Effects of melatonin,vitamin E and octreotide on lipid peroxidation during ischemia-reperfusion in the guinea pig retina

PURPOSE: The purpose of this study is to provide evidence that free radical damage is a component of retinal ischemia-reperfusion (I/R) injury, and to determine whether melatonin, vitamin E and octreotide can protect the retina from this injury. METHODS: The right eyes of 50 male guinea pigs weighing 500-600 g were used. The animals were randomly assigned to group 1 (control), group 2 (I/R), group 3 (melatonin + I/R), group 4 (vitamin E + I/R) and group 5 (octreotide + I/R). Groups 3, 4 and 5 received four subcutaneous injections with a 6-h interval for a total daily dose of 10 mg/kg melatonin, 150 mg/kg vitamin E and 22 microg/kg octreotide. The first dose of each substance was administered 5 minutes before retinal ischemia, which was induced for 1.5 hours, followed by reperfusion for 24 hours. All three substances were repeated for 6, 12 and 18 hours during reperfusion. The animals were killed at 24 hours of reperfusion. Retinas were isolated and processed for the quantification of malondialdehyde (MDA). RESULTS: The compounds had the following relationships: melatonin more than vitamin E more than octreotide in preventing retinal damage by ischemia-reperfusion. All three gave significantprotection against the formation of MDA (10.4+/-2.3, 12.4+/-2.4, 13.9+/-1.5 nmol/100 mg tissue wet weight, respectively) compared to the control (3.7+/-1.3 nmol/100 mg tissue wet weight) and I/R groups (22.7+/-6.2 nmol/100 mg tissue wet weight). CONCLUSIONS: This study demonstrates the inhibitory effect of melatonin, vitamin E and octreotide on MDA levels during retinal ischemia-reperfusion injury.     

Protective Effects of Vitamin E Forms (Alpha-tocopherol, Gamma-tocopherol and d-alpha-tocopherol Polyethylene Glycol 1000 Succinate) on Retinal Edema During Ischemia–reperfusion Injury in the Guinea Pig Retina

Purpose: The purpose of this study is to provide evidence that free radical damage is a component of retinal ischemia–reperfusion (I/R) injury, and to determine whether alpha-tocopherol, gamma-tocopherol and d-alpha-tocopherol polyethylene glycol 1000 succinate (TPGS) can protect the retina from this injury. Methods: The right eyes of 40 male guinea pigs weighing 500–600 g were used. The animals were randomly assigned to group 1 (control), group 2 (I/R), group 3 (I/R plus alpha-tocopherol), group 4 (I/R plus gamma-tocopherol) and group 5 (I/R plus TPGS). Groups 3, 4 and 5 received four subcutaneous injections at six-hour intervals for total dosage of 800 IU/kg alpha-tocopherol, 1000 IU/kg gamma-tocopherol and 750 IU/kg TPGS, respectively. The first dose of each substance was administered 5 minutes before retinal ischemia. Retinal ischemia was induced for 90 minutes, then followed by reperfusion for 24 hours. Injections of three substances were repeated at 6, 12 and 18 hours during reperfusion. The animals were killed at 24 hours of reperfusion. Sagittal sections of 4 μm were cut and stained with hematoxylin and eosin for light microscopic evaluation. The average thickness (edema) of the inner plexiform layer for each eye was measured in sagittal sections near the optic nerve and expressed in microns. Results: All the three substances showed statistically significant protection against the formation of retinal edema during ischemia–reperfusion injury. The mean thickness of the inner plexiform layer were 15.0, 25.44, 19.81, 21.38 and 20.88 μm in control, I/R, I/R plus alpha-tocopherol, I/R plus gamma-tocopherol and I/R plus TPGS groups, respectively. The results showed that the thickness of the inner plexiform layer in group 1 (control) was significantly lower than the other groups (p<0.001). The inner plexiform layer was thicker in the I/R group than with I/R plus alpha-tocopherol (p<0.001), I/R plus gamma-tocopherol (p<0.001) and I/R plus TPGS (p<0.01). The inner plexiform layer was not thicker in the I/R plus TPGS group than in the I/R plus alpha-tocopherol and I/R plus gamma-tocopherol groups. Compared to the I/R plus alpha-tocopherol group, the inner plexiform layer was significantly thicker in the I/R plus gamma-tocopherol group (p<0.01). Conclusions: The results from these experiments indicate that vitamin E forms have protective effects on the retina during retinal ischemia–reperfusion injury, but, the effects of alpha-tocopherol and TPGS appear to be much greater than that of gamma-tocopherol.     

