Download Microscopic techniques for application in experimental pathology of

Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
Microscopic techniques for application in experimental pathology of the
middle and inner ear
A. Trinidad1, J.R. García-Berrocal1, J. Nevado2 and R. Ramírez-Camacho1
1 Department of Otorhinolaryngology, Hospital Universitario Puerta de Hierro-Majadahonda, Universidad Autónoma de
Madrid, C/ Manuel de Falla 1, 28222, Majadahonda, Madrid, Spain
2 Department of Medical Genetics, Hospital Universitario La Paz, Universidad Autónoma de Madrid, Pº de la Castellana,
261, 28046 Madrid, Spain
Experimental ear research is crucial for the understanding of intimate mechanisms of hearing physiology and ear diseases.
While studies in humans are limited by anatomical features, studies in experimental models of disease allow for a wide
range of electrophysiological, analytical and morphological determinations. In the case of morphological analysis many
microscopy techniques are useful, from the conventional light microscopy stainings and immunohistochemical methods to
the most advanced electron microscopy and confocal laser scanning methods. This chapter shows application of light
microscopy, immunohistochemistry, transmission and scanning electron microscopy and environmental scanning electron
in the study of middle ear infections, biomaterials compatibility, inner ear damage and detailed anatomical features in
experimental models in the rat and the guinea pig.
Keywords inner ear, middle ear, immunohistochemistry, light microscopy, electron microscopy, hearing loss
1. Introduction
Hearing loss is a condition affecting humans worldwide. It is highly prevalent in the western countries due to increasing
ageing of population, industrial sources of intense noise and employment of drugs for severe diseases that may damage
the inner ear. Otitis media is an important health care pediatric problem all around the world and can also lead to
hearing deficits. Many questions regarding infection pathophysiology and control, prevention of damage by noise and
ototoxics and regenerating the ageing inner ear remain unknown. Approaches to the hearing organ in the past were
mainly based in morphological descriptions of temporal bones of patients at necropsies. Technical advances have
allowed detailed electrophysiological studies of the hearing in the living. However, studies in humans are limited by the
fact that the inner ear is enclosed in a bony capsule (the otic capsule) and sensory cells immersed in a fluid with a highly
specific electrolytic composition. This fact makes tissue sampling unattainable in the living. Therefore, extensive
research in middle and inner ear pathology is being done in animal models and will still be necessary in the future.
Animal models of ear disease allow a precise timing of the inflicted damage (whether toxic, traumatic or immunologic),
electrophysiological studies to determine effects over hearing function and finally detailed morphological studies of the
alterations caused.
The preferred species in hearing research are rodents: guinea pigs, rats, chinchillas, gerbils, mice and rabbits. The
preference for a particular model is dependent on the advantages that it may have in answering particular scientific questions.
Rat and chinchillas are extensively used in otitis media research. Rats are also employed in inner ear research as well as
mice (many inbreds are available) and guinea pigs, with an anatomically prominent cochlea that makes manipulation of
inner ear easier.
2. Light microscopy
2.1
Hematoxylin and eosin stain
This is the most commonly employed stain in medical diagnosis and still of great interest in ear research. It allows the
study of middle and inner ear structures in a variety of experimental pathology models such as experimental otitis and
biomaterial implantation. Destruction of middle ear ossicles of infectious origin is commonly treated with bone
reposition or implantation of prostheses made of polymers or metal. Rat has been widely employed to study interactions
between middle ear tissues and biomaterials in healthy ears [1], following obstruction of the Eustachian tube [2,3] and
in models of infection [4]. Conventional light microscopy stains provide information about reactive inflammation,
changes in bony structures, mucoperiosteal covering of middle ear cleft, integration of biomaterials, and other
pathological features in middle ear [5] and inner ear [6].
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Méndez-Vilas and J. Díaz (Eds.)
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d
l
Fig. 1 A Plastipore fragment implanted for 30 days in the middle ear of an animal inoculated with Pseudomonas aeruginosa. A
fibroepithelial interface (arrow) can be observed covering the prosthesis with penetration of fibrous tissue within the pores. Irregular
shape (d) and loss of some granules (l) can be observed (Hematoxylin&eosin x20).
