Experimental Branch Retinal Vein Occlusion Research Articles

Inner Retinal Changes in Acute Experimental BRVO Treated With Bevacizumab or Triamcinolone Acetonide.

Apoptosis is a key process in neural degeneration associated with retinal vascular diseases. Vascular endothelial growth factor (VEGF) antagonists, including bevacizumab, are used to treat macular edema in these diseases. As VEGF has a critical role in the preservation of retinal neuronal cells, this study investigates the effects of bevacizumab on neural damage in a pig model of branch retinal vein occlusion (BRVO) and compares it with triamcinolone acetonide (TA) which is reported to possess neuroprotective properties. Thirty-six pigs had a photothrombotic BRVO in both eyes. Six pigs were injected with bevacizumab in one eye and TA in the fellow eye, then they were sacrificed, the eyes enucleated, and retinas processed at 2, 6, 10, and 20 days, respectively, together with three pigs (six eyes) BRVO only and three normal pigs (six eyes). Neuronal degeneration (apoptosis) and associated inner retinal changes were determined by terminal deoxyynuclotidyl transferase dUTP nick-end labeling (TUNEL), histology, and immunohistochemistry for macrophages. TUNEL labeling showed significantly higher apoptosis rates in the ganglion cell layer (GCL) and the inner nuclear layer (INL) in the bevacizumab-treated compared with the TA-treated retinas at 2, 10, and 20 day time points after occlusion (P < 0.05). Pyknotic cells were significantly higher in the GCL in bevacizumab-treated eyes at 6, 10, and 20 days and in the INL at 2 days compared to TA-treated retinas (P < 0.05). Macrophage infiltration was seen at all time points in both untreated and treated retinas with an absence of significance between bevacizumab- and TA-treated retinas (P > 0.05). Neurodegeneration in the BRVO acute phase is exacerbated by current standard treatments for BRVO. These results may have implications for the timing and treatment type. In the acute phase of BRVO, VEGF suppression with bevacizumab and to a lesser extent with triamcinolone exacerbates apoptosis in the inner retinal layers, which has implications for both the timing and choice of treatment.

Laser-Induced Porcine Model of Experimental Retinal Vein Occlusion: An Optimized Reproducible Approach

Retinal vein occlusion (RVO) is a frequent visually disabling condition. The management of RVO continues to challenge clinicians. Macular edema secondary to RVO is often recurrent, and patients typically require intravitreal injections for several years. Understanding molecular mechanisms in RVO is a key element in improving the treatment of the condition. Studying the molecular mechanisms in RVO at the retinal level is possible using animal models of experimental RVO. Most studies of experimental RVO have been sporadic, using only a few animals per experiment. Here, we report on 10 years of experience of the use of argon laser-induced experimental RVO in 108 porcine eyes from 65 animals, including 65 eyes with experimental branch retinal vein occlusion (BRVO) and 43 eyes with experimental central retinal vein occlusion (CRVO). Reproducibility and methods for evaluating and controlling ischemia in experimental RVO are reviewed. Methods for studying protein changes in RVO are discussed in detail, including proteomic analysis, Western blotting, and immunohistochemistry. Experimental RVO has brought significant insights into molecular changes in RVO. Testing intravitreal interventions in experimental RVO may be a significant step in developing personalized therapeutic approaches for patients with RVO.

Aflibercept Intervention in Experimental Branch Retinal Vein Occlusion Results in Upregulation of DnaJ Homolog Subfamily C Member 17.

Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) used to treat macular edema following branch retinal vein occlusion (BRVO). Despite well-documented efficacy, there is limited knowledge about proteome changes following aflibercept intervention in BRVO. Proteome changes may provide insights into mechanisms of action as well as aspects related to safety profile. In seven Danish Landrace pigs, BRVO was induced with a well-established experimental model of argon laser-induced BRVO. BRVO was induced in both eyes. Three days after the induced BRVO, aflibercept was injected intravitreally in the right eyes, while the left eyes received intravitreal isotonic saline water. Retinas were collected 15 days after the induced BRVO and analyzed with label-free quantification liquid chromatography tandem mass spectrometry (LFQ LC-MS/MS). Fourteen proteins were changed in expression following aflibercept intervention in the BRVO model. LFQ LC-MS/MS identified an upregulation of DnaJ homolog subfamily C member 17 (DNAJC17) (fold change = 6.19) and a modest downregulation of isoform 2 of the protein encoded by N-myc downstream regulated gene 2 (NDRG2) (fold change = 0.40). NDRG2 was unchanged by Western blotting. In the additional significantly regulated proteins, only discrete changes were observed (fold changes 0.52–1.59). Our study is the first to report an association between aflibercept intervention and the heat shock protein DNAJC17. Our results indicate that the role of heat shock proteins in the treatment of BRVO should be further explored.

