Rat Iris Research Articles

Targeted inhibition of FcRn reduces NET formation to ameliorate experimental ulcerative colitis by accelerating ANCA clearance.

Dysregulated immune responses have now been recognized as an essential stimulator of ulcerative colitis (UC). Neutrophil extracellular traps (NET) were reported as the potential factor in sustaining mucosal inflammation in UC. NET formation further induces antineutrophil cytoplasm autoantibodies (ANCA) that serve as a biomarker in determining the severity of UC, which have a long half-life due to neonatal Fc receptor (FcRn)-mediated recycling. This study aimed to explore the role of the ANCA-NET cycle in UC and evaluate the potential of targeting FcRn in UC treatment. Dextran sodium sulfate-induced mice and rat models were used in this study. anti-rat FcRn monoclonal antibodies were used to block FcRn function in vivo. Disease activity index (DAI) and histopathological score (HS) were estimated to characterize the inflammation severity of UC. Serum concentrations of IgG, ANCA, TNF-α, IL-1β and CRP were measured using specific ELISA kits. Colonic NET-associated protein (NAP) expression was determined by western blotting. Serum ANCA and colonic NAPs showed a positive correlation that varied with changes in serum inflammation-related indexes (IRI; including TNF-α, IL-1β, and CRP) and DAI and HS in mice with UC. Blockade of FcRn significantly reduced serum ANCA levels and colonic NAP expression and effectively decreased serum IRIs, DAI, and HS in rats with UC. Especially during the inflammation recurrence period, blockade of FcRn exerted even better therapeutic effects in rats with UC than salazosulfapyridine. Our results show that anti-FcRn therapy has benefits in UC treatment through reduced colonic NET formation by accelerating serum ANCA clearance.

Integrated Transcriptome Analysis of Iris Tissues in Experimental Autoimmune Uveitis.

Uveitis is a severe ocular inflammatory disease that affects the uvea and frequently results in visual impairment, even irreversible blindness. The current treatments for uveitis have exhibited adverse side effects. To find novel targets of this disease, we perform comparative transcriptome analysis using normal (n = 4) and experimental autoimmune uveitis (EAU) (n = 4) rat iris samples. We mainly focus on the expression profiles of mRNAs and long non-coding RNAs, and identify NOD-like receptor signaling pathway as the one that plays a key role in the pathological changes of the EAU irises. Our work demonstrates that the EAU iris transcriptome can be mined to uncover novel targetable pathways for uveitis. The molecules in NOD-like receptor signaling pathway could be novel therapeutic targets for autoimmune uveitis.

LY294002 attenuates inflammatory response in endotoxin-induced uveitis by downregulating JAK3 and inactivating the PI3K/Akt signaling

Context Uveitis is a prevalent inflammatory eye disease that damages the vision of patients and even leads to blindness. LY294002, an inhibitor of PI3K, was reported to suppress the inflammation and alleviate the progression of many diseases. However, the function of LY294002 in uveitis is unclear. Objective This study aimed to explore the function of LY294002 in endotoxin-induced uveitis (EIU). Materials and methods EIU rat models were established via a single intravitreal injection of LPS. At 24 h after LPS injection, the rats received LY294002 treatment for 14 days. The histopathology was observed by H&E staining. The concentration of proinflammatory cytokines in aqueous humor was tested by ELISA. The expression of proinflammatory cytokines in the iris ciliary body (ICB) and retina of EIU rats were detected by RT-qPCR. JAK3, PI3K, and Akt expression were assessed by RT-qPCR and western blotting. Translocation of Akt in rat retinal Müller cells (rMC-1) was evaluated by immunofluorescence staining. Results LY294002 alleviated ocular inflammation and decreased inflammatory cell infiltration in the anterior chamber, iris, ciliary body, vitreous cavity, and retina of EIU rats. LY294002 decreased the concentration of proinflammatory cytokines INF-γ, IL-17, IL-6, TNF-α, and IL-1β in aqueous humor and their expression in the ICB and retina of EIU rats. LY294002 downregulated JAK3 expression in EIU rats. LY294002 inhibited p-PI3K and p-Akt expression in EIU rats and restrained Akt translocation from cytoplasm to cell membrane in LPS-treated rMC-1 cells. Conclusion LY294002 ameliorates inflammation in EIU by downregulating JAK3 and inactivating the PI3K/Akt signaling.

