down the contents (degradation). The remnants pass out of the cell by exocytosis. b) Inflammatory Response This is an extremely complex and powerful response and this course only outlines some of the basic processes. The ’aim’ of the response is to inactivate or destroy invading micro‐organisms, remove the débris and prepare for healing processes. In favourable circumstances, normal function will be restored, but if tissue repair is incomplete, scar tissue may be formed with loss of function. The initiating step is the release of histamine from mast cells (‘tissue basophils’). Histamine has two effects on blood vessels: vasodilation, resulting in increased blood flow; increased permeability, allowing leucocytes and plasma proteins to enter the tissues. The leucocytes will begin phagocytosis etc. The proteins include complement, antibodies and clotting proteins (especially fibrinogen). The influx of proteins will cause oedema (see Body Fluids). The characteristic symptoms of inflammation are redness, swelling, tenderness and pain. The first three are all accounted for by the influx of blood and the oedema. Pain is caused by local distension and mediating molecules released locally. c) Chemical Responses Interferon is produced by cells infected with any virus. It protects other cells against viral attack. In addition, it also slows cell division and enhances Natural Killer (NK) cells and cytotoxic T cells (below), properties which are used in cancer therapy. d) Natural Killer cells Attack any virus‐infected cell and damage the cell membrane so that they to swell up and burst (lysis). Note that both Interferon and NK cells will attack any virus‐infected cell, so it is a non‐specific response. e) Complement System This is a class of molecules with many complex functions. The main function in the context of this course is recognition of micro‐organisms and lysis (similar to NK cells). This reinforces many other inflammatory responses (hence the name!). I
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5. Adaptive Immunity (T cells and B cells) This is the response to specific agents which have been previously encountered. It is mediated by lymphocytes (B cells and T cells). B cells produce antibodies which circulate in the blood: a humoral or antibody–mediated response. T cells act directly on damaged cells: a cell‐mediated response. Both types of lymphocyte recognise foreign molecules (antigens) by means of antigen receptors on their surfaces. Each receptor binds only one antigen, conferring specificity. However, there are millions of lymphocytes recognising millions of antigens so the system has diversity. Exposure to an antibody results in the production of clones of cells which recognise the specific antigen. a) Humoral/Antibody‐Mediated Immunity (B cells) An antigen binds to B cells and stimulates them to divide into clones of plasma cells and memory cells. Plasma cells generate large amounts of the specific antibody. They are quite short‐lived. The antibodies are secreted into the blood (hence ‘humoral’) as immunoglobulins (Ig). IgM and IgG are responsible for most immune responses. IgA is found in the secretions (saliva, mucus etc). IgE targets parasites. Memory cells persist indefinitely. Memory cells are long‐lived so that another exposure to the antigen causes a rapid proliferation of the specific clone of memory cells and large numbers of antibody molecules are produced in a few hours. (Recall that lymphocytes recognise ‘self’ and ‘non‐self’ so the system has self‐tolerance.) b) Cell‐Mediated Immunity (T cells) Antigen with MHC is required to activate T cells. Macrophages are the principal antigen‐presenting cells. Division and differentiation of activated T cells gives rise to cytotoxic T cell, helper T cells, and suppressor T cells. Cytotoxic T cells kill infected cells by lysis (see above). Helper T cells are about 70% of all T cells (see AIDS below) and are probably the key regulating cells of specific immune responses. They enhance the activity of cytotoxic T cells as well as phagocytic activity of macrophages etc. They also stimulate the development of B cells into plasma cells. Suppressor T cells secrete cytokines that suppress activity of B cells, as well as the above types of T cell. They also inhibit phagocytosis. This is one of the self‐limiting mechanisms of the immune system. Suppressor T cells control autoimmunity, and prevent self‐attack in diseases such as arthritis, multiple sclerosis, crohns disease, diabetes, asthma and others. 6. Vaccination Adaptive Immunity can be natural or artificial, subdivided into active and passive mechanisms. Natural immunity arises actively by exposure to infection and passively by transfer of antibodies via the placenta or colostrum (first maternal milk). Artificial immunity (vaccination) arises actively by immunisation with an antigen that initiates the immune system against the antigen (active immunisation) and passively by the direct transfer of antibodies from another animal (passive immunisation). Thus, the mechanisms of Adaptive Immunity allow us to create vaccination. In simple terms, exposure to a foreign agent (antigen) such as a virus will generate B cells/memory and T cells that allows the immune system to amount an immune response. These B cells/memory cells and T cells are long lived and ‘remember’ they have seen this particular foreign agent (virus) before. This allows for a rapid immune defence upon subsequent exposures, in other words B cells and T cells I