Octreotide reduces ischaemia-reperfusion injury in the retina

PURPOSE: To investigate the role of octreotide on retinal lipid peroxidation and histopathological changes during ischaemia/reperfusion (I/R). METHODS: Three groups of seven pigmented guinea pigs each were formed. These represented a control group, an ischaemia group and an ischaemia/octreotide group. One eye of each animal was selected for histopathological evaluation and the other for biochemical assay. Bilateral pressure-induced retinal ischaemia was instigated for 90 min and was followed by 24 hours of reperfusion. Animals in the ischaemia/octreotide and ischaemia groups received either 10 micro g/kg of octreotide or saline, repeated five times at 6-hourly intervals, with the first dose administered 15 min prior to the ischaemic insult. Retinal malondialdehyde (MDA) levels and the thickness of the retinal layers were measured. These were compared with equivalent measurements of the control group. RESULTS: The mean MDA level increased in the ischaemia group (p < 0.01) but not in the octreotide group (p > 0.05). Significant increases in the thickness of the overall retina (p < 0.01), inner retina (p < 0.05) ganglion cell layer (p < 0.01) inner plexiform layer (p < 0.01) and inner nuclear layer (p < 0.01) were observed in the ischaemia group. No significant difference in thickness was found in any of the layers in the ischaemia/octreotide group. CONCLUSION: Octreotide reduces the increases in retinal MDA levels and retinal thickness observed during I/R.     

Interleukin-6 in retinal ischemia reperfusion injury in rats

PURPOSE: To study the role of interleukin (IL)-6 after retinal ischemia-reperfusion (I/R) injury in rats. METHODS: Intraocular pressure of adult male Lewis albino rats was raised to create retinal ischemia for 1 hour. Retinal reperfusion was reestablished, and the animals were killed at various time points after the injury. Their eyes were enucleated and processed for immunohistochemistry to detect IL-6 and ED-1 (a marker of microglial/phagocytic cells), enzyme-linked immunosorbent assay (ELISA) of IL-6 protein, and semiquantitative real-time RT-PCR for IL-6 mRNA. The neuroprotective effect of IL-6 was evaluated by giving intravitreal injections of 150 or 300 ng rat recombinant IL-6 to eyes immediately after I/R injury and counting cresyl violet-stained retinal ganglion cell layer cells (RGCLCs) and fluorochrome-labeled retinal ganglion cells (RGCs) on flat preparations of retinas at 7 days. RESULTS: IL-6-positive cells appeared after I/R injury in the inner plexiform layer (IPL) and the inner nuclear layer (INL). Their numbers were significantly higher 18 hours after the injury, and most of these cells were also ED-1 positive. ELISA showed noticeable increases in endogenous retinal IL-6 protein levels 8 hours after I/R injury. Semiquantitative real-time RT-PCR showed significant increases in endogenous retinal IL-6 mRNA levels between 2 and 18 hours. Exogenously added IL-6 prevented between 50% and 70% of RGC loss after I/R injury. CONCLUSIONS: IL-6 is upregulated after retinal I/R injury, and its expression by microglia/phagocytic cells may protect RGC layer neurons from I/R injury. Exogenously added IL-6 protects the inner retina after I/R injury.     

Aprotinin reduces ischemia-reperfusion injury in the retina of guinea pigs

PURPOSE: The aim of this study was investigate the role of aprotinin on retinal lipid peroxidation and histopathological changes during ischemia/reperfusion (I/R) of guinea pigs. METHODS: Three groups of seven pigmented guinea pigs each were formed: a control (group 1), ischemia/saline (group 2) and ischemia/aprotinin (group 3). One eye of each animal was selected for histopathological evaluation and the other for biochemical assay. Bilateral pressure-induced retinal ischemia was instigated for 90 min and was followed by 24 hours of reperfusion. Animals in the ischemia/aprotinin and ischemia/saline groups received either 20,000 KIU/kg of aprotinin or saline, repeated four times at 6-hour intervals, with the first dose administered 5 min prior to the ischemic insult. The animals were killed at 24 hours of reperfusion. Retinal malondialdehyde (MDA) levels and the thickness of the inner plexiform layers were measured. RESULTS: The level of MDA in group 1 was significantly (p<0.001) lower than the other groups. The mean MDA level in group 2 was significantly (p<0.01) higher than in group 3. The inner plexiform layer in group 1 was significantly (p<0.001) thinner than in the other groups. The mean thickness of the inner plexiform layer in group 2 was significantly (p<0.01) higher than in group 3. CONCLUSIONS: These data indicate that intraperitoneally administrated aprotinin has a protective effect against I/R injury in the retina of guinea pig as evidenced by reduced retinal MDA level and retinal thickness.     

