Immunolocalization of myocilin protein in the anterior eye of normal and primary open-angle glaucomatous dogs.

OBJECTIVE: The presence of myocilin was investigated in a colony of Beagles, a canine model for inherited primary open-angle glaucoma (POAG). The myocilin protein was localized in the normal and glaucomatous canine eyes by immunohistochemistry and immunocytochemistry. METHODS: Paraffin- and plastic-embedded specimens from the anterior uveas of 10 Beagles with inherited glaucoma (3 months to 13 years old) and 6 age-matched normal dogs were sectioned, and were then incubated with primary antibody, rabbit polyclonal antihuman MYOC IgG, overnight at 4 degrees C. Specimens were incubated with secondary antibody with one of the following: biotinylated link followed by peroxidase-labeled streptavidin and then by substrate-chromogen for light microscopy; fluorescent marker Texas red; or 18 nm colloidal gold-labeled goat antirabbit IgG for transmission electron microscopy. RESULTS: With normal, pre- and early glaucomatous canine specimens, cell membranes of smooth muscle cells of the iris and ciliary body stained positively, as well as most resident stromal and vascular endothelial cells. The cytoplasm of cells within the nonpigmented ciliary epithelium of the ciliary body processes stained intensely, being weaker along the pars plana. Trabecular meshwork (TM) cells and surrounding extracellular matrix labeled, as well as the sclera adjacent to the angular aqueous plexus. In specimens with moderate and advanced glaucoma, greater intensity of staining was observed within TM cells and adjacent sclera, and portions of the nonpigmented epithelium of the ciliary processes. Fibrinous material labeled intensely within the posterior chamber. CONCLUSIONS: Myocilin in the normal and glaucomatous canine eye was successfully immunolocalized. These findings with regard to the normal eye are nearly identical to those previously reported in humans, and support the original hypothesis that there is an increase in both accumulation and localization of myocilin in glaucomatous canine eyes. It also supports the possibility that changes in the activity of myocilin within the aqueous humor outflow pathway of individuals with spontaneous glaucoma are associated with the rise of intraocular pressure and subsequent development of this disease, but may not be the primary event in the initial raise in intraocular pressure in POAG in the Beagle.

A new model of glaucoma filtering surgery in the rat.

PURPOSE: The most common reason for long-term failure of glaucoma filtering surgery (GFS) is scarring of the external filtering "bleb" tissues. The identification of the factors that mediate this process, as well as the development and initial testing of new therapies to limit scarring is enhanced by the use of appropriate animal models. The standard animal model for studying GFS is the rabbit but newer investigative tools that examine changes induced in biologic systems at a genetic level have made development of a rat model desirable. METHODS: Glaucoma filtering surgery was performed on 20 Sprague-Dawley rats by introducing a 30-gauge silicone cannula through a penetrating scleral tunnel, under a limbal-based conjunctival flap and suturing the conjunctiva closed. Identical GFS was performed on 3 additional rats, which underwent histologic evaluation at days 2, 5, and 11, following surgery.Fistulizing surgery was also performed on 6 Sprague-Dawley rats, for comparison, by creating a full-thickness needle sclerostomy under a limbal-based conjunctival flap and suturing the conjunctiva closed. RESULTS: Following the cannula GFS, well-elevated filtering blebs formed and these gradually failed over the course of 8 to 13 days. Needle tract sclerostomy filtering blebs formed at the site of the fistulizing surgery but rapidly failed over the course of 2 to 3 days. CONCLUSION: Cannulated filtering surgery in the rat provides a longer lasting and more predictable model than needle tract sclerostomy for studying wound healing following GFS and may facilitate the study of induced changes at the gene level.

Comparative morphology of the tapetum lucidum (among selected species).

