Quantitative analysis of the optic nerve of the horse (Thoroughbred)

Three optic nerves (L1, R2, R3) 12-18 mm behind the eyeball of the horse (Thoroughbred) were investigated quantitatively under light and electron microscopes. Thin sections at the thickness of 0.35 microm were cut, stained by toluidine blue and observed under the light microscope. The areas of the optic nerve and the axon bundles were 20.03 +/- 1.04 and 16.59 +/- 0.79 mm2 (mean +/- SD, n=3), respectively. The axon numbers for optic nerve L1, R2 and R3, estimated from light micrographs, were about 481 x 10(3), 543 x 10(3), and 494 x 10(3), respectively. Axons of optic nerve L1 were also counted from electron micrographs and the total number of 488 x 10(3) was received. Furthemore, axon diameters of optic nerve L1 were also measured from electron micrographs. The diameter of a circle with the same peripheral length as an axon, was regarded as its diameter. The medullary sheath of the axon was not included during measuring. Altogether 5,744 axons were measured and axon diameters were in a range of 0.23-12.69 microm, with a mean of 2.56 +/- 1.45 microm (mean +/- SD). A regional difference of axonal diameters was found across the optic nerve: the mean diameter of axons in the centrodorsal region (2.28 microm) was the smallest, and had significant difference with those in several peripheral regions (P<0.05).     

Age-related changes in rat optic nerve: morphological studies

Age-related changes of the optic nerve were studied in 3-month-old (young), 12-month-old (adult) and 24-month-old (aged) male Sprague-Dawley rats. Cross sections of the intracranial portion of the optic nerves of animals of different age groups were stained with haematoxylin-eosin and examined under a light microscope at low and high magnification. Other sections were stained with crystal violet for demonstration of glial cells. A third group of sections were stained immunohistochemically to detect glial fibrillary acidic protein (GFAP) which is a marker for localizing and characterizing astrocytes. All morphological results were subjected to the quantitative analysis of images and to statistical analysis to identify significant morphometrical data. Tissue protein concentrations were determined on homogenized fragments of optic nerve. Our results demonstrate the following age-related changes: (1) increase of the optic nerve sheaths (meningeal membranes); (2) increased number of astrocytes; (3) increase of areal density of GFAP immunoreactivity; (4) increased diameter and area of the optic nerve; (5) decreased number of nerve fibres; (6) decreased-size of nerve fibres and (7) decrease of the nerve fibres/meningeal membrane ratio from 3:1 to 1:1. Moreover, the protein amount does not change with age. The rat optic nerve, therefore, appears sensitive to ageing processes and can be considered as a useful model for the studies on neuronal ageing.     

Peculiar bundles of filaments in Leydig cells of the lesser mouse deer (Tragulus javanicus): an ultrastructural study

Leydig cells of lesser mouse deer (Tragulus javanicus) testes were observed using light and transmission electron microscopies. Sexually mature lesser mouse deer were obtained in East Malaysia. The testes were perfused with 5% glutaraldehyde, postfixed with 1% OsO4, dehydrated in ethanol and embedded in Araldite. The semithin sections were cut, stained with toluidine blue and observed under light microscopy. The ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a JEM-1200 transmission electron microscope. As a result, two types of filament bundles were frequently recognized in Leydig cells, but not in other testicular cells. These bundles were clearly seen at even a light microscopic level. One type was bundles of actin filaments (approximately 5 nm in diameter). These structures were found not only in the cytoplasm but also in the nucleus. The other type was bundles of intermediate filaments (approximately 10 nm in diameter). These structures were found only in the cytoplasm. The existence of filament bundles has never been reported in the testicular cells of another mammalian species. Thus, while bundles of actin and intermediate filaments are specifically present in the Leydig cells of the lesser mouse deer, their functions are still unclear.     

