Short chain fatty acids inhibit corneal inflammatory responses to TLR ligands via the ocular G-protein coupled receptor 43.

PURPOSE: Short chain fatty acids (SCFAs) produced by gut microbiota are known to play primary roles in gut homeostasis by immunomodulation partially through G-protein coupled receptors (GPR) 43. Using mouse models of TLR ligand induced keratitis, we investigated whether SCFAs and GPR43 play any regulatory roles in the pathogenesis of inflammatory responses in the eye. METHODS: Both human and mouse eyes were labeled with a specific antibody for GPR43 and imaged by a laser scanning confocal microscope. Corneal cups from naïve C57BL/6J (B6) and GPR43 knockout (KO) mice were stimulated with TLR ligands in the presence or absence of sodium butyrate overnight and then processed for RT-PCR assay for expression of GPR43 and cytokines. Keratitis was induced by Poly I:C in wild type (WT) B6, GPR43KO and chimeric mice and the disease severity was evaluated by the corneal fluorescein staining test, and infiltrating cell staining and calculating in corneal whole mount. RESULTS: GPR43 is expressed in both human and mouse eyes and the expression is bidirectionally regulated by TLR ligands and butyrate. Butyrate significantly inhibited inflammation caused by several TLR ligands such as Poly I:C, Flagellin, and CpG-ODN (TLR-3, 5 and 9 agonists, respectively) in WT, but not GPR43KO, mice. Butyrate inhibition of TLR-induced keratitis is mediated by the GPR43 expressed in tissue but not hematopoietic, cells. CONCLUSIONS: This is the first report to demonstrate of the protective effect of SCFAs on microbial keratitis, and the dynamic expression and anti-inflammatory function of GPR43 in the eye. SCFAs can modulate inflammation and immunity in the eye through GPR43.

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aß, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

Yap1 is required for maintenance of adult RPE differentiation.

Nuclear YAP1 plays a critical role in regulation of stem cell proliferation, tissue regeneration, and organ size in many types of epithelia. Due to rapid turnover of most epithelial cell types, the cytoplasmic function of YAP1 in epithelial cells has not been well studied. The retinal pigment epithelium (RPE) is a highly polarized epithelial cell type maintained at a senescence state, and offers an ideal cell model to study the active role of YAP1 in maintenance of the adult epithelial phenotype. Here, we show that the cytoplasmic function of YAP1 is essential to maintain adult RPE differentiation. Knockout of Yap1 in the adult mouse RPE caused cell depolarization and tight junction breakdown, and led to inhibition of RPE65 expression, diminishment of RPE pigments, and retraction of microvilli and basal infoldings. These changes in RPE further prompted the loss of adjacent photoreceptor outer segments and photoreceptor death, which eventually led to decline of visual function in older mice between 6 and 12 months of age. Furthermore, nuclear ß-catenin and its activity were significantly increased in mutant RPE. These results suggest that YAP1 plays an important role in active inhibition of Wnt/ß-catenin signaling, and is essential for downregulation of ß-catenin nuclear activity and prevention of dedifferentiation of adult RPE.

Anatomic Studies of the Miniature Swine Cornea.

The domestic swine eye resembles the human eye both anatomically and physiologically. Xenotransplantation of the swine cornea to humans in need of full keratoplasty shows promise as a potential therapeutic strategy to restore vision in individuals with advanced corneal disease, especially those residing in developing nations. That said, we characterized the morphology of corneas from miniature swine, which are smaller in size, easier to handle, and more cost-effective compared to domestic swine. Eyes (N = 15) were harvested from miniature swine from different age groups: 1 month (N = 3), 2 month (N = 3), 4 month (N = 3), 8 month (N = 3), as well as 24 month old adult domestic swine (N = 3). They were immediately submerged in fixative and processed for histological examination at the light and transmission electron microscopic level. Gross anatomic measurements of the cornea were significantly less (P value ≤ 0.05) in miniature swine versus domestic swine. Corneal strata exhibited morphological characteristics similar to the domestic swine cornea. Adult miniature swine corneas show similar overall corneal thickness at 8 months of age versus domestic swine. Miniature swine exhibit similar corneal morphology with the domestic pig and humans, with the exception of Bowman's layer, which is absent in pigs. Therefore, miniature pigs may be a useful resource of corneal tissue for humans in need of full keratoplasty, as well as serve as a large eye model for ophthalmology residents to develop surgical skills and for development and testing of ocular therapeutic strategies that translate to humans. Anat Rec, 301:1955-1967, 2018. © 2018 Wiley Periodicals, Inc.

