Retinal Degeneration In Rats Research Articles

Transplantation of bone-marrow derived mesenchymal stem cells to treat rat retinal degeneration

Objective To observe the effects of subretinal transplantation of rat mesenchymal stem cells (rMSCs) on Sodium Iodate (SI)-induced retinal degeneration.Methods One hundred and twenty Brown-Norway (BN) rats were divided into three groups including SI injection group,rMSCs transplantation group and normal control group,each with 40 rats.The retinal degeneration was induced by caudal vein injection of SI.The retinal pigment epithelium(RPE)and neural retinal were evaluated by ocular fundus photograph,fluorescein fundus angiography (FFA),electroretinogram (ERG) and histological approach,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling).CM-Dilprelabeled primary rMSCs were transplanted into the subretinal space of Sl-induced rats.The survival,integration,and differentiation of rMSCs were observed between 14 day to 60 day after the transplantation.Results The rat retinal function was gradually reduced afterl4 days of SI injection,with a time-dependent manner.After the RPE cells were damaged,the outer segments of photoreceptors became disrupted and shortened until karyopyknosis.The nuclear morphology and positive TUNEL labeling indicated that the death of photoreceptor cells was apoptosis.After rMSCs transplantation,CM-DiI labeled donor cells were observed to be scattered in the subretinal space and expressed RPE cell markers.Average amplitude of bwave and Ops (oscillation potential) in ERG improved 27.80%,59.38% respectively after rMSCs transplantation.Conclusions Transplanted rMSCs can survive in subretinal space and differentiate into RPE cells,thus cure SI- induced retinal degeneration. Key words: Stem cells/cytology; Myeloid progenitor cells/transplantation; Mesenchymal stem cell transplantation/methods; Retinal diseases/diagnosis; Animals,laboratory

Photoreceptor Protection by Adeno-Associated Virus–Mediated LEDGF Expression in the RCS Rat Model of Retinal Degeneration: Probing the Mechanism

Lens epithelium-derived growth factor (LEDGF) is upregulated in response to stress and enhances the survival of neurons in the retina and optic nerve, as well as a wide range of other cells, such as fibroblasts and keratinocytes. Photoreceptor protection was investigated in the RCS rat retinal degeneration model after Ledgf delivery with an adeno-associated virus (AAV) and the mechanism of protection explored. Thirty-six RCS and nine P23H rats had bilateral subretinal injections of AAV-Ledgf in one eye and buffer in the contralateral eye as the control. Retinal function was evaluated 8 weeks later by the electroretinogram and compared with photoreceptor cell layer count. LEDGF mRNA and protein levels and mRNA levels of known stress-related factors were compared in treated and control retinas to explore the mechanism of LEDGF protection. Nine RCS rats were treated with adenovirus-heat shock protein 27 (Ad-HSP27) and examined for protection. Significant photoreceptor protection was evident functionally and morphologically in 65% to 100% of the RCS rats treated at early ages of up to 7 weeks. Cell protection was more prominent in the superior retinal hemisphere which has a slower natural degeneration rate in untreated eyes. Although many of the heat shock proteins and other stress-related genes showed significant elevation in the AAV-Ledgf-treated eyes, all increases were approximately twofold or less. Transduction of retinal cells with Ad-HSP27 also resulted in photoreceptor protection. AAV-Ledgf elicited no photoreceptor functional protection in P23H rhodopsin transgenic rat retina. Chronic LEDGF treatment via AAV-Ledgf administration gave successful protection of photoreceptors in the RCS rat retina and retarded cell death by about 2 weeks. Induction of heat shock proteins also gave photoreceptor protection. However, compelling evidence was not found that LEDGF protection was associated with upregulation of heat shock proteins.