南极企鹅外周血细胞初步研究

2012年8—10月,在天津海昌极地海洋世界采用Wright—Giemsa’s染色和Natt—Herrick’s染色方法研究了南极企鹅（Pygoscelisantarcticus）外周血细胞。研究表明,外周血液分类为：红细胞、白细胞和血栓细胞。其中自细胞包括：异嗜性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞和单核细胞。测得红细胞平均大小（长径短径）为15．32μm×7．83μm,异嗜性粒细胞直径为（11．98±0．97）μLm,嗜酸性粒细胞直径为（11．61±0．78）μm,嗜碱性粒细胞直径为（11．87±0．69）μm,淋巴细胞直径为（9．93±1．29）Ixm,单核细胞直径为（15．04±0．93）μm,血栓细胞直径为（6．96±0．79）μm。统计红细胞总数为（1．85±0．17）×1012／L,白细胞总数为（6．37±1．68）×109／L。     

Morphology and quantification of sheep pineal glands at pre‐pubertal,pubertal and post‐pubertal periods

The pineal gland is a neuroendocrine organ associated with photoperiodic regulation in mammals. The aim of this study was to evaluate the pineal gland at the pre‐pubertal, pubertal and post‐pubertal periods by means of morphology and stereology. The study examined at total of 24 ovine pineal glands collected from healthy female Akkaraman breed. Thick sections (40 μm) were cut and treated with synaptophysin. Following each thick section, six consecutive sections at a thickness of 5 μm were cut. Each thin section was stained with one of the following dyes: Crossman’s modified triple dye, glial fibrillary acidic protein (GFAP), melatonin marker, periodic acid–Schiff, Von Kossa and AgNOR. The pineal gland volume was measured using Cavalieri’s method. The optical fractionator was used to estimate the total number of pinealocytes. The percentage of parenchyma and connective tissue and degree of vascularization were estimated by the area fraction fractionator method. The pineal gland volumes in the pre‐pubertal, pubertal and post‐pubertal groups were 7.53 ± 1.715 mm³, 11.20 ± 1.336 mm³ and 17.75 ± 1.188 mm³, respectively (p < .5). The number of pinealocytes in the pre‐pubertal, pubertal and post‐pubertal groups was 3,244,000 ± 228,076, 4,438,000 ± 243,610, 7,381,766 ± 406,223, respectively (p < .05). The glands of the post‐pubertal group contained the highest amount of connective tissue (11.49 ± 2.103%; p < .5) and the largest GFAP staining area (p < .05). The melatonin staining density was the highest in the pubertal group. The density of lipofuscin staining was higher in the pubertal and post‐pubertal groups.     

Anatomical Studies of the Accessory Nerve in the Indian Buffalo (Bubalus bubalis)*

This is a study of the origin, formation, course, distribution, the fascicular number, their area and arrangement, total fibre count, density and diameterspectra of the accessory nerve in the Indian buffalo. The radices spinales are located from the level of C₁ to midlevel of C₆cord segments. The radices craniales arise in series from the medulla oblongata caudal to those of the vagus nerve. The accessory nerve contains 7931 myelinated fibres arranged in 31 fascicles. Of these 41 % have an external diameter of 10–12 μm. The greatest density of fibres viz. 1781/mm² is obtained in the anastomosing ramus from the second cervical spinal nerve. The least value (377/mm²) is found in its ventral ramus. However, the dorsal ramus appears to have a larger cross-sectional area.     

