HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.

Neuroprotection can be achieved by preventing apoptotic death of postmitotic cells. Apoptotic death can occur by either a caspase-dependent mechanism, involving cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), and caspase-9, or a caspase-independent mechanism, involving apoptosis-inducing factor (AIF). HIV protease inhibitors (PIs) avert apoptosis in part by preventing mitochondrial outer membrane permeabilization (MOMP), but the precise mechanism by which they work is not known. Here, we evaluated the impact of the PIs in a mouse model of retinal detachment (RD) in vivo and in murine primary retinal cell cultures in vitro. Oral administration of the PIs nelfinavir and ritonavir significantly inhibited photoreceptor apoptosis, while preventing the translocation of AIF from mitochondria to the nucleus as well as the activation of caspase-9. RD-induced photoreceptor apoptosis was similarly inhibited in mice carrying hypomorphic mutations of the genes encoding AIF or Apaf-1. Nelfinavir attenuated apoptosis as well as mitochondrial release of AIF and cytochrome c, and subsequent activation of caspase-9 in vitro, in photoreceptor cultures exposed to starvation or monocyte chemoattractant protein-1-stimulated (MCP-1-stimulated) macrophages. Our results suggest that the MOMP inhibition by PIs involved interruption of both caspase-dependent and caspase-independent apoptosis pathways and that PIs may be clinically useful for the treatment of diseases caused by excessive apoptosis.

In vivo imaging of endothelial injury in choriocapillaris during endotoxin-induced uveitis.

Early detection of ocular inflammation may prevent the occurrence of structural damage or vision loss. Here, we introduce a novel noninvasive technique for molecular imaging and quantitative evaluation of endothelial injury in the choriocapillaris of live animals, which detects disease earlier than currently possible. Using an established model of ocular inflammation, endotoxin-induced uveitis (EIU), we visualized the rolling and adhesive interaction of fluorescent microspheres conjugated to recombinant P-selectin glycoprotein ligand-Ig (rPSGL-Ig) in choriocapillaris using a scanning laser ophthalmoscope (SLO). The number of rolling microspheres in the choriocapillaris peaked 4-10 h after LPS injection. The number of the accumulated microspheres peaked 4 h after LPS injection in the temporal choriocapillaris and 4 and 36 h after LPS injection in the central areas around the optic disk. Furthermore, we semiquantified the levels of P-selectin mRNA expression in the choroidal vessels by reverse transcription-PCR and found its pattern to match the functional microsphere interactions, with a peak at 4 h after LPS injection. These results indicate that PSGL-1-conjugated fluorescent microspheres allow specific detection of endothelial P-selectin expression in vivo and noninvasive assessment of endothelial injury. This technique may help to diagnose subclinical signs of ocular inflammatory diseases.

Vascular adhesion protein-1 blockade suppresses choroidal neovascularization.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis.

Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age-related macular degeneration. Vascular adhesion protein-1 (VAP-1), a cell surface and soluble molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP-1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP-1 in an established model of ocular inflammation, the endotoxin-induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half-maximal inhibitory concentration (IC(50)) of 0.007 microM against human and 0.008 microM against rat SSAO, while its IC(50) against the functionally related monoamine oxidase (MAO) -A and MAO-B is >10 microM. In the retina, VAP-1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP-1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P-selectin expression. Our data suggest an important role for VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation. VAP-1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases.

Identification of resident and inflammatory bone marrow derived cells in the sclera by bone marrow and haematopoietic stem cell transplantation.