Mo-derived perivascular macrophage recruitment protects against endothelial cell death in retinal vein occlusion

BackgroundTo decipher the role of monocyte-derived macrophages (Mφs) in vascular remodeling of the occluded vein following experimental branch retinal vein occlusion (BRVO).MethodsThe inflammation induced by laser-induced BRVO on mice retina was evaluated at different time points by RT-PCR looking at inflammatory markers mRNA level expression, Icam-1, Cd11b, F4/80, Ccl2, and Ccr2 and by quantification of Iba1-positive macrophage (Mφ) density on Iba1-stained retinal flatmount. Repeated intraperitoneal EdU injection combined with liposome clodronate-induced monocyte (Mo) depletion in wildtype mice was used to differentiate Mo-derived Mφs from resident Mφs. Liposome clodronate Mo-depleted wildtype mice and Ccr2-deficient mice were used to evaluate the role of all CCR2+ and CCR2neg Mo-derived Mφs on EC apoptosis in the occluded vein.Resultscd11b, ICAM-1, F4/80, Ccl2, and Ccr2 mRNA expression were increased 1, 3, and 7 days after vein occlusion. The number of parenchymal (parMφs) and perivascular (vasMφs) macrophages was increased 3 and 7 days after BRVO. The systemic depletion of all circulating Mos decreased significantly the BRVO-induced parMφs and vasMφs macrophage accumulation, while the deletion of CCR2+-inflammatory Mo only diminished the accumulation of parMφs, but not vasMφs. Finally, apoptotic ECs of the vein were more numerous in fully depleted, liposome clodronate-treated mice, than in Ccr2−/− mice that only lack the recruitment of CCR2+ inflammatory Mos.ConclusionsBRVO triggers the recruitment of blood-derived parMφs and vasMφs. Interestingly, vasMφs accumulation was independent of CCR2. The observation that the inhibition of the recruitment of all infiltrating Mφs increases the vein EC apoptosis, while CCR2 deficiency does not, demonstrates that CCR2neg Mo-derived vasMφs protect the ECs against apoptosis in the occluded vein.

Dexamethasone intravitreal implant downregulates PDGFR-α and upregulates caveolin-1 in experimental branch retinal vein occlusion

A dexamethasone (DEX) intravitreal implant (OZURDEX) provides an effective treatment of inflammation secondary to branch retinal vein occlusion (BRVO). Retinal proteome changes which mediate the beneficial effects of the implant remain poorly understood. To study retinal proteome changes in BRVO following an intervention with a DEX implant this study combined an experimental model of BRVO with proteomic techniques. In eight Danish Landrace pigs experimental BRVO was induced in both eyes using argon laser. After inducing BRVO a DEX implant was injected into the right eye of each animal while the left control eye was given an identical injection without an implant. Fifteen days after BRVO and DEX implant intervention the retinas were excised and analyzed with tandem mass tag based mass spectrometry. A total of 26 significantly changed proteins were identified. DEX intervention reduced the retinal levels of platelet-derived growth factor receptor-α (PDGFR-α) and vascular endothelial growth factor receptor 2 (VEGFR-2). DEX treatment resulted in increased levels of caveolin-1, peptidyl-prolyl cis-trans isomerase FKBP5 and transgelin. Changes in PDGFR-α and caveolin-1 were confirmed with immunohistochemistry. In BRVO treated with the DEX implant a strong reaction for caveolin-1 was observed in the innermost retinal layers. DEX implant intervention may inhibit PDGF signaling by decreasing the retinal level of PDGFR-α while an increased content of caveolin-1 may help maintain the integrity of the blood-retinal barrier.

