Induction of Ischemic Tolerance Protects the Retina From Diabetic Retinopathy
2011; Elsevier BV; Volume: 178; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.01.040
ISSN1525-2191
AutoresDiego C. Fernandez, Pablo H. Sande, Mónica S. Chianelli, Hernán J. Aldana Marcos, Ruth E. Rosenstein,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoDiabetic retinopathy is a leading cause of acquired blindness. Available treatments are not very effective. We investigated the effect of a weekly application of retinal ischemia pulses (ischemic conditioning) on retinal damage induced by experimental diabetes. Diabetes was induced by an intraperitoneal injection of streptozotocin. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 minutes; this maneuver started 3 days after streptozotocin injection and was weekly repeated in one eye, whereas the contralateral eye was submitted to a sham procedure. Diabetic retinopathy was evaluated in terms of i) retinal function (electroretinogram and oscillatory potentials), ii) integrity of blood-retinal barrier (by albumin–Evans blue complex leakage and astrocyte glial fibrillary acidic protein IHC), iii) optical and electron microscopy histopathologic studies, and iv) vascular endothelial growth factor levels (using Western blot analysis and IHC). Brief ischemia pulses significantly preserved electroretinogram a- and b-wave and oscillatory potentials, avoided albumin–Evans blue leakage, prevented the decrease in astrocyte glial fibrillary acidic protein levels, reduced the appearance of retinal edemas, and prevented the increase in vascular endothelial growth factor levels induced by experimental diabetes. When the application of ischemia pulses started 6 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies for diabetic retinopathy treatment. Diabetic retinopathy is a leading cause of acquired blindness. Available treatments are not very effective. We investigated the effect of a weekly application of retinal ischemia pulses (ischemic conditioning) on retinal damage induced by experimental diabetes. Diabetes was induced by an intraperitoneal injection of streptozotocin. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 minutes; this maneuver started 3 days after streptozotocin injection and was weekly repeated in one eye, whereas the contralateral eye was submitted to a sham procedure. Diabetic retinopathy was evaluated in terms of i) retinal function (electroretinogram and oscillatory potentials), ii) integrity of blood-retinal barrier (by albumin–Evans blue complex leakage and astrocyte glial fibrillary acidic protein IHC), iii) optical and electron microscopy histopathologic studies, and iv) vascular endothelial growth factor levels (using Western blot analysis and IHC). Brief ischemia pulses significantly preserved electroretinogram a- and b-wave and oscillatory potentials, avoided albumin–Evans blue leakage, prevented the decrease in astrocyte glial fibrillary acidic protein levels, reduced the appearance of retinal edemas, and prevented the increase in vascular endothelial growth factor levels induced by experimental diabetes. When the application of ischemia pulses started 6 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies for diabetic retinopathy treatment. Diabetic retinopathy (DR) is a leading cause of acquired blindness. The incidence of DR is rarely detected in the first few years of diabetes, but it increases to 50% by 10 years and to 90% by 25 years of diabetes. Although DR has long been recognized as a vascular disease, it is becoming increasingly clear that retinal cells are also affected by diabetes, resulting in dysfunction and degeneration of neuronal cells.1Kern T.S. Barber A.J. Retinal ganglion cells in diabetes.J Physiol. 