G
Fig. 2 Polymorphonucleated cells (arrow) in contact with a fragment of Gore-tex® (G) and penetrating the small pores of the
biomaterial only in the periphery. Sacrifice at 30 days after implantation and inoculation with Pseudomonas aeruginosa
(Hematoxylin&eosin x50)
Interesting anatomical features can also be visualized with this staining. A longitudinal section of the stapes, a middle
ear ossicle, can be seen in figure 3. This small bone is located in the oval window, opening into the basal turn of the
cochlea. The stapedial artery can be found between the crurae of the stapes in the rat model.
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
ea
c
c
f
Fig. 3 The whole structure of the stapes can be appreciated in this image, including the head (h), crurae (c) and footplate (f). The
stapedial artery is usually an embryological structure in the human and other mammals, but it persists in the rat after birth (ea). Note
the annular ligament between the footplate of the stapes and the rim of the oval window (arrows). Hyperplasic mucoperiosteum can
be seen surrounding the bony structures, as this image was taken from a rat inoculated with Pseudomonas aeruginosa 15 days before
(Hematoxylin&eosin x50).
2.2
Masson trichrome
Masson's trichrome is a three-colour staining protocol used for distinguishing the connective tissue in the samples. It is
used preferentially for analysing connective tissue changes in the middle ear in animal models of otitis media.
2.3
Immunohistochemistry
This technique allows identification and precise localization of specific proteins in the tissue sampled. In the case of
experimental inner ear pathology, this is a technique that has been employed in localizing specific markers of apoptosis,
neural tissue, different signaling molecules in regeneration processes, receptors, etc. As an example, it is possible to
study apoptotic changes caused in the rat cochlea after injection of cisplatin, an antitumoral drug widely employed in
the treatment of solid tumors and associated with hearing loss in a high percentage of treated patients [7]. Caspase-3
activity in different cell populations of the organ of Corti can be determined by immunohistochemistry. Figure 4 shows
increased expression of caspase-3, which is related to the intrinsic pathway of pro-apoptotic signalling within the rat
cochleae [8].
OHC
IC
Fig. 4 Immunostaining of active caspase-3 in the nuclei of inner ear
cells from rats 7 days after a single dose of cisplatin (5 mg kg−1).
Nuclear caspase-3 immunostaining is seen in several cell populations
after administration of cisplatin. OHCs, outer hair cells; IC, interdental
cells of the spiral limbus. Magnification × 400.
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Microscopy: Science, Technology, Applications and Education
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Méndez-Vilas and J. Díaz (Eds.)
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Fig. 5 Immunostaining of heat shock protein 70 (HSP-70) in the stria vascularis of the rat cochlea after cisplatin injection (arrows).
HSP-70 might be a reparative molecule expressed after cellular damage.
2.4
Cytocochleogram
A cytocochleogram is a graphic representation of the anatomical state of the hair cells along the complete width and
length of the organ of Corti from which morphometric information can be obtained. It was first described by Engstrom
and coworkers in 1966 for producing surface preparations of the organ of Corti and a 2-dimensional graphic
representation of damaged hair cells along the entire length of the basilar membrane [9]. It is then possible to relate
structural damage with the frequency-related localization. This was first performed manually and was time-consuming.
Some authors have described computer-assisted methods to assess hair cell damage and produce a graphical
representation [10].
3. Transmission Electron Microscopy (TEM)
This technique is capable of visualizing objects to the order of a few angstrom (10-10 m) therefore allowing the study of
very small details in the cell populations of the organ of Corti. Different cell types can be analysed as well as organelles
and cell junctions. Immunohistochemistry is also applicable to this technique. Similarly, ultrastructural studies of
explanted prostheses of middle ear can also be performed. Figure 6 shows an example of fibrocyte of the lateral wall of
guinea pig cochlea. Ultra-structural features suggest that this is a type IIb fibrocyte. These cells are located between root
cells in the external sulcus and type I fibrocytes and are related to active pumping of K+ to endolymph from perilymph
(Na/K ATPase) through external sulcus cells, which is crucial in electrophysiological processes of hearing function [11,
12].
