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.

Anatomic features of the cetacean globe.

OBJECTIVE: To provide measurements of globe dimensions and describe morphological characteristics of the cetacean globe with an emphasis on Bowman's layer and encapsulated sensory corpuscles (ESC) for available cetacean species. ANIMAL STUDIED: Cetacean globes housed at the Comparative Ocular Pathology Laboratory of Wisconsin from various odontocete and two mysticete species. PROCEDURE: Measurements were taken from formalin fixed globes and images of formalin fixed globes with embedded rulers. Histological sections of globes were used to count ESC and measure Bowman's layer. RESULTS: The horizontal diameter of the globe was longer than the vertical diameter. The posterior sclera was thick, causing the internal axial length (and therefore the optical axis) to be shorter than the vertical diameter. The cornea was composed of an epithelium, Bowman's layer, collagenous stroma, thin Descemet's membrane and endothelial layer. Bowman's layer was present in all specimens except one Kogia breviceps. The thickness was variable, with the acellular layer thickest in Tursiops truncatus and thinnest in Kogia sp. The iris was well vascularized and muscled while the ciliary body lacked musculature, but retained vasculature. Single and clustered ESC were found in the anterior uvea, sclera surrounding the anterior uvea, trabecular meshwork, or some combination of these locations. They were often regionally grouped and varied from 0 to 21. There were three species where no ESC were found, L. borealis, D. capensis, and S. bredanensis, but the presence of these corpuscles cannot be ruled as only one section of the globe was analyzed.

Light microscopic evaluation and scanning electron microscopic analysis of horse eyes following deep anterior lamellar keratectomy.

OBJECTIVE To describe the technique of deep anterior lamellar keratoplasty (DALK) with Descemet's membrane (DM) exposure in horse eyes. Also, to compare the efficacy and safety of viscodissection and big-bubble techniques for DALK. ANIMALS STUDIED Thirty-four ex vivo horse eyes. PROCEDURE Deep anterior lamellar keratoplasty was performed in 34 ex vivo horse eyes. Two groups (Group V--viscodissection--2% sodium hyaluronate; Group A--air--big-bubble) of 17 eyes were studied. Other than the substance used, the surgical technique was similar for both groups. Nonperforated eyes were submitted for light microscopic histologic evaluation and scanning electron microscopic (SEM) analysis. RESULTS Group V--Perforations occurred in 18% of the eyes during surgery. Light microscopy revealed exposure of DM in 28% of the eyes with mean thickness of the remaining stroma being 70.4 µm. Group A--Perforations occurred in 42% of the eyes. Light microscopy revealed exposure of DM in 60% of the eyes with mean thickness of the remaining stroma being 23.3 µm. No significant differences in safety, efficacy and thickness of the remaining stroma (including all eyes or excluding those with DM exposure) were observed. SEM of the surgical site revealed a more even surface in those eyes with DM exposure compared to eyes with thicker remaining stroma in both groups. CONCLUSIONs We describe two DALK techniques (viscodissection and big-bubble) for use in horses. No significant differences in safety, efficacy and thickness of the remaining stroma were observed. However, a nonsignificant trend toward the big-bubble technique being more efficacious but less safe was observed.

Prevention of ocular scarring after glaucoma filtering surgery using the monoclonal antibody LT1009 (Sonepcizumab) in a rabbit model.

