Correlating retinal function and amino acid immunocytochemistry following post-mortem ischemia

We wanted to determine the characteristics associated with electrophysiological and neurochemical changes secondary to ischemic insult as well as correlate these electrophysiological and neurochemical changes. A Ganzfeld source was used to elicit electroretinograms in anesthetized adult Sprague-Dawley rats. Following baseline recordings, one eye was removed for control quantitative amino acid immunocytochemistry, and ischemic insult was induced by cervical dislocation. Following the induction of ischemia, a single electroretinogram signal was collected at 1, 2, 4, 6, 8, 16, 32 or 64 min, after which the eye was removed for immunocytochemistry. The post-receptoral b-wave was undetectable after 1 min post-ischemia, whereas phototransduction declined more gradually and persisted for up to 16 min post-mortem. Both phototransduction saturated amplitude and sensitivity decayed with a similar time course (tc=3.06 (2.73, 3.48) versus 3.29 (2.61, 4.62)min). Significant elevation of amino acid neurotransmitter levels was not observed until 6 min post-mortem. Between 8 and 16 min post-ischemia, glutamate and GABA were significantly accumulated in neurons and Müller cells (p<0.05). Beyond 16 min, the neurotransmitter elevation in neurons and Müller cells was relatively attenuated. Aspartate immunoreactivity was significantly elevated at 4 and 6 min post-ischemia in neurons, prior to a change in any other amino acid. Moreover, of the amino acids assessed the post-ischemic change in aspartate immunoreactivity showed the best correlation with phototransduction decay (r2=0.68). Our findings show that complete impairment of phototransduction coincides with the accumulation of amino acid neurotransmitter. The correlation of aspartate immunoreactivity and phototransduction provides evidence of heightened glutamate oxidation during ischemic insult.     

视网膜缺血性损伤中兴奋性氨基酸的变化

目的　观察视网膜缺血性损伤后视网膜和房水中谷氨酸 (glutamate,Glu)、天冬氨酸 (aspartate ,Asp)的含量变化。方法　采用血管结扎方法建立家兔视网膜缺血模型 ,正常兔作为对照组 ,观察缺血 30min、6 0min和 75min组视网膜和房水中Glu和Asp含量及形态学不同时间的动态变化。结果　缺血组视网膜Glu含量分别为 (5 .2 0± 1.6 0 )μmol·L-1、(4.2 1± 0 .96 ) μmol·L-1、(2 .11± 0 .6 7) μmol·L-1,正常对照组视网膜Glu含量为 (9.2 9± 2 .5 5 ) μmol·L-1;缺血组视网膜Asp含量分别为 (1.86± 0 .4 9) μmol·L-1、(1.0 7± 0 .2 1) μmol·L-1、(1.0 6± 0 .2 1) μmol·L-1,正常对照组视网膜Asp含量为 (2 .35± 0 .5 4 ) μmol·L-1;发生缺血性损伤时 ,缺血组Glu和Asp的含量与正常对照组有显著差异 (P <0 .0 1) ,缺血组组间比较有显著性差异 (P <0 .0 1)。随缺血时间延长 ,视网膜内Glu、Asp含量逐渐减少 ,房水中Glu、Asp含量逐渐增加。视网膜神经细胞持续丢失 ,以内颗粒层明显。结论　视网膜缺血时大量Glu、Asp释放入细胞外液 ,提示兴奋性氨基酸的神经毒性作用介导视网膜缺血性损伤。     

Pharmacological activation of mGlu2/3 metabotropic glutamate receptors protects retinal neurons against anoxic damage in the goldfish Carassius auratus