Effect of microglia suppression on branch retinal vein occlusion in mice

PurposeActivation of retinal microglia seems to have an influence on the outcome of retinal degenerative diseases. Recent studies show that systemic colony stimulating factor‐1 receptor (CSF‐1R) inhibition ablates microglia in the mouse central nervous system. However, the role of microglia inhibition in ischemic retinal disease remains unclear. The present study aims at investigating the effect of microglia depletion in vivo in experimental branch retinal vein occlusion (BRVO).MethodsCx3cr1gfp/+ mice, specifically expressing green fluorescent protein (gfp) on microglia/macrophages, were fed with the CSF‐1R inhibitor PLX5622 in mouse chow for two weeks before laser‐induced BRVO. Following BRVO, one group of animals was continuously treated with PLX5622 for three more weeks (PLX5622 group), while a second group was switched to normal diet (PLX5622 cessation group). Anatomical changes in the retina and gfp+ cell distribution were monitored weekly with optical coherence tomography and scanning laser ophthalmoscopy autofluorescence imaging (AF), respectively. Every week, 2–3 animals of each group were euthanized for histology. Retinal whole mounts stained for iba‐1 (microglia/macrophages) and brn3a (ganglion cells) were compared to in vivo AF images.ResultsAccumulation of gfp‐expressing cells in the area affected by BRVO was observed shortly after the laser in both groups. However, this accumulation was less pronounced in the PLX5622 treated group. Surprisingly, ganglion cell numbers were significantly decreased in the PLX5622 cessation group compared to the PLX5622 Group.ConclusionsThe data provided here showed that CSF‐1R inhibition has a protective effect on ganglion cells after experimental BRVO. Thus, such inhibitiors may be a promising tool for modulation of retinal microglia and could be proven a useful target for ischemic retinal diseases.

Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.

To characterize retinal microglia activation and macrophage recruitment in experimental branch retinal vein occlusion (BRVO). Experimental BRVO was induced in Balb/c mice and histologic changes were studied. Tissue hypoxia was visualized using pimonidazole hydrochloride. Monocyte-derived retinal cells were quantified using histology and flow cytometry. To investigate the dynamics of invading blood-borne macrophages, chimera mice were generated using bone marrow grafts from Cx3cr1(gfp/gfp) mice to rescue lethally irradiated wild-type BALB/c mice. Longitudinal in vivo imaging was performed to monitor cell invasion. The levels of proinflammatory cytokines in the retina were quantified by quantitative real-time PCR. Histology showed disruption of tissue architecture and temporary swelling with marked hypoxia coinciding with increased VEGF-A and hypoxia inducible factor-1α (HIF-1α) expression and elevation of proinflammatory cytokines within 3 days after experimental BRVO, followed by thinning of the inner retinal layers at later time points. Proinflammatory cytokine levels were elevated. Activation of resident retinal microglia and recruitment of circulating macrophages in areas of hypoxic retina were evident early after the insult and peaked at day 7, remaining elevated for up to 28 days. Flow cytometry showed upregulation of CD68 and major histocompatibility complex class-II (MHC-II) expression at day 3, culminating at day 7. Experimental BRVO causes hypoxia and breakdown of the inner blood-retina barrier, followed by activation of microglia and invasion of macrophages from the systemic circulation. Consequently, treatments targeting microglia activation or macrophage recruitment might potentially mitigate the sequelae and attenuate degenerative changes induced by retinal vein occlusion.

Retinal proteome changes following experimental branch retinal vein occlusion and intervention with ranibizumab

Animal models of experimental branch retinal vein occlusion (BRVO) provide a unique opportunity to study protein changes directly in retinal tissue. Results from these experimental models suggest that experimental BRVO is associated with an upregulation of extracellular matrix remodeling and adhesion signaling processes. To study whether these processes could be blocked by inhibition of VEGF-A, a porcine model of experimental BRVO was combined with proteomic analyses. In six Danish Landrace pigs experimental BRVO was induced with argon laser in both eyes. After 24 h an injection of 0.05 mL ranibizumab was given in the right eyes of the animals while left eyes received an injection of 0.05 mL 9 mg/mL sodium chloride water. Retinas were dissected three days after BRVO and the retinal samples were analyzed with label-free quantification as well as tandem mass tag based proteomics. In retinas treated with ranibizumab five proteins exhibited statistically significant changes in content with both proteomic techniques. These five proteins, which were all decreased in content, included integrin β-1, peroxisomal 3-ketoacyl-CoA thiolase, OCIA domain-containing protein 1, calnexin and 40S ribosomal protein S5. As anti-integrin therapies are under development for inhibition of angiogenesis in retinal diseases it is interesting that inhibition of VEGF-A in itself resulted in a small decrease in the content of integrin β-1. The decreased content of integrin β-1 indicates that extracellular matrix remodeling and adhesion processes associated with BRVO are at least partly reversed through inhibition of VEGF-A.

Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

AimsBranch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.Methods and ResultsWe here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC) dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.ConclusionExperimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

Proteins involved in focal adhesion signaling pathways are differentially regulated in experimental branch retinal vein occlusion

Branch retinal vein occlusion (BRVO) is a common retinal vascular disease, but global protein changes following the condition remain largely unelucidated. To bring new insights into pathological processes and identify potential therapeutic targets, large-scale retinal protein changes following BRVO were studied by combining a porcine model of experimental BRVO with proteomic analysis by label-free liquid chromatography mass spectrometry. Among a total set of 1974 proteins, 52 significantly upregulated proteins and 10 significantly downregulated proteins were identified in retinas with BRVO after 15 days. Significantly upregulated proteins were involved in signaling pathways of focal adhesion via integrin and blood coagulation. Proteins involved in focal adhesion signaling included collagen α-2 chain, laminin subunit β-2, laminin subunit γ-1, lipocalin-7, nidogen-2, osteopontin, integrin-β, α-actinin-1, isoform 2 of α-actinin-1, talin-2 and filamin C. The identified proteins indicate that BRVO was associated with extracellular matrix remodeling processes. The present study identified focal adhesion signaling and ECM remodeling as important biological mechanisms to evaluate in the search for signaling pathways that promote neovascularisation and macular edema following BRVO.

Dye-Free Porcine Model of Experimental Branch Retinal Vein Occlusion: A Suitable Approach for Retinal Proteomics.

Branch retinal vein occlusion induces complex biological processes in the retina that are generated by a multitude of interacting proteins. These proteins and their posttranslational modifications can effectively be studied using modern proteomic techniques. However, no method for studying large-scale protein changes following branch retinal vein occlusion has been available until now. Obtainment of retinal tissue exposed to branch retinal vein occlusion is only available through experimental animal models. Traditional models of experimental branch retinal vein occlusion require the use of Rose Bengal dye combined with argon laser photocoagulation. The use of Rose Bengal dye is problematic in proteomic studies as the dye can induce multiple protein modifications when irradiated. This paper presents a novel technique for proteomic analysis of porcine retinal tissue with branch retinal vein occlusion combining a dye-free experimental model with label-free liquid chromatography mass spectrometry based proteomics.

Retinal Oxygen distribution in normal and pathological cases; animal models and humans

AbstractThe distribution of PO2 close to the vitreoretinal interface is heterogeneous, found higher near the arteriolar wall. Preretinal and transretinal PO2 profiles indicate that O2 diffusion from the arterioles affects the PO2 in the juxta‐arteriolar preretinal region. Both the preretinal and inner retinal PO2 recorded far from the vessels remain constant in all retinal areas. The oxygen tension (PO2) in the inner half of the retina remains largely unaffected by moderate changes in perfusion pressure. In addition a constant PO2 is observed during systemic PaO2 changes induced either by hyperoxia or hypoxia. However, an increase of PaCO2 (hypercapnia), as well as an intravenous injection of acetazolamide (carbonic anhydrase inhibitor) can both lead to an increase of preretinal PO2 due to retinal vessels dilatation. In the case of eyes with experimental branch retinal vein occlusion, PO2 values, within the inner retinal layers, are indicative of hypoxic conditions, whereas in adjacent unaffected retinal areas the PO2 remain normal. In diabetic patients undergoing vitrectomy, reports have shown a lowered vitreal POg in the affected retinal areas. Occlusion of the retinal circulation renders most of the inner retina anoxic.