PURPOSE: Excessive scarring leading to failure of the filtering bleb continues to be a major problem after glaucoma filtration surgery. This study examines the antifibrotic effects of the anti-S1P monoclonal antibody LT1009 (Sonepcizumab) in prolonging bleb survival in a rabbit model of glaucoma filtering surgery. METHODS: The frequency of LT1009 dosage was determined initially using an enzyme-linked immunosorbent assay assay measuring LT1009 eye tissue retention in 6 New Zealand White rabbits. A further 21 New Zealand White rabbits underwent glaucoma filtering surgery. Bleb tissues were observed and compared clinically and histologically. The duration of bleb elevation was compared among LT1009, balanced saline solution (BSS) negative control, and mitomycin-C (MMC)-positive control. RESULTS: The mean duration of bleb survival was 28.5±8.5 days for rabbits receiving injections of LT1009, 21.0±5.6 days for those receiving injections of BSS, and 33.8±5.6 days for rabbits receiving MMC. Analysis of variance with post hoc testing suggests a statistically significant trend of improvement in bleb duration for LT1009 when compared with BSS controls. Nonpainful, upper eyelid edema was noted after 5 injections of LT1009, which resolved over a 10-day period. MMC eyes developed avascular conjunctivas with areas of thinning and sparse cellularity, whereas the conjunctiva of LT1009 and BSS eyes remained relatively normal. CONCLUSIONS: The monoclonal antibody LT1009 demonstrated a longer duration of bleb elevation than BSS control without adverse conjunctival effects associated with MMC. However, after multiple doses LT1009 use was associated with short-term upper eyelid edema.

Comparison of single versus multiple injections of the protein saratin for prolonging bleb survival in a rabbit model.

PURPOSE: We compared the anti-fibrotic effects of single versus multiple postoperative injections of saratin following glaucoma filtration surgery (GFS) in the rabbit model. METHODS: The experiment was in two parts. To determine the optimal frequency for postoperative therapy, seven New Zealand White (NZW) rabbits received an injection of saratin under the superior conjunctiva bilaterally, and ocular tissue concentration was determined using Western blot and bicinchoninic acid (BCA) assay. Next, 32 additional NZW rabbits underwent filtration surgery and received either single or multiple-dose saratin treatments. Mitomycin-C (MMC) and balanced saline solution (BSS) treatment acted as positive and negative controls, respectively. RESULTS: Rabbits receiving only one perioperative saratin injection had a mean bleb survival time of 29.8 ± 5.3 days, while those receiving multiple (either 3 or 5+) injections of saratin had mean bleb survival times of 26.3 ± 8.1 and 26.4 ± 4.2 days, respectively. Analysis of variance with post-hoc testing showed the single injection group had a statistically favorable effect on bleb survival duration compared to BSS controls and was not significantly different from MMC. The conjunctivas of the saratin-treated rabbits did not show the thinning or avascularity that was seen in the MMC treatment group. Rabbits receiving more than three injections of saratin suffered temporary conjunctival redness and two rabbits had upper eyelid edema. CONCLUSIONS: A single postoperative injection of saratin was able to prolong the duration of bleb elevation when compared to BSS controls. Additional treatments of saratin seemed to reduce effectiveness and caused short-term eye inflammation.

Prevention of ocular scarring post glaucoma filtration surgery using the inflammatory cell and platelet binding modulator saratin in a rabbit model.

CLINICAL RELEVANCE: Late complications can occur with use of current antimetabolites to prevent scarring following glaucoma filtration surgery (GFS). Safer, more targeted, anti-fibrosis agents are sought. OBJECTIVES: The protein saratin has been shown to exhibit anti-fibrotic and anti-thrombotic properties in response to injury, but had not been used for glaucoma surgery. The goal of this study was to compare the efficacy of saratin with that of the widely accepted mitomycin-C (MMC) in prolonging bleb survival following GFS in the rabbit model. Two saratin delivery routes were compared; a single intraoperative topical application versus a combination of intraoperative topical application with two additional postoperative injections. METHODS: Twenty-four New Zealand White rabbits underwent GFS and received either intraoperative topical saratin, intraoperative topical saratin plus two injections on post-operative days 4 and 8, balanced saline solution (BSS), or MMC. The bleb tissues and their elevation durations were compared based on clinical and histological findings. RESULTS: Rabbits receiving topical+injections of saratin had a mean bleb survival of 33.6±8.5 days, significantly higher than the negative BSS controls, which averaged 17.4±6.0 days (p = 0.018). No improvement over BSS was seen for rabbits receiving topical saratin only (15.5±4.8 days, p = 0.749). Rabbits receiving saratin did not develop bleb avascularity and thinning associated with MMC treatment and there were no apparent clinical signs of toxicity. CONCLUSIONS: Treatment with a single intraoperative topical application plus two additional postoperative injections significantly prolonged bleb elevation comparable to MMC, but without toxicity; however, topical application alone was ineffective.