2008; 586: 4401-4408Crossref PubMed Scopus (325) Google Scholar Vision loss occurs due to chronic hyperglycemia, vascular damage and leakage, edema, capillary basement membrane thickening, neovascularization, hemorrhage, and ischemia.2Kowluru R.A. Chan P.S. Oxidative stress and diabetic retinopathy.Exp Diabetes Res. 2007; 2007: 43603-43614PubMed Google Scholar Although the pathogenesis of DR is highly complex and not fully understood, VEGF is recognized as a major contributor to the development of DR.3Zhang X. Bao S. Hambly B.D. Gillies M.C. Vascular endothelial growth factor-A: a multifunctional molecular player in diabetic retinopathy.Int J Biochem Cell Biol. 2009; 41: 2368-2371Crossref PubMed Scopus (50) Google Scholar Despite the attempts to control blood glucose levels, many diabetic patients are affected by DR, which progresses to more severe forms of disease. Because available treatments are not very effective, it is imperative to develop better approaches for DR prevention and treatment. Unraveling which are the most critical mechanisms is unlikely to be achieved in studies limited to the clinically observable retinal changes in human DR. Far more detailed and invasive studies are required, preferably in a readily available animal model.4Antonetti D.A. Barber A.J. Bronson S.K. Freeman W.M. Gardner T.W. Jefferson L.S. Kester M. Kimball S.R. Krady J.K. LaNoue K.F. Norbury C.C. Quinn P.G. Sandirasegarane L. Simpson I.A. JDRF Diabetic Retinopathy Center GroupDiabetic retinopathy: seeing beyond glucose-induced microvascular disease.Diabetes. 2006; 55: 2401-2411Crossref PubMed Scopus (628) Google Scholar The streptozotocin-induced diabetes in rats shows many of the retinal alterations associated with human DR.5Yu P.K. Yu D.Y. Alder V.A. Su E.N. Cringle S.J. Intracellular structures of retinal vascular endothelium in normal and early diabetic rats.Aust N Z J Ophthalmol. 1998; 26: S53-S55Crossref PubMed Scopus (11) Google Scholar, 6Wei M. Ong L. Smith M.T. Ross F.B. Schmid K. Hoey A.J. Burstow D. Brown L. The streptozotocin-diabetic rat as a model of the chronic complications of human diabetes.Heart Lung Circ. 2003; 12: 44-50Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 7Kern T.S. In vivo models of diabetic retinopathy.in: Duh E.J. Diabetic retinopathy. Humana Press, Totowa, NY2009: 137-156Google Scholar Therefore, this model could be a useful tool for studying the pathogenic mechanisms involved in DR, as well as for developing new therapeutics. The retinal vessel tight junctions protect the vessels from leaking, but sustained hyperglycemia could damage tight junctions and the vessels could become leaky, allowing fluid or blood to seep into the retina, thus resulting in retinal swelling.8Harhaj N.S. Antonetti D.A. Regulation of tight junctions and loss of barrier function in pathophysiology.Int J Biochem Cell Biol. 2004; 36: 1206-1237Crossref PubMed Scopus (446) Google Scholar Because of progressive dysfunction, the capillaries die prematurely, leading to ischemia,9Aiello L.P. Gardner T.W. King G.L. Blankenship G. Cavallerano J.D. Ferris 3rd, F.L. Klein R. Diabetic retinopathy.Diabetes Care. 1998; 21: 143-156PubMed Google Scholar, 10Frank R.N. On the pathogenesis of diabetic retinopathy: a 1990 update.Ophthalmology. 1991; 98: 586-593Abstract Full Text PDF PubMed Scopus (145) Google Scholar which, in turn, induces irreversible morphologic and functional changes that result in blindness. Ischemic retinopathy develops when retinal blood flow is insufficient to match the metabolic needs of the retina, one of the highest oxygen-consuming tissues. Although there is no effective treatment against retinal ischemic injury, it is possible to activate an endogenous protection mechanism that prevents retinal ischemic damage by ischemic preconditioning (IPC).11Roth S. Li B. Rosenbaum P.S. Gupta H. Goldstein I.M. Maxwell K.M. Gidday J.M. Preconditioning provides complete protection against retinal ischemic injury in rats.Invest Ophthalmol Vis Sci. 1998; 39: 777-785PubMed Google Scholar, 12Roth S. Endogenous neuroprotection in the retina.Brain Res Bull. 2004; 62: 461-466Crossref PubMed Scopus (47) Google Scholar Ischemic preconditioning requires a brief period of ischemia applied before ischemic injury, which does not produce any damage per se, and triggers yet incompletely described mechanisms that result in tolerance to the subsequent severely damaging ischemic event.13Gidday J.M. Cerebral preconditioning and ischaemic tolerance.Nat Rev Neurosci. 