Don Samuelson

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

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.

Transmission electron microscopy studies in an experimental model of canine atopic dermatitis

Impairment of skin barrier function has been hypothesized in canine atopic dermatitis (AD). In this prospective, controlled study, the ultrastructure of the upper epidermal layers was investigated using an experimental model of canine AD. Seven atopic Beagles sensitized to Dermatophagoides farinae and four healthy Beagles were used as controls. Both normal and atopic dogs were challenged with D. farinae for 3 days. Clinical signs were scored and skin biopsies were taken from the inguinal area before and 3 days after allergen exposure. Samples were processed to enhance lipid visibility and evaluated by Transmission Electron Microscopy. Emphasis was placed on evaluation of the lipid lamellae (LL), and lamellar bodies (LB) of the stratum corneum.After allergen challenge, atopic Beagles developed severe pruritic dermatitis while no skin lesions were noted in the controls. Ultrastructurally, before allergen challenge, atopic Beagles displayed focally severe abnormalities in LL organization and wider intercellular spaces containing abnormal lipid material. In atopic Beagles, LBs were frequently found inside corneocytes while this finding was not observed in the controls. After allergen challenge, further increase of intercellular spaces was observed in the stratum corneum of atopic Beagles while no appreciable changes were observed in the normal dogs. Intercellular spaces in atopic Beagles were filled with abundant amounts of abnormal lipid material and highly disorganized LL. It is concluded that baseline differences in the ultrastructure of the skin exist between normal and experimentally sensitized atopic Beagles and that these changes are aggravated by allergen challenge and the resulting flare-up of dermatitis.

Chronic toxicity of dichloroacetate: Possible relation to thiamine deficiency in rats

The chronic use of dichloroacetate (DCA) for diabetes mellitus or hyperlipoproteinemias has been compromised by neurologic and other forms of toxicity. DCA is metabolized to glyoxylate, which is converted to oxalate and, in the presence of adequate thiamine levels, to other metabolites. DCA stimulates the thiamine-dependent enzymes pyruvate dehydrogenase and alpha-ketoacid dehydrogenase. We postulated that the neurotoxicity from chronic DCA administration could result from depletion of body thiamine stores and abnormal metabolism of oxalate, a known neurotoxin. For 7 weeks, rats were fed ad lib. Purina chow and water or chow plus sodium DCA (50 mg/kg or 1.1 g/kg) in water. A portion of the DCA-treated animals also received intraperitoneal injections of 600 micrograms thiamine three times weekly or 600 micrograms thiamine daily by mouth. Thiamine status was assessed by determining red cell transketolase activity and, in a blinded manner, by recording the development of clinical signs known to be associated with thiamine deficiency. At the 50 mg/kg dose, chronic administration of DCA showed no clinical toxicity or effect on transketolase activity. At the 1.1 g/kg dose, however, DCA markedly increased the frequency and severity of toxicity and decreased transketolase activity 25%, compared to controls. Coadministration of thiamine substantially reduced evidence of thiamine deficiency and normalized transketolase activity. Inhibition of transketolase by DCA in vivo was not due to a direct action on the enzyme, however, since DCA, glyoxylate, or oxalate had no appreciable effects on transketolase activity in vitro. After 7 weeks, plasma DCA concentrations were similar in rats receiving DCA alone or DCA plus thiamine, while urinary oxalate was 86% above control in DCA-treated rats but only 28% above control in DCA plus thiamine-treated animals. No light microscopic changes were seen in peripheral nerve, lens, testis, or kidney morphology in either DCA-treated group, nor was there disruption of normal sperm production in the DCA-treated group. We conclude that stimulation by DCA of thiamine-requiring enzymes may lead to depletion of total body thiamine stores and to both a fall in transketolase activity and an increase in oxalate accumulation in vivo. DCA neurotoxicity may thus be due, at least in part, to thiamine deficiency and may be preventable with thiamine treatment.

Immunohistochemical evaluation of filaggrin polyclonal antibody in atopic and normal beagles.