Periocular anterior adnexal anatomy and clinical adnexal examination of the adult Asian elephant (Elephas maximus).

Formalin preserved ocular-associated anterior adnexa tissues from five necropsied Asian elephants (Elephas maximus) were dissected with attention to the palpebrae, conjunctiva, nictitating membranes, nasolacrimal ducts, and periocular glandular tissues. Gross and histologic examination revealed that lacrimal and tarsal glands were not present. Evidence of the lacrimal drainage apparatus, including lacrimal punctae or any remnant of lacrimal sacs, was also absent. In contrast, well-developed sebaceous glands associated with accessory hairs along the palpebrae were exceptionally abundant. Mixed-secreting accessory lacrimal glands were noted in the deep stroma posterior to the tarsus of both palpebrae and the gland of the nictitating membrane. Apparently, the Asian elephant has developed a novel tear system in the absence of lacrimal and tarsal (meibomian) glands. Clinical examinations and bacterial cultures of the visible periocular tissues were performed on eight living adult Asian elephants to confirm the postmortem anatomic findings and provide guidance to the clinician during examination of the elephant conjunctiva.

Microanatomy of the anterior uveoscleral outflow pathway in normal and primary open-angle glaucomatous dogs.

OBJECTIVE: To compare the morphology of the uveoscleral (US) outflow pathway in normal and glaucomatous canines. ANIMALS STUDIED: 10 normal beagles, 10 beagles with inherited primary open-angle glaucoma, 4 cocker spaniels with spontaneous glaucoma. PROCEDURES: Formalin-preserved globes were sectioned tangentially and sagittally and treated with H&E, Masson's trichrome, or elastin stains or analyzed by immunohistochemistry to visualize smooth muscle actin. Tissues associated with the US pathway were observed and compared using light microscopy. RESULTS: Tangentially oriented sections clearly revealed spaces for the transport of aqueous humor at the junction of the posterior iridocorneal angle (ICA) and anterior ciliary body musculature (CBM). Within the external anterior-most of the US pathway, the supraciliary space, distinct connective tissue cords and smooth muscle pegs fastened the ciliary body to the adjacent sclera. Compared to normal controls, glaucomatous eyes developed a robust scleral elastic sheath at the junction between the posterior ICA and the anterior CBM. In advanced glaucomatous beagles and cocker spaniels, a large amount of melanophores were seen in the US pathway and surrounding vasculature. Within the C8M of glaucomatous specimens, the smooth muscle bundles appeared fewer and separated by elastic-rich ECM. Structures of the US pathway changed little with age. CONCLUSIONS: The anterior portion of the canine US pathway is well defined and appears to be altered little with age. However with glaucoma, changes of the US pathway were associated with its the elastic components, as well as the accumulation of melanophores. Collectively, these changes may have an effect on US outflow and, subsequently, aqueous humor dynamics.

Examination of the anterior uveoscleral pathway in domestic species.