The effects of prostaglandins and endocannabinoids on iris arterial vascularization in Wistar rats - Experimental analysis

Introduction: The iris vascular supply originates in the anterior and long posterior ciliary arteries. The endothelium influences local blood flow by releasing endothelium relaxing and contracting substances. From a functional perspective, the ocular vascular tonus adjustment is humoral and neural dependent. Objectives: The present article aims to evaluate the possible implications of topical administration of selective COX2 and nonselective COX inhibitors generically named nonsteroidal anti-inflammatory drugs (NSAIDs) and their possible interactions with the endocannabinoid system and the way they could interfere with the vascular tone at the level of ocular iris territory in Wistar rats. Materials and methods: Experimental protocol on Wistar rats was performed in accordance with present laws regarding animal welfare and ethics in animal experiments (Directive 86/ 609EEC/ 1986; Romanian Law 205/ 2004; Romanian Laws 206/ 2004, 471/ 2002 and 9/ 2008; Romanian Order 143/ 400). The studied substances were instilled topically under general anesthesia, and images of the rat iris vessels were captured over a period of 10 minutes. The obtained images were further analyzed using an appropriate hardware and software program. Results: The nonselective NSAIDs induced vascular dilation in the iris vessels, while the selective COX2 inhibitors determined a variable degree of vasoconstriction. Conclusion: In view of the results of this experiment and the added evidence found in literature, we consider that further research will show the potential benefits for the additional use of NSAIDs in ocular pathology, otherwise unaffected by this medication until the present time (for example, glaucoma treatment).

Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System Reduces Neovascularisation in Rats

Background: Age-related macular degeneration (AMD) is a progressive retinal disorder, characterised by imbalanced pro- and anti-angiogenic signals, leading vision loss. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy on primary cells with stable expression of human pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Methods: Rat retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) were co-transfected with pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats and transfected primary cells (tRPE and tIPE) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinase 2 and 9 (MMP-2/9) and microglial/macrophage markers were measured and evaluated. Findings: The animals injected with tRPE/IPE cells significantly showed a reduction in the leakage area at 14 days while at 7 days only tIPE cells showed a significant reduction. The CNV area at 7 days was reduced with tRPE/IPE cells. There was a significant increase in PEDF and PEDF/VEGF ratio with tRPE cells along with a reduction in the MMP-2 activity. Activated microglial fluorescence intensity increased in all groups analyzed. Interpretation: Our data demonstrate that a single administration of SB100X-engineered RPE/IPE cells release enough hPEDF to favour an anti-angiogenic environment that ultimately reduces CNV suggest that ex vivo non-viral gene therapy could be an effective and safe therapeutic option for angiogenic retinal diseases. Funding: This work was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134 and Fundacion Jesus Gangoiti Barrera. LGG received a predoctoral grant from the Asociacion de Amigos de la Universidad de Navarra. Declaration of Interest: A. Garcia-Layana is consultant in Thea, Allergan, Bayer, Novartis and Roche Laboratories. Z. Izsvak and Z. Ivics are co-inventors on several patents on Sleeping Beauty transposon technology. Ethical Approval: The study was performed according to the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Committee for Animal Research of the University of Navarra (protocol approval number 023-13).