Effect of Glial Cell Line-Derived Neurotrophic Factor on Retinal Function after Experimental Branch Retinal Vein Occlusion

The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs. Electrophysiological examination of the retina was performed in 20 pigs with standard and four-frame mfERG 4 weeks after induced BRVO and intravitreal injection of GDNF or vehicle. BRVO was induced by intraocular diathermia of the superior retinal vein. Inner retinal function was measured by analysis of the four-frame mfERG (iN1) and outer retinal function with standard mfERG (P1). In GDNF-treated BRVO eyes, P1 and iN1 amplitudes (P = 0.51 and 0.78) or implicit times (P = 0.08 and 0.99) did not differ from those in healthy fellow eyes. After vehicle injection, P1 and iN1 amplitudes of BRVO eyes were significantly lower than in the healthy fellow eye (P = 0.022 and 0.013). The log ratios of mfERG amplitudes between experimental and healthy fellow eyes were calculated (BRVO/healthy). GDNF improved the ratios of the four-frame mfERG (1.29 [0.88-1.88]) compared with vehicle (0.32 [0.21-0.50], P < 0.001). Equally, GDNF improved the ratios of the standard mfERG; GDNF (0.75 [0.51-1.10]) and vehicle (0.42 [0.27-0.63], P = 0.048). GDNF appears neuroprotective on retinal electrophysiological function after BRVO. The efficacy and safety of GDNF remain to be investigated in primate eyes.

Effects of intravitreal injection of anti‐TNFα and anti‐VEGF in experimental branch retinal vein occlusion in rats

AbstractPurpose To investigate the effect of intravitreal injection of anti‐TNFα and anti‐VEGF antibodies on the inner blood retinal barrier (iBRB) in a rat model of experimental branch retinal vein occlusion (BRVO). Claudin‐5, a tight junction protein seen in the retinal vascular wall was examined in order to evaluate the iBRB integrity.Methods BRVO was induced in the right eye of Long Evans rats, using laser photocoagulation. The left eye served as control. The intravitreal injection with either anti‐TNFα or anti‐VEGF was performed in the right eye of each animal, 6 hours after BRVO. Two days after laser application, animals were sacrificed and retinas were harvested. The expression and distribution of claudin‐5 were examined by immunofluorescent staining of flat‐mounts under confocal microscopy.Results Confocal micrographs showed that claudin‐5 is specifically expressed on the cell boundaries of retinal vessels. Two days after laser induced BRVO, the distribution of claudin‐5 in retinal veins of non‐treated rats was weaker, rougher and less linear compared to the images seen in control animals. The staining of claudin‐5 in retinal veins of rats treated with anti‐VEGF or anti‐TNFα was stronger, more linear and uniform.Conclusion The anti‐TNFα and anti‐VEGF drugs seem to partially restore the integrity of the iBRB which may contribute to the reduction of macular edema. Ongoing experiments using RT‐PCR will determine the effect of those drugs in the expression of genes that influence the development of macular edema after BRVO.

Oxygen distribution in the retina

AbstractPurpose To evaluate the distibution of the preretinal and transretinal partial pressure of oxygen(PO2) in normal and ischemic retinas.Methods Evaluation of either the preretinal or intraretinal oxygen partial pressure (PO2) distribution, using oxygen sensitive microelectrodes, in normal or ischemic retinal conditions.Results The distribution of PO2 close to the vitreoretinal interface is heterogeneous, being higher near the arteriolar wall. Preretinal and transretinal PO2 profiles indicate that O2 diffusion from the arterioles affects the PO2 in the juxta‐arteriolar preretinal region. Both the preretinal and inner retinal PO2 recorded far from the vessels remain constant in all retinal areas. The oxygen tension (PO2) in the inner half of the retina remains largely unaffected by moderate changes in perfusion pressure, systemic PaO2 changes occuring during either hyperoxia or hypoxia. However, an increase in PaCO2 (hypercapnia), as well as an intravenous injection of acetazolamide (carbonic anhydrase inhibitor) both lead to an increase in preretinal PO2 due to dilation of the retinal vessels. In eyes with experimental branch retinal vein occlusion, PO2 values within the inner retinal layers are indicative of hypoxic conditions, whereas adjacent areas appear to remain normal. In diabetic patients undergoing vitrectomy a lowered vitreal PO2 in the affected retinal areas has also reported.Occlusion of the retinal circulation renders most of the inner retina anoxic.Conclusion Thanks to the autoregulatory ability of the retinal circulation, the oxygen tension (PO2) in the inner half of the retina, remains largely unaffected during physiological stimuli. In both normal and inner retinal ischemic conditions,the oxygen supply from the choroid is sufficient to support only the photoreceptor inner segments.