OBJECTIVE: To investigate the uveoscleral (US) pathway in the normal eyes of four domestic spp.: the cat, pig, cow and horse by examining the comparative anatomical structure of anterior US pathway. ANIMALS STUDIED: Four cats, ten pigs, four cows, eight horses. PROCEDURES: Formalin-preserved specimens from anterior uveas of the cat, pig, cow and horse were embedded and serially sectioned sagittally and tangentially and stained with H&E, Masson's trichrome, smooth muscle actin immunolabel, or elastin stain. RESULTS: Spaces between the endings of the outer anterior ciliary body musculature (CBM) formed avenues for the beginning of the US pathway and varied in the amount of extracellular matrix (ECM) material being most developed in the pig. In the cow, other anterior muscle bundles attached the CBM to the sclera concomitant with the presence of an anterior elastic sheath. In the horse, these muscle bundles were connected to branching connective tissue trabeculae within the US pathway that were attached radially to the sclera. In the cat, muscle bundles were more abundant and formed a fine meshwork of trabecular associations with the posterior ICA. Supraciliary development was most pronounced in the horse and least in the pig. CONCLUSION: All species possessed clearly developed and unique US pathways. The outermost muscle bundles of the CBM appeared to have close interaction with the US pathway and the degree of these muscle associations differed with species. The species specific anatomical variations within the US pathway could play a pivotal role in the variability of aqueous outflow along this pathway.

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.

Light and electron microscopic evaluation of canine corneal endothelium following CO2 photokeratotomy.

OBJECTIVE: To determine using light and scanning electron microscopy if treatment with CO2 photokeratotomy alters the corneal endothelium in healthy dogs. PROCEDURE: Eight surgery laboratory dogs were determined to be free of ocular abnormalities. Under general anesthesia, the left eye of each dog was treated in a quadrant from 12 to 3 o'clock with the CO(2) laser in a defocused mode. The right eye served as a control. There were four treatment groups, each with 2 dogs: group 1 (2 W, 0.1 J/s, 0.8 mm tip), group 2 (3 W, 0.3 J/s, 0.8 mm tip), group 3 (2 W, 0.04 J/s NovaScan), group 4 (3 W, 0.06 J/s, NovaScan). The 0.8 mm tip delivered a power density of 382 W/cm(2) or 573 W/cm(2), at 2 or 3 watts respectively. The NovaScan handpiece delivered a power density of 30 W/cm(2) or 40 W/cm(2), at 2 or 3 Watts respectively. Following euthanasia, right and left corneas including a 2-mm scleral rim were harvested and fixed in commercial grade Karnovsky's fixative. One piece of cornea was processed routinely, embedded in Embed 812 resin, sectioned at 1 um, stained with toluidine blue and evaluated with the light microscope. A separate piece of each cornea was routinely processed and examined with a JEOL 6400 scanning electron microscope (SEM) at 20 KV. RESULTS: No changes in endothelial cell morphology were detected by light microscopy in the sections examined. SEM indicated normal endothelial cell morphology in control eyes with presumed artifactual changes at the corneal free margin (4/8 eyes). Multiple punctate to linear regions of endothelial cell destruction were observed in 6/8 laser-treated corneas. A significant increase in corneal thickness ranging from 1.90 to 37.28% was observed in all laser treated corneas. This increase in thickness correlated linearly with the degree of endothelial damage. Ultrastructural findings also correlated with postoperative clinical findings. CONCLUSION: CO2 laser photokeratotomy alters corneal endothelial cell morphology and thickness.

Unravelling the skin barrier: a new paradigm for atopic dermatitis and house dust mites.

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease caused by complex interactions between genetics and environmental factors. In human beings, impairment of the skin barrier is demonstrated and thought to be responsible for enhanced penetration of allergens and increased risk for allergic sensitization. Once inflammation is triggered, further impairment of the skin barrier occurs, leading to self-perpetuating cycles of sensitizations. Canine AD appears to share many similarities with the human counterpart, clinically and immunologically. It is hypothesized that a primary defect of skin barrier function also exists in subsets of atopic dogs (e.g. in an experimental model using high IgE-producing beagles), particularly in young dogs, and in sites predisposed to the development of lesions. This impairment is present in clinically normal skin, worsens with development of lesions and can be quantified by measurement of transepidermal water loss. Therefore, the distribution of lesions in AD may be linked to a primary skin barrier defect in those sites and not simply due to contact with allergens, and increased susceptibility to penetration of allergen may exist early in life. Ultrastructurally, transmission electron microscopy reveals that clinically normal skin in atopic dogs has abnormalities in lamellar body secretion and extracellular lamellar bilayer structure when compared with normal dogs. Development of lesions worsens these changes (e.g. widening of intercellular spaces, release of lamellar bodies, and disorganization of lipid lamellae). It is proposed that the paradigm of canine AD as primarily due to immunologic aberration ('inside/outside') should be shifted to include a primary defect in barrier function ('outside/inside').