电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用

目的　探讨电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用。方法　大鼠随机分为缺血再灌注组、电刺激组和假手术组。观察视网膜形态学改变 ;用NADPH黄递酶组织化学染色法 (NADPH NDP)观察视网膜内诱导型一氧化氮合酶 (iNOS)的表达 ;采用TUNEL法检测视网膜细胞凋亡情况。结果　 (1)缺血再灌注组的内视网膜层 (包括内核层、内丛状层、节细胞 )、神经纤维层和内界膜厚度增加 ,尤其是内丛状层厚度明显高于假手术组 (t=3 6 80 ,P <0 0 1) ;电刺激组的内视网膜厚度与假手术组相比 ,差异无显著意义 (t=1 0 6 4 ,P >0 0 5 ) ;(2 )光镜观察可见缺血再灌注组有明显的细胞核染色质致密浓缩、核碎裂等改变 ,电刺激组仅见少量核浓缩及碎裂 ;(3)电刺激组的iNOS阳性的神经节细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=3 32 6 ,P <0 0 1) ;(4)电刺激组大鼠发生凋亡的视网膜细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=4 0 38,P <0 0 1)。结论　电刺激大鼠小脑顶核对缺血再灌注所导致的视网膜组织损伤具有保护作用。     

Protective effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats

BACKGROUND: A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent ischemia and reperfusion. We performed a study to investigate the effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats. METHODS: Ten Wistar albino rats were divided into two groups of five animals (10 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). In both groups, the vertebral arteries were occluded bilaterally with an electric needle coagulator under an operating microscope. Forty-eight hours later the rats were reanesthesized, and both common carotid arteries were clamped to interrupt blood flow.The duration of ischemia was 30 minutes. The clamp was then removed to enable reperfusion for 4 hours. The animals were killed by decapitation, and retinal sections were evaluated under light and electron microscopy.The signs of ischemia-reperfusion injury (cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and apoptotic cell count) were recorded. RESULTS: Light microscopy of retinal sections from rats in the ischemic preconditioning group showed a well-preserved retinal structure. The mean thickness values (and standard deviation [SD]) for the inner nuclear layer (104.0 microm [2.54 microm] vs. 49.0 microm [ 10.83 microm]) and inner plexiform layer (134.8 microm [10.13 microm] vs. 88.5 microm [17.46 microm]) were significantly higher in the control group than in the preconditioning group (p = 0.009), indicating increased retinal thickness in the former group due to tissue edema resulting from ischemia-reperfusion injury.The mean mononuclear cell count (6.67 [SD 1.97] vs. 2.5 [SD 1.0]) and apoptotic cell count (18.2 [SD 5.7] vs. 5.3 [SD 1.0]) were significantly higher in the control group than in the preconditioning group (p = 0.002), indicating an inhibitory effect of ischemic preconditioning on leukocyte infiltration and apoptotic cell death. INTERPRETATION: Ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina.     

A pilot study of Fourier-domain optical coherence tomography of retinal dystrophy patients

PURPOSE: To characterize the macular anatomy of retinal dystrophy eyes using high-speed, high-resolution, Fourier-domain optical coherence tomography (FD-OCT). DESIGN: Case-control study. METHODS: Retinal dystrophy patients and normal age- and gender-matched controls underwent FD-OCT imaging using the RTVue (Optovue Inc., Fremont, California, USA). Vertical and horizontal 8-mm scans of 1024 lines/cross-section were obtained. Based on boundaries manually drawn on computer displays of OCT cross-sections, the thicknesses of the retina, inner retinal layer (IRL), and outer retinal layer (ORL) were averaged over both 5-mm (macular) and 1.5-mm (foveal) regions centered at the fovea. The IRL was the sum of nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) thicknesses. Total retinal thickness (RT) was measured between the internal limiting membrane (ILM) and the retinal pigment epithelium. ORL thickness was calculated by subtracting IRL thickness from RT. RESULTS: Fourteen patients (three retinitis pigmentosa, two cone-rod degeneration, two Stargardt disease, and seven normal controls) underwent FD-OCT imaging. Mean foveal RT was 271.3 +/- 23.3 microm for controls and 158.4 +/- 47.1 microm for retinal dystrophy patients (P < .001). Mean macular RT was 292.8 +/- 8.1 microm for controls and 199.1 +/- 32.6 microm for retinal dystrophy patients (P < .001). Mean macular ORL was 182.9 +/- 4.7 microm for controls and 101.3 +/- 18.7 microm for retinal dystrophy patients (P < .001); mean macular IRL was 109.9 +/- 6.4 microm for controls and 97.9 +/- 20.7 microm for retinal dystrophy patients (P = .06). CONCLUSION: Eyes with retinal dystrophy had a small (11%) decrease in macular IRL and severe (45%) decrease in macular ORL compared to normal controls.     