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allow the immune system to ‘adapt’ and ‘remember’ previous exposures. Now imagine we take a small protein fragment of a virus ‘X’ (i.e. let’s say a part of the viral coat), which has no ability to cause infection. The B cells and T cells amount an immune response against this virus ‘X’ protein coat fragment. Upon infection of virus ‘X’ the immune system is already ‘adapted’ and amounts a fast response to the protein coat fragment of virus ‘X’ and thus destroy the virus. 7. Immune Disorders Autoimmune diseases occur when the immune system fails to recognise ‘self’ and reacts against the normal tissues of the body. Examples are Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS). Autoimmune Deficiency Disease Syndrome (AIDS) caused by the human immunodeficiency virus (HIV), which specifically attacks Helper T cells, the key regulatory cells of the immune system. This leads to impaired immune function and hence increased susceptibility to infection, most notably pneumonia, TB and Kaposi’s sarcoma. Infection of brain neurones leads to dementia. 8. Self Test Questions 1. 2. 3. List the key features of innate and adaptive immunity List the stages of phagocytosis. List the characteristic symptoms of inflammation and explain each of them in terms of changes to blood vessels. I
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LESSON 15: BLOOD PATHOPHYSIOLOGY 1. Objectives To be able to: a) explain different types of anaemias and relate pathology to blood picture: definitions , types, causes, mechanisms b) explain examples of haemoglobinopathy (thalassaemia, sickle‐cell disease) Reading See McCann et al., (2009) 2. Introduction A working definition of anaemia is a ‘reduced oxygen‐carrying capacity of the blood’. Since oxygen‐carrying capacity depends mainly on the total mass of haemoglobin (Hb), we may consider that it depends upon the number of red blood cells (rbc) and the concentration of Hb per cell (MCH). Note that this may be considered as a compartment model: the red cell population is a compartment subject to addition and loss (‘input and output’). The size of the population is the resultant of the those two forces. (Factors affecting Hb production and function can be considered separately.) Hence Anaemias can be considered in terms of rbc number which depends upon: a) Reduced cell production For example by death of stem cells by radiation or toxins; impaired Vitamin B12 availability (required for DNA synthesis). b) Increased cell loss There are many causes but the final pathway is almost always the same, namely the decreased flexibility of the rbc membrane leading to removal in the spleen. 3. Clinical Causes of Anaemia The first is the only one affecting Hb production. Causes a‐d impair rbc production; e‐f increase rbc loss. a) Nutritional Anaemias arising from dietary deficiency of a factor, usually iron. The reduced availability of iron results in smaller, fewer rbc with reduced Hb (hypochromic) b) Pernicious anaemia Caused by lack of intrinsic factor from gastric mucus which protects B12 from digestion. This results in failure to absorb B12 which is required for DNA production. Hence proliferation (especially of marrow stem cells) is impaired. Treatment is injection of B12, bypassing defective mechanism. Cells look large (macrocytic), odd shapes and fewer. c) Aplastic The marrow fails to produce enough rbc, even though there are no deficiencies of any essential components. Example are the effects of toxic I
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chemicals, radiation (kills stem cells), invasion of marrow by cancer cells (e.g. leukaemia). Larger cells. Marrow depleted of cells with no abnormal cells. d) Renal Failure of EPO production, usually only in end‐stage renal disease. e) Haemorrhagic Blood loss may be acute (e.g. large haemorrhage) or chronic (low‐grade haemorrhage, excessive menstrual bleeding). Hidden blood loss should be sought in diagnosing anaemias. f) Haemolytic Rupture of circulating rbc. E.g. some auto‐immune conditions and malaria and sickle‐cell anaemia. In all these conditions, the rbc membrane becomes weak leading to haemolysis. Cells are hypochromic, macrocytic. (Thalassaemia). 4. Haemoglobinopathies Haemoglobinopathies are a subgroup of haemolytic anaemias and are characterised by defects in haemoglobin (Hb). These include Thalassaemia and Sickle‐Cell Disease. a) Thalassaemia Thalassemia is a quantitative defect in Hb. Normally the α and β globin chains are synthesised in equal numbers. In thalassaemia there is an imbalance and the excess unpaired chains precipitate onto the rbc membrane causing a loss of flexibility and hence increased cell loss. b) Sickle‐Cell Disease Sickle‐Cell Disease is a qualitative defect in Hb, a single amino acid is substituted. In hypoxic conditions the Hb molecule and hence he rbc change to the characteristic sickle shape. They may block small vessels causing tissue damage and pain. The trait confers some resistance to malaria. Again, there is reduced flexibility of rbc membrane and hence increased rbc loss. I
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INSTRUCTIONS FOR LABS PLEASE READ THESE INSTRUCTIONS CAREFULLY 1. General & Lab Policy ‐
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No bag policy in Labs; DO NOT bring bags into lab Switch Mobiles off (not silent) Lab Computers are NOT for personal use Be punctual – late arrival will be noted and you will miss the aims of the lab Clean up when asked at the end of class to avoid long queues When working in groups, discuss (quietly) your observations 2. Work through the slides making notes ‐
