Newborn Rat Retina Research Articles

Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina.

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3- immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.

Prostaglandin E2 promotes pathological retinal neovascularisation via EP4R-EGFR-Gab1-AKT signaling pathway

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Inhibitory effect of Houttuynia cordata Thunb on LPS-induced retinal microglial activation.

To identify the effect of Houttuynia cordata Thunb (HCT) on lipopolysaccharide (LPS)-induced microglial activation and investigate its possible molecular mechanisms. The primary retinal microglial cells were cultured from the retinas of newborn Sprague-Dawley rats and exposed to LPS, and/or HCT with different concentrations. The survival ability of retinal microglia cells was tested by standard MTT method. BrdU cell proliferation assay was used to evaluate the proliferation of retinal microglia. Inflammatory factors in the culture supernatants, including TNF-α, iNOS and IL-1β, were measured using ELISA. Microglia cells' migration was determined with Transwell migration assay. The total p38-MAPK and phosphorylation of p38-MAPK (p-p38-MAPK) were detected with Western blot. Primary retinal microglia in culture exposed to LPS to induce microglia activation. Pretreatment with HCT significantly inhibited the LPS-induced cell proliferation, but not the cell viability. LPS induced inflammatory reaction in microglia and cell migration. HCT significantly reduced LPS-stimulated release of pro-inflammatory factors and decreased the number of migrating cells substantially in a concentration-dependent manner. Moreover, the protein levels of p-p38 MAPK were identified as the up regulation and co-treatment with HCT obviously inhibited the upregulation of p-p38 MAPK, but had no effect on the levels of total p38-MAPK. The data suggest that HCT inhibits LPS-induced retinal microglial activation via suppression of the p-p38-MAPK. HCT may be used for the treatment of ocular diseases characterized by over-activated microglia.

Prostaglandin E2 Promotes Pathological Retinal Neovascularization via EP4R-EGFR-Gab1 Signalling Pathway

Background: Proliferative retinopathies, such as retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are major causes of visual impairment and blindness in industrialized countries. The rat oxygen-induced retinopathy (OIR) model is a well-established animal model for forms of pathological angiogenesis. PGE2, the major product of cyclooxygenases, is implicated in cellular proliferation, angiogenesis and migration via prostanoid receptor EP4. The aim of this study was to investigate the role of PGE2 and its EP4 receptor (EP4R) in the promotion of retinal neovascularization. Methods: In an OIR-induced rat model and a streptozotocin (STZ)-induced PDR model of pathological retinal angiogenesis, rats received an intravitreal injection of PGE2, Cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Findings: Intravitreal injection of PGE2 and Cay10598 significantly accelerated pathological retinal angiogenesis in OIR-induced rat retina and endothelial tip-cell formation in newborn rat retina. Treatment with PGE2 or Cay10598 also accelerated retinal NV in STZ-induced diabetic rats, which was ameliorated in rats pretreated with AH23848. In addition, pretreatment of human retinal microvascular endothelial cells (hRMECs) with AH23848 attenuated PGE2- or Cay10598- induced proliferation and migration by repressing the EGFR/Gab1/Akt signalling network. Interpretation: These results indicate that PGE2/EP4R mediated hRMECs proliferation by promoting the activation of the EGFR/Gab1/Akt signalling pathway and that this network is thus a potential therapeutic target for pathological intraocular angiogenesis. Funding: This work was supported by grants from the National Natural Science Foundation of China (no. 81770941 and 81800845), Jiangsu Key Medical Disciplines (ZDXKC2016008), Technology Development Fund (CSE12N1701), Wuxi Eminent Medical Talents (JCRC005 and QNRC077) and Young project of Wuxi health and Family Planning Commission (Q201707). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: A written informed consent was obtained from all the participants before the study, and the study was approved by the hospital ethics committee. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP), a Novel Secretagogue, Regulates Secreted Morphogens in Newborn Rat Retina.

Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment. Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot. Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor. Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.

Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures.

Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina.

Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Signal via Phospholipase C Pathway to Block Apoptosis in Newborn Rat Retina.