大鼠视网膜缺血-再灌注损伤后P38丝裂原活化蛋白激酶和半胱氨酸天冬氨酸蛋白酶表达的变化及尼莫地平对二者的影响

目的探讨L型钙通道阻滞剂尼莫地平对大鼠视网膜缺血一再灌注损伤的保护作用及其对细胞信号转导通路的影响。方法Wistar大白鼠95只,随机分为4组。A组正常（空白）对照组5只,B组视网膜缺血-再灌注组（实验对照组）30只,C组视网膜缺血-再灌注＋尼莫地平组（实验组）30只,D组低压灌注组（假手术组）30只,以前房灌注升高眼压的方法制备大鼠视网膜缺血-再灌注模型,分别于再灌注发生后2、6、12、24、72、168h各处死5只大鼠,石蜡包埋后切片。以原位杂交方法检测各时间点视网膜P38丝裂原活化蛋白激酶类（P38MAPK）mRNA表达情况,以免疫组化法检测视网膜半胱氨酸天冬氨酸蛋白酶3（caspase-3）表达情况。结果于Metamorph软件上处理,取平均吸光度值作统计分析。结果视网膜缺血-再灌注损伤后,P38MAPK和caspase-3表达增强。视网膜P38MAPK mRNA原位杂交信号位于节细胞层和内核层的细胞核内,正常视网膜只有少量表达。P38于B组缺血-再灌注后,6h表达即明显增加,至12h达到顶峰,持续至24h,72h后逐渐下降。C组使用尼莫地平后,P38表达趋势与B组相同,但表达水平下降。caspase-3表达情况与P38相似。2h开始见内核层细胞核散在阳性,6h后节细胞层内核层细胞核阳性数增加,至24h达到顶峰。C组caspase-3表达趋势同B组,阳性细胞数及着染深度均小于B组。对平均吸光度值行统计学分析表明：P38MAPK mRNA表达,在6、12、24、72h,B组与A、C组,C组与A、D组间差异有统计学意义。caspase-3表达,除0h、168h这两个时间点外,其他各时间点A、D组与B组,A、D组与C组,B组与C组间差异均有统计学意义。A组与D组间差异无统计学意义。结论P38 MAPK和caspase-3均参与了视网膜缺血-再灌注损伤中视网膜神经细胞信号的转导,尼莫地平通过下调P38MAPK和caspase-3的表达而实现对视网膜的保护作用。（中华眼科杂志,2006,42：435-442）     

Nitric oxide and octreotide in retinal ischemia-reperfusion injury

This experimental study was performed to investigate the role of ischemia–reperfusion injury on retinal nitric oxide activity and to determine whether octreotide, the synthetic analogue of natural somatostatin, modifies the nitric oxide activity during retinal ischemia–reperfusion in a quinea pig model. Three groups of seven pigmented male quinea pigs were formed; Control, Ischemia and the Ischemia/Octreotide groups. 90 minutes of pressure-induced retinal ischemia and 24 h of reperfusion were established in the ischemia and ischemia/octreotide groups. Saline for the ischemia group and 50 g/kg of octreotide for the ischemia/octreotide group were administered intraperitoneally five times with 6-h intervals. At the end of the reperfusion period both eyes of the animals of the three groups were enucleated. One eye of each animal was randomly selected for biochemical assay and the other for histopathological analysis. Retinal nitrate levels were measured and histopathological changes were evaluated in the groups. The mean retinal nitrate levels of the control, ischemia and ischemia/octreotide groups were 157.6±25.2, 106.4±20.1 and 96.4±17.7 mol/l, respectively. Nitrate levels decreased significantly both in the ischemia (p<0.01) and ischemia/octreotide (p<0.01) groups versus control. In the ischemia group, retinal histopathological changes, which were different from the control group, were prominent edema, polymorphonucleated leukocytes infiltration and vacuolated spaces in the inner retina. No significant change was observed in the histopathological specimens of the ischemia/octreotide group. Significant increase in the thickness of the inner plexiform layer of the retina of the ischemia group was observed versus the control and ischemia/octreotide groups (p<0.01 and p<0.01, respectively).The thickness of the inner plexiform layer of the retina of the ischemia/octreotide group did not change versus the control group. It was concluded that nitric oxide activity decreased during retinal ischemia–reperfusion and, although octreotide prevented the histopathological damage, it could not ameliorate the nitric oxide activity of the retina.This study was presented in part at the 23rd Congress of the European Society of Ophthalmology.     