Effect of anti‐VEGF drugs and steroids on the inner blood‐retinal barrier, after experimental BRVO in rats: preliminary results

AbstractPurpose To evaluate the effect of anti‐VEGF drugs and steroids on the inner blood‐retinal barrier (BRB) function after experimental branch retinal vein occlusion (BRVO) in rats. The integrity of the barrier is related to the expression of occludins by retinal endothelial cells.Methods Laser photocoagulation was applied to retinal veins of the right eye of 4 Long‐Evans rats, to induce BRVO. The left eye was used as control. One day after the occlusion, the right eye of each animal was injected intravitreally with either bevacizumab (n=2) or triamcinolone (n=2). The animals were sacrificed 7 days later; all retinas were harvested and prepared for indirect immunohistochemistry, using an anti‐ occluding primary antibody. The specimens were examined using a confocal microscope and images were acquired.Results In normal retinas, occludins appeared as a dense, well‐organised, fluorescent reticulum along the vascular wall. In eyes with BRVO, the structure of occludins was disrupted; irregular linear fluorescence appeared along occluded veins, while absence of fluorescence suggested down‐regulation of occludin expression. Occludin distribution was improved in animals having received treatment post BRVO.Conclusion The administration of bevacizumab and triamcinolone post BRVO seems to improve the function of the inner BRB. This could explain macular edema reduction after the use of these drugs. These preliminary results are part of an ongoing experiment, which will include the use of combination therapy in the management of BRVO.

Vasomotor Effect of Intravitreal Juxta-arteriolar Injection ofl-Lactate on the Retinal Arterioles after Acute Branch Retinal Vein Occlusion in Minipigs

To investigate the effect of L-lactate on retinal arteriolar diameter after acute branch retinal vein occlusion (BRVO) in minipigs. Thirteen eyes of 13 minipigs were evaluated, with the animals under general anesthesia. BRVO was induced by a standard method of argon laser endophotocoagulation. Two hours after BRVO, an intravitreal, juxta-arteriolar microinjection of 50 μL L-lactate 0.5 M (pH 7.4) was performed in nine eyes. Four eyes received a microinjection of 50 μL of the solvent (pH 7.4) that was used to prepare the solution of L-lactate and served as controls. Retinal arteriolar diameter changes were measured using a retinal vessel analyzer. Overall (n = 13), 2 hours after BRVO, there was a 9.0% ± 1.4% decrease in the retinal arteriolar diameter in the affected ares compared to baseline (P < 0.001). An increase of 26.2% ± 8.2% (P = 0.004) of the arteriolar diameter was evidenced 5 minutes after L-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before L-lactate microinjection. Thereafter, the vasodilatory effect of L-lactate persisted and remained significant until the end of the study period (27.7% ± 7.8% at 30 minutes) compared with the diameter before L-lactate microinjection (P = 0.002). Microinjection of the solvent alone (n = 4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1). These findings demonstrate a significant arteriolar vasodilation after intravitreal juxta-arteriolar L-lactate microinjection in eyes with experimental BRVO in the affected areas. L-lactate microinjection can reverse the arteriolar vasoconstriction that occurs in acute experimental BRVO.

Electrophysiological Consequences of Experimental Branch Retinal Vein Occlusion in Pigs and the Effect of Dorzolamide