Morphometry of cervical spinal cord in cat using design-based stereology

The spinal cord harbours nerve fibres that facilitate reflex actions and that transmit impulses to and from the brain. The cervical spinal cord is an area of particular interest in medicine and veterinary due to frequent pathologic alterations in this region. This study describes the morphometric features of the cervical spinal cord in cat using design-unbiased stereological methods. The cervical spinal cords of four male cats were dissected and samples were taken according to systematic uniform random sampling. Each sample was embedded in agar and cut into 60-µm thick sections and stained with cresyl violet 0.1% for stereological estimations. The total cervical spinal cord volume obtained by the Cavalieri estimator was 2,321.21 ± 285.5 mm³. The relative volume of grey matter and white matter was 23.8 ± 1.3% and 76.1 ± 1.3%. The dorsal horn and ventral horn volume were 12.3 ± 1.2% and 11.4 ± 0.7% of the whole cervical spinal cord. The volume of central canal was estimated to 3.8 ± 1 mm³. The total number of neurons was accounted 3,405,366.2 ± 267,469.4 using the optical disector/fractionator method. The number of motoneurons and interneurons was estimated to be 1,120,433.2 ± 174,796.7 and 2,284,932.9 ± 127,261.5, respectively. The average volume of the motoneurons and interneurons was estimated to 1980 µm³ and 680 µm³, respectively, using the spatial rotator method. This knowledge of cat spinal cord findings may serve as a foundation as a translational model in spinal cord experimental research and provide basic findings for diagnosis and treatment of spinal cord disorders.     

Regional Specialisation of the Ganglion Cell Density in the Retina of the Native Duck (Anas platyrhynchos) of Bangladesh

In this study, retinal whole‐mount specimens were prepared and stained with 0.1% cresyl violet for the ganglion cell study in the native duck (Anas platyrhynchos). The total number, distribution and size of these cells were determined in different retinal regions. The mean total number of ganglion cells was 1 598 501. The retinal area centralis had the highest ganglion cell density with 11 200 cells/mm². Number of ganglion cell bodies was the highest in temporal area, followed by dorsal, nasal and ventral areas. Ganglion cell size ranged from 5.25 to 80 μm². In the temporal and nasal region, most of the cells were ranged from 15 to 25 μm², and in the dorsal and ventral region, most of the cells were ranged from 12 to 25 μm². There was a marked trend for the retinal ganglion cell size to increase as the population density decrease towards the periphery. A population of small ganglion cells persisted into the central area just above the optic disc and the largest soma area was in the ventral zone of the retina. Thus, the specialisation of ganglion cell densities and their sizes support the notion that the conduction of visual information towards the brain from all regions of the retina is not uniform, and the central area is the fine quality area for vision in native duck.     

Comparative retinal ganglion cell and optic nerve morphology

The optic nerve is divided in four regions: intraocular, intraorbital, intracanalicular, and intracranial. The vertebrate retinal ganglion cells are classified by morphology, physiology and soma size. Species differences and similarities occur with retinal ganglion cells. Alpha retinal ganglion cells have large somata, large dendritic fields, large-diameter axons, and are most dense in the peripheral retina. Beta retinal ganglion cells have smaller diameter somata, smaller dendritic fields, small diameter axons, and predominate in the central retina. Gamma retinal ganglion cells are a heterogenous class of cells and have small diameter axons, and slow axon conduction velocities. The spatial distribution and organization of the retinal ganglion cells extends retinotopically through the nerve fiber layer, optic nerve, optic chiasm, optic tract, lateral geniculate nucleus, and visual cortex. The retinal nerve fiber layer thickness decreases from the optic disk toward the periphery of the retina. The retrobulbar optic nerve axon counts and axon density vary by species, with larger nerves having higher axon counts. Decussation of the optic nerve axons at the optic chiasm varies with 100% decussation in most birds and fish, 65% in cats, 75% in dogs, 80–90% in large animals, and 50% in primates. Centrifugal axons also occur in the optic nerve and may represent a method by which the brain can influence retinal activity.     

Spatial relationships among the cellular tapetum,visual streak and rod density in dogs

The dog visual system is well suited to dim light conditions due to rod-dominated retina and the reflective tapetum. The topographical distributions of rods and thickness of the tapetum of the dog were quantified in retinal whole mounts stained with thionine, and spatial relationships among the tapetum, rod density and visual streak of high ganglion cell density were elucidated. The relationship between the retina and tapetum was analyzed in parasagittal sections stained with thionine or hematoxylin-eosin. The tapetum was thick in its center, and the thickest part consisted of 9 to 12 tapetal cell layers. Rod density ranged from 200,000 to 540,000/mm². Maximum rod density was found in the area dorsal to the visual streak, and the density in that area was significantly higher than the rod density in the visual streak and accorded spatially with the thickest part of the tapetum. The horizontal visual streak was found over the horizontal line through the optic disc in the temporal half and extended slightly into the nasal half. The central area of the highest density of ganglion cells was approximately located midway between the nasal and temporal ends of the visual streak. The visual streak was located within the tapetal area, but ventrally to the thick part of the tapetum.     