Y27632对急性视网膜缺血再灌注大鼠视网膜组织形态学的影响

研究Rho激酶抑制剂Y27632 对视网膜缺血再灌注损伤大鼠视网膜组织形态学的影响。方法：实验研究。将60只SD大鼠随机分为4组，每组15只：正常对照组（正常组）、急性缺血再灌注损伤组（IRI组）、0.9%氯化钠溶液对照组（生理盐水组）、Y27632治疗组（Y27632组）。再灌注损伤后24 h（10只）和168 h（5只）处死各组动物，行HE染色、ADP酶染色检查，光镜下观察大鼠视网膜组织病理学变化及视网膜厚度变化。数据采用单因素方差分析。结果：正常组大鼠视网膜结构清晰，三层细胞结构排列整齐。IRI组于再灌注24 h后视网膜厚度增加，内外丛状层组织疏松，视网膜节细胞、内外核层细胞水肿明显、排列紊乱，视网膜节细胞减少。168 h后，视网膜水肿消退、厚度变薄、呈萎缩状，神经节细胞及内外核层细胞数量减少，在视网膜前和神经纤维层可见毛细血管。再灌注 24 h后，IRI组视网膜厚度较正常组增加（P=0.005），Y27632组视网膜厚度低于生理盐水组（P=0.032）。再灌注168 h后，IRI组视网膜厚度低于正常组（P<0.001），Y27632组视网膜厚度较生理盐水组增加（P=0.025）。正常组大鼠视网膜血管自视乳头发出，向四周呈放射状均匀分布，毛细血管网结构清晰。再灌注24 h后，IRI组视网膜血管管径变细，走行较僵直，分支减少，视乳头周围及中周部视网膜可见大片无灌注区，无灌注区周围可见新生血管芽渐成网状。Y27632组可见视乳头周围及中周部视网膜局部无灌注区形成，无灌注区周围可见新生血管。后极部4PD无灌注区面积明显小于IRI组及生理盐水组。结论：Y27632玻璃体腔注射可以减轻视网膜缺血再灌注早期的视网膜水肿，减少视网膜神经节细胞的凋亡，减少视网膜新生血管生成，减轻再灌注晚期的视网膜萎缩，具有视神经保护作用。     