AIMS: To characterise bone marrow derived cells in the sclera under normal and inflammatory conditions, we examined their differentiation after transplantation from two different sources, bone marrow and haematopoietic stem cells (HSC). METHODS: Bone marrow and HSC from green fluorescent protein (GFP) transgenic mice were transplanted into irradiated wild-type mice. At 1 month after transplantation, mice were sacrificed and their sclera examined by histology, immunohistochemistry (CD11b, CD11c, CD45), and transmission and scanning electron microscopy. To investigate bone marrow derived cell recruitment under inflammatory conditions, experimental autoimmune uveitis (EAU) was induced in transplanted mice. RESULTS: GFP positive cells were distributed in the entire sclera and comprised 22.4 (2.8)% (bone marrow) and 28.4 (10.9)% (HSC) of the total cells in the limbal zone and 18.1 (6.7)% (bone marrow) and 26.3 (3.4)% (HSC) in the peripapillary zone. Immunohistochemistry showed that GFP (+) CD11c (+), GFP (+) CD11b (+) cells migrated in the sclera after bone marrow and HSC transplantation. Transmission and scanning electron microscopy revealed antigen presenting cells among the scleral fibroblasts. In EAU mice, vast infiltration of GFP (+) cells developed into the sclera. CONCLUSION: We have provided direct and novel evidence for the migration of bone marrow and HSC cells into the sclera differentiating into macrophages and dendritic cells. Vast infiltration of bone marrow and HSC cells was found to be part of the inflammatory process in EAU.

Characterization of cytokine responses to retinal detachment in rats.

PURPOSE: Photoreceptor apoptosis is associated with retinal detachment (RD) induced photoreceptor degeneration. Previously, we demonstrated the importance of caspase activation for RD-induced photoreceptor death in a rat model of RD. However, extracellular signals that precede the activation of caspases and photoreceptor degeneration remain unclear. The aim of this study is to characterize the molecular and cellular responses that occur after RD. The expression of cytokines, chemokines, and growth factors were examined in a rat model of RD. METHODS: RD was induced in adult rats by subretinal injection of sodium hyaluronate. Retinal tissues were collected at various times (1, 3, 6, 24, and 72 h) after the induction of detachment. To screen for expressional changes in response to RD, major candidates for cytokines, chemokines, and growth factors were broadly examined by quantitative real time polymerase chain reaction (QPCR). To identify the cellular sources of the expressed genes, cells from various layers of the retina were obtained using laser capture microdissection (LCM), and their mRNAs were isolated. Protein expression was quantified by immunohistochemistry and Enzyme Linked-Immuno-Sorbent Assay (ELISA). To assess the potential of early response genes after RD to induce photoreceptor degeneration, exogenous recombinant proteins were subretinally injected and the photoreceptor cell death was assessed using a TdT-dUTP terminal nick-end labeling (TUNEL) assay at 24 h after RD. RESULTS: At 72 h after RD a significant increase in mRNA levels for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemotactic protein-1 (MCP-1), and basic fibroblast growth factor (bFGF) were detected in the neural retina. LCM revealed increased expression of mRNA for bFGF and MCP-1 in all retinal layers, though bFGF was especially evident in the outer nuclear layer (ONL) and MCP-1 in the inner nuclear layer (INL). TNF-alpha was increased in the ONL and the INL, and IL-1beta was increased in the ganglion cell layer. Time course experiments showed that TNF-alpha, IL-1beta and MCP-1 increased within 1 h after RD, while bFGF was increased by 24 h. Increased protein expression for TNF-alpha, IL-1beta, and MCP-1 was demonstrated by ELISA at 6 h after RD. Immunohistochemistry showed TNF-alpha and bFGF expression in the whole retina, with IL-1beta specifically expressed in astrocytes and MCP-1 in Müller cells. Subretinal administration of MCP-1 significantly increased TUNEL-positive cells in the ONL 24 h after RD, while injection of vehicle control, TNF-alpha, or IL-1beta showed no effect. CONCLUSIONS: Retinal glial cells, including astrocytes and Müller cells, are a major source of cytokine induction after RD. The increased expression and release of MCP-1 may be an important cause of photoreceptor degeneration associated with RD. This study helps to understand the mechanisms of RD-induced photoreceptor degeneration. Our results may provide new therapeutic targets to prevent photoreceptor degeneration following RD.

Alteration of leukocyte-endothelial cell interaction during aging in retinal microcirculation of hypertensive rats.