Mertk Drives Myosin II Redistribution during Retinal Pigment Epithelial Phagocytosis

Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Misregulation of rhodopsin phosphorylation and dephosphorylation found in P23H rat retinal degeneration

To examine rhodopsin (Rho) functions in P23H rat, kinetics of Rho regeneration and dephosphorylation were investigated by spectrophotometric analysis and immunofluorescence labeling method using specific antibodies toward phosphorylated 334Ser or 338Ser site. Rho dephosphorylation at both sites was extremely delayed in P23H retina as compared to normal ones. Kinetics of Rho regeneration was not altered between normal and P23H rats under dark adaptation. Next, to study the effects of several Ca(2+)channel blockers on this model, retinal function and morphology were evaluated. Among them, nilvadipine showed a significant protective effect against P23H retinal degeneration. Neurotrophic factor, fibroblast growth factor-2 and Arc, known to suppress the apoptosis in the central nervous system, were significantly upregulated upon administration of nilvadipine. The present study indicates that misregulation of Rho phosphorylation may be involved as an important step in retinal degeneration of P23H and administration of nilvadipine may be a potential therapeutic agent for the retinal degenerations.

Transplantation of marrow stromal cells to rescue retinal cells in SI-induced retinal degeneration in rats

Transplantation of marrow stromal cells to rescue retinal cells in SI-induced retinal degeneration in rats

Photopic ERG Negative Response from Amacrine Cell Signaling in RCS Rat Retinal Degeneration

The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat.

BDNF-treated retinal progenitor sheets transplanted to degenerate rats: Improved restoration of visual function

The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60 days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a “low light” intensity of 1 cd/m 2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects—either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs—still needs to be elucidated.

Microarray expression analysis of the early N-methy- N-nitrosourea-induced retinal degeneration in rat

The study was undertaken to investigate the gene expressions in N-methy- N-nitrosourea (MNU)-induced rat retinal degeneration (RD) by performing microarray analysis of retinal RNA at 12 h. All rats were randomly divided into a normal group, a 12 h model group and a 24 h model group. Rats in the two model groups received a single intraperitoneal injection of 40 mg/kg body weight of MNU, while those in the normal group were injected with equivalent volume of physiological saline. After 12 h and 24 h of the injection, rats in each respective group were sacrificed, respectively. One eye of each animal was used for hematoxylin and erosin (H&E) staining, and fresh retinas of the other eye of each animal in the both normal group and 12 h model group were used to extract total RNA, which was analyzed by microarray and real time RT-PCR. Retinal histological alteration was found in the 24 h model group. There were 75 genes differently expressed (ratio ≥2.0), including 64 genes up-regulated and 11 genes down-regulated. Seven genes were assayed by real time RT-PCR and demonstrated the same alteration tendency as in microarray analysis. These genes that expressed differently mainly involved signal transduction, development, immune and defense, and apoptosis, etc. The major pathways were MAP-kinase signaling pathways, Toll-like receptor signaling pathway and apoptosis pathway involved. The results suggest that there are significant changes of gene expression in the early stage of MNU-induced RD. These microarray results provide clues to understand the molecular pathways underlying photoreceptor degeneration and indicate directions for future studies.

Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo

The purpose of this study is to characterize the co-expression of nestin—a neuroectodermal stem cell and a reactive glial marker—with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all- trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1–4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C α (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCα, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.