N-乙酰-5-羟色胺对视网膜缺血-再灌注损伤大鼠视网膜 Fas、FasL 蛋白表达的影响

目的　探讨N-乙酰-5-羟色胺（N-acetylserotonin，NAS）对视网膜缺血-再灌注损伤（retina ischemia-reperfusion injury，RIRI）大鼠视网膜Fas、FasL蛋白表达的影响。方法　取健康成年Sprague Dawley大鼠54只，将大鼠随机分为正常组（6只）、RIRI组（24只）与NAS组（24只）；采用高眼压法建立大鼠RIRI模型，依据造模后不同时间点将RIRI组与NAS组大鼠又分为6 h、12 h、24 h及72 h四个亚组。NAS组于造模前30 min腹腔注射NAS（5 mg·kg^-1），RIRI组腹腔注射等剂量的生理盐水。通过HE染色在光学显微镜下观察各组大鼠视网膜形态学变化，并记录各组大鼠视网膜厚度及视网膜神经节细胞数，采用免疫组织化学染色法检测NAS对RIRI大鼠视网膜Fas、FasL蛋白表达的影响。结果　HE染色显示，正常组大鼠视网膜各层细胞分界清晰，形态正常，神经细胞排列整齐；RIRI组大鼠再灌注后6 h视网膜各层出现水肿，以神经节细胞层及内核层较显著，神经节细胞数较正常组减少；随后视网膜水肿进一步加重，神经节细胞继续减少；NAS组大鼠在再灌注后6 h、12 h、24 h 视网膜水肿程度较 RIRI组轻，NAS组在再灌注后72 h视网膜厚度较 RIRI组厚，NAS组各时间点神经节细胞数均较 RIRI组多，差异均有统计学意义（均为P＜0.05）。免疫组织化学染色显示，正常组几乎未见 Fas⁺细胞。再灌注后6 h，RIRI组视网膜神经节细胞及内核层开始出现少量 Fas⁺细胞；再灌注后12 h，RIRI组视网膜 Fas⁺细胞表达逐渐增多；再灌注后24 h视网膜Fas⁺细胞数达到高峰，棕色阳性染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后 72 h 视网膜 Fas⁺细胞较再灌注后 24 h 减少。NAS组在再灌注后6 h、12 h、24 h、72 h 视网膜 Fas⁺细胞数均较 RIRI组各时间点减少，再灌注后24 h，Fas⁺细胞数达较高水平，随后下降，差异均有统计学意义（均为P＜0.05）。正常组视网膜可见 FasL 全层低表达。RIRI组再灌注后 6 h，视网膜神经节细胞层和神经纤维层存在少量 FasL⁺细胞；再灌注后12 h FasL蛋白表达逐渐增多；再灌注后24 h FasL⁺细胞数达高峰，可见深棕色的细胞膜及细胞质染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后72 h FasL蛋白的阳性表达逐渐减少。NAS组再灌注后6 h、12 h、24 h、72 h 视网膜FasL⁺细胞数均少于 RIRI组各时间点阳性细胞数，差异均有统计学意义（均为P＜0.05）。结论　NAS可通过抑制RIRI大鼠视网膜细胞Fas、FasL蛋白的表达，减轻缺血再灌注对大鼠视网膜细胞造成的损伤。     

Effect of gestational nicotine treatment on newborn rat retina: a histopathological and morphometric analysis

BACKGROUND: Smoking is a significant risk factor in several debilitating and fatal diseases. It has been implicated in bilateral tobacco-toxic and Leber's hereditary optic neuropathies. Although it has been demonstrated that smoking has a cumulative effect on retinal and optic nerve functions and causes diffuse and localised retinal sensitivity decrease in healthy chronic heavy smokers, the affected retinal layer has not been identified and there is no experimental study investigating the effect of nicotine exposure during gestation on the newborn rat retina. PURPOSE: This experimental investigation evaluated histologically the influence in vivo of maternal nicotine treatment during pregnancy on the newborn rat retina. Different dosages of the test compound simulated the range of low, moderate, and heavy smokers in humans. METHODS: Experimentally naive, adult female Wistar-albino rats weighing 200-250 g were mated with adult male rats over 2 days for copulation in the proportion of two females for every male animal. After confirming pregnancy with vaginal smear method, 40 gravid rats (dams) were then randomly assigned into four equal groups (three experimental and one control; n = 10 in each). On day 9 of gestation, groups 1, 2, and 3 experimental dams were treated with intraperitoneal (i.p.) (-)-nicotine tartrate at doses of 0.5, 1, and 2 mg kg body weight-1 day-1, respectively during pregnancy from gestational day 9-21. Group 4 control dams were given i.p. saline solution daily for the same period. After normal delivery, the newborn litters were sacrificed at postnatal day 1 or day 30. The eyes were enucleated for histopathologic and morphometric analysis of the retinas. Nicotine-induced neuronal changes were measured by morphometric analyses on cell counts of ganglion cell layer (linear cell density in number per unit length of retina) and thickness of the various retinal layers. RESULTS: The litters in control group 4, and experimental groups 1 and 2 had normal retinal findings. On the other hand, morphometric analysis of retinal sections in experimental group 3 eyes demonstrated a 20.7% decrease in the number of surviving ganglion cells (40.7 +/- 2.0) compared with controls (51.3 +/- 1.1; p < 0.001). The thickness of whole retina (126.6 +/- 5.4 microm) was also reduced by 13.5% compared with controls (146.3 +/- 4.5 microm; p = 0.007). The main site of retinal atrophy was the inner plexiform layer (30.1 +/- 1.6 microm vs 43.5 +/- 1.3 microm; p < 0.001) with almost no change in the other retinal layers. CONCLUSIONS: Gestational nicotine treatment induces marked changes in the organisation of the developing retina in newborn rats histopathologically. Quantitative morphometric analysis clearly demonstrated that the two most affected structures were the retinal ganglion cells and the inner plexiform layer, both of which are supplied by central retinal artery.     