2006; 7: 437-448Crossref PubMed Scopus (640) Google Scholar It was shown that IPC affords the retina a greater degree of protection against ischemic damage than any known neuroprotective agent.11Roth S. Li B. Rosenbaum P.S. Gupta H. Goldstein I.M. Maxwell K.M. Gidday J.M. Preconditioning provides complete protection against retinal ischemic injury in rats.Invest Ophthalmol Vis Sci. 1998; 39: 777-785PubMed Google Scholar Moreover, clinical studies support the effectiveness of IPC in the brain of humans with transient ischemic attacks.14Wegener S. Gottschalk B. Jovanovic V. Knab R. Fiebach J.B. Schellinger P.D. Kucinski T. Jungehülsing G.J. Brunecker P. Müller B. Banasik A. Amberger N. Wernecke K.D. Siebler M. Röther J. Villringer A. Weih M. MRI in Acute Stroke Study Group of the German Competence Network Stroke: Transient ischemic attacks before ischemic stroke: preconditioning the human brain? a multicenter magnetic resonance imaging study.Stroke. 2004; 35: 616-621Crossref PubMed Scopus (263) Google Scholar Although IPC confers robust neuroprotection in different models of ischemia, its clinical utilization is mostly limited because the onset of retinal ischemia is largely unpredictable, in contrast to the onset of reperfusion that could be more predictable. In this vein, another endogenous form of ischemic protection, in which a short series of repetitive cycles of brief ischemia/reperfusion are applied immediately at the onset of reperfusion, termed postconditioning, has been reported in several tissues.15Na H.S. Kim Y.I. Yoon Y.W. Han H.C. Nahm S.H. Hong S.K. Ventricular premature beat-driven intermittent restoration of coronary blood flow reduces the incidence of reperfusion-induced ventricular fibrillation in a cat model of regional ischemia.Am Heart J. 1996; 132: 78-83Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 16Zhao H. Sapolsky R.M. Steinberg G.K. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats.J Cereb Blood Flow Metab. 2006; 26: 1114-1121Crossref PubMed Scopus (148) Google Scholar, 17Pignataro G. Meller R. Inoue K. Ordonez A.N. Ashley M.D. Xiong Z. Gala R. Simon R.P. In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning.J Cereb Blood Flow Metab. 2008; 28: 232-241Crossref PubMed Scopus (179) Google Scholar We have shown that 7-minute ischemia applied 5 minutes after the reperfusion onset induces an almost complete protection of retinas exposed to ischemic injury.18Fernandez D.C. Bordone M.P. Chianelli M.S. Rosenstein R.E. Retinal neuroprotection against ischemia-reperfusion damage induced by postconditioning.Invest Ophthalmol Vis Sci. 2009; 50: 3922-3930Crossref PubMed Scopus (38) Google Scholar On the basis of the highly effective protection induced by IPC and postconditioning against an acute ischemic episode, the aim of this work was to analyze the effect of brief ischemia pulses on retinal damage induced by experimental diabetes. Male Wistar rats (300 ± 50 g) were housed in a standard animal room with food and water ad libitum under controlled conditions of humidity and temperature (21°C ± 2°C) and under a 12-hour light/dark cycle (lights on at 7:00 AM). All animal procedures were in strict accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For diabetes induction, a single intraperitoneal streptozotocin injection (60 mg/kg in 0.1 mol/L citrate buffer, pH 4.5) was performed. Control rats received an equal volume of citrate buffer. Animals were examined 72 hours after injections using a glucose meter (Contour TS; Bayer, Buenos Aires, Argentina), and those with blood glucose levels >350 mg/dL were considered diabetic. For the assessment of body weight and blood glucose levels, a group of diabetic animals with intact eyes was included. Rats were monitored weekly, and the weight and plasma glucose levels were determined. Animals were anesthetized with ketamine hydrochloride (150 mg/kg) and xylazine hydrochloride (2 mg/kg) administered intraperitoneally. After topical instillation of proparacaine, the anterior chamber was cannulated with a 30-gauge needle connected to a pressurized bottle filled with sterile saline solution. Rats were submitted to different treatments depicted in Figure 1. Three days after vehicle or streptozotocin injection, retinal ischemia was induced by increasing intraocular pressure to 120 