OBJECTIVES: The phenomenon of 'eye-shine' is seen in a variety of animal species, and is generally thought to be related to the presence of an intraocular reflecting structure, the tapetum lucidum. The tapetum lucidum is a biologic reflector system that is a common feature in the eyes of vertebrates. It normally functions to provide the light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation, thereby enhancing visual sensitivity at low light levels. The tapetum lucidum is presented here according to a classification based on the location, as well as the composition, of this reflective layer. Finally, the physical and chemical properties, as well as the origins of the different tapeta lucida, are discussed and compared. METHODS: The anatomic and biochemical aspects of the tapetum lucidum in various vertebrates are examined. Morphologic observations were made from paraffin and plastic embedded specimens. Specimens were treated with traditional stains and observed by light and transmission electron microscopy. RESULTS: Some species (primates, squirrels, birds, red kangaroo and pig) do not have this structure and they usually are diurnal animals. In vertebrates, the tapetum lucidum exhibits diverse structure, organization and composition. Therefore, the retinal tapetum (teleosts, crocodilians, marsupials, fruit bat), the choroidal guanine tapetum (elasmobranchs), the choroidal tapetum cellulosum (carnivores, rodents, cetacea), and the choroidal tapetum fibrosum (cow, sheep, goat, horse) are described. CONCLUSIONS: The tapetum lucidum represents a remarkable example of neural cell and tissue specialization as an adaptation to a dim light environment and, despite these differences, all tapetal variants act to increase retinal sensitivity by reflecting light back through the photoreceptor layer. These variations regarding both its location and structure, as well as the choice of reflective material, may represent selective visual adaptations associated with their feeding behavior, in response to the use of specific wavelengths and amount of reflectance required.

Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs.

PURPOSE: To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. METHODS: Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age-matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate-chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. RESULTS: Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. CONCLUSIONS: The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.

Metallothionein expression protects against carbon tetrachloride-induced hepatotoxicity, but overexpression and dietary zinc supplementation provide no further protection in metallothionein transgenic and knockout mice.

Metallothionein and zinc have been implicated in cellular defense against a number of cytotoxic agents. With respect to the free radical-generating hepatotoxin carbon tetrachloride, conclusions about a defensive role were reached from in vitro studies, in vivo studies using inducers of metallothionein and studies using injections of pharmacological amounts of zinc. Metallothionein knockout (null) and metallothionein transgenic mice are more direct models to examine the effects of metallothionein expression on induced cytotoxicity. Similarly, zinc presented via the diet is a more physiological model than that presented via injection. We examined whether metallothionein-overexpressing mice or metallothionein knockout mice had altered sensitivity to carbon tetrachloride and whether supplemental dietary zinc reduced sensitivity to carbon tetrachloride in these genotypes. Metallothionein knockout mice produced no metallothionein and were unable to sequester additional hepatic zinc in response to elevated dietary zinc. Hepatotoxicity, as measured by serum alanine aminotransferase activity, histological analyses and hepatic thiol levels, was greater in the knockout mice than in controls 12 h after carbon tetrachloride treatment but not at later time points (up to 48 h). In contrast, metallothionein-overexpressing mice produced more metallothionein and sequestered more liver zinc than control mice, but hepatotoxicity was similar between genotypes. Supplemental dietary zinc had no effect on hepatotoxicity with either genotype. These data suggest metallothionein null mice were more susceptible to carbon tetrachloride-induced hepatotoxicity than were control mice. However, neither metallothionein overexpression nor supplemental dietary zinc provided further protection.

Microanatomy of facial vibrissae in the Florida manatee: the basis for specialized sensory function and oripulation.

Sirenians, including Florida manatees, possess an array of hairs and bristles on the face. These are distributed in a pattern involving nine distinct regions of the face, unlike that of any other mammalian order. Some of these bristles and hairs are known to be used in tactile exploration and in grasping behaviors. In the present study we characterized the microanatomical structure of the hair and bristle follicles from the nine regions of the face. All follicles had the attributes of vibrissae, including a dense connective tissue capsule, prominent blood sinus complex, and substantial innervation. Each of the nine regions of the face exhibited a distinct combination of these morphological attributes, congruent with the previous designation of these regions based on location and external morphological criteria. The present data suggest that perioral bristles in manatees might have a tactile sensory role much like that of vibrissae in other mammals, in addition to their documented role in grasping of plants during feeding. Such a combination of motor and sensory usages would be unique to sirenians. Finally, we speculate that the facial hairs and bristles may play a role in hydrodynamic reception.

Rose bengal positive epithelial microerosions as a manifestation of equine keratomycosis.