In human atopic dermatitis (AD), impairments in skin barrier function are emphasized and hypothesized to increase risk of allergic sensitization. Filaggrins, crucial proteins for keratinization, are decreased in lesional and nonlesional human atopic skin. As canine AD shares numerous similarities with the human counterpart, this study aimed to evaluate a polyclonal antibody against human filaggrin in atopic beagles sensitized to house dust mites (HDM) and normal healthy dogs. The effects of HDM exposure on immunostaining and clinical signs were evaluated in both groups. Positive immunohistochemical staining with anti-filaggrin antibody was evaluated both objectively and subjectively by two blinded investigators. Pearson correlation test showed significant correlation between objective and subjective scores, both at baseline and after allergen exposure (r = 0.80; P = 0.0017 and r = 0.75; P = 0.013 respectively). Analysis of variance showed significant effect of time (P = 0.01) with immunostaining being higher in baseline samples than after HDM exposure. It also showed a significant group x time interaction (P = 0.02) with immunostaining not changing significantly over time in atopic dogs, while decreasing in normal dogs after HDM exposure. An independent t-test showed that, at baseline, atopic beagles had significantly less positive immunostaining than controls (P = 0.009) and that, after HDM exposure, there was no significant difference between groups. No correlation existed between clinical scores and immunostaining. In atopic dogs immunostaining was characterized by faint granular staining, while normal samples showed discrete intense staining. Moreover, immunostaining was present in all epidermal layers in many samples, suggesting cross-reactivity of the antibody used with other epidermal proteins besides filaggrin.

AN OUTBREAK OF FUNGAL DERMATITIS AND STOMATITIS IN A FREE-RANGING POPULATION OF PIGMY RATTLESNAKES (SISTRURUS MILIARIUS BARBOURI) IN FLORIDA

Between September 1997 and March 1998, a severe skin, eye, and mouth disease was observed in a population of dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), at the Lake Woodruff National Wildlife Refuge in Volusia County, Florida (USA). Three affected pigmy rattlesnakes were submitted for necropsy. All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues. Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomoris methenamine silver. Samples of lesions were cultured for bacteria and fungi. Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp. While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found. In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site. Masses from six of these snakes were biopsied in the field. Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains. Analysis of a database representing 10,727 captures in previous years was performed after the 1998 outbreak was recognized. From this analysis we determined that 59 snakes with clinical signs similar to those seen during the 1998 outbreak were documented between 1992 and 1997. This study represents the first documented report of a mycotic disease of free-ranging snakes.

Histomorphometry of the optic nerves of normal dogs and dogs with hereditary glaucoma.

The beagle dog with hereditary primary open-angle glaucoma, unlike other animal models of human glaucoma, possesses a slowly progressive, sustained elevation of intraocular pressure. The effects of this insidious elevation in intraocular pressure on the axons of the optic nerves of three beagles at early stages of glaucoma and two beagles with advanced signs of glaucoma were compared to the optic nerves of four age-matched normal dogs. Plastic embedded optic nerve cross-sections (1 micron) 1 mm posterior to the lamina cribrosa were osmicated and stained with Toluidine Blue. Axons from 0.2 to > 2.0 microns in diameter were counted and measured in 16 cross-sectional regions of equal size within the whole optic nerve using a computerized image analysis system. The mean optic nerve axon diameters in the normal, early glaucomatous, and advanced glaucomatous dogs were 1.53, 1.25 and 1.13 microns respectively. The average total optic nerve axon count in the normal dogs was 148,303. Approximately 16% of the total axonal fibers were counted in each nerve. The counts of optic nerve axons 2.0 microns or greater in diameter were reduced by up to 60% in the central regions of the optic nerves of affected beagles. The large diameter axons of the peripheral optic nerve of the beagle dogs with glaucoma were more resistant to the elevated intraocular pressure. The counts of axons > 0.6 to 0.8 micron in diameter were significantly increased in glaucomatous beagles.