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Work your way through all the slides Use low‐power objective first when examining a slide to give an overview of the section and help to orientate you quickly After examining object at high power, return to low‐power; this will help to fix the structure in your mind It is vital that you make your own notes for each slide Draw representative areas and make notes of classification and structure/function. Concentrate on area specified in lab notes. Practical classes form an important learning situation to discuss problems. Attempt and understand answers to questions; don’t just copy answers Check that you have met the learning outcomes for the class 3. Using Digital SlideBox ‐
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Go to Digital SlideBox: http://trinity‐dsb.slidepath.com/dsb/login.php Register as per instructions given in the Info & Registration Slides; after doing this log onto SlideBox Click onto the relevant folder Click onto the first slide Note the following tools for using SlidePath o Select Slide Allows you to select the slides for this lab, start with the first slide and work your way through in order o View Options Make sure that the three tools are selected: Annotations, Annotation Information and Slide Overview o Navigator Controls Allows you to move around the slide and also magnify into the slide. You can also do this by double‐clicking into the area of interest. Clicking the middle of the directional tool (i.e. the circle) takes you back to the main view o Annotation Information This Annotation box is VERY IMPORTANT. You should click onto each on the Titles. These are numbered in the same order as the Lab Notes. Work your way through these annotations making sure that you have viewed each annotation carefully. MAKE YOUR OWN DIAGRAMS AND NOTES. I
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4. Practical Books ‐
Each student must keep a file of labelled drawings. This is most important at the beginning of course so that difficulties can be spotted as they arise. A loose‐leaf ring‐binder is most convenient. 5. Binocular microscopes ‐
Binocular microscopes may be available for you to practise. You will be informed when these are available. 6. Safety & Lost Property ‐
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Always wear a lab coat. Gloves will be supplied where required. Look after your belongings and do not let them clutter the floor. Note carefully the fire exits are and learn assembly point for the building. If required to evacuate follow instructions of supervisor or demonstrator. The Department and University cannot be responsible for personal property. Lost Property is usually stored in Laboratory in which it is found; apply to Mr Aidan Kelly LAB ASSESSMENTS Please note that there will be assessments associated with each of your laboratory classes. These assessments will be given at the end of each lab. I
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LABS: MED: HHD: PHYSIOLOGY: HISTOLOGY LAB ONLY HISTOLOGY LAB ONLY ALL LABS YOU SHOULD PRINT OUT THESE SHEETS AND BRING THEM TO THE LAB I
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LEARNING OUTCOMES OVERALL 1. to relate structure to function 2. to identify tissue components: epithelia, connective tissue, nerve and muscle 3. to understand the basis of the classification of tissue components 4. to recognise several types of secretory cell and relate structure and function 5. to recognise the circulating blood cells EPITHELIUM, SECRETION & SKIN To be able to: 1. describe methods of histology preparation and effects on tissue 2. explain different stains, with special reference to cytoplasmic reaction 3. classify epithelia and describe patterns of proliferation and differentiation 4. relate cytological & histological types of epithelia & secretory cells to function 5. describe the histology and functions of skin Example Questions: 1. Name and describe the shape & arrangement of cells in lining epithelium 2. State how epithelium is adapted to serve its function in its organ/structure 3. Relate pattern of staining (H&E) in pancreatic acinar cells to their function 4. Describe the function of mucous/serous cells EXCITABLE TISSUES To be able to: 1. distinguish the three types of muscle. 2. relate skeletal muscle histochemistry to fibre type and physiology 3. recognise neurons and their processes. 4. know the structure of synapses, especially the motor endplate, and to be able to relate structure to function. Example Questions: 1. Muscle a) What type of muscle is present in this organ/structure? b) State what is visible that identifies it as this particular type of muscle. c) How is this type of muscle innervated? 2. Nerve cell a) Describe and Name the cell. b) What is its function? CONNECTIVE TISSUE (CARTILAGE, BONE, BLOOD) To be able to: 1. outline classification, components and function of connective tissues. 2. describe structure and function of bone and its constituent cells. 3. outline general processes and steps in intracartilaginous ossification. 4. identify circulating blood cell types, know their frequencies and functions. 5. outline principles of formation and differentiation of circulating blood cells. Example Questions: 1. In what respect is hyaline cartilage atypical of its Basic Tissue type? 2. Name cells involved in bone formation. Give their functions and locations within developing bone. 3. Apply this question to all cells you encounter in blood smears a) Describe nucleated cell in field; b) Name the cell; c) What is its function? 4. Name stem cell from which all blood cells are derived. Where is it found? 5. Outline general pattern of proliferation of haemopoietic stem cell and its descendants.