Motion Correction in Optical Resolution Photoacoustic Microscopy

In this paper, we are proposing a novel motion correction algorithm for high-resolution OR-PAM imaging. Our algorithm combines a modified demons-based tracking approach with a newly developed multi-scale vascular feature matching method to track motion between adjacent B-scan images without needing any reference object. We first applied this algorithm to correct motion artifacts within one three-dimensional (3D) data segment of rat iris obtained with OR-PAM imaging. We then extended the application of this algorithm to correct motions to obtain vasculature imaging in the whole mouse back. In here, we stitched five adjacent 3D data segments (large field-of-view) obtained while changing the focus of OR-PAM differently for each subarea. The results showed that the motion artifacts of both large blood vessels and microvessels could be accurately corrected in both cases. Compared to the manually stitching method and the traditional SIFT algorithm, the algorithm proposed in this paper has better performance in stitching adjacent data segments. The high accuracy of the motion correction algorithm makes it valuable in OR-PAM for high-resolution imaging of large animals and for quantitative functional imaging.

Three-dimensional Hessian matrix-based quantitative vascular imaging of rat iris with optical-resolution photoacoustic microscopy in vivo.

For the diagnosis and evaluation of ophthalmic diseases, imaging and quantitative characterization of vasculature in the iris are very important. The recently developed photoacoustic imaging, which is ultrasensitive in imaging endogenous hemoglobin molecules, provides a highly efficient label-free method for imaging blood vasculature in the iris. However, the development of advanced vascular quantification algorithms is still needed to enable accurate characterization of the underlying vasculature. We have developed a vascular information quantification algorithm by adopting a three-dimensional (3-D) Hessian matrix and applied for processing iris vasculature images obtained with a custom-built optical-resolution photoacoustic imaging system (OR-PAM). For the first time, we demonstrate in vivo 3-D vascular structures of a rat iris with a the label-free imaging method and also accurately extract quantitative vascular information, such as vessel diameter, vascular density, and vascular tortuosity. Our results indicate that the developed algorithm is capable of quantifying the vasculature in the 3-D photoacoustic images of the iris in-vivo, thus enhancing the diagnostic capability of the OR-PAM system for vascular-related ophthalmic diseases in vivo.

Mitochondria in smooth muscle cells of viscera.

The spatial density of mitochondria was studied by thin-section electron microscopy in smooth muscles of bladder, iris and gut in mice, rats, guinea-pigs and sheep. Morphometric data included areas of muscle cell profiles (~6,000 muscle cells were measured) and areas of their mitochondria (more than three times as many). The visual method delivers accurate estimates of the extent of the chondrioma (the ensemble of mitochondria in a cell), measuring all and only the mitochondria in each muscle cell and no other cells. The digital records obtained can be used again for checks and new searches. Spatial density of mitochondria varies between about 2 and 10% in different muscles in different species. In contrast, there is consistency of mitochondrial density within a given muscle in a given species. For each muscle in each species there is a characteristic mitochondrial density with modest variation between experiments. On the basis of data from serial sections in the rat detrusor muscle, mitochondrial density varies very little between the muscle cells, each cell having a value close to that for the whole muscle. Mitochondrial density is different in a given muscle, e.g., ileal circular muscle, from the four mammalian species, with highest values in mouse and lowest in sheep; in mice the mitochondrial density is nearly three time higher that in sheep. In a given species there are characteristic variations between different muscles. For example, the bladder detrusor muscle has markedly fewer mitochondria than the ileum, and the iris has markedly more.

Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery.

Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

Topical administration of Metamizole and its implications on vascular reactivity in Wistar rats- Experimental research.