Structure and Function of Embryonic Rat Retinal Sheet Transplants

Purpose: To evaluate retinal sheet transplants in S334ter-line-3 retinal degenerate rats by comparing visual responses recorded electrophysiologically with morphology based on light and electron microscopy. Methods: S334ter-line-3 retinal degenerate rats (n = 7) received retinal sheet transplants between postnatal days 28 and 31. The donor tissue was derived from transgenic embryonic day 19 (E19) rat retinae expressing human placental alkaline phosphatase (hPAP). Fresh retinal sheets were gently transplanted into the subretinal space of the left eye with the help of a custom-made implantation tool. Selected rats (n = 5) were subjected to electrophysiologic evaluation of visual responses from the superior colliculus about 84–121 days after surgery. Transplanted eyes were processed for light microscopy (LM) and electron microscopy (EM) evaluations. Results: All the transplanted rats that were evaluated for visual responses in the brain showed responses to very low light stimulation (−3.42 to −2.8 log cd/m2) of the eye in a small area of the superior colliculus corresponding with the placement of the transplant in the host retina. Histologic evaluation showed that most of the transplants contained well-laminated areas with correct polarity in the subretinal space. Inside the transplant areas, rosettes of photoreceptors with inner and outer segments were found. In the laminated areas, the outer segments of photoreceptors were facing the host retinal pigment epithelium (RPE). Immunohistochemical evaluation of hPAP donor cells revealed areas with specific staining of the transplants in the subretinal space. Electron microscopic evaluation showed a glial demarcation membrane between the host and the transplant, however, processes originating from the transplant were observed inside the host retina. Conclusions: Sheets of E19 rat retina transplanted into the subretinal space of S334ter-line-3 rats survived without immune rejection and continued to show visual function when tested after 3 months. Well-developed photoreceptors and many synapse types were seen within the transplants. hPAP staining showed a certain degree of integration between the host retina and the transplant suggesting that transplanted photoreceptors contributed to the restored light sensitivity.

Behavioral evaluation of visual function of rats using a visual discrimination apparatus

A visual discrimination apparatus was developed to evaluate the visual sensitivity of normal pigmented rats (n=13) and S334ter-line-3 retinal degenerate (RD) rats (n=15). The apparatus is a modified Y maze consisting of two chambers leading to the rats’ home cage. Rats were trained to find a one-way exit door leading into their home cage, based on distinguishing between two different visual alternatives (either a dark background or black and white stripes at varying luminance levels) which were randomly displayed on the back of each chamber. Within 2 weeks of training, all rats were able to distinguish between these two visual patterns. The discrimination threshold of normal pigmented rats was a luminance level of −5.37±0.05logcd/m2; whereas the threshold level of 100-day-old RD rats was −1.14±0.09logcd/m2 with considerable variability in performance. When tested at a later age (about 150 days), the threshold level of RD rats was significantly increased (−0.82±0.09logcd/m2, p<0.03, paired t-test). This apparatus could be useful to train rats at a very early age to distinguish between two different visual stimuli and may be effective for visual functional evaluations following therapeutic interventions.

Protective effect of Ginkgolide B on N-methyl-N-nitrosourea induced retinal degeneration in rat

目的 观察不同剂量的银杏内酯B(GB)对N-甲基-N-亚硝脲(MNU)诱导的SD大鼠视网膜变性的保护作用.方法 取出生后46 d的SD大鼠86只,随机抽取6只为正常对照组;80只分为4大组,每组20只.随机分为模型对照组,GB大、中、小剂量组.各组每次4只分别于24 h、48 h、3、5、7 d行右眼闪光视网膜电图(ERG)检查,并通过视网膜形态学分析测量中心视网膜的外视网膜厚度.结果 ERG正常组a波振幅为(105.4±16.8)μV,b波振幅为(292.6±19.6)μV.模型对照组24 h后,a波消失.GB各剂量治疗组a波消失时间分别为2、3、3 d,小、中剂量组b波消失时间分别为5 d和7 d,大剂量组7 d时b波振幅下降为正常的8%.中心视网膜厚度检查:正常组大鼠中心视网膜的外视网膜厚度为(98.4±1.8)μm.MNU处理7 d模型对照组外视网膜厚度为(17.8±2.1)μm,而GB各剂量治疗组分别为(25.2±2.7)、(38.3±2.3)、(45.8±2.3)μm.结论 GB对MNU诱导的实验性大鼠视网膜变性所致的大鼠视网膜光感受器细胞损伤和视功能损害具有保护作用,并且这种作用呈剂量依赖性。

Effects of electroacupuncture on retinal nerve growth factor and brain-derived neurotrophic factor expression in a rat model of retinitis pigmentosa