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DIGITAL SLIDEBOX HISTOLOGY LAB 1. EPITHELIUM, SECRETION & SKIN Classify surface epithelium, secretory cells and muscle in each of the slides. Make notes on relationship of structure (cytology) and function in each case. SLIDES 1 Duodenum from Kitten (H&E Stain) 1. Simple columnar epithelium: Single layer of cells with microvilli (not seen): nuclei are in a line in lower half of layer. 2. Pale mucus‐secreting goblet cells: Scattered among columnar cells. 3. Duodenal (Brunner's) glands: Lie inside outer muscle coat; composed of pale, vacuolated cells with rounded nuclei near bases of cells. They produce a mucus containing enzymes. 4. Outer muscle coat: Consists of two layers of smooth muscle (small spindle‐shaped cells with single central nucleus). 5. Villus and Lumen: Formed by the arrangement of the epithelial cells. Q1: What is the function of the microvilli (‘brush border’) Q2: Which structural features of these secretory cells are shared by serous and mucous cells, respectively? SLIDES 2 Neck Block from Rabbit (H&E and Shorr Stain) Oesophagus 1. Stratified squamous epithelium: Several layers of cells, becoming progressively flattened as they near the surface. Nuclei are rounded and dense at base and become large and pale then small and dense (pyknotic) as cells fill with keratin. A layer of keratin flakes (squames) may be present on the surface (keratinisation). There is a thin, broken layer of smooth muscle (muscularis mucosae) just below the epithelium. 2. Outer muscle coat consists of skeletal muscle: Large fibres with many nuclei at the periphery. In longitudinal section, cross‐striations may be seen. ‐ Additional Slide: Now go to Shorr Stain Slide. Smooth Muscle Striations: Focus into this area and note the cross‐striations in the muscle fiber. Q3: What is rate of proliferation of epithelial cells in oesophagus? (How do you know? What is significance of this rate of proliferation?) Trachea 3. Pseudostratified columnar ciliated epithelium: Cilia are small, hairlike processes at surface [cf. microvilli in duodenum]. All cells do not reach surface so nuclei are at different levels. 4. Hyaline cartilage: Deep (external) to layer of connective tissue is a layer of hyaline cartilage with cells (chondrocytes) in spaces (lacunae), scattered in the matrix. 5. Also view the following: Chondrocytes, Cells in Mitosis, and the Cilia. ‐ This slide was treated with vincristine (VCR) to arrest mitosis in metaphase. ‐ Additional Slide: Now go to Trachea (Rat) H&E slide. Note the above structure and cells in this additional slide. Q4: What is advantage of pseudostratified epithelium? Q5: How can solid cartilage grow? I
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SLIDES 3: Ureter from Cat (H&E Stain) 1. Transitional epithelium: several layers of cells. Ureter is surrounded by white adipose tissue. The outer muscle coat consists of two layers of smooth muscle. 2. WAT: The ureter is surrounded by a large amount of WAT Q6: What is rate of proliferation of epithelial cells? How do you know? What is significance of this rate of proliferation? SLIDES 4 Tongue from Human (H&E and Shorr Stain) 1. Stratified squamous epithelium: Similar to oesophagus 2. Skeletal muscle: Similar to oesophagus. Note the bundles of muscle fibres in a variety of directions to allow movement of tongue. Example of longitudinal and transverse section muscle fiber are shown. ‐ Additional Slide: Now go to Shorr Stain Slide. Smooth Muscle Striations: Focus into this area and note the cross‐striations in the muscle fiber. 3. Fat cells: Note the clear apperance of these cells. The fat has been dissovled out in the preparation of this section, namely during the ethanol dehydration process. Clusters (acini) of secretory cells lie beneath epithelium. 4. Mucous cells: Are pale and vacuolated due to unstained mucus [cf. duodenum]; nuclei are flattened at bases of cells. 5. Serous cells and Serous Acinus: Serous cells are bluish due to RNA for protein synthesis. The nuclei are more rounded and not squashed at bases of cells. The bundle of serous cells can be seen arranged in an acinus. Q7: Which type of secretory cells lies deeper? Q8: How are the muscle fibres of tongue arranged? Why? SLIDES 5 Salivary gland Serous from Cat (H&E Stain) 1. Duct: Surrounded by simple cuboidal epithelial cells 2. Acinus: The bundle of serous cells can be seen arranged in an acinus. Compare these cells and structures to the tongue. Q9: How does epithelial type vary with duct size? Salivary gland Mucous from Cat (H&E Stain) 1. Duct: Surrounded by stratified (layered) cuboidal epithelial cells. Compare this to the simple cuboidal epithelial cells in other ducts. 2. Acini: Three acinii are seen. Note the arrangement of the cells with the nucleus of each cell at the base. SLIDES 6 Pancreas from Rabbit (Azan Stain) 1. Exocrine (dark) portions. The basal part of exocrine cells is blue (basophilic) reflecting RNA activity for protein (enzyme) synthesis. 2. Endocrine (pale) portions. The pale cells are endocrine cells. I