Progression of Pro23His Retinopathy in a Miniature Swine Model of Retinitis Pigmentosa.

PURPOSE: We characterize the progression of retinopathy in Filial 1 (F1) progeny of a transgenic (Tg) founder miniswine exhibiting severe Pro23His (P23H) retinopathy. METHODS: The F1 TgP23H miniswine progeny were created by crossing TgP23H founder miniswine 53-1 with wild type (WT) inbred miniature swine. Scotopic (rod-driven) and photopic (cone-driven) retinal functions were evaluated in F1 TgP23H and WT littermates using full field electroretinograms (ffERGs) at 1, 2, 3, 6, 9, 12, and 18 months of age, as well as the Tg founder miniswine at 6 years of age. Miniswine were euthanized and their retinas processed for morphologic evaluation at the light and electron microscopic level. Retinal morphology of a 36-month-old Tg miniswine also was examined. RESULTS: Wild type littermates reached mature scotopic and photopic retinal function by 3 months, while TgP23H miniswine showed abnormal scotopic ffERGs at the earliest time point, 1 month, and depressed photopic ffERGs after 2 months. Rod and cone photoreceptors (PR) exhibited morphologic abnormalities and dropout from the outer nuclear layer at 1 month, with only a monolayer of cone PR somata remaining after 2 months. The retinas showed progressive neural remodeling of the outer retina that included dendritic retraction of rod bipolar cells and glial seal formation by Müller cells. The TgP23H founder miniswine showed cone PR with relatively intact morphology exclusive to the area centralis. CONCLUSIONS: The F1 Tg miniswine and the TgP23H founder miniswine exhibit similar retinopathy. TRANSLATIONAL RELEVANCE: TgP23H miniswine are a useful large-eye model to study pathogenesis and preservation cone PRs in humans with retinitis pigmentosa.

Two-Step Reactivation of Dormant Cones in Retinitis Pigmentosa.

Most retinitis pigmentosa (RP) mutations arise in rod photoreceptor genes, leading to diminished peripheral and nighttime vision. Using a pig model of autosomal-dominant RP, we show glucose becomes sequestered in the retinal pigment epithelium (RPE) and, thus, is not transported to photoreceptors. The resulting starvation for glucose metabolites impairs synthesis of cone visual pigment-rich outer segments (OSs), and then their mitochondrial-rich inner segments dissociate. Loss of these functional structures diminishes cone-dependent high-resolution central vision, which is utilized for most daily tasks. By transplanting wild-type rods, to restore glucose transport, or directly replacing glucose in the subretinal space, to bypass its retention in the RPE, we can regenerate cone functional structures, reactivating the dormant cells. Beyond providing metabolic building blocks for cone functional structures, we show glucose induces thioredoxin-interacting protein (Txnip) to regulate Akt signaling, thereby shunting metabolites toward aerobic glucose metabolism and regenerating cone OS synthesis.

Prenatal Exposure to Curcumin Protects Rod Photoreceptors in a Transgenic Pro23His Swine Model of Retinitis Pigmentosa.