Licht- und elektronenmikroskopische Untersuchungen an der Blut-Milch-Schranke der laktierenden Milchdrüse des Rindes

Light and electron-microscopic investigation of the blood-milk-barrier in lactating cow udders This paper reports a light and electron microscope (TEM) study of the blood-milk-barrier in the lacting mammary gland of the cow. The morphometric analysis of thick sections showed a significant difference between the posterior left quarter and the both right quarters of the udder. The distance between the endothelium of the capillary in the perialveolar connective tissue and the basal membrane of the secretory epithelium measures 2.33 μm. The thick sections used here have some important advantages over paraffin-embedded sections (cut at 5 μm), especially in connection with morphometric investigations. The electronmicroscope observations revealed the following remarkable structures in the blood-milk-barrier:

• 1 The endothelium of the capillaries in perialveolar connective tissue contains many pinocytotic vesicles and possesses pseudopodic structures.
• 2 The myoepithelial cells do not appear continuously and are therefore only indirectly related to the barrier.
• 3 The basal system contains special folds of epithelial cells.

     

Demonstration of pseudorabies viral antigen in thick and ultrathin tissue sections of young pigs using an immunogold method

Pseudorabies (Aujeszky's) virus antigens were labeled in thick and ultrathin tissue sections of young pig brain and liver tissue, using an indirect immunogold method. Antigens were tagged with 20 nm gold particles. Controls proved the specificity of the reaction in paraffin sections and ultrathin epoxy sections. Immunogold staining was compared with immunoperoxidase staining in paraffin sections. In ultrathin sections stained with the immunogold method, the gold particles were present on viral nucleocapsids and viral envelopes, as well as on a number of other intracellular structures. These included the inner nuclear membrane, the nucleoplasm, intranuclear filaments, the endoplasmic reticulum, and free cytoplasmic polyribosomes. Gold particles were absent on mitochondria and microtubules. In paraffin sections, immunogold labeling for pseudorabies virus antigen was less sensitive than immunoperoxidase staining. Immunogold staining of ultrathin tissue sections can yield additional information on virus-host cell interactions.     

Spectral domain optical coherence tomography (SD-OCT) assessment of the healthy female canine retina and optic nerve

Objective To provide normative data for canine whole retinal thickness (WRT), nerve fiber layer thickness (NFL), photoreceptor layer thickness (PR), and outer nuclear layer thickness (ONL) using spectral domain optical coherence tomography. Animal studied: Twelve healthy adult intact female beagles. Procedure Horizontal volume scans through the area dorso‐temporal from the optic nerve (superior retina), and the area ventro‐temporal from the optic nerve (inferior retina) were used to evaluate the thickness of retinal NFL, PR, ONL, and WRT. Peripapillary circular scans were used to evaluate NFL thickness. Statistical analyses were performed to compare the thickness of the individual layers between the superior and inferior retina (paired t‐test). One‐way analysis of variance (ANOVA) was used to compare the thickness of peripapillary NFL between the superior, inferior, temporal and nasal quadrants of the circle scan. Results The WRT, PR, and NFL thickness were greater in the superior than in the inferior retina (198.7 ± 9.6 μm vs. 164.4 ± 6.4 μm, P < 0.0001; 95.5 ± 6.5 μm vs. 78.8 ± 7.4 μm, P < 0.0001; and 26.4 ± 1.6 μm vs. 25.0 ± 1.9 μm, P = 0.0236, respectively). No statistical difference was found between the ONL thickness of the superior and inferior retina (50.1 ± 6.4 μm vs. 44.3 ± 3.6, P = 0.0578). Peripapillary NFL thickness showed a similar tendency as the linear scans, with the superior quadrant having the greatest thickness (91.26 ± 7.0 μm) and the inferior quadrant being the thinnest (76.42 ± 9.2 μm) (P < 0.001). Conclusions Results of our in vivo studies showed significant differences between thickness values for the superior (tapetal) and inferior (nontapetal) retinal regions.     