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SLIDES 7 Acinar, Serous & Mucous cell diagrams ‐ View diagrams of acinar, serous & mucous cells as seen in the electron microscope. Recognise components of cells and relate them to appearance of cells in light microscope and to their specific secretions. Q10: Distinguish ducts and secretory units. Why is there intense staining of the acinar cells, refer to their ultrastructure? SLIDES 8 Thyroid gland from Rat (H&E Stain) 1. The three sections shown were isolated with animals treated with: [1] Thyroid hormone (TH) (right); [2] vehicle (middle); [3] an inhibitor of TH synthesis (left) SLIDES 9 Skin of scalp from Human (H&E Stain) 2. Hair Follicle. The shaft of the hair grows in the follicle. Transverse section. ‐ Additional Slide: Now go to Ear (Ox) Stained with Orcein. Note the hair shaft that can be seen in the follicle and also the cartilage in the ear of the Ox. Also note the elastic fibres. Q11: What is nature of other dark‐stained structures in section? 3. Keratin. The cells of the outer layers of the epidermis die, lose their nucleus and become keratinised 4. Sebaceous Glands. A collection of glands can be seen. The oily produce is reflected in the cytoplasm first as small droplets which gradually fuse and constitute the product of the gland. The cells perish during the secretion. 5. Layers. Note the Keratin, Epidermal, and Dermal layers. SLIDES 10 Glabrous Skin of finger pulp from Human (H&E and Sudan Stain) 1. Keratin Layer. Note the deeper layer of keratin on the finger compared to the scalp, for protection. 2. Epidermal Layer. Compare and conrast the thickness of this layer with that found in the skin of the scalp 3. Dermal Layer. 4. Dermal Papilla. Sensory receptors are expressed in the dermal papilla ‐ Additional Slide: Now go to Skin Stained with Sudan/Heamtoxylin Stain. Note the orange coloured fat cells stained with Sudan Stain. EXCITABLE TISSUE SLIDES 11 Spinal cord smear from Ox (Methylene blue Stain) 1. Cell Body: Methylene blue stain shows only the basophilic (blue) granules and the nucleolus 2. Nerve Fibers: The cytoplasm is extended into processes (axon, dendrites) ‐ Additional Slide: Now go to Spinal cord tissue H&E (from Kitten) .Identify and draw the large neurones. Compare their appearance in this slide with that in the Methylene blue Stain. Q12: What is the significance of the prominent nucleolus? Q13: What do the large blue granules represent? I
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SLIDES 12 Transverse Section (TS) of nerve trunk from Rabbit Osmic acid stain SLIDE 1. Myelin Sheath: The fatty component of myelin is stained by osmic acid stain. Compare with H&E stain, where this is not observable. 2. Nerve cell: Nerve cell is encircled by a single schwann cell in the peripheral nevous system. In the central nervous system, nerve cells are myelinated by one or more oligodendrocytes. 3. Nerve Bundles: Also view the nerve bundles 4. Connetive Tissue Fat The fat in the surrounding tissue is also stained by the Osmic acid stain. H&E stained SLIDE 5. Axon and Cell bodies: In this slide the alcohol dehydration has removed the fat content. The myelin and fat globules in the connective tissue are not seen in this H&E stained section. The Axon nerve fibre and cell body are clearly seen. ‐ Additional Slide: Now go to the Ultrastructure of neurons and synapses Diagram as seen in the electron microscope. SLIDES 13 Heart from Sheep (H&E Stain) 1. Cardiac muscle cells: Examine cardiac muscle cells in TS and LS. The are usually cut in oblique section. The cells are much larger than smooth muscle cells (e.g. duodenum) and each has a central nucleus (occasionally two). Striations are present but rarely visible. The purple masses within muscle cells are parasites 2. Pale conducting (Purkinje) fibres: Lie just beneath the endocardium 3. Also view the following: Endocardium and Parasites ‐ Additional Slide: Now go to the Motor Endplate Diagram as seen in the electron microscope, which shows neuronal innervation of the skeletal muscle. ‐ Compare and contrast the 3 types of muscle seen in the Heart, Ureter (see above) and Tongue (see above). CONNECTIVE TISSUE (CARTILAGE, BONE, BLOOD) Review loose irregular connective tissue in all slides with lining epithelia SLIDES 14 Developing bone from Rabbit 1. Developing bone: Identify the epiphysis. Identify osteoclasts, osteoblasts and osteocytes and review their functions. Look for fibrocartilage at the musculotendinous junction ‐ Now view the diagram of bone formation. Distinguish the steps in the process of intracartilaginous ossification. I
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SLIDES 15 Circulating blood cells diagrams 1. Blood Types: Take note of the blood types. Draw your own flowchart of the different cells 2. Granulocytes and Agranulocytes Classifications: Review the blood cell classification (granulocytes and agranulocytes). 3. Blood Cell Frequencies. Note the bigger white blood cells compared to the red blood cells. Calculate the % of white compared to red blood cells. Q14: How do the results compare with expected values and what are the potential sources of error?). I
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IMMUNOCYTOCHEMISTRY LAB 1. Organisation of this practical In this laboratory you will learn the process of immunocytochemistry (also called immunostaining). Immunocytochemistry is the name given to immunostaining of proteins in cell culture, while immunohistochemistry is the name given to immunostaining of protein in cell tissue. This technique has many uses in research, allowing to specifically stain a protein of interest and thus examine its tissue, cellular and subcellular distribution, as well as total protein expression levels. For example, in a diseased cell or after treatment with a drug, the protein of interest may move from the cell to the nucleus or may show increased protein expression levels, giving insights into its function and mechanisms of regulation. The process of immunocytochemistry takes several days to carry out. Parts of the experiment have been prepared in advance. Arrange yourselves into groups of 3 persons. Due to the number of steps it is important that you work relatively quickly. The approximate time allocation for the major steps of this practical is as follows: 2. The Protocol LAB 4 Premade: aspirate media & wash once with 1ml PBS (5 min) Premade: inc. 1ml ice cold methanol x 5min @ RT 1. wash 2 x 1ml PTx buffer (500µl 10% Triton X‐100 in 50ml PBS) (10 min) 2. block 30 min x RT in 1ml blocking buffer 1ml 100% Normal Goat Serum (5% final; stock in ‐20˚C) 1ml 10% BSA (0.5% final; stock 0.5g/5ml H2O 10% stock, Sigma A788 RIA Grade, Fraction V) 18ml 0.1% Triton X‐100/PBS 3.