Aim: The aim of this paper was to describe the possible implications of topical (ocular) administration of Metamizole on vascular reactivity of the iris in Wistar rats. No other study regarding its topical use was found. Methods: Male adult Wistar rats were anaesthetized with Ketamine 100 mg /kg body weight - injected intraperitoneally - while maintaining spontaneous respiration and the blink reflex. After selecting the area of interest (long posterior ciliary artery - LPCA), manual adjustments of the image magnitude, clarity, and brightness were made, and the experiment began. The image recording lasted 10 minutes. Results: Metamizole induced a slight vasoconstriction that started with the initial moment for all the doses used. After the topical administration of Metamizole, we did not observe an increase of the vascular diameter of LPCA in a dose dependent manner. The saline solution used as a negative control did not modify the vessel diameter. Conclusions: Metamizole (dipyrone) is a non-opioid drug, which is commonly used in human and veterinary medicine. It is the most popular first-line analgesic in various populations. In some cases, this agent is still incorrectly classified as a non-steroidal anti-inflammatory drug. The high analgesic efficacy of metamizole, as well as its spasmolytic effect, makes it a very important pharmaceutical agent that could be used in the therapy of various eye disorders in humans and in animals. Abbreviations: COX = Cyclooxygenase; LPCA = Long Posterior Ciliary Artery; PRP = panretinal photocoagulation; PDR = proliferative diabetic retinopathy; Sec = second(s); VSPR = very severe non proliferative diabetic retinopathy.

Use of antibiotic‐resistance‐free plasmids for non‐viral transfection of pigment epithelium‐derived factor in rat primary cells

AbstractPurpose We aim to assess the pigment epithelium‐derived factor (PEDF) production in cells after transfection with plasmids free of antibiotic resistance markers (pFAR4) in a non‐viral Sleeping Beauty transposon system (SB100X).Methods ARPE‐19 cell line and primary rat iris and retinal pigment epithelial (IPE and RPE) cells were transfected with SB100X transposase and PEDF transposon encoding pFAR4 plasmids (1:16 ratio). Control cells were transfected with SB100X and pFAR4‐Venus plasmids. Supernatants were taken every 24 hours for kinetics. The effect of transfecting different amount of cells was also analyzed. PEDF production was tested by Western blot and ELISA.Results A cumulative PEDF secretion over time in ARPE‐19 and primary cells was shown, reaching almost 3‐fold higher PEDF production in ARPE‐19 cells. Control cells did not express recombinant PEDF, but showed fluorescence. The transfection of 10,000 cells clearly showed higher PEDF expression than cells that were transfected as a group of 70,000 and subsequently divided.Conclusion Rat primary cells are efficiently transfected and produced PEDF as it happens in ARPE‐19 cell line. Merging of the SB100X and pFAR4 technologies led to increased PEDF expression and improved safety by avoiding the transfer and potential integration of antibiotic resistance genes. This study demonstrates the feasibility of our approach based on ex vivo transfection of a low number of primary autologous pigment epithelial cells.

Spectral degree of polarization uniformity for polarization-sensitive OCT

Depolarization of light can be measured by polarization-sensitive optical coherence tomography (PS-OCT) and has been used to improve tissue discrimination as well as segmentation of pigmented structures. Most approaches to depolarization assessment for PS-OCT – such as the degree of polarization uniformity (DOPU) – rely on measuring the uniformity of polarization states using spatial evaluation kernels. In this article, we present a different approach which exploits the spectral dimension. We introduce the spectral DOPU for the pixelwise analysis of polarization state variations between sub-bands of the broadband light source spectrum. Alongside a comparison with conventional spatial and temporal DOPU algorithms, we demonstrate imaging in the healthy human retina, and apply the technique for contrasting hard exudates in diabetic retinopathy and investigating the pigment epithelium of the rat iris.

