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.     

Ocular vascular endothelial growth factor levels in diabetic rats are elevated before observable retinal proliferative changes

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF levels in ocular tissue of 6-, 12-, 18- and 28-week-old Goto-Kakizaki (GK) rats, a well-known model of non-insulin-dependent diabetes, were evaluated by highly sensitive ELISA. VEGF concentrations in the GK rat as well as in non-diabetic Wistar rat significantly decreased from the age of 6 weeks to 18 weeks. However, although VEGF concentrations in the Wistar rat continued to fall significantly from 18 to 28 weeks of age, the levels were maintained between 18 and 28 weeks of age in GK rats. Levels were significantly different between the GK and Wistar rats at 28 weeks of age. Results of immunohistochemical studies of the eyes of Wistar and GK rats at 28 weeks of age suggest diffuse distribution of this cytokine in cells of neural origin. Weak to moderate VEGF immunoreactivity was exhibited mainly in the ganglion cell layer, inner plexiform layer and inner/outer nuclear layers in rats with and without diabetes. However, in the retinal optic nerve fiber layer, retinal pigment epithelium and choroid, strong VEGF immunoreactivity was noted only in the GK rat. In conclusion, increased VEGF production in certain ocular tissue, similar to that in humans, is observed quite early, at least before the appearance of observable retinal changes in the diabetic GK rat. This also suggests that the GK rat can be used as a model of initial or latent phase diabetic retinopathy.     

Ganglion cell loss in RCS rat retina: a result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium

In the dystrophic Royal College of Surgeons (RCS) rat retina, there is a progressive loss of photoreceptors. As a result, the retinal circulation becomes apposed to the retinal pigment epithelium (RPE) and neovascular formations develop. RPE and inner nuclear layer cells migrate along these vessels towards the retinal ganglion cell (RGC) layer. The retinal layers gradually become disrupted, and some of the RGC axon bundles involute into the retina. These bundles are always associated with blood vessels, and there is evidence of axon damage where they juxtapose. In wholemount preparations of dystrophic retinae (> or =6 months of age), abrupt changes are observed in the trajectory of RGC axon bundles, where they are crossed by circumferential vessels. Degenerative profiles can be seen at these locations. Visualisation of RGCs with Fluoro-gold shows wedge-shaped sectors in the dystrophic retina devoid of labelling, initially in the ventral retina but later spreading dorsally. It is hypothesised that the vessels supplying the neovascular formations contract and pull surface vessels into the retina, thus displacing any axon bundles that lie beneath them into the inner plexiform layer. The contractility may be an intrinsic property of the vessels or it may be conferred by the cells migrating along them. Axonal transport becomes blocked at the points of tension, thereby causing retrograde degeneration of the parent RGCs. Because RGC loss is also a feature of human retinitis pigmentosa, the RCS rat may provide a model to test interventions devised to prevent such loss following photoreceptor degeneration. This model also may be useful for testing methods designed to control blood vessel and matrix formation.     

Promotion of healthy lifestyle: knowledge and attitudes of primary care physicians

Preretinal neovascularization and chronic retinal oedema are the two major sight-threatening complications that can occur during diabetic retinopathy. Ocular neovascularization is strongly associated with retinal ischaemia, and growth factors have been implicated in its pathogenesis. The ischaemic retina is assumed to secrete growth factors that stimulate residual vessels to proliferate. Interest has focused on basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta) and more recently vascular endothelial cell growth factor (VEGF). Histologic studies have demonstrated the presence of growth factor proteins and receptors and/or their mRNA, mainly VEGF, PDGF, and bFGF, in preretinal membranes of patients with proliferative diabetic retinopathy. Elevated intravitreal levels of IGF-1 and VEGF correlating with neovascular activity have been found in some patients. However, a direct causal relationship between ischaemia, growth factors and neovascularization has not been clearly demonstrated despite considerable research work. To date, the growth factor correlating most closely with neovascularization is VEGF. As many growth factors seem to be produced during the neovascular process, their specific inhibition probably will have limited effects. Laser photocoagulation of the retina has proved beneficial for regression of new vessels, probably through destruction of the ischaemic retina producing neovascular growth factors, and is currently the only treatment for proliferative diabetic retinopathy. Inhibition of IGF-1 by somatostatin analogs has produced unsatisfactory results. Other vascular inhibitors are currently being studied.     