Glutamate induced cell death mechanisms gained considerable attention lately as excessive release of extracellular glutamate was reported to cause neurodegeneration in brain areas including the retina. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to provide neuroprotection through anti-apoptotic effects in the glutamate-model and also in other degeneration assays. Although PACAP is known to orchestrate complex intracellular signaling primarily through cAMP production, the mechanism that mediates the anti-apoptotic effect in glutamate excitotoxicity remains to be clarified. To study this mechanism we induced retinal neurodegeneration in newborn Wistar rats by subcutaneous monosodium-glutamate injection. 100pmol PACAP and enzyme inhibitors were administered intravitreally. Levels of caspase 3, 9, and phospho-protein kinase A were assessed by Western blots. Changes in cAMP levels were detected employing a competitive immunoassay. We found that cAMP blockade by an adenylyl-cyclase inhibitor (2',4'-dideoxy-adenosine) did not abrogate the neuroprotective effect of PACAP1-38. We show that following intravitreal PACAP1-38 treatment cAMP was unaltered, consistent with the inhibitor results and phospho-protein kinase A, an effector of the cAMP pathway was also unaffected. On the other hand, blockade of the alternative phosphatidylcholine-specific PLC pathway using an inhibitor (D609CAS) abrogated the neuroprotective effects of PACAP1-38. Our results highlight PACAP1-38 ability in protecting retinal cells against apoptosis through diverse signaling cascades. It seems that at picomolar concentrations, PACAP does not trigger cAMP production, but nonetheless, exerts a significant anti-apoptotic effect through PLC activation. In conclusion, PACAP1-38 may signal via both AC and PLC activation producing the same protective outcome.

Bevacizumab Reduces Neurocan Content and Gene Expression in Newborn Rat Retina In Vitro

Extracellular matrix (ECM) and cellular membrane proteoglycans (PGs) play important roles in neural differentiation and cell adhesion. Vascular endothelial growth factor, an important signal protein in vascular and retinal neural cell development, is retained in the ECM due to its high affinity for PG. Bevacizumab, an anti-VEGF agent, has been extensively used for treating retinal diseases in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on neurocan, phosphacan, and syndecan-3 PG levels in newborn rat retina. Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 48 hours. Immunohistochemical staining was assessed against neurocan, phosphacan, and syndecan-3. Proteoglycan content was quantified based on the intensity of immunohistochemical labeling. Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. The results from the treatment and control groups were compared. No significant difference in the staining intensity and mRNA expression of phosphacan and syndecan-3 was observed between the groups. However, a significant decrease in neurocan content and mRNA expression was observed in bevacizumab-treated retinal explants compared with controls. Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Interaction of copolymer‐1‐activated T cells and microglia in retinal ganglion cell protection

The interaction of copolymer-1-activated T cells and microglia on retinal ganglion cells survival in vitro was explored. Copolymer-1-specific T cells were made by repeated copolymer-1 stimulation of T cells, microglia were isolated from the retinas of newborn rats, and then, they were co-cultured (the experimental group) for 48 h. Retinal ganglion cells were collected from the retinas of adult rats, purified, and then, the supernatants from different groups were added. After 72 h, terminal-deoxynucleotidyl transferase-mediated d-UTP nick end labelling analysis was used to observe retinal ganglion cell apoptosis, and reverse transcription polymerase chain reaction was used to test messenger RNA expression of Caspase-3 and Caspase-8. The levels of cytokines, including insulin-like growth factor-1, brain-derived neurotrophic factor, tumour necrosis factor-α and interleukin-10, in the supernatants were examined by enzyme-linked immunosorbent assay to explore the possible mechanisms undergoing. After 72 h, the mean retinal ganglion cell apoptosis rate in the experimental group was the lowest (25.36%) among the groups. The messenger RNA expression of Caspase-3 and Caspase-8 in this group was significantly lower than that of the control groups (both P < 0.05). The secretion of interlekin-10 and brain-derived neurotrophic factor, insulin-like growth factor-1 and tumour necrosis factor-α in the supernatant of the experimental group were higher than that of the control groups (both P < 0.05) after co-culture. The interaction of copolymer-1-specific T cells with microglia could reduce retinal ganglion cell apoptosis. The related immune mechanisms were complicated. Upregulation of brain-derived neurotrophic factor and insulin-like growth factor-1, and the balance of some pro-inflammatory and anti-inflammatory cytokines may be involved in this protective autoimmunity.