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remove blocking buffer inc. 30 min x RT with 1˚y Ab in blocking buffer wash 3 x 0.5ml PTx buffer (15 min) inc. 30 min x RT with 2˚y Ab in blocking buffer (1:200 dilution), plus Hoescht nuclear stain (1:1000). wash 3 x 0.5ml PTx buffer (15 min) carefully remove coverslip from well (5 min) place inverted onto glass slide with a drop of ANTIFADE (contains 10% glycerol) go round with nail polish and then visualize (Lab 5) (5 min) LAB 5 You will go to the confocal microscope in your group. Each group will have a chance to view their results for 20 mins: 11. description of confocol setup (10 min) 12. view cell staining results on confocal (10 min) ‐ what is the blue stain? ‐ how does Hoescht stain work? ‐ what is the red or green stain? Please now return to the lab for the in‐course assessment. I
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3. Principles of antibody staining The principles of immunostaining a specific protein in cells is as follows: 1. Primary antibody (1oy) Primary antibodies specifically bind to the protein of interest 2. Secondary antibody (2oy) Secondary antibodies bind to the 1oy Ab. Secondary antibodies can be coupled to many types of tags – fluorescent molecules, dyes or enzymes. Fluorescent molecules emit at various wavelengths 1. Blue : absorption at 440nm, emission at 490nm 2. Green : absorption at 490nm, emission at 570nm 3. Red : absorption at 620nm, emission at 780nm 3. Fluorescent microscopy The protein‐1oy‐2oy fluorescent complex can be viewed under a fluorescent microscope, a confocal microscope or a two‐photon microscope. 4. Species specificity of antibodies Species specificity of antibodies is important. There are two important properties of an antibody: 1. the protein the antibody recognises 2. the species the antibody was made in Key points about antibodies are: 1. 1oy Abs: that recognise the protein of interest are normally created in rabbit or mouse, i.e. anti‐rabbit or anti‐mouse antibodies respectively; sometimes 1oy Abs can also be made in guinea‐pig, rat or even sheep. 2. 2oy Abs: that recognise anti‐rabbit or anti‐mouse antibodies are normally created in sheep, goat or donkey, i.e. anti‐sheep, anti‐goat or anti‐donkey antibodies respectively. Once isolated these 2oy Abs can be coupled to fluorescent molecules. Fluorescent tag
Figure 1. Cartoon depiction of the protein‐
2oy Anti-mouse goat Ab
10y‐20y complex. For example, an antibody that recognises Tubulin made in mouse is known as an anti‐
Tubulin mouse antibody. An antibody that 1oy Anti-Tubulin mouse Ab
recognises a Mouse Antibody made in goat is known as an anti‐Mouse goat antibody. Protein
Figure 2. Importance of getting the 10y and 20y Ab species to match. It is important to use the right species combination of antibodies, i.e. a 2oy anti‐
Mouse sheep or goat antibody will not bind to a 1oy anti‐Tubulin rabbit antibody. A 2oy anti‐
Rabbit sheep or goat antibody will bind to a 1oy anti‐Tubulin rabbit antibody. Fluorescent tag
2oy Anti-mouse goat Ab
1oy Anti-Tubulin rabbit Ab
Protein
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5. Polyclonal and Monoclonal antibodies. Antibodies can be monoclonal or polyclonal: 1. Polyclonal Antibodies: Antibodies are made by injecting an antigen (i.e. purified protein or peptide fragment) into an animal (e.g. rabbit). The rabbit’s immune response (B cells / plasma cells) creates a series of antibodies that recognise various parts of the protein or peptide fragment i.e. the n‐terminal, c‐terminal or internal parts of the protein or peptide. Up to 100 different antibodies can be made in the animal that recognise various parts of the same protein or peptide. This mixture of antibodies is given the name ‘polyclonal antibody’, which can be extracted from the serum of the immunised animal. 2. Monoclonal Antibodies: An antibody that recognises a specific part of a specific protein can be encoded in a cell line, which generates a single antibody. This is known as a ‘monoclonal antibody’. The cells used to create monoclonal antibodies are normally mouse cells. Thus monoclonal antibodies are usually mouse antibodies. I
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HAEMATOLOGY LAB Safety: Lab coats must be worn in this class Blood is a potentially dangerous substance, therefore you must observe all the normal safety precautions when handling it. White coats must be worn and you are provided with disposable gloves. You must dispose of these and all other disposables in the receptacles provided at the end of the practical. 1. Learning Outcomes This lab will allow you to measure in your own blood some of the parameters which may be helpful in diagnosis. The lab debriefing session will look at the results in detail and also consider pathophysiological changes. To be able to: 1. describe and carry out the method of obtaining and handling a blood sample, including SAFETY implications 2. describe normal variations in blood values and limits of normal range: explain how to measure or derive the following haematological parameters in principle and practice and explain their significances and list their normal values ‐ Haemoglobin Concentration, Haematocrit, Red Cell Count, Mean Corpuscular Haemoglobin Concentration, Mean Corpuscular Volume and Mean Corpuscular Haemoglobin 3. explain testing of blood groups: describe how to determine the ABO group and Rhesus group of a blood sample in principle and practice and the explain the implications of mismatching 4. explain an example of a blood group pathology: how to estimate the Osmotic Fragility of red blood cells in principle and practice and explain its significance 5. correctly derive units in which all above parameters are usually expressed. 