Comparative study of astrocytes in human and rabbit retinae

Immunohistochemical location of Glial Fibrillary Acidic Protein (GFAP) was used to compare the morphology of astrocytes in vascularized and partially vascularized retinae (human and rabbit, respectively). Astrocytes in human and rabbit retinae were found in the same regions as the blood vessels. These cells in partially vascularized retinae differed from those in vascularized retina in several respects. Firstly, there were six morphological types in rabbit retina and only two in human retina. Secondly, in rabbit retinae there were astrocytes only related to blood vessels called "perivascular astrocytes" which were absent in human retinae. Thirdly, the astrocytes were located in the nerve fiber layer and ganglion cell layer in both types of retinae, but in human retinae these cells could also be seen in the internal nuclear layer. These observations demonstrate that there are many differences between astrocytes in human and rabbit retina, suggesting that rabbit retina could be used with caution as an experimental model in comparative studies with human retina.     

Localization of vascular endothelial growth factor in human retina and choroid

OBJECTIVE: To examine the distribution and relative levels of vascular endothelial growth factor (VEGF) in the nondiabetic and preproliferative diabetic human retina and choroid. METHODS: Immunohistochemical localization was performed on frozen sections from cryopreserved postmortem human tissue using a polyclonal antibody against VEGF and a streptavidin peroxidase system. Eyes from 5 subjects without diabetes and 8 subjects with diabetes were examined and analyzed using a 7-point immunohistochemical grading system. RESULTS: In subjects without diabetes, weak or no VEGF immunoreactivity was associated with retinal blood vessels. In subjects with diabetes, we found significantly increased immunoreactivity in the retinal vascular endothelium and blood vessel walls. Vascular endothelial growth factor immunoreactivity was also associated with intravascular leukocytes in subjects with and without diabetes. In the choroid of subjects without diabetes, immunoreactivity was almost exclusively associated with intravascular leukocytes, whereas in diabetic subjects, immunoreactivity was localized within choriocapillaris endothelium, choroidal neovascular endothelium, and migrating retinal pigment epithelium cells. CONCLUSIONS: The observed increase in VEGF immunoreactivity in the diabetic retina and choroid suggests that VEGF may contribute to 2 well-documented events during retinopathy: increased vascular permeability and angiogenesis.     

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.     

Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate

PURPOSE: The purpose of the study is to determine the effect of exogenous vascular endothelial growth factor (VEGF) on the primate retina and its vasculature. METHODS: Ten eyes of five animals were studied. Physiologically relevant amounts of the 165 amino acid isoform of human recombinant VEGF were injected into the vitreous of six healthy cynomolgus monkey eyes. Inactivated human recombinant VEGF or vehicle was injected into four contralateral control subject eyes. Eyes were assessed by slit-lamp biomicroscopy, tonometry, fundus color photography, fundus fluorescein angiography, light microscopy, and immunostaining with antibodies against proliferating cell nuclear antigen and factor VIII antigen. RESULTS: All six bioactive VEGF-injected eyes developed dilated, tortuous retinal vessels that leaked fluorescein. Eyes receiving multiple injections of VEGF developed progressively dilated and tortuous vessels, venous beading, edema, microaneurysms, intraretinal hemorrhages and capillary closure with ischemia. The severity of the retinopathy correlated with the number of VEGF injections. None of the four control eyes exhibited any abnormal retinal vascular changes. The endothelial cells of retinal blood vessels were proliferating cell nuclear antigen positive only in the bioactive VEGF-injected eyes. CONCLUSION: Vascular endothelial growth factor is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies, such as hemorrhage, edema, venous beading, capillary occlusion with ischemia, microaneurysm formation, and intraretinal vascular proliferation.     

In vitro evaluation of the long-body On-X bileaflet heart valve

AIM: To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes. METHODS: Immunohistochemical localisation of VEGF, using antibodies raised against VEGF165 and VEGF121,165,189, was carried out on specimens of normal human retina (n = 15), diabetic retinas ((a) with no overt retinopathy (n = 19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n = 6), (c) with active proliferative retinopathy (n = 6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n = 15)), excised diabetic fibrovascular membranes (n = 19), and non-diabetic fibrocellular membranes (n = 7). The degree and pattern of immunostaining was recorded. RESULTS: In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF165 antibody was generally confined to endothelial cells adn perivascular regions while the VEGF121,165,189 antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined. CONCLUSIONS: This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.     