Purpose To describe the clinical appearance of corneal epithelial cell microerosions associated with keratomycosis in the horse. METHODS: Retrospective clinical study. RESULTS: Multifocal, punctate, superficial corneal opacities with positive rose bengal retention were noted in six horses with presumed 'viral keratitis'. Faint fluorescein staining was also present in three cases. Equine herpesvirus tissue culture inoculation was negative for a cytopathic effect in three cases. Aspergillus (n = 3), Curvularia (n = 1), and an unidentified fungus (n = 1) were cultured in five horses, and hyphae found on corneal cytology from the sixth. Mixed bacterial infections were present in three eyes. The eyes of two horses with Aspergillus progressed to deep melting corneal ulcers that required surgical therapy. The microerosions remained superficial, but persistent in the other four eyes. Natamycin was utilized topically in all six horses. Transmission electron microscopy from case 6 revealed mucin layer disruption, an intact corneal epithelial cell layer, and fungal attachment to degenerating epithelial cells. The visual outcome was positive in all six horses, although healing was prolonged (48.5 +/- 14.5 days on average in the horses with no surgery; 62 days on average in the two horses that required surgery). CONCLUSIONS: Complete removal or full-thickness penetration of the corneal epithelial cell barrier may not be necessary to allow fungal adherence and initiation of keratomycosis in the horse. Prior to colonization and invasion of the horse cornea, fungi may induce changes in the mucin layer of the tear film that result in or are associated with rose bengal positive microerosions of the superficial corneal epithelium. Horses with painful eyes, and eyes with superficial, multifocal corneal opacities should have their corneas stained with both fluorescein and rose bengal as fungal microerosions may stain weakly, or not at all, with fluorescein, and may thus be mistaken for presumed 'viral keratitis' of the horse.

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa.

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.

Glutamine synthetase: a key enzyme for intestinal epithelial differentiation?

BACKGROUND: We have previously shown that glutamine synthetase protein and mRNA are concentrated in the crypt region of the rat small intestine and that the activity of this enzyme is highest around the time of weaning. This anatomical location and time of peak activity are sites and periods of active enterocyte differentiation. This led to our current hypothesis that glutamine synthetase is important in the differentiation of enterocytes. METHODS: To test our hypothesis, we treated Caco-2 cells with physiologic (0.6 mM) glutamine concentrations in cell culture medium. The experimental group was treated with methionine sulfoximine, an irreversible glutamine synthetase inhibitor, and the control group with phosphate buffered saline. Three standard and well-defined markers of intestinal differentiation-sucrase-isomaltase activity, microvillus formation, and electrical impedance in transwell plates-were compared between the two groups. RESULTS: The methionine-sulfoximine-inhibited group was found to have lower sucrase-isomaltase activity, a lower density of microvilli, and lower electrical impedance values over time compared with the control group. CONCLUSION: The experimental group was found to be less differentiated by all three markers of differentiation. Therefore, glutamine synthetase is important for Caco-2 cell differentiation.

The influence of aging and low zinc nutrition on the choroid in the pig: I. The melanocyte.

The effects of low zinc nutrition and aging on central choroidal melanocytes were examined in the pig. Three populations of pigs (young, pregnant and aged), were maintained on either control (C) or low zinc (LZ) diets. Twenty-five weanling boars were sacrificed at 2, 4, 8, 10, and 12-month intervals, and nine pregnant sows and eight aged sows were sacrificed after a 6-month interval. Melanocytes of the central choroid were morphologically and morphometrically examined. The melanocyte was found to be conservative in its form, which was mostly elliptical longitudinal profile, throughout the different age populations that were fed the C diet. Morphometric observations revealed that this cell type increased in size in the oldest animals, having been 40% greater than that in the younger two populations. However, the overall percentage area occupied by melanocytes remained the same throughout all age groups. In the animals that were fed the LZ diets, a large subpopulation of choroidal melanocytes was oval to round in shape in the pregnant and aged groups. Many members of this subpopulation possessed less opaque pigment than the elliptically shaped cell. Measurements of the size and percentage area occupied in these oldest groups increased significantly. In addition to the change of size, shape and melanin opaqueness, unusually large melanosomes were consistently observed in the pregnant and aged LZ groups. Low zinc nutrition had a remarkable age-related impact on the usually quiescent melanocyte.