We examined the expression of mGlu2/3 metabotropic glutamate receptors in the retina of the goldfish Carassius auratus. mGlu2/3 receptors were expressed in all retinal layers internal to the photoreceptor layer, particularly in the outer and inner nuclear layers. Although the goldfish brain is able to tolerate prolonged periods of anoxia, we examined whether anoxia could induce retinal damage. Three hours of anoxia induced in the retina the development of apoptotic cell death, as assessed 48 h later by TUNEL staining. TUNEL-positive cells were particularly found in the inner and outer nuclear layers, and were also present in the ganglion cell layer. Pharmacological activation of mGlu2/3 receptors by systemic injection of LY379268 (0.5 mg/kg, i.p., 15 min before the onset of anoxia) substantially protected retinas against anoxia-induced cell death. In contrast, systemic injection of the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p., 15 min before the onset of anoxia), significantly amplified cell death. Finally, as mGlu2/3 receptors are implicated in the control of extracellular glutamate concentrations, we examined the stimulation of glutamate release in isolated goldfish retinas. Depolarizing medium containing 30 mM KCl led to a significant increase in glutamate release, which was substantially reduced by LY379268. We conclude that activation of mGlu2/3 receptors may provide a major defensive mechanism against ischemic/anoxic retinal damage.     

Retinal amino acid neurochemistry in health and disease

Advances in basic retinal anatomy, genetics, biochemical pathways and neurochemistry have not only provided a better understanding of retinal function but have also allowed us to link basic science to retinal disease. The link with disease allowed measures to be developed that now provide an opportunity to intervene and slow down or even restore sight in previously ‘untreatable’ retinal diseases. One of the critical advances has been the understanding of the retinal amino acid neurotransmitters, related amino acids, their metabolites and functional receptors. This review provides an overview of amino acid localisation in the retina and examples of how retinal anatomy and amino acid neurochemistry directly links to understanding retinal disease. Also, the implications of retinal remodelling involving amino acid (glutamate) receptors are outlined in this review and insights are presented on how understanding of detrimental and beneficial retinal remodelling will provide better outcomes for patients using strategies for the preservation or restoration of vision. An internet‐based database of retinal images of amino acid labelling patterns and other amino acid‐related images in health and disease is located at http://www.aminoacidimmunoreactivity.com .     

The action of inhibitory neurotransmitters, γ-aminobutyric acid and glycine may distinguish between the area centralis and the peripheral retina in cats

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine, and of their antagonists, bicuculline and strychnine, were compared between ganglion cells from the central and peripheral retinae of optically intact eyes in barbiturate-anaesthetised cats. The visual response of on-cells was inhibited by GABA and enhanced by bicuculline. The visual response of off-cells was inhibited by glycine and enhanced by strychnine. The sensitivity of cells to the transmitters was lower in the peripheral retina than in the area centralis, whilst the sensitivity to the antagonists was similar in both regions of the retina. Cells from the area centralis were inhibited by either GABA or glycine, but never both. Cells from the periphery were less selective and were inhibited by both transmitters.     

谷氨酸对视网膜缺血的损害机制和相关治疗策略

视网膜缺血损害的机制复杂,大量的研究资料表明视网膜在缺血及再灌注时谷氨酸的释放量增加,谷氨酸的兴奋性毒性作用在缺血视网膜的病理发展中起到了重要的作用,是引起缺血视网膜神经元死亡的重要因素.通过减少谷氨酸的释放,应用谷氨酸受体拮抗剂限制谷氨酸的生物活性等,可以阻止或减轻缺血视网膜的损害.虽然到目前为止临床上还未出现治疗视网膜缺血的有效药物,但对谷氨酸的损害机制和相关治疗策略的实验性研究,为治疗该疾病提供了广阔的前景.     