Noninvasive assessment of vascular structure and function in conscious rats based on in vivo imaging of the albino iris

Experimental techniques allowing longitudinal studies of vascular disease progression or treatment effects are not readily available for most animal models. Thus, most existing studies are destined to either study individual time points or use large cohorts of animals. Here we describe a noninvasive technique for studying vascular disease that is based on in vivo imaging of the long posterior ciliary artery (LPCA) in the iris of albino rats. Using a slit-lamp biomicroscope, images of the LPCA were taken weekly in conscious normotensive Wistar Kyoto rats (WKY, n = 10) and spontaneously hypertensive rats (SHR, n = 10) for 10 wk. Using imaging software, we found that lumen diameter was significantly smaller and the wall-to-lumen (W/L) ratio larger in SHR than in WKY. Wall thickness was not different. Blood pressure correlated with the W/L ratio. Histology of the abdominal aorta also revealed a smaller lumen diameter and greater W/L ratio in SHR compared with WKY. Corneal application of the muscarinic receptor agonist pilocarpine elicited a dose-dependent vasodilation of the LPCA that could be antagonized by inhibition of nitric oxide synthase, suggesting that the pilocarpine response is mainly mediated by endothelium-derived nitric oxide. Consistent with endothelial dysfunction in SHR, pilocarpine-induced vasodilation was greater in WKY rats than in SHR. These findings indicate that in vivo imaging of the LPCA allows assessment of several structural and functional vascular parameters in conscious rats and that the LPCA responds to disease insults and pharmacologic treatments in a fashion that will make it a useful model for further studies.

A novel technique to study the time course of morphological and functional vascular responses to hypertension in conscious rats

Assessment of morphological vascular responses to exposure to cardiovascular risk factors in experimental animals requires ex vivo experiments that do not allow assessment of the time course of vascular alterations in individual subjects. We used a slit‐lamp biomicroscope (resolution <1 μm) to photograph the long posterior ciliary artery (LPCA) of the iris in conscious normotensive Wistar Kyoto rats (WKY, n=10) and spontaneously hypertensive rats (SHR, n=10) on regular‐salt diet (RS) and in SHR (n=10) on high‐salt diet (HS). The same segment along the LPCA was imaged in consecutive weekly imaging sessions and an imaging software was used to determine the wall to lumen (W/L) ratio. After 4 weeks systolic blood pressure (SBP) did not change in WKY (115±5 vs. 111±5 mmHg) and SHR‐RS (148±7 vs. 155±3 mmHg) but increased significantly in SHR‐HS (139±5 mmHg vs. 166±6 mmHg, p<0.05). The time course of the changes in W/L ratio of the LPCA mirrored the time course of the SBP changes. While W/L ratio did not change in WKY‐RS (+12±10%) and SHR‐RS (+18±17%), there was a 39±14% increase in W/L ratio in SHR‐HS. Since the LPCA was not dilated pharmacologically the W/L ratio assessed in this study may reflect the combination of morphologic alterations and changes in vascular tone. In vivo imaging of the LPCA may allow assessment of the time course of morphological and functional vascular responses to hypertension in conscious rats. (Supported by the American Heart Association)

Biocompatibility of chemically cross-linked gelatin hydrogels for ophthalmic use

Biocompatibility is a major requirement for the development of functional biomaterials for ophthalmic applications. In this study, we investigated the effect of cross-linker functionality on ocular biocompatibility of chemically modified gelatin hydrogels. The test materials were cross-linked with glutaraldehyde (GTA) or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and were analyzed using in vitro and in vivo assays. Primary rat iris pigment epithelial cultures were incubated with various gelatin discs for 2 days, and the cellular responses were monitored by cell proliferation, viability, and pro-inflammatory gene and cytokine expression. The results demonstrated that the cells exposed to EDC cross-linked gelatins had relatively lower lactate dehydrogenase activity, cytotoxicity, and interleukin-1beta and tumor necrosis factor-alpha levels than did those to GTA treated samples. In addition, the gelatin implants were inserted in the anterior chamber of rabbit eyes for 12 weeks and characterized by clinical observations and scanning electron microscopy studies. The EDC cross-linked gelatin hydrogels exhibited good biocompatibility and were well tolerated without causing toxicity and adverse effects. However, a significant inflammatory reaction was elicited by the presence of GTA treated materials. It was noted that, despite its biocompatibility, the potential application of non-cross-linked gelatin for local delivery of cell and drug therapeutics would be limited due to rapid dissolution in aqueous environments. In conclusion, these findings suggest ocular cell/tissue response to changes in cross-linker properties. In comparison to GTA treatment, the EDC cross-linking is more suitable for preparation of chemically modified gelatin hydrogels for ophthalmic use.