To study the electrophysiological consequences of experimental branch retinal vein occlusion (BRVO) in pigs and the effect of dorzolamide. BRVO was induced in 16 pigs by diathermia. At 4 weeks animals were examined with multifocal electroretinography (mfERG) before and after dorzolamide or vehicle. The direct component P1 (outer retina) and indirect component iN1 (inner retina) were analyzed. Ophthalmoscopy, fundus photography, and fluorescence angiography were performed. BRVO eyes displayed signs of retinal damage and ischemia on ophthalmoscopy, fundus photography, and fluorescence angiography. mfERGs were affected by surgery; amplitude ratios (BRVO/healthy) were less than one (P1 = 0.30 [0.20-0.45]; iN1 = 0.35 [0.23-0.54]), and implicit time ratios were above one (1.04 [1.03-1.06] and 1.03 [1.02-1.05)]. In healthy eyes, iN1 amplitudes after treatment normalized to baseline (after/before) were lower in dorzolamide-treated animals than in the vehicle group (P = 0.05). After dorzolamide iN1 amplitude ratios (BRVO/healthy) were significantly higher than after vehicle (P = 0.01) and were not significantly different from one (0.97 [0.74-1.26]), indicating that the iN1 amplitudes in BRVO eyes were not different from those in healthy eyes after dorzolamide. BRVO in pig eyes examined by mfERG is a promising model for testing new treatment strategies in retinal ischemia. The local effects of BRVO are detectable on the mfERG and can be altered by dorzolamide. The decreased iN1 amplitudes caused by dorzolamide in healthy eyes were not seen in BRVO eyes possibly because of an increase in preretinal oxygen tension and improved function of the ischemic retina counteracting the effect of inner retinal acidification.

Vasomotor effect of intravitreal juxta‐arteriolar injection of L‐lactate on the retinal arterioles following acute branch retinal vein occlusion in minipigs

Abstract Purpose After branch retinal vein occlusion (BRVO), the retinal territory affected by the occlusion becomes hypoxic and the arterioles crossing this territory are often constricted. The purpose of the current experimental animal study is to investigate the effect of intravitreal juxta‐arteriolar microinjection of L‐lactate on the retinal arteriolar diameter following acute BRVO in minipigs. Methods Under general anesthesia, 9 eyes of 9 minipigs were evaluated. Argon laser endo‐photocoagulation was used to create BRVO. Two hours after BRVO, an intravitreal juxta‐arteriolar microinjection of 50 µl L‐lacate 1 mM (pH=7.4) was performed. The procedure was recorded in real time and retinal arteriolar diameter changes were measured in vivo using a Retinal Vessel Analyzer. Results Two hours after BRVO, the retinal arteriolar diameter decreased from 210 ± 16 AU to 183 ± 14 AU in the affected territories (p=0.04). Five minutes following L‐lactate juxta‐arteriolar microinjection, the retinal arteriolar diameter increased to 241 ± 16 AU (p&lt; 0.01). The vasodilatory effect of L‐lactate persisted and remained significant till the end of the study period (243 ± 15 AU at 30 min, p&lt;0.01). Conclusion We measured a significant vasodilation after intravitreal juxta‐arteriolar L‐lactate microinjection in eyes with experimental BRVO. L‐lactate microinjection can be of potential interest in acute BRVO, reversing the arteriolar vasoconstriction, thus leading to increase of the retinal arteriolar blood flow and possibly to improvement of the retinal cellular function in the occluded territory.

Analysis of gene expression in acute ischemic neuroretinas : a genome‐wide screen discriminating occlusion (BRVO) versus laser effects in rats

Abstract Purpose Identification of genes differentially regulated in rat neuroretinas submitted to an experimental acute branch retinal vein occlusion (BRVO), to a laser treatment, or to no treatment at all. Methods We have developed an in vivo experimental model of venous occlusion by photodynamic thrombosis in rat retinas. After anaesthesia, a sodium fluorescein solution was injected in rat tail 15 minutes before laser treatments. To induce ischemia in tested retina, venous sites adjacent to the optic nerve head were photocoagulated with an argon laser. In one group of tested animals, the retina was exposed to laser treatment at sites located between major vessels. As this treatment may have an effect upon choroidal blood flow, control eyes were not subjected to laser treatment. RNAs were isolated from the neuroretina 30 minutes post treatments, and processed for Affymetrix gene‐chip analysis. Results Genome‐wide screen enabled us to identify 308 and 348 genes which were up‐ or down‐regulated, respectively, by BRVO and laser treatment only. When we compared the transcriptomes of retinas subjected to vessel occlusion or laser treatment to the control one, we found that the expression profiles of, respectively, 116 and 126 genes were specifically modified. The majority of the up‐ and down‐regulated genes encode proteins involved in different aspects of early stress response, neuroprotection, inflammation and apoptosis. Conclusion Our microarray analysis revealed changes in gene expression bearing similarities to gene expression results from other ischemia models. Furthermore, it revealed that laser treatment may have an unreported impact on retina’s metabolism.