The aim of this study was to investigate the effect of electroacupuncture (EA) on the progression of retinal degeneration in rats affected by inherited retinitis pigmentosa (IRP) and to correlate this event with the retinal expression of neurotrophins. Thirty-day-old Royal College of Surgeons (RCS) rats were exposed to 25-min-long daily sessions of low-frequency EA for 11 consecutive days. Control-untreated and EA-treated rats were sacrificed 1 h after the last EA session, and their retina removed for biochemical, molecular, and immunohistochemical analyses. Our data revealed that daily sessions of low-frequency EA for 11 days to RCS rats during a critical developmental stage of retinal cell degeneration cause an increase of retinal nerve growth factor (NGF) and NGF high-affinity receptor (TrkA) expression; and increase of outer nuclear layer (ONL) thickness; and enhanced vascularization. These findings suggest the possible beneficial effects of EA treatment in the development of IRP-like retinal degeneration of RCS rats and that the mechanism through which EA might exerts its action on the regulation of NGF and brain-derived neurotrophic factor (BDNF) and/or their receptors in retinal cells.

Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft–host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft–host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.

The LEI/L-DNase II pathway is activated in light-induced retinal degeneration in rats

Retinal death induced by light seems to be a caspase-independent process. In this work we investigate the LEI/L-DNase II pathway, a caspase-independent pathway, in light-induced retinal degeneration in Fischer rats. Measurement of DNase activity in total retinal extracts of light exposed Fischer rats was performed by analysing a plasmid degradation on an agarose gel. The same method was used to measure the in vitro activity of recombinant LEI (reticulocyte lysate) after incubation with calpains. L-DNase II activity is observed in retinal extracts of light exposed Fischer rats and increases with time illumination. In this apoptotic death, the activation of calpains has been shown. Here we show that L-DNase II activation is not catalized by calpains. The present study indicates that the LEI/L-DNase II may be a possible pathway activated during photoreceptor apoptosis in light-induced retinal degeneration but that this pathway is not directly activated by calpains.

Optokinetic test to evaluate visual acuity of each eye independently

A previously described optokinetic testing apparatus [Nat. Neurosci. 5 (2002) 53] was modified to measure vision in each eye separately for evaluation of monocular treatments. This apparatus consists also of a striped rotating drum. Ca. 170° of the drum are illuminated from outside and ca. 190° of the drum move behind a stationary black wall. The rat sits unrestrained in the drum center in a tube so that one eye is unexposed to the rotating stripes. Normal pigmented and retinal degenerate transgenic S334ter-3 rats were tested with the original and the modified apparatus. The usefulness of this method was tested in retinal degenerate rats with retinal transplants in one eye. In retinal degenerate animals, the amount of time (seconds) spent for head-tracking tended to be higher with the original method, possibly due to simultaneous stimulation of both eyes. In rats with retinal transplants, visual responses were significantly preserved in transplanted eyes at late stages of retinal degeneration. In conclusion, contributions from the fellow eye to the optokinetic tracking response can be limited by this testing modification, which is useful for evaluation of treatment effects to one eye.

Identification of novel genes and altered signaling pathways in the retinal pigment epithelium during the Royal College of Surgeons rat retinal degeneration

Shed photoreceptor outer segments (POS) are phagocytosed by RPE cells in a circadian manner. The homozygous deletion of the c- mer gene abolishes the ingestion phase of this phagocytosis in the Royal College of Surgeons (RCS) rat strain, which in turn leads to the death of photoreceptor cells. We identified RPE transcripts for which the expression is modulated by the abrogation of POS phagocytosis. A microarray approach and the differential display (DDRT–PCR) technique revealed 116 modulated known genes, 4 modulated unknown genes, and 15 expressed sequenced tags (ESTs) corresponding to unknown genes. The microarray and DDRT–PCR analyses detected alterations in signaling pathways such as the phosphatidylinositol 3-kinase–Akt–mTOR pathway and the DLK/JNK/SAPK pathway. The abrogation of POS phagocytosis caused a decrease in endomembrane biogenesis and altered endocytosis, exocytosis, transcytosis, and several metabolic and signaling pathways in RCS RPE cells. We also found differential levels of transcripts encoding proteins involved in phagocytosis, vesicle trafficking, the cytoskeleton, retinoic acid, and general metabolism.