葛根素对大鼠视网膜缺血再灌注损伤的保护作用

目的观察葛根素(puerarin)对大鼠视网膜缺血再灌注的保护作用及机制。方法成年Wistar大鼠随机分成对照组、缺血再灌注未治疗组、缺血再灌注葛根素治疗组。采用前房灌注液体形成高眼压而建立RIR模型。治疗组在缺血前30min给予大鼠腹腔内注射葛根素。缺血60min后恢复血流。光镜观察各组视网膜内层厚度以及浸润入视网膜的中性粒细胞数目、神经节细胞数变化;免疫组化法检测Caspase-3蛋白在各组视网膜中的表达。结果葛根素治疗组再灌注6h以后各时间段视网膜内层厚度均较未治疗组视网膜缺血再灌注厚,早期视网膜内层水肿增厚,晚期视网膜神经节细胞数目减少及视神经纤维层萎缩变薄,神经节细胞数目多于未治疗组,而视网膜中的中性粒细胞数目少于未治疗组;Caspase-3蛋白于再灌注后24h达到高峰,但各时间段治疗组表达强度均较未治疗组明显减弱。结论葛根素对视网膜缺血再灌注损伤有治疗作用,抑制缺血再灌注损伤后的炎症反应和Caspase-3蛋白的表达是其可能的保护机制。     

Effects of intraperitoneal vitamin E, melatonin and aprotinin on leptin expression in the guinea pig eye during experimental uveitis

PURPOSE: To observe ultrastructural changes and leptin expression in the guinea pig eye during experimental uveitis (EU) and the effects of vitamin E, melatonin and aprotinin on leptin expression. METHODS: Thirty male guinea pigs were randomly classified into five groups. Group 1 was the control group. Groups 2, 3, 4 and 5 received intravitreal injections of bovine serum albumin (BSA) to induce EU. At the same time on the third day, groups 3 (EU + vitamin E), 4 (EU + melatonin) and 5 (EU + aprotinin) received intraperitoneal vitamin E (150 mg/kg), melatonin (10 mg/kg) and aprotinin (20,000 IU/kg), respectively. On the sixth day, histopathological and clinical scoring of inflammation were performed, and leptin expression was investigated in the retina, choroid, sclera, episclera and cornea, and compared. RESULTS: There was a remarkable increase in leptin expression in the retina, choroid, sclera and episclera in the EU group. Leptin expression in the treatment groups was similar to that in the control group. At light and electron microscopic levels, ganglion cells were oedematous and inner plexiform layer thickness had increased in the EU group retinas. Oedema was decreased in the treatment groups. Comparison of the EU and treatment groups revealed significant differences histopathologically and clinically. CONCLUSION: Experimental uveitis causes an increase in leptin expression in the retina, choroid, sclera and episclera of guinea pigs. Vitamin E, melatonin and aprotinin inhibit this increase. Leptin seems to be closely related to ocular inflammation.     

小檗胺对兔急性高眼压视网膜损伤保护作用的实验研究

目的探讨小檗胺(berbamine,BER)对急性高眼压下视网膜损伤的保护作用及其机制。方法 96只大耳白兔随机分6组:正常对照组,阴性对照组,阳性对照组(VER),BER A、B、C组,后5组建立兔缺血-再灌注损伤模型,术后分时段观察视网膜形态、厚度的变化,并测量视网膜细胞内Ca2+含量的变化。结果阳性对照组,BER A、B、C组视网膜早期水肿较轻,晚期视网膜内层厚度明显厚于阴性对照组,术后168h BER C组视网膜内层厚度为(104.82±0.87)μm、阳性对照组为(105.14±0.92)μm,与正常对照组(105.94±0.66)μm相比,差异均无统计学意义(均为P>0.05)。造模后0h视网膜细胞中Ca2+含量开始升高,随再灌注时间延长呈逐渐增高趋势。BER组随再灌注时间的延长及药物浓度的增加,细胞内Ca2+含量逐渐减少,至术后168h,BER C组Ca2+含量为(45.35±0.48)μg·g-1,与正常对照组(44.61±0.84)μg·g-1相比,差异无统计学意义(P>0.05)。结论视网膜细胞内Ca2+超载,参与了视网膜缺血-再灌注损伤,BER通过抑制Ca2+内流从而保护视网膜细胞,并呈浓度依赖性。     

Neuroprotective Effects of Granulocyte Colony-Stimulating Factor on Ischemia-Reperfusion Injury of the Retina