The influence of aging and low zinc nutrition on the choroid in the pig: II. The melanosome.

The effects of low zinc nutrition and aging on central choroidal melanosomes were examined in the pig. Melanosomes of central choroidal melanocytes were morphologically and morphometrically examined in three populations of pigs (young, pregnant and aged), that were maintained on either control (C) or low zinc (LZ) diets. In C groups, the typical melanosomes decreased in size with age, although a subpopulation of larger melanosomes occurred in the oldest group. In contrast, the melanosomes of the animals on LZ diets increased in size significantly in the adult groups. The melanosomes in the pregnant and aged groups were 65% and 30-40% greater than those of the age-matched C groups. Extremely large melanosomes were frequently encountered in adult LZ choroidal melanocytes. Melanogenesis of these large bodies included the formation of one or more outer shells. Fusion of adjacent large melanosomes was also observed. Melanolysosomal-like bodies were observed, particularly among the pigmented cells in the large blood vessel region of C and LZ adults. Melanin dynamics, i.e. its production and breakdown, occurred within choroidal melanocytes throughout much of the lifespan of the pig. This dynamic was greatly influenced by low zinc nutrition, resulting in unusual and aberrant melanin activity.

Comparison of Optisol-GS and neomycin-polymyxin B-gramicidin ophthalmic solution for corneal storage in the dog.

The objective of the research was to compare the efficacy of Optisol-GS (OGS, Bausch & Lomb Surgical, Irvine, CA, USA) with triple antibiotic ophthalmic solution (neomycin-polymyxin B-gramicidin, NPG; Bausch & Lomb, Tampa, FL, USA) in preserving the viability of corneal endothelial cells. The study subjects were thirty young to middle-aged dogs with no gross corneal pathology that had been euthanized by pentobarbital overdose for reasons unrelated to this project. Corneal tissues were harvested, analyzed, and randomly assigned to treatment groups: one of two media (OGS or NPG), and one of five storage times (1, 7, 14, 21, or 35 days). Six corneas were stored in each medium for each time period. Corneal endothelial cell viability was evaluated pre- and poststorage by vital staining (trypan blue and alizarin red S), and endothelial cell morphology was evaluated with scanning electron microscopy. Storage in NPG caused significant loss (100%) of endothelial cells after all storage times. OGS storage maintained a high level of endothelial cell viability up to 21 days (98.9% +/- 1.3% viability). A significant decrease in percentage viability was also found for OGS-stored corneas between 21 and 35 days, when endothelial cell viability decreased to 61.4% +/- 45.9%. The conclusions are that NPG storage at -20 degrees C is a very poor choice of media for corneal tissue banking if graft clarity is the goal. Storage in Optisol-GS at 4 degrees C for up to 21 days resulted in significantly higher percentages of viable endothelial cells. Optisol-GS storage should facilitate corneal preservation for canine keratoplasty patients.

Photoreceptor density of the domestic pig retina.

The spatial distribution and densities of photoreceptors in seven whole-mounted porcine retinas were studied and maps illustrating photoreceptor topography were constructed. Total photoreceptor densities ranged from to 83 000 to 200 000 cells/mm2, with a mean of 138 500 cells/mm2. Cone densities ranged from 39 000 (area centralis) to 8500 cones/mm2 (peripherally), with a mean of 16 400 cones/mm2. Rod:cone ratios ranged from 3:1 centrally to 16:1 peripherally, with a mean ratio of 8:1. Averaged photoreceptor densities are greatest (166 000 cells/mm2) within the central inferior retina, and regional differences in rod:cone ratios were found. Cone densities are increased in a broad region dorsal to the optic disk, extending both nasally and temporally. This region is believed to represent the area centralis. Cone densities gradually decrease and taper towards the periphery and inferior retina as rod:cone ratios increase. In addition to the many anatomic and ultrastructural similarities to the human eye, this study illustrates similarities within the photoreceptor mosaic of these two species and supports the use of the pig retina as a model for human/animal research.

Use of the Internet for long-term clinical follow-up.