Dietary docosahexaenoic acid protects against N-methyl- N-nitrosourea-induced retinal degeneration in rats

The effect of dietary intake of specific types of fatty acids on retinal degeneration due to N-methyl- N-nitrosourea (MNU)-induced photoreceptor cell apoptosis was evaluated. Fifty-day-old female Sprague–Dawley rats were given a single intraperitoneal injection of 50 mg kg −1 body weight of MNU, and were then switched to one of five different diets containing the following fatty acids at the following weight percentages: 10% linoleic acid (LA); 9·5% palmitic acid (PA) and 0·5% LA; 9·5% eicosapentaenoic acid (EPA) and 0·5% LA; 4·75% EPA, 4·75% docosahexaenoic acid (DHA) and 0·5% LA; or 9·5% DHA and 0·5% LA. When rats developed MNU-induced mammary tumors with a diameter of ≧1 cm, or at the termination of the experiment (20 weeks after MNU injection), retinal tissue samples were obtained and examined. Incidence and severity of retinal damage were compared by histologic examination. MNU-induced retinal degeneration was prevented in rats fed the diet containing 9·5% DHA (4·75% DHA was less effective), whereas it was accelerated in rats fed the 10% LA diet. Over the course of the 20-week experimental period, the fatty acid composition of serum reflected differences in dietary fatty acids. The present results indicate that a diet containing 9·5% DHA can counteract MNU retinotoxicity in the rat retina. DHA may play a role in protection against MNU-induced photoreceptor cell apoptosis in the rat retina.

Study of pharmacological effects of nilvadipine on RCS rat retinal degeneration by microarray analysis

In our recent study, we found that the Ca 2+ antagonist, nilvadipine caused significant preservation of photoreceptor cells in The Royal College of Surgeons (RCS) rats [Invest. Ophthalmol. Vis. Sci. 43 (2002) 919]. Here, to elucidate the mechanisms of nilvadipine-induced effects we analyzed altered gene expression of 1101 genes commonly expressed in rodent by DNA microarray analysis in the retinas of nilvadipine-treated and untreated RCS rats and SD rat. In the total number of genes, the expression of 30 genes was altered upon administration of nilvadipine to RCS rats, including several genes related to the apoptotic pathway and other mechanisms. Remarkably, neurotrophic factors, FGF-2 and Arc, known to suppress the apoptosis in the central nervous system, were up-regulated. These changes were also confirmed by real-time quantitative (Taqman) RT-PCR and Western blot analysis. Therefore, our present data suggested that administration of nilvadipine to RCS rats increases the expression of endogenous FGF-2 and Arc in retina, and potentially has a protective effect against retinal degeneration.

Retinal transplantation--advantages of intact fetal sheets.

Retinal transplantation aims to prevent blindness and to restore eyesight, i.e., to rescue photoreceptors or to replace damaged photoreceptors with the hope of reestablishing neural circuitry. Retinal donor tissue has been transplanted as dissociated cells or intact sheets. A promising experimental paradigm is the subretinal transplantation of sheets of fetal retina with or without its attached retinal pigment epithelium (RPE) into recipient rats with retinal degeneration. As long as healthy RPE either from the host or from the graft is present, such transplants can develop lamination resembling a normal retina. Different methods have been used to demonstrate transplant/host connectivity. In two different rat retinal degeneration models, visually evoked responses can be demonstrated in an area of the superior colliculus corresponding to the placement of the transplant in the retina. In summary, sheets of fetal retina can morphologically repair an area of a degenerated retina, and there is evidence to suggest that transplants form synaptic connections with the host and restore visual responses in blind rats.