2. Class Results As you obtain results, add them to the spreadsheet of Class Results for your sex. You should also compare your results with normal values and discuss any deviations with the demonstrator. All results and their implications will be discussed in the next lecture/seminar. 3. Blood collection and treatment At the start of the practical a demonstrator/phlebotomist will collect a sample of venous blood from your arm which will be put into a plastic vial containing an anticoagulant such as EDTA for use in this practical. Write your name on this vial. Gently invert 10 times to mix the blood and anticoagulant. Do not shake it vigorously. Blood cells settle out of suspension quite quickly. Therefore it is essential that you mix the blood immediately before you withdraw each sample for analysis. I
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4. Your Data Records Complete a copy as your personal record and hand in a copy before leaving the lab. Group JS / Dip Ex (circle one) Name _______________________________ Date _______________________________ Sex _______________________________ 1. BLOOD GROUP ABO _________________ 2. HAEMATOCRIT (PCV) Rhesus (+/‐) _________________ Reading 1. _________________ Reading 2. _________________ Mean _________________ Reading 1. _________________ 3. HAEMOGLOBIN Reading 2. 4. RED BLOOD CELL COUNT 5. SEDEMENTATION RATE (ESR) 6. OSMOTIC HAEMOLYSIS First evident Complete 7. CORPUSCULAR – MCV 8. CORPUSCULAR – MCHC 9. CORPUSCULAR – MCH _________________ g/dL _________________ x 1012/L _________________ mm/hr _________________ % NaCl _________________ % NaCl _________________ fL _________________ g/dL RBC _________________ pg/RBC Compare your readings with the normal values 1.
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What is a ‘normal value’? What are the sources of variation of individual parameters, compared to normal values? Consider the implications of each of your observations. How might these observations change in the following conditions: haemolytic anaemia, haemorrhage? I
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Test 1 – Blood Groups You must start with test for Rhesus Group because it takes 1.5 hr to incubate. When blood from a donor is used for transfusion it must be compatible with the patient. It is necessary to ascertain group, or type. There are many varieties of blood groups, but only two are clinically important: ABO and Rhesus (Rh) systems. 1. RHESUS GROUPS Red cells of some people carry a surface antigen named D. This antigen is also carried by red cells of Rhesus monkey, hence the name. This blood is typed Rh‐
positive. Those people whose red cells do not possess antigen‐D are Rh‐negative. Approx. 7% of population are Rh‐negative. The surface antigen‐D genes are carried on different chromosomes from ABO antigens and are expressed independently. They differ from ABO groups in that antibodies do not occur normally in the plasma. The antibody (anti‐D) only occurs as result of wrongly infusing a Rh‐
negative person with Rh‐positive blood. Experimental Method (Rhesus Typing) A test tube is required because slide method is not sensitive for Rh typing. 1. Open cap containing your blood sample – it will be stiff so be careful! 2. Dilute 1 drop of blood with 19 drops of isotonic saline (i.e. 1 in 20 dilution). 3. Take 1 drop of this blood suspension into a separate tube 4. Add 1 drop of anti‐D serum to it. 5. Label this tube and incubate for 1.5 hours at 37°C. 6. Read for presence of agglutination. Agglutination means antigen‐D is present and red cells are Rh‐positive. Non‐agglutination denotes Rh‐
negative red blood cells (approx. 7% of population). 2. ABO GROUPS There are 4 main groups — A, B, AB & O named according to antigens (agglutinogens) carried by red cells. In group A, blood cells have antigen A, and plasma/serum the antibody anti‐B. In group B blood cells carry antigen B and plasma/serum antibody anti‐A. Blood of group AB has both antigens A and B on cells and no antibody in plasma/serum. The group O blood has neither A or B antigens on cells but has both anti‐A and anti‐B antibodies in plasma/serum. The group can be ascertained from cells or from serum or plasma. To avoid difficulties due to clotting, serum is used rather than plasma. Generally cells are used but it is advisable to do it both ways whenever possible. Experimental Method (Grouping Cells) Determine group of your own cells. You are provided with following: (i) anti‐A serum, (ii) anti‐B serum, (iii) 0.9% NaCl (saline), (iv) glass slide marked into three compartments, (v) test‐tube, and (vi) pipette 1. Put 3 drops saline into empty test‐tube and add 1 drop of blood. Gently mix. 2. Now, using pipette, put a drop of this cell suspension in centre of each of compartment on divided slide 3. To left‐hand compartment add drop of anti‐A 4. To right‐hand add drop of anti‐B 5. No serum is added to centre compartment as control. Add a drop of saline 6. Rock slide and watch for appearance of agglutination in compartments 7. If agglutination appears in left your blood is group A. If in right your blood is group B. If in both you are group AB. If in neither you are group O. I