mm Hg for exactly 5 minutes, as previously described.18Fernandez D.C. Bordone M.P. Chianelli M.S. Rosenstein R.E. Retinal neuroprotection against ischemia-reperfusion damage induced by postconditioning.Invest Ophthalmol Vis Sci. 2009; 50: 3922-3930Crossref PubMed Scopus (38) Google Scholar With this maneuver, complete ocular ischemia was produced, characterized by the loss of the electroretinogram (ERG) b-wave and the cessation of flow in retinal vessels, determined by funduscopic examination. The contralateral eye was cannulated without raising intraocular pressure (sham procedure). During ischemia pulses, animals were kept normothermic with heated blankets. This maneuver was repeated weekly until week 10 (Figure 1). In a group of nondiabetic animals, only one eye was subjected weekly to 5-minute ischemia, whereas the contralateral eye remained intact. In another set of experiments, ischemia pulses started after 6 weeks of diabetes induction and continued until week 12. Electroretinographic activity was assessed before and weekly after diabetes induction, as previously described.18Fernandez D.C. Bordone M.P. Chianelli M.S. Rosenstein R.E. Retinal neuroprotection against ischemia-reperfusion damage induced by postconditioning.Invest Ophthalmol Vis Sci. 2009; 50: 3922-3930Crossref PubMed Scopus (38) Google Scholar Briefly, after 6 hours of dark adaptation, rats were anesthetized under dim red illumination. Phenylephrine hydrochloride and tropicamide were used to dilate the pupils, and the cornea was intermittently irrigated with balanced salt solution to maintain the baseline recording and to prevent keratopathy. Rats were placed facing the stimulus at a distance of 20 cm. All recordings were completed within 20 minutes and animals were kept warm during and after the procedure. A reference electrode was placed through the ear, a grounding electrode was attached to the tail, and a gold electrode was placed in contact with the central cornea. A 15-W red light was used to enable accurate electrode placement. This maneuver did not significantly affect dark adaptation and was switched off during the electrophysiologic recordings. Electroretinograms were recorded from both eyes simultaneously, and 10 responses to flashes of unattenuated white light (5 ms, 0.2 Hz) from a photic stimulator (light-emitting diodes) set at maximum brightness (6 cd s/m2 without filter) were amplified, filtered (1.5-Hz low-pass filter, 1000 high-pass filter, notch activated), and averaged (Akonic BIO-PC, Buenos Aires, Argentina). The a-wave was measured as the difference in amplitude between the recording at onset and the trough of the negative deflection, and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Runs were repeated 3 times with 5-minute intervals to confirm consistency. Mean values from each eye were averaged, and the resultant mean value was used to compute the group means ± SEMs of a- and b-wave amplitude. The mean peak latencies and peak-to-peak amplitudes of the responses from each group of rats were compared. Oscillatory potentials (OPs) were assessed using the same photic stimulator with a 0.2-Hz frequency and filters of high (300 Hz) or low (100 Hz) frequency. The amplitudes of the OPs were estimated by measuring the heights from the baseline drawn between the troughs of successive wavelets to their peaks. The sum of three OPs was used for statistical analysis. The ERGs were registered before ischemia pulses. Rats were anesthetized and intracardially perfused with 150 mL of saline solution followed by 300 mL of a fixative solution containing 4% formaldehyde in 0.1 mol/L phosphate buffer (pH 7.2). Then, eyes were carefully enucleated, immersed for 24 hours in the same fixative, and embedded in paraffin. Eyes were sectioned (5 μm) along the vertical meridian through the optic nerve head. Sections were stained with hematoxylin-eosin and were analyzed by masked observers. Microscopic images were digitally captured with a microscope (6-V halogen lamp, 20 W, equipped with a stabilized light source, Nikon Eclipse E400; Nikon, Abingdon, VA) attached to a digital camera (Coolpix s10; Nikon). For morphometric analysis, digitalized images captured