PURPOSE: Rhodopsin localization and rod photoreceptor (PR) morphology is altered in embryonic transgenic (Tg) Pro23His (P23H) miniswine. At birth, the Tg P23H swine retina lacks rod driven signaling. Curcumin, a neuroprotective food additive, has been shown to rescue Tg P23H rat rod PRs and promote normal trafficking of rhodopsin. We tested the hypothesis that prenatal exposure to curcumin would prevent PR morphological changes in Tg P23H miniswine retinae. METHODS: A domestic sow was inseminated with semen from a Tg P23H miniswine founder. Her daily diet was supplemented with curcumin (100 mg/Kg body weight) from embryonic (E) day 80 to E112. The same diet without curcumin was fed to a second inseminated control sow. At E112, 2 days before parturition, both sows were euthanized. Their embryos were harvested, genotyped, and their eyes enucleated and prepared for morphological evaluation. RESULTS: In all pigs, we measured mean outer retinal thickness, localization of rhodopsin, and rod PR morphology. Curcumin-treated Tg P23H swine embryonic retinas were similar to WT. Untreated Tg P23H embryonic retinas show significant degenerative effects; their outer retina was thinner, rod PR morphology was abnormal, and rhodopsin was mislocalized to the outer nuclear layer (ONL). CONCLUSIONS: These data support a role for curcumin as a neuroprotective agent that prevents/delays morphological abnormalities associated with rod PR degeneration in this Tg P23H swine model of retinitis pigmentosa (RP). TRANSLATIONAL RELEVANCE: Curcumin, a Food and Drug Administration-approved dietary supplement, may arrest/delay PR degeneration if ingested by individuals at risk for developing RP.

A Pro23His mutation alters prenatal rod photoreceptor morphology in a transgenic swine model of retinitis pigmentosa.

PURPOSE: Functional studies have detected deficits in retinal signaling in asymptomatic children from families with inherited autosomal dominant retinitis pigmentosa (RP). Whether retinal abnormalities are present earlier during gestation or shortly after birth in a subset of children with autosomal dominant RP is unknown and no appropriate animal RP model possessing visual function at birth has been available to examine this possibility. In a recently developed transgenic P23H (TgP23H) rhodopsin swine model of RP, we tracked changes in pre- and early postnatal retinal morphology, as well as early postnatal retinal function. METHODS: Domestic swine inseminated with semen from a TgP23H miniswine founder produced TgP23H hybrid and wild type (Wt) littermates. Outer retinal morphology was assessed at light and electron microscopic levels between embryonic (E) and postnatal (P) day E85 to P3. Retinal function was evaluated using the full field electroretinogram at P3. RESULTS: Embryonic TgP23H rod photoreceptors are malformed and their rhodopsin expression pattern is abnormal. Consistent with morphological abnormalities, rod-driven function is absent at P3. In contrast, TgP23H and Wt cone photoreceptor morphology (E85-P3) and cone-driven retinal function (P3) are similar. CONCLUSIONS: Prenatal expression of mutant rhodopsin alters the normal morphological and functional development of rod photoreceptors in TgP23H swine embryos. Despite this significant change, cone photoreceptors are unaffected. Human infants with similarly aggressive RP might never have rod vision, although cone vision would be unaffected. Such aggressive forms of RP in preverbal children would require early intervention to delay or prevent functional blindness.

Cone photoreceptors develop normally in the absence of functional rod photoreceptors in a transgenic swine model of retinitis pigmentosa.

PURPOSE: Human and swine retinas have morphological and functional similarities. In the absence of primate models, the swine is an attractive model to study retinal function and disease, with its cone-rich visual streak, our ability to manipulate their genome, and the differences in susceptibility of rod and cone photoreceptors to disease. We characterized the normal development of cone function and its subsequent decline in a P23H rhodopsin transgenic (TgP23H) miniswine model of autosomal dominant RP. METHODS: Semen from TgP23H miniswine 53-1 inseminated domestic swine and produced TgP23H and Wt hybrid littermates. Retinal function was evaluated using ERGs between postnatal days (P) 14 and 120. Retinal ganglion cell (RGC) responses were recorded to full-field stimuli at several intensities. Retinal morphology was assessed using light and electron microscopy. RESULTS: Scotopic retinal function matures in Wt pigs up to P60, but never develops in TgP23H pigs. Wt and TgP23H photopic vision matures similarly up to P30 and diverges at P60 where TgP23H cone vision declines. There are fewer TgP23H RGCs with visually evoked responses at all ages and their response to light is compromised. Photoreceptor morphological changes mirror these functional changes. CONCLUSIONS: Lack of early scotopic function in TgP23H swine suggests it as a model of an aggressive form of RP. In this mammalian model of RP, normal cone function develops independent of rod function. Therefore, its retina represents a system in which therapies to rescue cones can be developed to prolong photopic visual function in RP patients.

