Upregulation of Semaphorin 3A and the associated biochemical and cellular events in a rat model of retinal detachment.

BACKGROUND: Retinal detachment, as a result of injury or disease, is a severe disorder that may ultimately lead to complete blindness. Despite advanced surgical repair techniques, the visual acuity of patients is often limited. We investigated some of the biochemical and morphological alterations following experimental retinal detachment in laboratory animals. METHODS: Unilateral retinal detachment was induced in male Wistar rats; contralateral untreated eyes served as a control. Approximately half of the retinal area was detached by a sub-retinal injection of 5 mul Saline. The incidence and extent of the retinal detachment was evaluated using MRI analysis and fundus images. The retinas were collected at intervals of 24 hours, 7, 14 and 28 days following the procedure. Using Western blot and immunohistochemical analysis, the expression levels of Semaphorin3A, Neuropilin1, GAP43 and NF-H were studied. In addition, morphological changes in Müller and microglial cells were examined. TUNEL staining was used to assess apoptosis. RESULTS: We found that the expression level of Semaphorin3A was up-regulated and reached its peak at two time points: 24 hours and 14 days after surgery. A similar pattern of expression was found for Neuropilin1. TUNEL-positive cells, indicating apoptotic processes, were evident 24 hours post retinal detachment and increased after 7 days. On the other hand, GAP43 expression was up-regulated 14 days after retinal detachment, and further intensified 28 days post-surgery. Microglial cells were activated shortly after detachment and concentrated mostly at the inner plexiform layer. GFAP staining revealed hypertrophy of Müller cells. CONCLUSIONS: The biochemical and morphological changes suggest that apoptosis as well as axonal regrowth take place following retinal detachment. Collectively, these findings may explain the limited success following repair surgery in terms of visual acuity and physiological function of the retina. Our study may open a new approach for treatment of early phase retinal detachment, as well as improve post-operative care that may, in turn, improve the functional result of the surgery. In addition, further study is required on several other factors that may affect visual acuity, such as size and location of the detached area and the time lapse between detachment and surgery.

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism.

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2(-/-) are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2(+/-) mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.

Functional damage to inner and outer retinal cells in experimental glaucoma.

PURPOSE: To investigate the cellular sources underlying the functional damage observed by multifocal electroretinography (mfERG) responses of glaucomatous eyes of monkeys. METHODS: First- and second-order (K1 and K2, respectively) mfERG responses of three normal and three experimentally induced glaucomatous eyes of cynomolgus monkeys were measured at two different levels of luminance. Retinal contributors to the responses were isolated by intravitreal injections of pharmacological agents that suppress specific retinal cells. gamma-Aminobutyric acid (GABA) and glycine were administered to block inner retinal function, followed by 2-amino-4-phosphonobutyric acid (APB), to block ON-bipolar cells. RESULTS: An inner retinal component removed by GABA and glycine was found in both the normal and glaucomatous eyes. However, it was attenuated in the latter, correlating with changes observed in the baseline K1 responses. Delays in the latency of outer retinal components were found in the responses of the glaucomatous eyes. K2 responses were dominated by an inner retinal contribution and were diminished in the responses of glaucomatous eyes. The outer retina responded to increased luminance with a shorter implicit time. A distinct wave part of the inner retinal component responded to increased luminance with increased amplitudes. CONCLUSIONS: The integration of the retinal sources forming the mfERG response was compared between normal and glaucomatous monkey eyes. Both inner and outer retinal functions were aberrant in the responses of the glaucomatous eyes, with the attenuation of the inner retinal function more conspicuous. Nevertheless, glaucomatous eyes retained certain inner retinal activity, despite the advanced stage of disease. K2 responses were more sensitive to glaucomatous changes than were K1 responses.

The effect of contrast and luminance on mfERG responses in a monkey model of glaucoma.

PURPOSE: To evaluate the effect of contrast and luminance attenuation on the multifocal electroretinogram (mfERG) responses of normal and glaucomatous eyes of cynomolgus monkeys. METHODS: Nine adult male cynomolgus monkeys with unilateral experimentally induced glaucoma were used. Hypertension-induced damage was confirmed by tomography of the optic disc. mfERGs were recorded with five different stimulus contrasts and/or luminance settings. The first-order and the first slice of second-order responses were analyzed. RESULTS: Waveforms of normal and glaucomatous eyes differed in shape and amplitude. Second-order responses contributed to first-order responses of the signals in the normal eyes, but made a negligible contribution to the signals in the glaucomatous eyes. Contrast and luminance attenuation affected both first- and second-order responses. The differences between signals in normal and glaucomatous eyes were sufficiently large for an unsupervised clustering algorithm to achieve accurate segregation. CONCLUSIONS: The observations in this study indicate that outer and inner retinal generators participate in first-order mfERG responses and that both inner and outer retinal contributors respond to contrast and luminance changes in stimulus. The hypertension-induced changes in the mfERG furthermore suggest damage to both inner and outer retina.

Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology.

PURPOSE: ERG and histologic changes were investigated in normal rabbits after intravitreal implantation of encapsulated cell technology (ECT) devices releasing ciliary neurotrophic factor (CNTF). METHODS: Fifteen adult New Zealand White albino rabbits had ECT devices secreting CNTF at 22, 5, or 0 ng/d implanted in the superior temporal quadrant of the left eye. The low dose has been shown to produce substantial rescue of photoreceptors in the rcd1 canine model of retinal degeneration. Right eyes were untreated. Ganzfeld dark- and light-adapted ERGs and clinical observations were performed at 5, 15, and 25 days after implantation. Rod a-waves and rod and cone b-waves and outer nuclear layer (ONL) morphology were evaluated at 25 days. RESULTS: Clinical examination showed minimal changes in a few CNTF-treated eyes, including vitreous membranes and engorgement of iris vessels at day 25. Retinas appeared normal. CNTF did not significantly affect the rod a- or b-waves, although the b-wave amplitude tended to be larger in CNTF-treated retinas at low flash intensities. The cone b-wave amplitude was significantly reduced in high-dose eyes at some flash intensities. The ONL area in high-dose eyes was significantly greater because of increased thickness than in fellow retinas. ONL cell size was significantly increased, and staining density decreased in CNTF-treated retinas. CONCLUSIONS: CNTF, given by intravitreal ECT device at doses that protect photoreceptors in a canine model of retinal degeneration (5 ng/d), did not adversely affect either rod or cone ERG function of normal rabbit retina. The cone ERG was more sensitive to suppression being reduced, at low flash intensities, by 22 ng/d. Dose-related changes in the ONL and photoreceptor cell nuclei did not represent a toxic effect, because they were not associated with deficits in the rod ERG over a broad range of intensities.