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Test 2 – Heamatocrit Value (Packed cell volume, PCV) Experimental Method (Micro Method) 1. Don’t forget to mix blood gently inverting 8‐10 times before taking sample 2. Fill two micro‐haematocrit tubes with blood, about three‐quarters full, by capillary action, holding them nearly horizontal 3. If capillary doesn’t fill you can create a partial vacuum by pressing the end of a (gloved) finger over free end of the tube while other is still in blood 4. Seal empty end of each tube and place sealed ends down in racks provided 5. Keep capillary tube horizontal all times, even when filling from your vial or sealing it. Only tilt it to vertical when you are placing in rack 6. Write your name in corresponding spaces on sheet 7. Your tubes will be placed in a centrifuge and spun for 5 min 8. Read length of red cell column as a ratio of length of total blood column using reader illustrated. 9. Reading Haematocrit. Put capillary tube in groove on clear Perspex slider so that bottom of column of blood is on 0% line. Slide cursor to left or right so top of plasma is on 100% line. Use lever on left to move grey line until it intersects boundary between red cells and plasma. Read haematocrit from scale on right. Test 3 – Estimation of Heamoglobin Experimental Method (Hemoximeter Method) 1. Fill two hemoximeter tubes approximately 2/3 full with blood 2. When apparatus is ‘ready’ carefully attach tube to apparatus receptor area 3. DO NOT try to remove surplus blood from outside of tube 4. Press ‘Aspirate’ button 5. When apparatus accepts sample, close shutter, await result (g Hb/dL blood) 6. Do not apply second tube until ‘ready’ signal appears Test 4 – Red Cell Count Demonstration: A demonstrator will take your blood sample to Coulter counter and give you a print‐out of results. Compare your estimates with print‐out and normal value. You will need some of the values to derive other measures (below) Test 5 – Erythrocyte Sedimentation Rate (ESR) Demonstration: In many diseases plasma protein level is abnormal. Fibrinogen precipitation on red cell membranes may result in increased adhesiveness between adjacent cells. The test is non‐specific. A Westegren tube containing anticoagulant I
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will be filled with blood at start of lab. Note rate of sedimentation (mm/hour) after 1 hr. Normal values are 3 ‐ 8 mm/hr, although 30 mm/hr is within normal limits. Test 6 – Osmotic Fragility of Red Blood Cells Demonstration: Although red cells require osmotic environment, they offer some resistance to disintegrating by hypotonic solutions. Thus a slight lowering of osmotic pressure will not cause bursting (haemolysis). Fragility tests measure degree of resistance to hypotonic environment, a function of membrane flexibility. Red cell suspensions are made in varying saline concentrations. In iostonic or near‐
isotonic solutions cells (opaque red) will settle leaving clear liquid above. At a lower saline concentration haemolysis occurs and released haemoglobin stains liquid above. Complete haemolysis is evident as a stained liquid without opaque cells. At room temperature, red blood cells begin to haemolyse at 0.5% NaCl (0.9% is isotonic). Haemolysis is complete at 0.2% NaCl. At 37°C resistance to haemolysis is lower. Express haemolytic state as complete (C), partial (P) or none (N). Tube % NaCl Haemolytic State (C, P, N) 1 1.0 2 0.8 3 0.6 4 0.4 5 0.2 1. 2. 3. In which tube did haemolysis begin? In which tube is it complete? Discuss importance of haemolytic environment of the red blood cells. Test 7 – Calculating Values a) Mean Corpuscular Value (MCV) MCV =
PCV
RBC (/L) x 100
fL
[1 femtolitre (fL) = 10‐15 L] b) Mean Corpuscular Haemoglobin Concentration (MCHV) The average concentration of haemoglobin in red cells is calculated from haemoglobin and packed cell volume (haematocrit) and expressed as g/dL of red blood cells. (d/L is unit used in clinical practice.) MCHC =
=
grams haemoglobin in 100 ml blood
volume of red cells in 100 ml blood
Hb
x 100 g/dL
PCV
x 100 g/dL c) Mean Corpuscular Haemoglobin (MCH) MCH =
Hb x 10
RBC (/L)
pg Hb/RBC [1 picogram (pg) = 10‐12 g] I
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REVISION: MOCK EXAM PAPER (JS Physiology only) 1. Learning Outcomes In this lab your will be provided with a mock‐exam laboratory session. 2. The Mock Exam ATTEMPT ALL QUESTIONS. Time allowed: 2 hours NOTES 1. 2. 3. 4. 5. 5. As a guide to time, the proportion of total marks available for each question is given. ALL answers should be given in the answer sheets provided. Return ALL the Question and Answer sheets Insert your Student No. on ALL pages of the answer sheet ‘Describe’ is not the same as ‘Name’. If ‘Name’ is its official name Each question refers to the previous one, unless it obviously doesn’t. THIS EXAMINATION IS HELD UNDER COLLEGE EXAMINATION RULES 3. The Exam Paper This will be provided on the day of the lab. The exam will be divided into two parts. PART 1: SLIDES [20 Marks] Part 1 will be composed of a number of questions related to the slides on SlideBox. You will be asked to identify the organ, structure and/or cell type and its function. In general the answers to these questions only require 1‐2 words. You should avoid spending time on long winded answers. Each question will be worth 1 mark and the total marks for part 1 will be equal to 20 marks. PART 2: QUESTIONS [20 Marks] Part 2 will be composed of a questions related to the lecture notes and the histology module in general. You will be asked to describe a process or a group of cells and their function. You may also be asked to label a structure or provide classification of cell types. These are short‐answer questions, often requiring a list of cells, functions, or steps of processes. Each question will be worth 3‐5 marks and the total marks for part 2 will also equal to 20 marks. [END] I
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