Inhibition of PVR with a tyrosine kinase inhibitor, dasatinib, in the swine.

PURPOSE: We tested the efficacy of dasatinib, a Food and Drug Administration (FDA)-approved tyrosine kinase inhibitor, to prevent proliferative vitreoretinopathy (PVR). METHODS: The effect of dasatinib on RPE sheet growth was determined by measuring enlargement of cultured RPE sheets in the presence or absence of dasatinib. Epithelial-mesenchymal transition (EMT) of RPE cells was assessed by expression of S100A4. A scratch wound assay, cell number count, and type I collagen contraction assay were used to examine the effect of dasatinib on migration, proliferation, and extracellular matrix (ECM) contraction, respectively. Our swine model of experimental PVR with green fluorescent protein-positive (GFP+) RPE cells was used to assess the efficacy of dasatinib in preventing traction retinal detachment (TRD) caused by PVR. Full-field electroretinography and histologic examination were used to determine the retinal toxicity of dasatinib. RESULTS: Dasatinib prevented RPE sheet growth, cell migration, proliferation, EMT, and ECM contraction in a concentration-dependent manner. 0.1 µM dasatinib inhibited nearly 80% of vitreous fluid-stimulated collagen gel contraction. Dasatinib also prevented TRD caused by PVR in vivo. Only 1/11 eyes had a TRD in the presence of dasatinib, while all 11 controls eyes had a TRD. Dasatinib did not cause any detectable toxicity of the retina. CONCLUSIONS: Dasatinib significantly inhibited PVR-related RPE changes in vitro and prevented TRD in an experimental PVR model in the swine without any detectable toxicity. Our data suggested that dasatinib may be effective in the prevention of PVR.

Iodoacetic acid, but not sodium iodate, creates an inducible swine model of photoreceptor damage.

Our purpose was to find a method to create a large animal model of inducible photoreceptor damage. To this end, we tested in domestic swine the efficacy of two chemical toxins, known to create photoreceptor damage in other species: Iodoacetic Acid (IAA) and Sodium Iodate (NaIO(3)). Intravenous (IV) administration of NaIO(3) up to 90 mg/kg had no effect on retinal function and 110 mg/kg was lethal. IV administration of IAA (5-20 mg/kg) produced concentration-dependent changes in visual function as measured by full-field and multi-focal electroretinograms (ffERG and mfERG), and 30 mg/kg IAA was lethal. The IAA-induced effects measured at two weeks were stable through eight weeks post-injection, the last time point investigated. IAA at 7.5, 10, and 12 mg/kg produce a concentration-dependent reduction in both ffERG b-wave and mfERG N1-P1 amplitudes compared to baseline at all post-injection times. Comparisons of dark- and light-adapted ffERG b-wave amplitudes show a more significant loss of rod relative to cone function. The fundus of swine treated with ≥10 mg/kg IAA was abnormal with thinner retinal vessels and pale optic discs, and we found no evidence of bone spicule formation. Histological evaluations show concentration-dependent outer retinal damage that correlates with functional changes. We conclude that NaIO(3,) is not an effective toxin in swine. In contrast, IAA can be used to create a rapidly inducible, selective, stable and concentration-dependent model of photoreceptor damage in swine retina. Because of these attributes this large animal model of controlled photoreceptor damage should be useful in the investigation of treatments to replace damaged photoreceptors.

Anatomical evidence of photoreceptor degeneration induced by iodoacetic acid in the porcine eye.

