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Mathematics for Biologists
19.11.99
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Mathematics for Biologists
Part Biology
Morphometric - Stereologic Analyses
of Lung Tissues
Protocol
Jan. 31st 1998
Headed by: Dr. Sänger
Handed in by:
Maricela Yip (Mat-#: 9424495)
Salzburg, January 31st 1998
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biophysics.sbg.ac.at/home.htm
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Morphologic-stereologic investigation of epithelial cilia cell, macrophage cell and alveoli in human lungs:
Introduction:
The lung’s main function is to allow for efficient
exchange of gases (O2 and CO2) between the
surrounding air and the blood. For this purpose
the air is led through the bronchi into a large
volume made up of many small chambers, the
alveoli. Similarly blood flows through arteries
into a dense capillary network which is
contained in the alveolar walls and is then
collected and conducted back to the heart by
veins.
Respiratory passages:
When a mammal breaths in, air enters the
respiratory system through the mouth or nose.
The air is warmed and humidified by the moist
mouth or the nasal cavity which are located
posteriorly to the pharynx. The pharynx
branches into a pair of tubes; one the
esophagus, leads to the stomach, while the
other, the wind pipe, or trachea, is the air-way
leading to the lungs. At the anterior end of the
trachea, lies the larynx, housing the vocal cords.
Just above the opening to the larynx, is a flap of
tissues called the epiglottis, which normally
closes off the larynx during swallowing and thus
prevents food from accidentally entering the
lungs. Ventilation of lungs is brought about
passively by the intercostal muscle of the ribcage, which is connected via the pleura and
pleural cavity and the diaphragm.
Lungs:
The trachea branches into two hollow passage
ways called bronchi, each of which enters a
lung. Finer and finer branchings of these tubes
create an inverted tree, with thousands of narrow
airways, or bronchioles, that eventually leads to
millions of tiny, bubble-shaped, sacs, called
alveoli.
It is in the alveoli, that gas-exchange takes place.
The terminal bronchioles, the respiratory
bronchioles, the alveolar ducts, and the alveolar
sacs, constitute the respiratory portion of the
lungs. Many of the cells that line the larger
airways produce a sticky mucus ideally suited
to the capturing inhaled dirt-particles or
microorganisms. This mucus is continuously
cleared from the bronchi by the beating of cilia,
which sweep the mucus and any trapped debris
up toward the pharynx, where they can be
swallowed or expelled.
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Alveolus: (L. small cavity)
Gases are transferred across the thin-walled alveoli found in the region distal to the terminal bronchioles, termed
acini. The airways leading to the terminal bronchiole constitute the non-respiratory portion of the lungs. Alveoli in a
joining acini are interconnected by a series of holes, the pores of kohn, allowing the collateral movement of air,
which may be a significant factor in gas distribution during lung ventilation.
Each alveolus is surrounded by blood capillaries, and the inside of each tiny pouch is lined with a moist layer or
epithelial cells. At those places where the wall of a capillary lies near the outer wall of an alveolus, O2 easily diffuses
out of the alveolus and into red blood cells squeezing down the center of the narrow capillary. Meanwhile, CO2
leaves the blood, diffusing out of the capillary and entering the alveolus. From the alveolus, it is expelled to the
outside with the next exhalation.
The lung-wall tension depends on the properties of the alveolar wall and the surface tension at the liquid-air
interface. The explanation for the relatively low surface tension of the liquid lining the lungs is the presence of
surfactant, lipo-protein complexes that bestow a very low surface tension in the liquid-air interface. Lung surfactant
not only reduce the effort associated with breathing but also help prevent alveoli from collapsing.
Finally a few words about the tabacco plant itself:
Tobacco (Nicotiana tabacum) is a hardy C3 plant that grows up to 2m tall and produces very large leaves and spikes
of pretty, usually pink, flowers. As a natural defense, tobacco leaves and stems produce various compounds that
discourage insects and other predators. Among them is a bitter-tasting nitrogen-containing compound, an alkaloid
called nicotine.
The cigarettes, cigars, pipe and chewing tobacco made from the tobacco plant seem to be addictive. This is because
the nicotine in tobacco leaves causes a strong psychologically dependence on the taste and feel of tobacco.
Besides that, analysis of smoke from burning tobacco has shown to contain over 4000 separate compounds, including
DDT, arsenic, nitrosamines, and formaldehyde, all known carcinogens. Similar tests of chewing tobacco reveals
traces of three
additional carcinogens: cadmium, uranium, polonium. One of the most damaging compounds in
tobacco smoke remains as the poisonous gas carbon monoxide (CO).