PURPOSE: Hypertension, one of the more common chronic diseases affecting the elderly, has been reported to influence leukocyte-endothelial cell interaction. The leukocyte-mediated inflammatory process contributes to age-related changes in vessels. This study was designed to evaluate age-related changes in leukocyte-endothelial cell interaction in the hypertensive rat retina. METHODS: Male spontaneous hypertensive rats (SHR; 1.5, 3, 6, 12, and 20 months of age) and age-matched Wistar-Kyoto rats (WKY) were used. The number of accumulated leukocytes was counted in sections of flat-mounted retinal tissue. The expression of intercellular adhesion molecule-1 (ICAM-1) and CD18 (the common beta-chain of ICAM-1 ligands) was evaluated. Retinal thickness was evaluated histologically. RESULTS: The number of accumulated leukocytes and the expression of ICAM-1 and CD18 increased in the aged retina. The number of leukocytes that accumulated and the expression of CD 18 were significantly higher in the SHR group than in the WKY group (P < 0.01). In addition, retinal thickness decreased with age. CONCLUSION: Leukocyte-endothelial cell interaction increased in the aged retina and these changes were more severe in SHR retina than in WKY retina. This increased interaction was first observed at 3 months, a relatively young age.

Intravitreal injection of corticosteroid attenuates leukostasis and vascular leakage in experimental diabetic retina.

PURPOSE: Recently, intravitreal injection of corticosteroids has been in wide use as a treatment for diabetic macular edema, and the outcomes have been favorable. However, the exact mechanism remains unclear. The hypothesis for the current study was that intravitreal corticosteroids may improve diabetic retinal edema by amelioration of blood-retinal barrier (BRB) breakdown, by inhibiting leukocyte stasis (leukostasis). METHODS: Diabetes was induced in 6-week-old male Long-Evans rats by intraperitoneal injection of streptozotocin (75 mg/kg). Three weeks after induction of diabetes, intravitreal injection of dexamethasone (40 microg/10 microL) was performed. At 2 days after intravitreal injection, accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and BRB breakdown was evaluated by measurement of retinal vascular permeability. The mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1 in the retina were also studied. RESULTS: The number of leukocytes accumulated in the retina, once increased in the diabetic group, was decreased by 31.6% (P = 0.0001) after dexamethasone injection. The level of BRB breakdown, also elevated in the diabetic group, was suppressed by 61.1% (P = 0.0046) after dexamethasone injection. The level of ICAM-1 mRNA expression and its protein, upregulated in the diabetic group, were downregulated by dexamethasone treatment by 70.0% (P < 0.0001) and 56.4% (P = 0.0003). CONCLUSIONS: Intravitreal injection of corticosteroids improves diabetic retinal edema through inhibiting leukocyte recruitment in the diabetic retina.

Thrombin inhibitor reduces leukocyte-endothelial cell interactions and vascular leakage after scatter laser photocoagulation.

PURPOSE: Macular edema is one of the most serious adverse effects after retinal scatter laser photocoagulation. It has been suggested that the inflammatory reaction after photocoagulation may be involved in the pathogenesis of macular edema. This study was designed to evaluate quantitatively the inhibitory effects of argatroban, a direct thrombin inhibitor, on leukocyte-endothelial cell interactions and vascular permeability after scatter laser photocoagulation. METHODS: Argon laser photocoagulation was performed in one half of the retina in pigmented male rats (n = 114). Argatroban was administered just before scatter laser photocoagulation. In the other half of the retina, AO leukocyte fluorography was used to evaluate in vivo leukocyte rolling along the retinal vein and accumulation in the retinal capillary bed. The expressions of P-selectin and intercellular adhesion molecule (ICAM)-1 were evaluated by reverse transcription-polymerase chain reaction. Retinal vessel permeability was quantified by using fluorescein isothiocyanate (FITC)-conjugated dextran. RESULTS: Scatter laser photocoagulation caused significant inflammatory leukocyte-endothelial cell interactions in the nonphotocoagulated half of the retina. Treatment with argatroban suppressed leukocyte-endothelial cell interactions. The maximum number of rolling and accumulating leukocytes was reduced by 46.6% (P < 0.01) and 51.4% (P < 0.01), respectively. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P < 0.05). Retinal vascular permeability was also suppressed significantly (P < 0.05). CONCLUSIONS: Argatroban suppressed leukocyte-endothelial cell interactions and blood-retinal barrier breakdown after scatter laser photocoagulation, suggesting that argatroban prevents postlaser macular edema.

Argatroban attenuates leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia.