Fig. 6 View of a type II fibrocyte in the lateral wall of guinea
pig cochlea by means of a transmission electron microscope
(x5000).
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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Fig. 7 Gap junctions between fibrocytes. Gap junctions are considered to play
an important role in ions exchange in the organ of Corti.
4. Scanning Electron Microscopy (SEM)
SEM is a commonly employed high resolution technique that allows topographical study of surfaces. It provides
spectacular images of biological and non-biological tissues and therefore is a popular technique in the field of ear and
hearing research. Immunohistochemical methods are available for this technique.
Panoramical views of the cochlea are available with this technique for determining the areas with better conservation
of the organ of Corti (figure 8) and ulterior visualization at higher magnifications (figure 9). This allows anatomical
studies in healthy organs as well as determination of pathological changes after inflicting a damage to the inner ear of
immunological or toxic nature [13]. Changes in sensory cells as well as supporting cells can be observed. In fact,
supporting cells are believed to play a more active role in inner ear homeostasis and function than traditionally
considered [14, 15]. In middle ear pathology, SEM allows visualization of cholesteatoma debris, ciliated cells in the
respiratory mucosa, etc.
a
b
Fig. 8 Panoramic view of the cochlea of guinea pig from base (b) to apex (a) with SEM. The cochlear portion was separated from
the rest of the temporal bone and the bony covering removed under surgical microscope. Despite a careful manipulation during this
process, the organ of Corti can be unintentionally destroyed in some portions, so a panoramic view allows the researcher to identify
the preserved regions (arrows) for subsequent magnification and detailed study.
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Microscopy: Science, Technology, Applications and Education
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Méndez-Vilas and J. Díaz (Eds.)
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Fig. 9 In this closer view of the sensory hair cells the
three rows of outer hair cells (OHC) can be observed with
the characteristic V-shaped stereocilia. The single row of
inner hair cells is separated from the OHC by the tunnel of
Corti (original magnification x1800).
Fig. 10
The monocellular layer of the Reissner membrane can be fully appreciated in this image.
Fig. 11 Closer view of the inner hair cells (IHC) row of the organ of Corti after immunomediated damage in a guinea pig model of
immuno-mediated disease of the inner ear. Balloon-like protrusions correspond to “bleb” formation in the cuticular region of IHC.
Blebs have been have been associated with cell injury, although they have also been observed in healthy cells during particular stages
of the cell cycle and play a role during embryogenesis.
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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5. Environmental Scanning Electron Microscopy
Environmental scanning electron microscopy (ESEM) is a descendant of the conventional scanning electron microscopy
that allows visualization of biological samples in their natural state. This technique is capable of imaging samples
without vacuum in the sample chamber and therefore biological samples do not need dehydration and conductive
coating for visualization, thus avoiding distortion of structures and artifacts. ESEM can be applied to the study of
biofilms, bacterial communities enclosed in a self-produced matrix or glycocalyx that protect them from biocidal
attacks. Biofilms have been associated with infectious diseases of the middle and inner ear and may explain difficulty to
eradicate middle ear infections in some patients. Experimental models of biofilm infection in the middle ear are being
developed and ESEM plays a crucial role in identifying presence of biofilms and analysing glucocalyx features (figure
12).
Fig. 12 Micrograph of a fragment of middle ear mucosa with
colonization by biofilms of Pseudomonas aeruginosa (*) in an
experimental model of biofilm infection in the rat. Note the
smooth surface of non-colonized mucosa surrounding the biofilm
(original magnification 2000x, pressure 4.4 Torr).
6. Confocal Laser Scanning Microscopy
This microscopy technique is currently considered a gold standard in the study of biofilms. It allows the differentiation
between living and death cells withing the biofilms as well as identification of species by use of different dyes.
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