Iodoacetic acid (IAA) induces photoreceptor (PR) degeneration in small animal models, however, eye size and anatomic differences detract from the usefulness of these models for studying retinal rescue strategies intended for humans. Porcine eyes are closer in size to human eyes and have a rich supply of rod and cones. This study investigated whether IAA also produced PR degeneration in the porcine retina, whether the damage was preferential for rods or cones, and whether IAA induced remodeling of the inner retina. Pigs were given a single i.v. injection of IAA and were euthanized 2-5 weeks later. Eyes were enucleated and immersed in fixative. Forty-six eyes were studied: Control (n = 13), and from pigs that had received the following IAA doses: 5.0 mg/kg (n = 7); 7.5 mg/kg (n = 10); 10.0 mg/kg (n = 6); 12.0 mg/kg (n = 6). Tissue was retrieved from four retinal locations: 8 mm and 2 mm above the dorsal margin of the optic disc, and 2 mm and 8 mm below the disc, and was processed for conventional histology, immunohistochemistry, and transmission electron microscopy. At 5.0 mg/kg IAA produced mild, variable cell loss, but remaining cells exhibited normal features. At doses above 5.0 mg/kg, a dose-dependent reduction was observed in the length of PR inner and outer segments, and in the number of PR nuclei. Specific labeling revealed a massive dropout of rod cell bodies with relative sparing of cone cell bodies, and electron microscopy revealed a reduction in the number of PR synaptic terminals. Mild dendritic retraction of rod bipolar cells and hypertrophy of Müller cell stalks was also observed, although the inner nuclear layer appeared intact. The porcine IAA model may be useful for developing and testing retinal rescue strategies for human diseases in which rods are more susceptible than cones, or are affected earlier in the disease process.

Relationships between increased aqueous outflow facility during washout with the changes in hydrodynamic pattern and morphology in bovine aqueous outflow pathways.

Previous studies suggest that the structural correlate for the increased outflow facility (C) during washout in the bovine eye is separation between the inner wall (IW) and underlying juxtacanalicular connective tissue (JCT). However, how these structural changes affect hydrodynamic patterns of outflow during washout has not been studied. We hypothesize that an increase in the outflow facility during washout is associated with an increase in the effective filtration area (EFA) of aqueous outflow, which is regulated by a loss of the connectivity between the IW and JCT. To test this hypothesis, the relationship between C and the hydrodynamic patterns of outflow as well as the morphological changes in JCT and IW during the washout were investigated. Ten bovine eyes were perfused at 15 mmHg with Dulbecco's PBS + 5.5 mM glucose (DPBS) for 30 min to establish stable baseline C. After measuring baseline C, five eyes (short-duration group) were perfused with 0.5 mL DPBS containing 0.002% microspheres (0.5 microm) to trace the hydrodynamic pattern of outflow. Five other eyes (long-duration group) were perfused for 3 h to elicit a significant washout effect followed by subsequent perfusion of the same volume (0.5 mL) of microspheres to map out the outflow pattern after washout. All eyes were then perfusion-fixed. Anterior segments were sectioned and prepared for confocal and light microscopy. Total length (TL) and filtration length (FL) of the IW were measured in > or =15 images/eye to calculate percent effective filtration length (PEFL = FL/TL) while TL and length exhibiting JCT/IW separation (SL) were measured in > or =13 images/eye to calculate percent separation length (PSL = SL/TL). In long-duration eyes, C increased 170.5 +/- 21.3% (mean +/- SEM, 1.55 +/- 0.24 vs 4.13 +/- 0.55 microl/min/mmHg, p = 0.001) above baseline. Pre-fixation C (4.13 +/- 0.55 microl/min/mmHg) in long-duration was 1.6-fold greater than that (2.14 +/- 0.61 microl/min/mmHg; p = 0.042) in short-duration. A more uniform tracer labeling was observed in the JCT/IW of long-duration eyes compared to short-duration. PEFL was 2.3-fold larger (52.82 +/- 6.06 vs. 22.2 +/- 6.0%; p = 0.007) and PSL was 2.6-fold larger (54.2 +/- 6.0 vs. 20.5 +/- 1.3%; p = 0.004) in long-duration eyes compared to short-duration. Data from all eyes revealed a positive correlation between PEFL and PSL (p = 0.02). Both PEFL and PSL demonstrated significant positive correlations with the relative increase in C due to washout (p < or = 0.05). An additional experiment was performed in which unequal volumes of tracer (0.5 and 1.0 mL) were perfused in paired eyes for both short- and long-duration (N = 2 for each condition) to examine the affect on PEFL. No significant change in PEFL was found in eyes perfused with 0.5 and 1.0 mL within the same group. These data support our hypothesis that separations between the IW and JCT result in an increase in the EFA that then influences C. Altogether, these data suggest that outflow hydrodynamics and the tissue structure work together to regulate outflow resistance.