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Effects of tabacco smoke in human cells:
Paralyzed Cilia: Tobacco smoke can paralyze the cilia, the microscopic hairlike projections
from cells lining the airways of the human respiratory tract. Without these continuously beating cilia, germs and
particles of foreign matter can enter the lungs and cause irritation and infection. The lungs of a smoker and his
respiratory passageways compensate by producing more mucus, which is expelled in a cough. Perennial coughing can
weaken the lungs and lead to chronic bronchitis.
Lung Cell Changes: Cadmium, nitrogen dioxide, and other substances in cigarette smoke
can rupture cells in the lungs' tiny, ballonlike air sacs, or alveoli. They can also prevent a cell's smooth endoplasmic
reticulum from producing normal amounts of surfactant. Both of these changes can contribute to permanent
shortness of breath and to lung diseases such as emphysema.
Disturbed Mitochondria: Smoke destroys the mitochondria's normal internal structure, and with it, their ability to
carry out the reactions of the Krebs cycle (an elementary process in cell respiration) and the electron transport chain.
Thus the cell is starved for ATP (energy carrier within the cell) energy and eventually dies.
DNA Damage: Many of the toxic compounds in tobacco can attack and damage DNA. Repair enzymes in the
nucleus attempt to fix these broken strands, and correct mismatched base pairs. However, continual exposure to the
toxins can lead to an accumulation of errors, which are implicated in the formation of cancerous tumors.
Nicotine and the Cell: Certain cells in the nervous and muscular system have receptor proteins in their membranes
that bind with nicotine, causing a different cell response to normal nerve signals, explaining how nicotine acts as a
stimulant.
Increased Carbon Monoxide: Smokers have elevated CO levels in their bloodstream. When CO combines with
hemoglobin, the pigment delivers less oxygen to the body's tissues, including the brain, and the person's ability to
think clearly is reduced.
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Immune Cell Changes: Researchers studying the white blood cells (macrophages) that patrol and protect the
airways and lungs have found an increase in the size and numbers of lysosomes within the cells and a decrease in
protein synthesis. The cells are apparently so busy ingesting foreign particles and the debris of damaged cells that
they can't grow and function properly. Immune cell disruption helps explain why smokers catch colds, flu, and
pneumonia more easily than nonsmokers, as well as experience increased cancer rates.
Further Consequences of tobacco use: Tobacco users have lowered resistance to colds and flu, and slower healing of
broken bones and other wounds. Female smokers tend to have more miscarriages and babies of lower birth weight.
Even in their teens and twenties, tobacco users tend to develop periodontal (gum) disease four times as often as
nonsmokers.
A smoker’s lungs
A non-smoker’s lungs
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1. Working Hypothesis:
Statistical evidence show that smokers are more likely to suffer from respiratory diseases and premature death than
nonsmokers. Morphologic-stereological approach should help to determine the following effects:
Hair-like cilia:
In a Nonsmokers lungs, the cilia protrude from the cells that line the trachea and bronchioles sweep mucus and debris
from the respiratory passageways.
In a smokers lungs, the cilia are paralyzed or broken by cigarette smoke. Debris can reach the lungs and accumulate,
blackening and clogging the delicate tissues lining the alveoli.
Stereological methods should show how the all over cilial-surface area is lower in smokers.
Lung Cell Changes: Cadmium, nitrogen dioxide, and other substances in cigarette smoke can rupture cells in the
lungs' tiny, ballonlike air sacs, or alveoli. They can also prevent a cell's smooth endoplasmic reticulum from
producing normal amounts of surfactants which further deteriorates the alveolar walls. These changes can contribute
to permanent shortness of breath and to lung diseases such as emphysema.
Stereology will help us to show the loss of alveolar surface area of a smoker lungs versus a nonsmoker.
Macrophage Cells: in white blood cells, lysosomes enlarge after smoking.
Macrophages exposed to cigarette smoke do show significant changes, lysosomes of these immunodefencee cells can
be used to show the modifications under stressed conditions; therefore, morphologic-stereological techniques can be
used to prove this correlation?
2. Components of the Structures, needed for stereologic investigation
The different components must be clearly separated and identifiable. Each parameter is obtained as the ratio of two
measurements, one estimating the size of the objects of interest (phase), the other the size of the space in which they
are contained (reference).