were processed using Scion Image for Windows software (Scion Corporation, Frederick, MD). The average total retina and retinal layer thicknesses (in micrometers) for each eye were measured, and the number of cells in the ganglion cell layer (GCL) per 200 μm was determined in the same topographic region of the retina. Measurements (×200) were obtained 1 mm dorsally and ventrally from the optic disc. For each eye, results obtained from four separate sections were averaged, and the mean of five eyes was recorded as the representative value for each group. Antigen retrieval was performed by heating sections at 90°C for 30 minutes in citrate buffer (pH 6.3) and then preincubated with 2% normal horse serum, 0.1% bovine serum albumin, and 0.4% Triton X-100 in 0.01 mol/L PBS for 1 hour. For vascular endothelial growth factor (VEGF) immunodetection, paraffin sections were treated with 0.3% H2O2 in PBS for 20 minutes (for blocking endogenous peroxidase activity) and incubated overnight at 4°C with a rabbit polyclonal anti-VEGF antibody (1:800; Calbiochem, La Jolla, CA). An immunohistochemical detection was performed using the LSAB2 System-HRP (Dako, Carpinteria, California), based on biotin-streptavidin-peroxidase, and visualized using 3,3′-diaminobenzidine as chromogen. For neuronal nuclear protein (NeuN) immunodetection, paraffin sections were incubated overnight at 4°C with a mouse monoclonal anti-NeuN antibody (1:200; Millipore, Bilerica, MA), and then an anti-mouse secondary antibody conjugated to Alexa Fluor 568 (1:500; Molecular Probes, Eugene, OR) was used. After immunostaining, nuclei were stained with the fluorescent dye DAPI. Measurements of cell number were performed as described above. For immunodetection of glial cells, rats were perfused as previously described. Perfusion pressure was kept low to minimize damage to vessels. The posterior eye cups were immersed in an ice-cold fixative for 4 hours. Retinas were carefully detached and flat-mounted with the vitreous side up in Superfrost microscope slides (Erie Scientific Company, Portsmouth, NH). Whole-mount retinas were incubated overnight at 4°C with a mouse monoclonal anti–glial fibrillary acidic protein (GFAP) antibody conjugated to Cy3 (1:400; Sigma Chemical Co., St. Louis, MO). Specimens were mounted with antifade medium (Vectashield; Vector Laboratories, Burlingame, CA) and viewed with a fluorescence microscope (BX-50; Olympus, Tokyo, Japan) mounted with a video camera (3CCD; Sony, Tokyo, Japan) attached to a computer running image analysis software (Optimus; Media Cybernetics, Silver Spring, MD). In all cases, comparative digital images from different samples were obtained using identical exposure time, brightness, and contrast settings. The retina was divided into four quadrants, and images from the central (peripapillary region) and peripheral area were obtained. The area occupied by GFAP-positive astrocytes was measured in a square area corresponding to 0.125 mm2 and expressed as a percentage of the total area. For each group, results obtained from four separate quadrants were averaged, and the mean of four eyes was recorded as the representative value. Regularly, some sections were treated without the primary antibodies to confirm specificity. Vascular permeability was analyzed by measuring albumin–Evans blue complex leakage from retinal vessels as previously described.19Ma N. Hunt N.H. Madigan M.C. Chan-Ling T. Correlation between enhanced vascular permeability, up-regulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria.Am J Pathol. 