Proteolytic Cleavage of Type I Collagen Generates an Autoantigen in Autoimmune Uveitis

This study was initiated to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats by melanin-associated antigen (MAA; 22-kDa fragment of type I collagen alpha2 chain) derived from rat iris and ciliary body (CB), to localize MAA within the eye, and to investigate the possible mechanism of MAA generation in vivo. The EAAU model replicates idiopathic human anterior uveitis. Lewis rats sensitized to rat MAA developed anterior uveitis, and EAAU induced by rat MAA can be adoptively transferred to naive syngenic rats by MAA-primed T cells. Animals immunized with rat MAA developed cellular immunity to the antigen. MAA was detected only in the iris and CB of the eye. Iris and CB were the major source of matrix metalloproteinase-1 (MMP-1) in the naive eye, and ocular expression of MMP-1 was up-regulated, whereas expression of tissue inhibitor of metalloproteinase 1 decreased before the onset of EAAU. These results demonstrated that EAAU can be induced by autologous MAA. Uveitogenic antigen is present only in the iris and CB of the eye, and the imbalance between MMP-1 and tissue inhibitor of metalloproteinase 1 may play a role in the generation of MAA in vivo. Collectively, the evidence presented here suggests that MAA is an autoantigen in EAAU. These observations may extend to idiopathic human anterior uveitis and facilitate the development of antigen-specific therapy.

Ghrelin expression in the iris and ciliary epithelium of fetal and newborn rats

Abstract Purpose: The presence of ghrelin was previously identified in the posterior epithelium of the iris and in the non‐pigmented ciliary epithelium. Ghrelin is also a potent relaxator of the iris sphincter muscle. Methods: Sprague‐Dawley rat fetuses were removed by caesarian section at 10.5, 11.5, 12.5, 13.5, 17.5, 20.5 dpc, and 15 day‐old animals (P15) were euthanized Specimens (heads or eyes) were paraffin embedded and processed for in situ hybridization (ISH). Digoxigenin‐labeled ghrelin probe was synthesized from a 247bp fragment of prepro‐ghrelin. Results: Ghrelin’s mRNA was expressed in the posterior epithelium of the iris of 17.5, 20.5 dpc fetuses and P15 animals. Furthermore, in 20.5 dpc fetuses and P15 animals there was also ghrelin expression in the non‐pigmented ciliary epithelium. Conclusions: Ghrelin is expressed during development of the rat’s eye from 17.5 dpc onward. Its expression is more evident in the posterior epithelium of the iris and ciliary epithelium. These findings could enrol a new function of ghrelin in the development of the anterior segment of the eye.

Localization and ontogeny of aquaporin-1 and -4 expression in iris and ciliary epithelial cells in rats

The precise localization of aquaporin (AQP)1 and AQP4 was studied in iris and ciliary epithelial cells, in both mature and developing rats, to elucidate the molecular mechanisms underlying aqueous humor balance. Anterior segments of eyes dissected from embryonic day (E)13, E15, E18, and E20, postnatal day (P)0, P7, and P14, and postnatal week 8 rats were subjected to immunofluorescence analysis with AQP isoform-specific antibodies. In adult rat eye, AQP1 was localized to the apical and basolateral plasma membranes of iris epithelial cell layers and of anterior ciliary non-pigmented epithelial (NPE) cells. Conversely, AQP4 was localized to the basolateral plasma membrane of NPE cells in ciliary epithelium and the posterior iris. Developmentally, AQP1 was detected as early as E15 in immature iris and ciliary epithelial cells, and expression persisted throughout development up to adulthood. In contrast, AQP4 was first observed at P7 in the developing pars plicata, and the AQP4-positive area gradually spread to cover the entire pars plicata as development proceeded. These findings indicate that both AQP1 and AQP4 contribute to aqueous humor secretion in the rat eye, thereby maintaining proper intraocular pressure. Moreover, AQP appears to play a major role in aqueous humor secretion in early eye development. This study thus provides a basis for understanding the molecular mechanisms of aqueous humor secretion in pathological and physiological conditions.