An ultrastructural study on the Leydig and Sertoli cells in the immature lesser mouse deer (Tragulus javanicus)

Leydig and Sertoli cells of the immature lesser mouse deer testes, obtained in East Malaysia, were observed using light and transmission electron microscopy (TEM). The testes were fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethanol, and embedded in Araldite M. Serial semi-thin sections were cut, stained with toluidine blue and observed using light microscopy. Serial ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and examined using TEM. As a result, ultrastructurally, two types of underdeveloped filament bundles were infrequently recognized in Leydig cells, but not in other testicular cells. One type was the underdeveloped bundles of actin filaments (approximately 5 nm in diameter), which were found in the nucleus of Leydig cells. The other type was the underdeveloped bundles of intermediate filaments (approximately 10 nm in diameter), which were found in the cytoplasm of Leydig cells. A multivesicular nuclear body (MNB)--specifically present in the Sertoli cell nucleus of ruminant testes--was infrequently observed. The MNB is situated in the vicinity of nuclear membrane, still in an underdeveloped stage.     

Gender differences in the rat corpus callosum: An ultrastructure study

Sexual dimorphism exists at all levels of the nervous system, from genetic, anatomical and system levels. The sexual dimorphism in the axonal content of the corpus callosum (CC) has always been controversial; hence, the aim of this study was to analyse the differences in total, myelinated and unmyelinated axons density of various regions of the CC between male and female rats. To assess that, six pairs of adult male and female rats were perfused and the CC was removed and sectioned. Four sections from different subregions of the corpus callosum that represent the genu, anterior body, posterior body, and splenium, were stained, and electron microscopic images were captured using stereological guidelines. Later, the axons density for each subregion was calculated and compared between males and females. The findings of the present study indicated region‐specific differences in the myelinated, unmyelinated or the ratio of myelinated/total axons in the CC between male and female rats.     

Ultrasonographic examination of the carpal canal in dogs

The aim of this study was to determine the course of the median nerve and its adjacent structures in the carpal canals of 8 healthy dogs by using high-frequency transducers. Before performing ultrasonography, the transverse and posteroanterior diameters as well as the perimeter of the carpus were measured at just proximal to the side of the carpal pad. The anatomical structures were then determined at two levels of the carpal canal, which were named the proximal and distal levels, on the transverse sonograms. The cross-sectional areas, perimeters and the transverse and posteroanterior diameters of the median nerve were measured at these levels. Although all the measurements were larger at the proximal level, significant differences between the proximal and distal levels were determined for the cross-sectional area, the perimeter and the transverse diameter of the median nerve. On the transverse sonogram, the deep digital flexor tendon was seen in almost the center of the carpal canal like a comma shape and also it had a small concavity on the caudal side. The superficial digital flexor tendon was seen as an ovoid shape on the transverse sonograms and it was located nearly at the posterior side of the carpal canal. Both tendons were seen as intermediate-grade echogenic structures. The median artery was located inside of the concavity of the deep digital flexor tendon. Also, the median nerve was seen at the posteromedial side of the median artery. As a result of this study, the cross-sectional areas of the median nerve ranged between 1.01-2.68 mm² at the proximal level and between 0.93-1.91 mm² at the distal level.     