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.

Immunocytochemical localization of smooth muscle actin-containing cells in the trabecular meshwork of glaucomatous and nonglaucomatous dogs.

PURPOSE: It has been recently demonstrated that trabecular meshwork (TM) cells within the canine iridocorneal angle (ICA) contain smooth muscle actin (smA) and possess contractile abilities that probably alter aqueous outflow. As the number of trabecular meshwork cells in glaucomatous canine eyes have been found to be less than those in age-matched nonglaucomatous eyes, we hypothesize that the sub-population of TM cells that contain smooth muscle actin will also decline with age. We also hypothesize that a greater loss of these cells will be observed in glaucomatous eyes than in nonglaucomatous eyes of the same age. In the present study the ICA of 17 glaucomatous and eight nonglaucomatous eyes were examined for the presence of smA-containing TM cells. MATERIAL AND METHODS: Five-micron sagittal sections of each whole globe were immunolocalized for smooth muscle actin. Positive and negative controls were performed concomitantly. RESULTS: Labeling was observed in the meshwork of 10 out of the 17 glaucomatous eyes, distributed across all of the age groups represented, including eyes with primary and secondary glaucoma. Smooth muscle actin labeling was observed in the TM of 7 out of 14 eyes with closed-angle glaucoma. Positive immunoreaction was observed in 3/3 eyes with open ICAs. Labeling of smooth muscle actin was observed in the anterior part of the meshwork in only 4 of the 17 glaucomatous eyes, each having had secondary glaucoma. There were no eyes in which label was observed exclusively in the anterior portion of the meshwork. Labeling was most consistently observed in the outer, posterior uveal TM and the inner, posterior corneoscleral TM. All of the eight nonglaucomatous eyes showed greater labeling in both area and intensity than the glaucomatous eyes of the same age. CONCLUSION: It was concluded that smooth muscle actin-cell loss is associated with age in canine eyes and that this loss is more severe in glaucomatous eyes.

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.

Three-dimensional reconstruction of the angioarchitecture of the ciliary body of the West Indian manatee (Trichechus manatus).

OBJECTIVE: To examine the angioarchitecture of the ciliary body in the West Indian manatee (Trichechus manatus), through the use of three-dimensional reconstruction. PROCEDURE: Specimens from West Indian manatee were preserved in 10% buffered formalin, embedded in paraffin, serial sectioned and stained by Masson trichrome for light microscopic three-dimensional reconstruction and evaluation. RESULTS: The network of blood vessels in the ciliary processes of the West Indian manatee is fed by the major arterial circle that lies mostly near the base of the iris. The branching arterioles give rise to a capillary-sinusoidal bed that extends internally along each process, emptying into two sets of veins, one being elevated. The elevated and nonelevated veins join posteriorly before emptying into the choroidal venous system. CONCLUSIONS: The angioarchitecture of the ciliary body of the West Indian manatee is clearly unique when compared to those previously examined in land mammals. Three-dimensional reconstruction of paraffin sections is an effective means to evaluate vascular patterns in ocular specimens, especially those unavailable for corrosion casting.

Comparative anatomy of the ciliary body of the West Indian manatee (Trichechus manatus) and selected species.