The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes.

Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 microM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 microm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in > or =16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in > or =16 micrographs/eye, and the average percent separation length (PSL=SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54+/-0.34 (+/-SEM) to 2.36+/-0.54 microL/min per mmHg (58.2+/-18.9%) while control eyes changed from 1.67+/-0.41 to 1.71+/-0.39 microl/min per mmHg (6.0+/-9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p=0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6+/-3.7%) compared to control eyes (18.2+/-4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3+/-3.6%) than in controls (20.8+/-2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p=0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p=0.01), a finding identical to what we reported to occur during the "washout" effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP.

Comparative studies between species that do and do not exhibit the washout effect.

Ocular perfusion studies from all non-human species performed to date consistently demonstrate a perfusion-volume-dependent increase in aqueous outflow facility known as the "washout" effect. However, this "washout" effect does not occur in human eyes. We have recently documented that, in bovine eyes, the washout associated increase in facility correlates with the extent of physical separation between the juxtacanalicular connective tissue (JCT) and the inner wall endothelium lining the aqueous plexus (the bovine equivalent of Schlemm's canal). We hypothesize that if washout truly correlates with inner wall/JCT separation then this separation should not occur in human eyes that do not exhibit the washout effect, even after prolonged perfusion. Eight enucleated human and eight bovine eyes were used in this study. Aqueous humor outflow facility was measured at 15 mmHg for long-duration (3 h) or short-duration (30 min to 1 h) perfusion (n=4 for each group). All eyes were perfusion-fixed at 15 mmHg, and examined morphologically with both light and electron microscopy. In bovine eyes, outflow facility increased 81% (p=0.049) from 1.06 +/- 0.06 microl/min per mmHg (mean+/-SEM) at baseline to 1.92 +/- 0.30 microl/min per mmHg after 3 h due to washout. The pre-fixation outflow facility in long-duration eyes (1.92 +/- 0.30 microl/min per mmHg) was 2-fold greater than pre-fixation facility in short-duration eyes (0.92 +/- 0.11 microl/min per mmHg; p=0.0387). In human eyes, washout was not observed; baseline outflow facility was similar between both groups (0.18 +/- 0.02 vs. 0.25 +/- 0.08 microl/min per mmHg; p=0.518); however, pre-fixation outflow facility in long-duration eyes showed a 40% decrease compared to baseline outflow facility in those same eyes (p=0.017, paired Student's t-test). In bovine eyes, significant expansion and rarefaction of the JCT and inner wall/JCT separation was much more prevalent in long-duration eyes, and data from all bovine eyes revealed a correlation between the extent of inner wall/JCT separation and the absolute value of outflow facility measured immediately prior to fixation (p=0.0024) as well as the washout-induced increase in outflow facility (p=0.0006). In human eyes, no significant morphologic differences were observed between long- and short-duration perfusion, with no observed change in inner wall/JCT separation or expansion between the two groups. Morphologic analysis revealed that the previously described "cribriform plexus" of elastic-like fibers was far more extensive in the JCT of human eyes, appearing to form numerous connections to the inner wall endothelium. The cribriform plexus appears to function as a mechanical tether that maintains inner wall/JCT connectivity in human eyes by opposing hydrodynamic forces generated during perfusion, potentially explaining the lack of washout in humans.