Asci of the Pezizales. IV. The Apical Apparatus of Thelebolus

Light- and electron-microscopic examinations were performed on the ascal walls of Thelebolus microsporus, T. crustaceus, T. polysporus, and T. stercoreus with special emphasis on the development, cytochemistry, and morphology of the apical apparatus formed in each species. In all species the ascus differs in size, shape, and number of ascospores, but the apical apparatus is notably similar. Wall structure most closely resembles that of the bitunicate ascus. Using Congo red and acid fuchsin for light microscopy and silver methenamine for electron microscopy, we observed that the ascal wall in T. polysporus and T. stercoreus has two layers, each with two strata. The inner layer of the wall in all species consists of microfibrils in a banded pattern. Major development of the apical apparatuses occurs during early ascosporogenesis, differing from that in operculate representatives. Ascal dehiscence occurs in a modified jack-in-the-box manner. Thelebolus does not belong in the Pezizales.

Long-term effect of retinal ganglion cell axotomy on the histomorphometry of other cells in the porcine retina.

PURPOSE To determine the effect of retinal ganglion cell axotomy on the thickness of inner plexiform, inner nuclear, and outer plexiform layers, as well as the densities of short- and middle-to-long-wavelength cones, in the porcine retina. METHODS Unilateral retinal ganglion cell axotomy was performed in seven domestic pigs by either surgical optic nerve section or peripapillary argon laser photocoagulation. Damage to the retinal vasculature was ruled out with fluorescein angiography. Histologic examination of the retinal tissue was performed nine months later. Cone densities were determined immunohistochemically with the anti-visual pigment antibodies COS-1 and OS-2. Image analysis of semithin retinal cross sections was used to measure the thickness of the retinal layers. The effect of axotomy was quantified by optic nerve axon counts and estimations of retinal ganglion cell counts. The data were compared between the eyes with axotomy and the contralateral normal eye using the nonparametric Wilcoxon rank sum test. RESULTS Treatment of the peripapillary retina with the argon laser resulted in a median decrease in axon counts and retinal ganglion cell density estimates of 31%. No optic nerve axons and cells resembling retinal ganglion cells were found in the eyes with transected optic nerves. There was no significant difference in either the thickness of any retinal layers or cone densities between axotomized and normal control eyes. CONCLUSION No signs of retrograde transsynaptic degeneration were observed in porcine retinas nine months after retinal ganglion cell axotomy.

Gastric and Colonic Zinc Transporter ZIP11 (Slc39a11) in Mice Responds to Dietary Zinc and Exhibits Nuclear Localization

Zinc transporters have been characterized to further understand the absorption and metabolism of dietary zinc. Our goal was to characterize zinc transporter Slc39a11 (ZIP11) expression and its subcellular localization within cells of the murine gastrointestinal tract of mice and to determine if dietary zinc regulates ZIP11. The greatest ZIP11 expression was in the stomach, cecum, and colon. Both Zip11 mRNA and ZIP11 protein were shown to be downregulated during dietary zinc restriction (<1 mg Zn/kg) in the murine stomach tissue but were unaffected in the colon. Acute repletion with zinc did not restore Zip11 mRNA levels in the stomach. Immunohistochemistry (IHC) revealed high ZIP11 levels in the lower regions of gastric glands and parietal cells of the stomach. IHC analysis of the colon showed a marked ZIP11 abundance within the cytoplasm of the colonic epithelial cells. IHC also showed an increase in ZIP11 expression in the colon during zinc restriction. There is a robust abundance of ZIP11 in the nuclei of cells of both stomach and colon. Our experiments suggest that when dietary zinc intake is compromised, the colon may increase zinc transporter expression to improve the efficiency for absorption via increased expression of specific zinc transporters, including ZIP11 and also zinc transporter Slc39a4. In conclusion, ZIP11 is highly expressed within the murine stomach and colon and appears to be partially regulated by dietary zinc intake within these tissues. ZIP11 may play a specialized role in zinc homeostasis within these tissues, helping to maintain mucosal integrity and function.