Expression of insulin-like growth factor-I gene in normal and diabetic rat eye

The expression of insulin-like growth factor I (IGF-I) gene was studied in both normal and diabetic rat eyes via in situ hybridization. The results revealed the regional expression of IGF-I gene in the rat eyes. The expression of IGF-I mRNA in the internal nuclear layer and ganglion cell layer is strong, in choroid, retinal pigment epithelium and external nuclear layer is moderate, in sclera is weak in degree and no such mRNA is detected in the cornea. The abundance of IGF-I mRNA in diabetic eye tissues is significantly (P < 0.05 or P < or = 0.01) higher than that in normal eye. These findings suggest that (1) functionally, the eye ball be considered to be an "IGF-I paracrine-autocrine system", and (2) the high expression of IGF-I indicate its initiation of diabetic retinopathy and its promotion of the progression of the lesion.     

Adenoid cystic carcinoma of the breast: value of histologic grading and proliferative activity

Using vitreous fluorophotometry and quantitative fluorescence microscopy the authors studied the permeability of the blood-retinal barrier (BRB) to fluorescein in control and in 8 days streptozotocin-induced diabetic rats. Vitreous fluorophotometry showed that fluorescein permeates BRB in control and in diabetic rats. However, in diabetic rats the permeability to fluorescein was significantly increased as compared to control rats. The vitreous penetration ratio (VPR) values for total and free fluorescein at 60 min, were higher for diabetic rats (231.2+/-12.9 min-1 for total fluorescein and 1299.24+/-58.0 min-1 for free fluorescein) than for control rats (95.5+/-3.5 min-1 for total fluorescein and 646.6+/-55. 0 min-1 for free fluorescein) (P<0.05). Quantitative confocal fluorescence microscopy confirmed these findings and identified the site of leakage across the BRB by comparing the relative importance of the fluorescein leakage across the outer and inner BRB. In control rats the fluorescence levels remained relatively low in the photoreceptor layer, next to the outer BRB but in the inner nuclear layer, next to the inner BRB reached values that were almost ten times higher. These results suggest that in retinas of control rats fluorescein penetrates predominantly through the inner BRB. In diabetic rats the fluorescence levels in the photoreceptor and in the inner nuclear layer were significantly increased as compared to the fluorescence levels in controls rats. Nevertheless, in the inner nuclear layer the fluorescence levels were also generally higher than the fluorescence levels at the photoreceptor layer. The rates of fluorescence levels between the inner nuclear layer and the photoreceptor layer were apparently 3:1, 60 min after the single intravenous injection of fluorescein. Also, the fluorescein penetration in the inner nuclear layer of the diabetic rats is higher than that observed in the inner nuclear layer of the control rats (P<0.001). These findings suggest that the permeability to fluorescein of both components of the BRB is increased 8 days after the induction of diabetes by streptozotocin and that the permeability of the retinal vasculature is preferentially affected.     

Transient neovascularisation of the frog retina during optic nerve regeneration

In conditions such as diabetic retinopathy, degenerative events in the retina are associated with neovascularisation. It is well established that a proportion of retinal ganglion cells die during optic nerve regeneration in the frog. The present study has determined whether neovascularisation takes place during this regenerative process. To do so, the pattern of blood vessels overlying the retinal ganglion cell layer was analysed in the frog Litoria (Hyla) moorei. We examined normal animals and those undergoing optic nerve regeneration following nerve crush. Blood vessels were visualised by perfusion with Indian ink and retinae were prepared as wholeamounts. In normal animals, the vascular tree was found to lie superficial to the nerve fibre layer and was more complex in regions overlying the area centralis and visual streak. After nerve crush, abnormal blood vessels transiently formed between the existing branches of the vascular tree. The new vessels were concentrated as an annulus centred on the optic nerve head and over the area centralis in midtemporal retina. The neovascularisation became most extensive between 6 and 10 weeks postcrush and disappeared by 12 weeks. The spatiotemporal sequence of neovascularisation suggests that it is triggered by accumulations of degenerating material formed as a proportion of the ganglion cells die during optic nerve regeneration.     

Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for nonendothelial targets for VEGF

Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.     

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.     

Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998

Prolonged hyperglycemia results in a number of diabetic complications, including retinopathy. Pericyte degeneration is one of the earliest histological changes observed in the development of diabetic retinopathy. Increased free radicals generated under hyperglycemia could damage the retina, which abounds in polyunsaturated fatty acids. In the current study, a severalfold increase in thiobarbituric acid-reactive substances was found in rat retina cultured in hyperglycemic medium, which decreased significantly when trolox, an amphipathic antioxidant, was included in the medium. To examine the contribution of oxidative stress in vivo, diabetic rats were fed trolox (0.4% in the diet) during the course of the experiments. After 5 mo of hyperglycemia, whole mounts of retinal vessels were prepared and endothelial cells (E) and pericytes (P) were counted. The ratio of E/P in the retinas obtained from normal rats, diabetic rats, and diabetic rats fed trolox were 1.74 +/- 0.186, 3.78 +/- 0.47, and 2.32 +/- 0.24, respectively. A significant restoration of pericytes by trolox suggests the involvement of oxidative injury during pericyte loss in diabetic retinopathy.     

Retinal hypoxia in long-term diabetic cats

PURPOSE: To determine whether the retina is hypoxic in early stages of diabetic retinopathy in cats and to correlate intraretinal PO2 with fluorescein angiographic and histologic alterations. METHODS: Intraretinal PO2 was measured with microelectrodes in three cats with long-standing diabetes (>6 years) that had been followed with fluorescein angiographs every 6 months. Average PO2 in the inner vascularized half of the retina was compared with similar measurements in 21 control animals. Photoreceptor oxygen consumption was also compared. The retinal vascular endothelium of the diabetic animals was stained for ADPase activity in flatmounts, and transverse sections were used to visualize microscopic alterations in vascular structure. RESULTS: PO2 in the inner half of the retina was abnormally low in the diabetic cats, 7.7+/-5.2 mm Hg (35 penetrations in 3 cats) versus 16.4+/-9.3 mm Hg in normal cats (85 penetrations in 21 cats) (P < 0.001). Oxygenation was almost normal in some regions of the diabetic retinas, but little evidence of oxygen supply from the retinal circulation was observed in other regions. Inner retinal hypoxia was present in areas with no detectable capillary dropout in fluorescein angiograms or flatmounts. The worst changes histologically were microaneurysms, leukocyte and platelet plugging of aneurysms and venules, and degenerating endothelial cells in capillary walls. These histologic abnormalities were confined to small regions, some of which could be positively correlated with markedly abnormal PO2 profiles. Photoreceptor oxygen utilization was not affected in two diabetic cats, but was below normal in one animal in which choroidal PO2 was low. CONCLUSIONS: This is the first direct demonstration of retinal hypoxia in early diabetic retinopathy, before capillary dropout was evident clinically. Hypoxia was correlated with endothelial cell death, leukocyte plugging of vessels, and microaneurysms.     

Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin

This study determined whether retinal degeneration during diabetes includes retinal neural cell apoptosis. Image analysis of retinal sections from streptozotocin (STZ) diabetic rats after 7.5 months of STZ diabetes identified 22% and 14% reductions in the thickness of the inner plexiform and inner nuclear layers, respectively (P < 0. 001). The number of surviving ganglion cells was also reduced by 10% compared to controls (P < 0.001). In situ end labeling of DNA terminal dUTP nick end labeling (TUNEL) identified a 10-fold increase in the frequency of retinal apoptosis in whole-mounted rat retinas after 1, 3, 6, and 12 months of diabetes (P < 0.001, P < 0. 001, P < 0.01, and P < 0.01, respectively). Most TUNEL-positive cells were not associated with blood vessels and did not colocalize with the endothelial cell-specific antigen, von Willebrand factor. Insulin implants significantly reduced the number of TUNEL-positive cells (P < 0.05). The number of TUNEL-positive cells was also increased in retinas from humans with diabetes. These data indicate that retinal neural cell death occurs early in diabetes. This is the first quantitative report of an increase in neural cell apoptosis in the retina during diabetes, and indicates that neurodegeneration is an important component of diabetic retinopathy.