Structure
Ciliated epithelial cell
vs
Cilia
Macrophage
vs
Lysosome
Alveolar ducts
vs
alveolus
Objects
Reference: A ciliated epithelial cell represents a structural unit.
Phase: Cilia, part of this structure, are the site of interest on which debris and
smoked gas affects them, here as the phase.
Reference: Macrophage is the direct immune defense organelle that ingest
and digest undesired material and debris that enters along with air into
the lungs, therefore considered to be the reference unit.
Phase: Lysosome are contained in the macrophage, are small membrane
bounded structures that contain a set of powerful hydrolytic enzymes,
which are capable of breaking down most organic materials, so that the
foreign body is quickly disassembled; i.e.: the phase.
Reference: Alveolar ducts as the pulmonary gas exchanger consist of at least
two alveoli of the same air duct system, hence, the reference.
Phase: Alveolus is a thin-walled, sacklike chamber in the vertebrate lung
where gas exchange takes place, the single structure within the unit.
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2.1. Determination of Parameters
The quantitative description of the structure is the density of the various components within the structure (the quantity
per unit volume).
Structure:
surface density
X
Hair cilia: in a 2-D, SEM picture, comparison of the size of
the surface between a nonsmoker hair cilia with a smoker hair
cilia reveals that a smoker’s epithelial cell is severely damaged
compared to a nonsmoker’s cilial epithelium; i.e.: ”surface” is
the criteria to look for.
Lysosome: the size of the lysosome of a smoker is
significantly larger than the nonsmoker’s; i.e.: the
”volumetric” content determines the effect.
X
Alveolus: in a smoker’s, the alveolar walls are almost lost,
resulting in shortness of breath and death; i.e.: all over surface
area determines efficiency in gas exchange.
volume density
X
2.2. Measuring Methods
Since 2-D cuts obtained by a series of microtomial cuts do not reveal the actual 3-D structure, indirect census
techniques like point, intersection, or number counting are used to rebuild the original structure
(the parameter of interest).
Structure:
Hair cilia: irregular patches of surface area of the epithelial
cell are better determined by counting the number of
intersecting lines that falls into the irregular patches.
Lysosome: the sizes of the lysosomes of the nonsmoker’s and
the smoker’s can be easily determined by this method.
Alveolus: the surface of alveolar walls can be easily
determined by counting the intersections that fall into the air
sacs of an alveolus.
surface density
intersection
counting
volume density
point counting
intersection
counting
2.4. Testgrid
The spatial distances between testlines should not exceed dimensions of objects of interest; i.e.:
• Cilium: spatial distance between testlines should be the size of average cilial length;
• Lysosome: spatial distance between testlines should be the average lysosomal size;
• Alveolus: spatial distance between testlines should be the distance of alveolar walls.
Structure
Hair cilia: Damaged cilia are better determined with a finer
testgrid, whereas intact cilia, with the coarse one; so that, we
can see the difference between the two SEM pictures.
Lysosome: Enlarged lysosomes in macrophages of smokers
are determined by a coarse testgrid, whereas the finer is more
suitable for the census of a non-smoker’s tissue.
Alveolus: A short-lined testgrid is probably best to use
because nonsmoker’s alveoli are far smaller (more walls in
between them) than of a smoker’s (increase surface area per
alveolus) due to the loss of alveolar walls.
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Testgrid
double square lattice
double square lattice
short-lined multipurpose testsystem
(M168)
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Healthy epithelial cilia
Damaged epithelial cilia
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Loss of alveolar walls
Normal alveolar walls
References:
• Stereological Methods Vol. 1, Ewald. R. Weibel, Academic Press London 1979, UK
• Animal Physiology 4th ed., Eckert, W.H. Freeman and Company New York 1997, USA
• The Nature of Life. 3rd ed., Postlethwait J.H., Hopson J.L, McGraw Hill, New York 1995, USA
• Zoology, Robert L. Dorit, Warren F. Walker, Robert D. Barnes, Saunders College Publishing, Orlando 1991, USA
• Medical Physiology, Ninth Edition, Arthur C. Guyton, John E. Hall, W.B.Saunders Company 1996, USA
• Cell and Tissue Ultrastructure, Patricia C. Cross, K. Lynne Mercer, W.H.Freeman and Company 1993, USA
• The pathway for oxygen, E. R. Weibel, Harvard University Press, 1984, USA
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