1996; 149: 1745-1762PubMed Google Scholar Briefly, animals were anesthetized and injected intracardially with a solution of Evans blue (2% wt/vol dissolved in PBS). Immediately after injection, animals turned visibly blue, confirming the dye uptake and distribution. After 40 minutes, animals were sacrificed and flat-mounted retinas were obtained as described above. Microphotographs were obtained using identical exposure time, brightness, and contrast settings. For each group, results were qualitatively analyzed by comparing four eyes for group. After anesthesia and thoracotomy, animals were perfused through the left ventricle with three perfusates, according to DePace,20DePace D.M. Distribution of intravascularly injected lanthanum ions in ganglia of the autonomic nervous system of the rat.J Auton Nerv Syst. 1984; 11: 339-347Abstract Full Text PDF PubMed Scopus (8) Google Scholar with minimal modifications. The initial perfusate was 100 to 150 mL of 1% NaNO3 in PBS, immediately followed by a 5-minute infusion with 300 to 350 mL of ionic lanthanum solution containing 40 mmol/L La(NO3)3.6 H2O, 80 mmol/L NaCl, 3.5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 1 mmol/L glucose at pH 7.4. Lanthanum nitrate solution was followed by an additional 10-minute perfusion with 300 to 350 mL of 2% glutaraldehyde and 4% paraformaldehyde prepared in a sulfate-salt solution containing 43 mmol/L NaLa(NO3)3.6H2O, 80 mmol/L NaCl, 3.5 mmol/L KCl, 1.0 mmol/L CaCl2SO4, 16 mmol/L NaHCO3, 10 mmol/L sodium acetate, 3.5 mmol/L KCl, 1.0 mmol/L CaCl2, 1.0 mmol/L MgCl2, 1.0 mmol/L glucose, 1.6 mmol/L Na2HPO4, 0.4 mmol/L NaH2PO4, and 33 mmol/L sucrose at pH 7.4. After perfusion, retinas were dissected out, and after several washings, tissue blocks were postfixed in 2% aqueous osmium tetroxide for 1 hour. Dehydration was accomplished by gradual ethanol series and tissue samples were embedded in epoxy resin. Semithin sections were stained with toluidine blue and ultrathin sections were stained with uranyl acetate and lead citrate. Afterward, sections were viewed and photographed using a transmission electron microscope (Zeiss EM 10 C; Zeiss, Oberkochen, Germany). For each group, results were qualitatively analyzed by comparing four eyes for group. Animals were sacrificed at 6 or 10 weeks after diabetes induction at 7 days after the sixth and 10th ischemic pulses, respectively. Moreover, VEGF levels were also assessed in animals that had been diabetic for 6 weeks and were sacrificed at 24 hours or 3 days after the sixth ischemia pulse (Figure 1). Retinas (one per condition) were homogenized in 150 μL of a buffer containing 10 mmol/L HEPES, 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L KCl, Triton 0.5% (vol/vol), pH 7.9, supplemented with a cocktail of protease inhibitors (Sigma Chemical Co.). After 15 minutes at 4°C, homogenates were gently vortexed for 15 seconds and centrifuged at 3000 × g for 10 minutes. Supernatants were used to determine protein concentration. Proteins (50 μg per sample) were separated in SDS, 12% polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes for 60 minutes at 15 V in a Bio-Rad Trans-Blot SD system (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 for 60 minutes at room temperature and then incubated overnight at 4°C with a rabbit anti-VEGF antibody (1:1000). Membranes were washed and then incubated for 1 hour with a horseradish peroxidase–conjugated secondary antibody (1:2000). Immunoblots were visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences). Autoradiographic signals were quantified by densitometry using ImageQuant software and adjusted by the density of β-actin. Protein content was determined by the method of Lowry et al,21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin Phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar using bovine serum albumin as the standard. Statistical analysis was made by a two-way analysis of variance followed by Tukey's test. Table 1 summarizes the average weight and blood glucose levels after the injection of vehicle or streptozotocin. At 3, 6, and 10 weeks after injections, a significant weight loss of streptozotocin-treated rats compared with vehicle-injected rats was observed. Blood glucose levels were significantly increased at these time points in streptozotocin-injected rats. The weekly application of 5-minute ischemia pulses did not change these parameters compared with diabetic animals without ischemia pulses.Table 1Average Body Weight and Blood Glucose Concentration Assessed at Different Time PointsAverage body weight, gAverage blood glucose concentration, mg/dLTime after vehicle or streptozotocin injectionControlDiabetes without ischemia pulsesDiabetes with ischemia pulsesControlDiabetes without ischemia pulsesDiabetes with ischemia pulses3 days315.6 ± 