Use of the Internet for patient-specific consultation across international boundaries has been demonstrated. This report describes the efforts of Baylor College of Medicine and NASA to conduct a telemedicine consultation with Moscow, Russia. Consultation between Russian and American physicians was performed over the Internet with a combination of real-time and store-and-forward techniques. The clinical focus involved a 65-year old Russian scientist who had undergone mitral valve replacement in the United States 5 years earlier. Development of new activity-related chest pain, dyspnea, and intermittent atrial fibrillation led to a consultation with his American cardiologist and cardiac surgeon. Real-time video was supplemented with telephone voice communication to overcome bandwidth limitations. Prior to the video link, the patient's recent history and clinical data were made available via the Internet using file transfer protocol (FTP). The patient's medications, new electrocardiographic findings, and activity status were reviewed. Specific clinical recommendations were made as a result of this telemedicine consultation. This case illustrates the technical factors, clinical implications, and confidentiality issues related to using the Internet for telemedicine consultations and demonstrates that the Internet may provide an alternative means for long-term clinical follow-up of patients.

Photoreceptor outer segments in aqueous humor from dogs with rhegmatogenous retinal detachments.

OBJECTIVE: To determine whether photoreceptor outer segments can be found in aqueous humor from dogs with rhegmatogenous retinal detachment (RRD). DESIGN: Case series. ANIMALS: 4 dogs with unilateral RRD, 2 dogs with bilateral RRD, 1 dog with unilateral non-RRD, and 1 dog with glaucoma without retinal detachment. PROCEDURE: Aqueous humor samples were fixed in 2% glutaraldehyde and examined by means of transmission electron microscopy. RESULTS: Outer segments were found in aqueous humor from 7 of 8 eyes with RRD but were not found in aqueous humor from dogs with non-RRD or glaucoma. CLINICAL IMPLICATIONS: Photoreceptor outer segments may move into the anterior chamber of eyes with RRD.

Immunohistochemical localization of cysteine-rich intestinal protein in rat small intestine.

Cysteine-rich intestinal protein (CRIP) is a LIM (cysteine-rich motif of leu-11, isl-1, and mec-3 genes) domain protein with a double zinc finger motif. The protein is abundantly expressed in the intestine, peritoneal macrophages, and peripheral blood mononuclear cells. The function of CRIP is not known. The purpose of this study was to determine the cellular distribution of CRIP in rat intestine, as an initial step toward eventual determination of a function. Immunohistochemical and immunogold labeling electron microscopy using a purified polyclonal rabbit antibody to a synthetic peptide representing a zinc finger domain of rat CRIP were carried out on sections of rat duodenum. Western blotting was used to detect signal specificity of the antibodies. These immunohistochemical and electron microscopy studies showed particularly high abundance of CRIP in the cytoplasmic granules of Paneth cells of the intestine. Some evidence of CRIP expression was also found in cells of the villus tip, but abundance was less than that found in the Paneth cells. The localization of CRIP in Paneth cells and its presence in mononuclear cells suggests that CRIP may be involved in host defense mechanisms and/or tissue differentiation/remodeling processes common to these cell types.

Differential expression of cysteine-rich intestinal protein in liver and intestine in CCl4-induced inflammation.

Cysteine-rich intestinal protein (CRIP) is a double zinc finger (LIM domain) protein that is developmentally regulated but has an unknown function. CRIP is highly expressed in the intestine, but expression is low in liver. To determine if CRIP expression is regulated under altered physiological status, we used CCl4-induced injury as a model to produce hepatic injury and systemic effects associated with inflammation. Since CRIP is a zinc finger protein and zinc decreases the hepatic response to CCl4, the effect of supplemental dietary zinc (300 mg/kg diet) was also examined. Our results show that this supplemental level of dietary zinc did not affect the index of hepatic injury (plasma alanine aminotransferase), indicating zinc did not have a protective effect. Liver CRIP mRNA increased with CCl4 and CRIP protein was shown by immunohistochemistry to be localized in hepatocytes near the vascular supply. In the intestine, CCl4 caused a transient decrease in CRIP mRNA, but supplemental dietary zinc treatment prevented this decrease. These current results show that CRIP expression changes in response to cellular damage due to acute hepatic injury and are consistent with a functional role for CRIP in proliferation, differentiation, or turnover.