兔眼视网膜脱离后视网膜谷氨酸含量与视网膜色素上皮细胞增殖的关系

目的研究兔眼孔源性视网膜脱离(retinal detachment,RD)后不同时程视网膜内谷氨酸(glutamate,Glu)含量变化及视网膜色素上皮(vetinal pigment epithelial,RPE)细胞增殖状况及其2者之间的关系.方法42只有色家兔,按时间随机分为6组8h,1、4、7、14及28d.任选1眼制备RD模型,另1眼为对照眼.各组采用高压液相色谱仪(HPLC)及流式细胞仪(FCM)分别对视网膜内Glu浓度及RPE细胞DNA进行定量检测,并寻找2者之间的相互关系.结果RD后8h,视网膜内Glu含量明显升高(4.68±1.22)μmol·g-1,与对照眼(1.87±0.81)μmol·g-1相比具有显著性差异(P＜0.001);但随着脱离时间的推移,至14d,视网膜内Glu含量明显回降(3.78±0.88)μmol·g-1,与对照眼(1.57±0.66)μmol·g-1相比无显著性差异(P＞0.05).RPE细胞的增殖指数(PI)在RD后8h明显增加(18.41%±0.22%),与对照眼(16.62%±0.69%)相比差异有显著性(P＜005);但28d组实验眼(16.70%±0.88%)与对照眼(15.41%±2.44%)的差异则无显著性意义(P＜0.05).且视网膜内Glu含量变化与RPE细胞的PI值呈显著正相关关系(r=0.436,P＜0.01).结论视网膜脱离后RPE细胞发生增殖与视网膜内Glu浓度异常增高有密切关系.     

Unusual presentation of ocular tuberculosis: multiple chorioretinitis,retinal vasculitis and ischaemic central retinal vein occlusion

A 54‐year‐old male patient presented with a sudden painless visual loss in his left eye. Ophthalmic examinations revealed panuveitis, ischaemic central retinal vein occlusion, multiple chorioretinitis and retinal vasculitis. The diagnosis of tuberculosis was confirmed with anterior tap analysis and QuantiFERON‐TB test. Anti‐tuberculosis treatment and intravitreal anti‐vasculature endothelial growth factor therapy were performed.     

Lipid and fatty acid profile of the retina, retinal pigment epithelium/choroid, and the lacrimal gland, and associations with adipose tissue fatty acids in human subjects

Accumulation of lipids within Bruch's membrane (BrM) and between BrM and retinal pigment epithelium (RPE) accounts for one of the biological changes associated with normal aging and may contribute to the development of age-related maculopathies. The origin of these lipids is still being actively investigated. The relative contribution of plasma lipids and lipids coming from the neural retina remains a matter of controversy. Low-density lipoproteins (LDLs) have been reported to significantly participate in the retina's lipid supply, after active remodeling within RPE. Meanwhile, RPE expresses the enzymatic machinery for synthesizing lipoprotein-like particles. The objective of this study was to establish associations between the fatty acid profile of the ocular structures and adipose tissue as a surrogate for the subjects' past dietary intake. Lipids and fatty acids were analyzed from the neural retina, retinal pigment epithelium (RPE)/choroid, the lacrimal gland, and adipose tissue, collected from 27 human donors (19 women, eight men) aged 59-95 years. DHA concentrations in the neural retina were positively associated with the concentrations in cholesteryl esters (CEs) from RPE/choroid and negatively associated with DHA concentrations in phospholipids (PLs) from RPE/choroid. DHA in orbital fat was positively associated with DHA in the lacrimal gland. No significant association was observed in the other ocular structures. Linoleic acid in orbital fat was positively associated with linoleic acid in the lacrimal gland, followed by the neural retina and CEs from RPE/choroid; it was slightly correlated with PLs from RPE/choroid. Other fatty acids that originate exclusively from the diet such as trans fatty acids were detected in orbital fat, the lacrimal gland, PLs, and CEs from RPE/choroid. DHA in the neural retina was poorly associated with its dietary intake, contrary to other fatty acids such as linoleic acid. Within this context, CEs may be important carriers of fatty acids entering the retina. Although epidemiological studies have reported the benefit of DHA in the prevention of age-related macular degeneration, the leading cause of blindness in Western countries, the relevance of supplementing patients with DHA is questioned.