9.4337.6 ± 10.0333.7 ± 8.4109.4 ± 6.4484.7 ± 16.5⁎P < 0.01 versus aged-matched control animals, by Tukey's test.476.0 ± 15.4⁎P < 0.01 versus aged-matched control animals, by Tukey's test.3 weeks404.3 ± 5.1320.0 ± 7.9⁎P < 0.01 versus aged-matched control animals, by Tukey's test.305.4 ± 5.2⁎P < 0.01 versus aged-matched control animals, by Tukey's test.110.3 ± 4.3498.1 ± 12.7⁎P < 0.01 versus aged-matched control animals, by Tukey's test.502.3 ± 12.9⁎P < 0.01 versus aged-matched control animals, by Tukey's test.6 weeks432.6 ± 4.7317.2 ± 11.1⁎P < 0.01 versus aged-matched control animals, by Tukey's test.306.7 ± 12.2⁎P < 0.01 versus aged-matched control animals, by Tukey's test.105.9 ± 5.3519.8 ± 16.5⁎P < 0.01 versus aged-matched control animals, by Tukey's test.528.3 ± 16.2⁎P < 0.01 versus aged-matched control animals, by Tukey's test.10 weeks461.2 ± 6.1307.8 ± 19.2⁎P < 0.01 versus aged-matched control animals, by Tukey's test.300.6 ± 18.5⁎P < 0.01 versus aged-matched control animals, by Tukey's test.117.5 ± 4.3583.4 ± 9.8⁎P < 0.01 versus aged-matched control animals, by Tukey's test.†P < 0.01 versus 3 days after streptozotocin injection, by Dunnett's test.596.3 ± 8.3⁎P < 0.01 versus aged-matched control animals, by Tukey's test.†P < 0.01 versus 3 days after streptozotocin injection, by Dunnett's test.The injection of streptozotocin induced a significant decrease in body weight and an increase in blood glucose levels. Ischemia pulses in streptozotocin-injected rats did not change these parameters. Data are given as mean ± SEM (n = 10 animals per group). No significant differences in the average body weight were observed in diabetic animals during the study. P < 0.01 versus aged-matched control animals, by Tukey's test.† P < 0.01 versus 3 days after streptozotocin injection, by Dunnett's test. Open table in a new tab The injection of streptozotocin induced a significant decrease in body weight and an increase in blood glucose levels. Ischemia pulses in streptozotocin-injected rats did not change these parameters. Data are given as mean ± SEM (n = 10 animals per group). No significant differences in the average body weight were observed in diabetic animals during the study. Figure 2A depicts the temporal course of the electroretinographic activity in control and diabetic animals without or with ischemia pulses. Representative ERG waveforms registered at different time points of diabetes are shown in Figure 2B. No ERG changes were noticed in control rats during the entire period of analysis. In diabetic eyes without ischemia pulses, a progressive decrease in the scotopic ERG a- and b-wave amplitude was observed, reaching significance at 7 and 5 weeks after injection of streptozotocin, respectively. In eyes submitted to ischemia pulses, the diabetes-induced decrease in ERG a- and b-wave amplitude was significantly prevented. The sum of OP amplitudes significantly decreased after 9 weeks of diabetes, compared with vehicle-injected animals, whereas ischemia pulses prevented the effect of diabetes (Figure 2C). No significant differences in the ERG between control eyes and eyes submitted to ischemia pulses were observed along the study (data not shown). No apparent morphologic differences were observed among groups. A morphometric analysis of retinal sections performed 10 weeks after streptozotocin injection revealed no differences in the total retina and retinal layers thickness, as well as in the number of cells in the GCL, as shown in Figure 3. In addition, NeuN-immunopositive neuron density in the GCL did not differ between retinas from diabetic animals submitted to a sham procedure or ischemia pulses (Figure 3). The blood retinal barrier (BRB) integrity was analyzed in flat-mounted retinas by the albumin–Evans blue-complex leakage method, 6 weeks after diabetes induction (Figure 4). In nondiabetic animals, the dye was exclusively observed within the vessel lumen of the complete superficial and deeper retinal vasculature, with very low background fluorescence levels. In diabetic animals submitted to a sham procedure, a generalized leakiness and focal dye leakage from the optic disc
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