Activation of c-Jun N-Terminal Kinase (JNK) during Mitosis in Retinal Progenitor Cells

Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.

A Precise Temporal Dissection of Monosodium Glutamate-Induced Apoptotic Events in Newborn Rat Retina In Vivo

Although L-glutamate is the main excitatory neurotransmitter in the retina, excess glutamate level triggers severe neuronal damages. Therefore, monosodium glutamate has been used to probe neurodegenerative mechanisms but precise toxicity schedule is not available in vivo. We report, for the first time, a temporal analysis of apoptotic processes induced by subcutaneously applied monosodium glutamate. We investigated the glutamate triggered subcellular processes over a time scale of 48 h in neonatal retina. We employed immunoblots to measure the level of activated apoptotic factors and immunocytochemistry to reveal the dying cells. Upregulation of active caspase-9 started at 3 h and peaked at 6 h post-injection. Activations of caspase-3, caspase-6 and caspase-7 consistent with their late-phase roles increased at 6 h post-injection. The apoptotic processes were terminated by 24 h post-injection. Caspase 12 and calpain-2 seemed unaffected by subcutaneous monosodium glutamate administration. Uniquely, we found that the ubiquitous calpain-1 is not expressed in newborn rat retina.

Expression and light sensitivity of clock genes Per1 and Per2 and immediate‐early gene c‐fos within the retina of early postnatal Wistar rats

Mammalian retina contains a circadian clock that is composed of components similar to those of the master circadian clock within the suprachiasmatic nuclei of the hypothalamus. The aim of the present study was to elucidate whether, when, and where the transcripts of the clock genes Per1 and Per2 and the immediate early gene c-fos are spontaneously expressed and/or induced by light in the newborn rat retina. At postnatal day 1 (P1), P3, P5, and P10, Wistar rat pups were released into constant darkness, and a 30-minute light pulse was administered during the subjective day or during the first or second part of subjective night. Gene expression was determined 30 minutes, 1 hour, 2 hours, and 4 hours after the light pulse by in situ hybridization followed by emulsion autoradiography. Endogenous expression of Per1 was detected in the neuroblastic retina, and Per2 expression was detected in the inner part of the neuroblastic retina from birth. Light pulses induced c-fos expression in ganglion cells from P1. Until P5, the cells were localized in the dorsal part of the retina, but, at P10, they were already distributed across the entire retinal circumference. Light pulses also induced the expression of c-fos and Per1 in the retinal pigment epithelium until P3, but not afterward. Expression of the Per2 gene was not photoresponsive until P10. These data demonstrate that the rat retina is light-sensitive immediately after birth. During early postnatal development, the spatial distribution of spontaneous and light-induced gene expression within the retinal layers changes gradually.

Further investigation of MSG induced apoptotic events and the pharmacological time window of PACAP in newborn rat retina

Further investigation of MSG induced apoptotic events and the pharmacological time window of PACAP in newborn rat retina

α-Crystallin Downregulates the Expression of TNF-α and iNOS by Activated Rat Retinal Microglia in vitro and in vivo

Purpose: Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of α-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia. Methods: Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without α-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method. Results: Activated microglia significantly upregulated TNF-α and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-α protein. Treatment with α-crystallin in vitro and in vivo downregulated their expression. Conclusion: The protective effect of α-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-α and iNOS.

Dissection of apoptotic events and pathways induced by MSG in newborn rat retina

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Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layers (in the neuroblast layer--NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retinal layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retinal is discussed.