Purpose: It has been reported that granulocyte colony-stimulating factor (G-CSF) provides neuroprotection in models in which neuronal cell death is induced. This research was designed to investigate the effects of G-CSF on neurodegeneration of the inner retinal layer in a rat model of ischemic reperfusion (I/R) injury. Materials and Methods: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 min in the left eyes of the rats. A sham operation was carried out on the right eyes. G-CSF (100 μg/kg/day in 0.3 ml saline) or the same volume of saline was intraperitoneally injected just before the operation and continued for 4 consecutive days (a total of 5 consecutive days). Morphological examinations, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed 7 days after I/R induction. The expression of phosphorylated AKT in the retina was examined by Western blot analysis and immunohistochemistry. Results: Cell loss in the ganglion cell layer was more significantly reduced in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats (20.3 vs. 6.6%). The inner retinal thickness ratios, such as the inner plexiform layer to the inner limiting membrane/outer nuclear layer and the inner nuclear layer/outer nuclear layer, were significantly better preserved in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats. TUNEL assays showed fewer apoptotic cells in the retinal sections of the I/R-induced eyes of the G-CSF-injected rats. The phosphorylation of AKT (p-AKT/AKT) was upregulated in the retinas of the I/R-induced eyes of the G-CSF-injected rats. Conclusion: Our results demonstrated that systemic injection of G-CSF can protect retinal ganglion cells and inner retinal layers from I/R injury. The effects could be associated with the activation of AKT.     

Diosmin protects rat retina from ischemia/reperfusion injury

Abstract Objective: Diosmin, a natural flavone glycoside, possesses antioxidant activity and has been used to alleviate ischemia/reperfusion (I/R) injury. The aim of this study was to clarify whether the administration of diosmin has a protective effect against I/R injury induced using the high intraocular pressure (IOP) model in rat retina, and to determine the possible antioxidant mechanisms involved. Methods: Retinal I/R injury was induced in the rats by elevating the IOP to 110?mmHg for 60?min. Diosmin (100?mg/kg) or vehicle solution was administered intragastrically 30?min before the onset of ischemia and then daily after I/R injury until the animals were sacrificed. The levels of malondialdehyde (MDA) and the activities of total-superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined 24?h after I/R injury. At 7 days post-I/R injury, electroretinograms (ERGs) were recorded, and the density of surviving retinal ganglion cells (RGCs) was estimated by counting retrograde tracer-labeled cells in whole-mounted retinas. Retinal histological changes were also examined and quantified using light microscopy. Results: Diosmin significantly decreased the MDA levels and increased the activities of T-SOD, GSH-Px, and CAT in the retina of rats compared with the ischemia group (P<0.05), and suppressed the I/R-induced reduction in the a- and b-wave amplitudes of the ERG (P<0.05). The thickness of the entire retina, inner nuclear layer, inner plexiform layer, and outer retinal layer and the number of cells in the ganglion cell layer were significantly less after I/R injury (P<0.05), and diosmin remarkably ameliorated these changes on retinal morphology. Diosmin also attenuated the I/R-induced loss of RGCs of the rat retina (P<0.05). Conclusion: Diosmin protected the retina from I/R injury, possibly via a mechanism involving the regulation of oxidative parameters.     

Protection of the retina from ischemia-reperfusion injury by L-carnitine in guinea pigs

PURPOSE: To investigate the efficacy of L-carnitine in preventing retinal injury followed by ischemia-reperfusion. METHODS: The eyes of 34 guinea pigs were used in this experiment. The guinea pigs were divided into two groups: the first group (n=17) was given L-carnitine intraperitoneally (500 mg/kg) and second group (n=17) received the same dose of saline solution. Under general anesthesia, peritomy was performed. Retro-orbital tissues were ligated for 90 minutes and ischemia was induced, followed by 4 hours of reperfusion. One of the enucleated eye was stained with hematoxylin and eosin (H&E) and retinal thicknesses were evaluated. Thiobarbituric acid reactive substances (TBARS) levels were determined in the retina of the other eye. RESULTS: Mean TBARS levels in retinal tissue were found lower in L-carnitine group (2.77 +/- 0.55 microM) than in the control group (6.57 +/- 1.19 microM), (p<0.01). On the other hand, mean retinal thickness was found to be increased in the control group (47.47 +/- 5.62 microm) when compared to the L-carnitine group (26.52 +/- 4.65 microm), (p<0.01). In correlation analysis, significantly positive relationships were found between retinal TBARS level and retinal thickness both in the control and L-carnitine groups (r=0.981, p<0.01 and r= 0.967, p<0.01 respectively). CONCLUSIONS: L-carnitine is effective in preventing retinal injury followed by ischemia-reperfusion.