Iris-Derived Cells from Adult Rodents and Primates Adopt Photoreceptor-Specific Phenotypes

The purpose of this study was to investigate the effects of various genes related to photoreceptor development on rodent and primate iris cells and the potential of iris cells as donor cells for retinal transplantation. Adult rat and monkey iris tissue were cultured in serum-free medium containing basic fibroblast growth factor. Gene deliveries of Crx, Nrl, NeuroD and some combinations (Crx-Nrl, Crx-NeuroD) were performed with recombinant retrovirus. Immunocytochemistry, Western blot analysis, RT-PCR, and intracellular recording were used to examine the expression of photoreceptor-specific phenotypes in the iris-derived cells after gene transfer, . Coculture of the iris-derived cells with embryonic retinal explant was conducted, to investigate the potential integration of these cells in coculture conditions. Misexpression of Crx induced adult rat iris cells to express several photoreceptor-specific antigens and transcripts, such as rhodopsin, recoverin, cGMP-gated channel, arrestin, interphotoreceptor retinal-binding protein, rhodopsin kinase, and NeuroD. In primates, a combination of Crx and NeuroD was needed to induce monkey iris-derived cells to adopt photoreceptor-specific phenotypes. Furthermore, the photoreceptor-like cells derived from both rat- and primate-iris tissues showed rod photoreceptor-specific electrophysiological response to light stimuli after Crx and Crx-NeuroD gene transfer, respectively. The results further showed that iris-derived cells integrated in the developing host retina in coculture conditions. Adult iris-derived cultured cells of both rodents and primates expressed photoreceptor-specific phenotypes by inductions of transcription factors. These iris-derived photoreceptor-like cells have electrophysiological characteristics of rod photoreceptors. Furthermore, they can integrate in the developing retina under coculture conditions.

The role of NGF uptake in selective vulnerability to cell death in ageing sympathetic neurons

We have examined the hypothesis that differences in nerve growth factor (NGF) uptake and transport determine vulnerability to age-related neurodegeneration. Neurons projecting to cerebral blood vessels (CV) in aged rats are more vulnerable to age-related degeneration than those projecting to the iris. Uptake of NGF was therefore examined in sympathetic neurons projecting from the superior cervical ganglion (SCG) to CV and iris in young and old rats by treating the peripheral processes of these neurons with different doses of I125-NGF. Total uptake of I125-NGF was reduced in old CV-projecting, but not iris-projecting, neurons. Numbers of radiolabelled neurons projecting to each target were counted in sectioned ganglia. The data showed age-related reductions in numbers of labelled neurons projecting to CV, but no change in numbers of neurons projecting to the iris. Calculation of uptake of I125-NGF per neuron unexpectedly showed no major age-related differences in either of the two neuron populations. However, uptake per neuron was considerably lower for young and old CV-projecting, compared to iris-projecting, SCG neurons. We hypothesized that variations in NGF uptake might affect neuronal survival in old age. Counts of SCG neurons using a physical disector following retrograde tracing with Fluorogold confirmed the selective vulnerability of CV-projecting neurons by showing a significant 37% loss of these neurons in the period between 15 and 24 months. In contrast, there was no significant loss of iris-projecting neurons. We conclude that vulnerability to, or protection from, age-related neurodegeneration and neuronal cell death are associated with life-long low, or high, levels of NGF uptake, respectively.