Regulation of cytokine signaling components in developing rat retina correlates with transient inhibition of rod differentiation by CNTF

Ciliary neurotrophic factor (CNTF) is known to inhibit the differentiation of rod photoreceptors from postmitotic precursor cells. During early postnatal development, photoreceptor precursors lose their responsiveness to CNTF. The underlying events causing this change in responsiveness are unknown. Moreover, whether rods express CNTF receptor alpha, a prerequisite for a direct response to the factor, is controversial. Since morphological studies have previously produced conflicting results, we have analyzed the expression of cytokine receptor components and potential ligands in the rat photoreceptor layer by real-time reverse transcription with the polymerase chain reaction after laser microdissection and by immunoblotting. Cytokine effects on rods were studied in explant cultures from newborn rat retina. CNTF receptor alpha (CNTFR alpha) and leukemia inhibitory factor receptor ss (LIFRss) were expressed in immature photoreceptors. Expression of the CNTF-specific alpha-subunit (but not of LIFRss) was downregulated specifically in the photoreceptor layer in parallel with the appearance of opsin-positive rods. The decrease of CNTFR alpha levels in explant cultures was closely correlated with the loss of precursor cell responsiveness to CNTF. Increasing the CNTF concentration in the culture medium led to prolonged CNTFR alpha expression and, concomitantly, to persistent inhibition of rod differentiation. Application of CNTF and LIF in vitro induced phosphorylation of STAT3. Inducibility of STAT3 activation by CNTF decreased with photoreceptor maturation, whereas the LIF effect persisted. Our results thus indicate that CNTF acts directly on photoreceptor precursors inhibiting their differentiation via activation of the JAK/STAT3 signal transduction pathway, and that this effect is temporally limited because of the downregulation of CNTFR alpha.

A role for CK2 upon interkinetic nuclear migration in the cell cycle of retinal progenitor cells

In developing retina, the nucleus of the elongated neuroepithelial cells undergoes interkinetic nuclear migration (INM), that is it migrates back and forth across the proliferative layer during the cell cycle. S-phase occurs at the basal side, while M-phase occurs at the apical margin of the retinal progenitors. G1 and G2-phases occur along the nuclear migration pathway. We tested whether this feature of the retinal cell cycle is controlled by CK2, which, among its many substrates, phosphorylates both molecular motors and cytoskeletal components. Double immunolabeling showed that CK2 is contained in BrdU-labeled retinal progenitors. INM was examined after pulse labeling the retina of newborn rats with BrdU, by plotting nuclear movement from basal to apical sides of the retinal progenitors during G2. The CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited the activity of rat retinal CK2, and blocked nuclear movement proper in a dose-dependent way. No apoptosis was detected, and total numbers of BrdU-labeled nuclei remained constant following treatment. Immunohistochemistry showed that, following inhibition of CK2, the tubulin cytoskeleton is disorganized, with reduced acetylated and increased tyrosinated tubulin. This indicates a reduction in stable microtubules, with accumulation of free tubulin dimers. The results show that CK2 activity is required for INM in retinal progenitor cells.

Retinal Growth Hormone in Perinatal and Adult Rats

Growth hormone (GH) mRNA and protein have recently been localized in the neural retina of embryonic chicks, in which exogenous GH promotes cell survival. GH is also expressed in the rat CNS, in which it has neuroprotective roles, although its presence in the rat neural retina is unknown and is the focus of the present study. GH immunoreactivity, to a 22-kDa protein, was present in extracts of fetal (embryonic day [ED]17) eyes and in extracts from the neural retinas of newborn pups, comparable to GH immunoreactivity in pituitary extracts. The GH immunoreactivity in the neural retina was widespread but was most intense in large rounded cells in the retinal ganglion cell (RGC) layer and in the optic fiber layer derived from the axons of the RGCs. A 693-bp cDNA was also generated by the RT-PCR of RNA extracted from the eyes of ED17 rats and from the neural retinas and eyes of newborn rats, when amplified in the presence of oligonucleotide primers for the rat GH cDNA. Expression of the GH gene in the neural retina was also shown by specific in situ hybridization of an antisense GH riboprobe to cells in the neural retina, particularly those in the RGC layers of fetal and adult rat eyes. These results demonstrate GH expression in the neural retinas of fetal, newborn, and adult rats, in which retinal GH might have neuroprotective roles.

The Peptides ADNF-9 and NAP Increase Survival and Neurite Outgrowth of Rat Retinal Ganglion Cells In Vitro

Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.