Ultrastructural,Sensory and Functional Anatomy of the Northern Elephant Seal (Mirounga angustirostris) Facial Vibrissae

Vibrissae (whiskers) play a key role in underwater orientation in foraging phocids through vibrotactile sensation processing. Our aim was to evaluate the structure of northern elephant seal (NES) vibrissae by means of light (LM) and transmission electron microscopy (TEM), in order to elucidate their function. Vibrissal follicles were processed using standardized laboratory methods and LM/TEM techniques. Individual follicular axonal numbers were counted and axonal diameter measured and averaged. NES have mystacial, rhinal, supraorbital and labial vibrissae. The vibrissal follicles are histologically subdivided into a ring, upper and lower cavernous sinuses (LCS). Each vibrissa is innervated by the deep vibrissal nerve. The average number of axons per large mystacial vibrissa is 1804 (±123), rhinal 985 (±241), supraorbital 1,064 (±204) and 374 (±65) in labial vibrissa. The entire vibrissal system carries an estimated 148 573 axons, and mystacial vibrissae alone have 125 323 axons. Axonal conduction velocity for each vibrissal type is 55.26 m/s for labial, 56.58 m/s for rhinal and 35.88 m/s for mystacial vibrissae. TEM and LM revealed a plethora of mechanoreceptors within the vibrissal follicles: Merkel cell‐neurite complexes, lanceolate and pilo‐Ruffini end organs. A vast number of sensory axons projecting from the entire vibrissal system indicate that the vibrissal sensory area takes up a large proportion of phocids’ somatosensory cortex. In conclusion, NES has highly sensitive and finely tuned vibrotactile vibrissal sense organs.     

Reference values for selected ophthalmic diagnostic tests and clinical characteristics of chinchilla eyes (Chinchilla lanigera)

Objective To establish reference values for the Schirmer tear test I (STT I), the phenol red thread tear test (PRTT), the intraocular pressure (IOP) with rebound tonometry, to determine the corneal sensitivity for healthy chinchillas, and to describe clinical aspects of normal chinchilla eyes. Animals One hundred and twenty‐two eyes of 61 healthy pet chinchillas of different age and gender were investigated. Procedures A full ophthalmic exam including slit lamp biomicroscopy, ophthalmoscopy, measurement of STT I, PRTT, determination of the corneal touch threshold (CTT), and the measurement of the IOP (TonoVet®) was performed. The normal appearance of the lid, the iris, the lens, the fundus, and the optic nerve disc was evaluated. Results The results of the STT I were very low and not reliable, and the measurement was discontinued. The median value of PRTT was 14.0 mm wetting/15 s (mean 14.6 ± 3.5 mm wetting/15 s). The median CTT was 32.5 mm (mean 31.2 ± 7.0 mm) respectively 1.2 g/mm² (mean 1.5 ± 0.9 g/mm²). The median IOP was 3.0 mmHg (mean 2.9 ± 1.8 mmHg). The predominating iris color was brown. The fundus pigmentation varied. Few lens alteration were seen in otherwise healthy chinchilla eyes. Most chinchillas had myelinated discs. Optic nerve cupping was present in 62% of the animals. Conclusion Because of the small amount of tears, the PRT test is recommended for tear measurements in chinchillas. The IOP in chinchillas seems to be quiet is low in comparison to other rodents.     

Examination of the rat eye at the early stage of development with osmium tetroxide staining

This communication describes the benefit of osmium tetroxide (OsO4) staining on the examination of the eye during the early stage of organogenesis of rat embryos. The embryos were obtained by laparotomy on embryonic day 12 (ED 12) and were stained with OsO4 for examination of the ocular tissues with a binocular stereo-microscope, light microscope and scanning electron microscope. At the binocular stereo-microscopic level, the invaginated lens placode, lens pit and optic cup were clearly distinguished. The osmium-stained lens placode and the optic cup were light brown and dark brown in color, respectively. Light microscopic examination revealed that OsO4 postfixation could provide superior paraffin-embedded embryonic sections. Scanning electron microscopic examination revealed the lens pit as a round opening between the lateral nasal prominence and maxillary prominence. Thus, a rapid technique by which the ocular tissues of rat embryos can be examined under a binocular stereo-microscope was developed. This OsO4 staining method will provide a useful tool for research on organogenesis and ocular development.