OBJECTIVE: To examine the anatomy of the ciliary body in the West Indian manatee (Trichechus manatus), paying close attention to its vascularization and to compare to those of its distant relative, the African elephant (Loxodonta africana), the amphibious hippopotamus (Hippopotamus amphibius) and the aquatic short-finned pilot whale (Globicephala macrorhynchus). PROCEDURE: Specimens from each species were preserved in 10% buffered formalin, and observed stereomicroscopically before being embedded in paraffin, sectioned and stained by Masson trichrome, hematoxylin and eosin, and periodic acid-Schiff for light microscopic evaluation. RESULTS: The network of blood vessels in the ciliary processes of the West Indian manatee appear to have an intricate pattern, especially with regard to venous outflow. Those of the elephant are slightly less complex, while those of the hippopotamus and whale have different vascular patterns within the ciliary body. Musculature within the ciliary body is absent within the manatee and pilot whale. CONCLUSIONS: In general, there appears to be a direct relationship between the increased development of vasculature and the loss of musculature within the ciliary bodies of the aquatic and amphibious mammals presently studied. Specifically, the ciliary body of the West Indian manatee has a comparatively unique construction, especially with regard to its vasculature.

Corneal vascularization in the Florida manatee (Trichechus manatus latirostris) and three-dimensional reconstruction of vessels.

The cornea of the Florida manatee is unique and unusual in its anatomy in that blood vessels have been found throughout. In all other animal species, this is considered a pathological condition impeding vision, and is usually caused by injury or trauma. The purpose of this study was to more clearly describe corneal vascularization by examining the architecture through three-dimensional reconstruction in order to find possible patterns in size, distribution, and location of blood vessels relative to gender, age, location, and season. Twenty-six eyes from 22 individuals were prepared for histologic examination and subsequent three-dimensional reconstruction. Every specimen examined had blood vessels in the cornea, comprising an average of 0.3% of total surface density (volume) of the cornea. No differences were found between individuals based on gender, age, and season. Environmental influences were not a significant factor either, which was not originally anticipated. The presence of vessels at the level of the anterior epithelium was surprising and it appeared that the vascularization was directed more anteriorly than was originally thought. The presence of blood vessels in a prenatal eye was also found. In all the eyes examined, no signs of injury or trauma could be observed. The presence of blood vessels appears to minimally impair vision based on their low density, size, and location. The association of vessels with the anterior epithelium and development of vessels within the fetus point to an evolutionary adaptation possibly due to the manatee's unique ability to move between water bodies.

Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis.

Zn homeostasis in animals is a consequence of avid uptake and retention, except during conditions of limited dietary availability, and/or factors such as parasites, which compete for this micronutrient or compromise retention by the host. Membrane proteins that facilitate Zn transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families. Because dietary recommendations are based on the balance between intestinal absorption and endogenous losses, we have studied Zn transporter expression of the murine intestinal-pancreatic axis to identify transporters that are likely to be involved in homeostatic control of Zn metabolism. Marked tissue specificity of expression was observed in Zn-depleted vs. Zn-adequate mice. As shown by quantitative PCR, Western blot analysis, and immunohistochemistry, intestinal Zip4 was markedly up-regulated in response to Zn-depletion conditions. The increased abundance of Zip4 is concentrated at the apical membrane of enterocytes. There are 16 ZnT and Zip transporters expressed in pancreas. Only two, ZnT1 and ZnT2 (both cellular Zn exporters), show a progressive down-regulation under Zn-depleted conditions. In Zn-adequate mice, ZnT1 is diffusely distributed in acinar cell cytoplasm and colocalizes with alpha-amylase but is not detected in pancreatic islets. In acinar cells during Zn depletion, ZnT1 is localized to the plasma membrane. Intestinal Zip4 up-regulation by Zn-depletion conditions is dampened in metallothionein knockout mice, suggesting that intracellular Zn pools influence these responses. The results show that Zn transporter expression in the intestinal-pancreatic axis is a component of the homeostatic regulation of this micronutrient.

Glutamine regulates Caco-2 cell tight junction proteins.

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.