BACKGROUND AND PURPOSE: Argatroban, a direct thrombin inhibitor, has been shown to reduce neural injury after transient cerebral ischemia. It has also been reported that this neuroprotective effect results from an anticoagulant function. This study was designed to evaluate quantitatively the inhibitory effects of argatroban on leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. METHODS: Retinal ischemia was induced for 60 minutes in male Long-Evans rats by temporary ligation of the optic sheath (n=342). Argatroban was administered just after induction of ischemia. Leukocyte and platelet behavior in the retinal microcirculation was then evaluated in vivo with scanning laser ophthalmoscopy. The expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) was evaluated by reverse transcription-polymerase chain reaction. After 10 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Treatment with argatroban suppressed leukocyte-endothelial cell interactions; the maximum numbers of rolling and accumulated leukocytes were reduced by 90.1% (P<0.05) and 58.7% (P<0.05), respectively, at 12 hours after reperfusion. Treatment with argatroban also suppressed platelet-endothelial cell interactions; the maximum numbers of rolling and adhering platelets were reduced by 91.8% (P<0.01) and 78.9% (P<0.01), respectively, at 12 hours after reperfusion. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P<0.01). Histologic examination demonstrated the protective effect of argatroban on ischemia-induced retinal damage (P<0.01). CONCLUSIONS: Argatroban treatment suppressed leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. This inhibitory effect on postischemic blood cell-endothelial cell interactions might partially contribute to its neuroprotective effects.

In vivo three-dimensional evaluation of leukocyte behavior in retinal microcirculation of mice.

PURPOSE: To evaluate new physiologic and three-dimensional methods for monitoring leukocyte behavior in mouse retina. METHODS: Endotoxin-induced uveitis (EIU) was produced in mice by footpad injection of lipopolysaccharide (LPS). Leukocytes were labeled with acridine orange (AO). Leukocyte rolling in the retinal microcirculation was evaluated in vivo with AO digital fluorography. The number of migrated leukocytes was counted in flatmounted retina. The behavior of leukocyte migration was observed three-dimensionally at the time of peak migration. After leukocytes were labeled with AO, the mice were perfused with rhodamine-labeled concanavalin A lectin to stain the vascular endothelium. Leukocyte migration into the retina was then monitored three-dimensionally with confocal microscopy, and the velocity of the migration was measured. RESULTS: Both leukocyte rolling and migration peaked at 48 hours after LPS injection. Leukocytes were seen to extravasate from the deeper capillary layers and to migrate toward the outer layer of the retina. The traveling velocity of extravasated leukocytes in retinal tissue was 2.0 +/- 0.1 microm/h. CONCLUSIONS: New methods have been demonstrated for the three-dimensional and quantitative evaluation of leukocyte behavior in mouse retina.

Platelets adhering to the vascular wall mediate postischemic leukocyte-endothelial cell interactions in retinal microcirculation.

PURPOSE: Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo. METHODS: Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia-reperfusion injury. CONCLUSIONS: This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Corneal thickness in eyes following pars plana lensectomy for congenital cataracts.

PURPOSE: To evaluate the corneal thickness following pars plana lensectomy for congenital cataracts. METHODS: The corneal thickness was measured in 24 eyes of 24 patients with congenital cataracts who had undergone pars plana lensectomy at a mean age of 24 +/- 32 (SD) months. The mean age at the time of our evaluation was 15 +/- 3 years. These measurements were compared with those in 15 eyes of an age-matched group of 15 normal volunteers. The central corneal thickness and endothelium were evaluated in both groups. RESULTS: The mean corneal thickness of the cataract-extracted eyes (592 +/- 47 micro m) was significantly greater than that of the controls (529 +/- 43 micro m; P < 0.001). There was no significant difference in the corneal endothelial cell count between cataract-extracted eyes (3420 +/- 715/mm(2)) and the controls (3182 +/- 358/mm(2); P = 0.49). However, there were significant differences in the frequency of hexagonally shaped endothelial cells (63% +/- 8.4%, cataract-extracted eyes; 70% +/- 7.7%, controls; P < 0.01), and in the coefficient of variation in the endothelial cell size (33 +/- 6.0, cataract-extracted eyes; 26 +/- 4.8, controls; P< 0.01). CONCLUSION: The central cornea of congenital cataract-extracted eyes was significantly thicker than that of controls.

