Regenerating sciatic nerve does not utilize circulating cholesterol

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.     

Anxiety and cognitive performance in adolescent women with disruptive behavior disorders

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and permeability-inducing factor that has been implicated in the pathogenesis of diabetic retinopathy. In the present study, the localization and magnitude of VEGF, VEGF receptor-1 (VEGFR-1), and VEGF receptor-2 (VEGFR-2) gene expression were examined in the eye of streptozotocin-induced diabetic rats using quantitative in situ hybridization. VEGF protein was also examined by immunohistochemistry. Abundant VEGF mRNA and protein were present in the retinae of control rats. In the retinae of diabetic rats, VEGF gene expression was increased compared with control animals (p = 0.001). The increase in VEGF mRNA was noted in the ganglion cell layer and inner nuclear layer but not in the pigment epithelium of the retina. VEGF was also detected in blood vessels, ciliary body, and lens epithelium in both control and diabetic rats. The distributions of VEGFR-1 and VEGFR-2 were similar in both control and diabetic rats. VEGFR-1 mRNA was present beneath the inner limiting membrane and in the ganglion cell layer, inner nuclear layer, outer plexiform layer, and outer limiting membrane of the retina; it was also detected in blood vessels, the ciliary body, and the cornea. The magnitude and distribution of ocular VEGFR-1 mRNA were not affected by experimental diabetes. Expression of VEGFR-2 mRNA was noted in the inner nuclear layer and pigment epithelium of the retina and in blood vessels. An increase in VEGFR-2 mRNA in the diabetic retina was restricted to the inner nuclear layer. The presence of VEGF and its receptors in the control retina suggests a physiologic role for VEGF within the eye. The changes in retinal expression of VEGF and VEGFR-2 in association with diabetes suggest a role for this pathway in diabetic retinopathy.     

Expression of vascular endothelial growth factor and its receptors in rats with protein-overload nephrosis

BACKGROUND: Based on the fact that vascular endothelial growth factor (VEGF) increases vascular permeability, it is speculated that VEGF might be involved in the development of proteinuria, although this remains unconfirmed. The production and site of action of VEGF remains unclear in nephrotic renal diseases. METHODS: Non-radioactive in situ hybridization was performed to examine the expression of VEGF mRNA and its receptors, flt-1 and KDR/flk-1, in a rat model of nephrosis induced by intraperitoneal injection of bovine serum albumin (BSA). Saline injected rats were served as control animals. RESULTS: Neither morphological changes nor deposition of immunoglobulin or complement were observed in our model. Proteinuria developed, reaching a maximum level in rats injected with BSA for 3 days, followed by persistent proteinuria until day 14. The expression of mRNA for VEGF and the two receptors was markedly upregulated in glomeruli of BSA-induced nephritis compared with the control group. VEGF mRNA was localized in glomerular cells, including cells in mesangium, visceral and parietal epithelial cells. In contrast, flt-1 mRNA and KDR/flk-1 mRNA were expressed on glomerular endothelial cells and cells in mesangium. The ratio of glomerular cells positive for VEGF mRNA and its receptors mRNA increased proportionately with the severity of proteinuria. Immunohistochemistry for ED-1 and proliferating cell nuclear antigen showed no significant increase in infiltrating macrophage or cellular proliferation. Conclusions: Our results suggest that altered glomerular expression of VEGF and its receptors is not associated with proliferation of endothelial cells, but rather with proteinuria in BSA-induced nephritis in rats. VEGF may play a different role in different renal diseases.     

Presence of ERK2 in rat retinal cells

PURPOSE: Extracellular signal-regulated kinase 2 (ERK2) participates in the phosphorylation cascade that is activated in the an early intracellular response to various hormones and growth factors. We examined the expression and distribution of the ERK2 protein and mRNA in the rat retina before and after light exposure. METHODS: Rats were held on a 12 hr light/dark cycle and their retinas were removed and examined either just before or 2 or 30 min after light exposure. The tissue was processed for Western blotting to evaluate the presence of the protein for ERK2, and for in situ hybridization to evaluate the mRNA of ERK2. RESULTS: The Western blotting method showed a strong specific staining of a 42 kDa protein band in the retinal samples. This band corresponded to the expected size of p42 MAP kinase (ERK2). In situ hybridization histochemistry showed an intense localization of ERK2 mRNA in the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL) of the retina. The intensity and distribution of these signals did not differ among the animals, regardless of exposure to light. CONCLUSIONS: While ERK2 may be involved in the signal transduction system activated in retinal cells by light exposure, its precise role remains to be defined.     

Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion

Immunoreactivity to beta-amyloid precursor protein (APP) was present in the inner plexiform, ganglion cell and optic fibre layers, as well as in blood vessels, at birth in normally developing rat retinas. In the inner plexiform layer immunoreactivity disappeared by postnatal day (P) 14. A small population of ganglion cells was immunoreactive at birth, but none were visible at P7. From P14 onwards, however, there was weak immunoreactivity in ganglion cells again, and strong staining in Müller glia. Retinas affected by neonatal optic tract lesions contained more immunoreactive ganglion cells at P4 than did controls, but by P14 there was a severe loss of ganglion cells. These observations are consistent with APP being involved in retinal differentiation, including maturation of glia and neurones, synaptogenesis and possibly neuronal survival.     