Pars plana vitrectomy for epiretinal membrane associated with sarcoidosis.

PURPOSE: To examine retrospectively the visual outcomes in patients undergoing vitrectomy for epiretinal membranes secondary to sarcoid uveitis. METHODS: Eleven consecutive patients (11 eyes) with epiretinal membrane and uveitis associated with sarcoidosis underwent pars plana vitrectomy. RESULTS: Nine eyes (82%) gained two or more lines of Snellen visual acuity at 1-12 months after surgery. However, 4 of these 9 eyes lost two or more lines of Snellen visual acuity by the final visit. Overall, 5 eyes (45%) had attained at least two Snellen lines of visual acuity improvement, 5 eyes (45%) were unchanged, and 1 eye (10%) had worsened by two lines at the final visit. Nine eyes (81%) achieved visual acuity of 20/40 or better by the final visit. Slit-lamp biomicroscopy and fluorescein angiography showed that cystoid macular edema had resolved in 4 of 7 eyes postoperatively; vitritis improved in all cases. Postoperative complications included cataract formation, glaucoma, and membrane recurrence. Subsequent surgeries consisted of cataract extraction in 2 eyes and membrane peeling in 1 eye. CONCLUSIONS: Pars plana vitrectomy appears to have a beneficial effect on restoring vision in eyes with epiretinal membrane and uveitis associated with sarcoidosis, but final visual acuity was limited by the development of cataract and membrane recurrence.

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Glaucoma secondary to pars plana lensectomy for congenital cataract.

BACKGROUND: To identify risk factors related to the onset of secondary glaucoma in eyes that have undergone pars plana lensectomy for congenital cataract. METHODS: We investigated intraocular pressure levels after pars plana lensectomy for congenital cataract in 80 eyes of 44 patients who were followed up for at least 36 months. Mean age at the time of pars plana lensectomy was 11+/-18 months. Mean follow-up period was 116+/-57 months. RESULTS: An elevation in intraocular pressure (>20 mmHg) was found in 21 eyes of 12 patients (26%). In 7 of these 21 eyes surgical treatment was required. The incidence of the coexistence of microcornea (or microphthalmos) with congenital cataract was significantly higher in the glaucoma group than that in nonglaucoma group. The final visual acuity in the nonglaucoma group was significantly better than that in the glaucoma group. CONCLUSION: Our results show the importance of careful follow-up of intraocular pressure after pars plana lensectomy for congenital cataract, especially when microcornea (or microphthalmos) is present.

In vivo evaluation of retinal injury after transient ischemia in hypertensive rats.

A number of studies have suggested that hypertension affects the pathogenesis of inflammatory reactions in various organs. The objective of this study was to evaluate the effects of hypertension on leukocyte-endothelial interactions after transient retinal ischemia. Transient retinal ischemia was induced for 60 minutes in spontaneously hypertensive rats (SHR) and in age-matched normotensive Wistar-Kyoto rats (WKY). At 4, 12, 24, 48, and 72 hours after reperfusion, flat-mount retinas were prepared to evaluate the density of leukocytes that had been accumulated in the retina. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression was studied by semiquantitative polymerase chain reaction and ICAM-1 protein levels were studied by enzyme-linked immunosorbent assay. At 14 days after reperfusion, the retinal damage and the effect of superoxide dismutase on the damage were evaluated histologically. In SHR, the number of accumulated leukocytes peaked at 48 hours after reperfusion, and it was upregulated to 5.2-fold, as compared with that of WKY (P<0.001). ICAM-1 mRNA expression and ICAM-1 protein levels were increased significantly in the ischemia-reperfused retina in SHR compared with WKY (P<0.05). Histological examination demonstrated marked increase in the retinal ischemia/reperfusion damage in SHR (P<0.01) and a significant amelioration of the damage by treatment with superoxide dismutase in SHR (P<0.05). Oxidative stress may thus be an important mechanism for the deterioration seen in ischemia/reperfusion injury in the SHR retina.

Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes.

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.