Estrogen receptor expression in bovine and rat retinas

PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis

Vascular endothelial growth factor (VEGF) can induce proliferation of sinusoidal endothelial cells. Its mRNA expression was increased in proliferating rat hepatocytes in primary culture. To clarify a role of VEGF in liver after necrosis, expressions of VEGF and its receptors were measured in the liver or liver cells isolated from rats after carbon tetrachloride intoxication. Hepatic VEGF mRNA expression increased later than 24 h after the intoxication and became prominent at 168 h when liver necrosis disappeared, while hepatic mRNA expressions of its receptors increased between 24 and 72 h. VEGF mRNA expression was increased in Kupffer cells, hepatic macrophages and stellate cells isolated from rats between 24 and 72 h after the intoxication and in hepatocytes at 168 h compared to those cells from normal rats. Immunohistochemical VEGF stains were comparable to such results. Vascular endothelial cells existed abundantly in the necrotic areas, and sinusoidal endothelial cells appeared following disappearance of the necrotic areas. VEGF mRNA expression in hepatocytes isolated from 70% resected liver was increased at 12 h after the operation and became marked between 72 and 168 h. Similar increase of hepatic VEGF expression was immunohistochemically seen. In conclusion, VEGF derives from nonparenchymal as well as parenchymal cells in rat liver after necrosis. The former might contribute to vascular endothelial cell proliferation and the latter to sinusoidal endothelial cell regeneration.     

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.     

Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human

Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity

PURPOSE: To assess the role of vascular endothelial growth factor (VEGF) in the feline model of retinopathy of prematurity (ROP). METHODS: Retinopathy of prematurity was induced in neonatal cats by raising them in an oxygen-enriched (70% to 80%) atmosphere for 4 days to suppress vessel formation and then returning them to room air for 3 to 27 days. In situ hybridization was used to detect the expression of VEGF and its high-affinity receptor, flk-1, in the retina of neonatal cats, and glial fibrillary acidic protein immunocytochemistry was used to assess astrocyte status. RESULTS: The expression of VEGF in the innermost layers of retina fell in hyperoxia and increased on return to room air. Vascular endothelial growth factor expression was transient; it was maximal where vessels were about to form, and it rapidly downregulated after vessels had formed. During the proliferative vasculopathy of ROP, VEGF expression was stronger than in the normally developing retina, and the astrocytes that normally express VEGF degenerated. After the degeneration of astrocytes, VEGF was expressed by neurones of the ganglion cell layer. flk-1 was expressed by intraretinal and preretinal vessels. Supplemental oxygen therapy reduced or eliminated the overexpression of VEGF expression, astrocyte degeneration, and formation of preretinal vessels. CONCLUSIONS: Regulation of VEGF by tissue oxygen mediates the inhibition of vessel growth during hyperoxia and the subsequent proliferative vasculopathy. Degeneration of retinal astrocytes creates conditions for the growth of preretinal vessels.     

Bleeding times and the antithrombotic effects of high-dose aspirin, hirudin and heparins in the rat

The authors described the use of scanning electron microscopy after cryofracture to analyze rat retinas after experimental ischemia-reperfusion sequence. In Sprague-Dawley albino rats, intraocular hypertony by cannulation of anterior chamber was performed for 1 h on one eye. The other eye served as control. After 48 h of reperfusion, retinas were dissected. They were frozen and fractured before ultrastructural analysis by a scanning electron microscope. In the ischemic eyes the thickness of the photoreceptors layer was reduced, through internal disorganization resulting in misalignment of rods. There was swelling of the outer segments, a loss of adhesion between segments and superficial necrosis. Scanning electron microscopy after cryofracture permitted an analysis of the external morphology of retinal cells and intercellular components, especially the outer layers.     

Differential localization and expression of alpha and beta isoenzymes of protein kinase C in the rat retina

Expression and cellular localization of three isoenzymes of Ca2+-dependent protein kinase C (PKCalpha, PKCbeta, and PKCgamma) in the adult rat retina were revealed by immunohistochemistry and in situ hybridization histochemistry with isoenzyme-specific antibodies and cRNA probes. Immunoreactivities and mRNA signals for PKCalpha were conspicuous in rod bipolar cells. A subgroup of amacrine cells expressed PKCalpha. The cells in the ganglion cell layer also displayed PKCalpha gene products. Positive immunoreactivities for PKCbeta were localized as stripe patterns in the inner plexiform layer, corresponding to the stratification levels of axon terminals of cone bipolar cells. The somata of cone bipolar cells expressed PKCbeta. Amacrine cells and retinal ganglion cells also displayed PKCbeta gene products. The results obtained by immunohistochemistry were confirmed with colocalization of mRNA signals for PKCalpha and PKCbeta on the somata. The cell membranes showed stronger immunoreactivities than did the cytoplasms for both PKCalpha and PKCbeta. Neither immunoreactivities nor mRNA signals for PKCgamma were detected in all retinal regions. The differential roles of Ca2+-dependent PKC isoenzymes could be revealed in physiological defined retinal neurons.     

c-fos gene expression in postnatal rat retinas with light/dark cycle

We examined the diurnal variation of c-fos gene expression during a 12:12 light/dark cycle in developing rat retinas by in situ hybridization histochemistry. c-fos Gene was not expressed before postnatal day 10 (P10) but was expressed on P15 in the outer nuclear layer throughout the dark period and in the inner nuclear layer and the ganglion cell layer during the light period. These results demonstrated that the earliest c-fos gene expression occurred between P11 and P15. The good correlation between the expression of c-fos gene and the genes coding for proteins involved in phototransduction, in terms of their diurnal variation and in their development, suggested that c-fos gene may play a role in the regulation of these genes in retinal cells during the light/dark cycle.