Mechanism Of Retina Edema And Minocycline's Effect On Retina Edema In The Early Diabetic Rat

Retina edema accounts for much of the visual dysfunction associated with many ocular disease such as central retinal vein occlusion, uveitis and diabetic retinopathy. In particular, diabetic macular edema (DME) is the most common cause of vision loss in this patient population. The fluid accumulation contributes to the degeneration of retinal neurons and to a decline in visual acuity, as it results in compression of neurons, nerve fibers, and vessels (which exacerbates ischemic conditions), and in an elongation of the routes for the diffusion of metabolic substrates and oxygen[2]. Accumulation of intraretinal fluid may be caused by intensified fluid leakage due to breakdown of the blood-retinal barrier (BRB) or by the damaged removal of fluid from the retinal tissue into the blood[3,4].Once the balance between the rate of fluid entry into the retinal tissue and the rate of fluid absorption from the retina was disturbed, retina edema may occur. Previous study has found that clinically significant diabetic macular edema develops only when (in addition to vascular leakage) the active transport mechanisms of the blood-retinal barrier are impaired[5]. Another study has also shown that anomalies in vessel permeability need to be accompanied by insufficient edema resolving mechanisms to cause retina edema[6]. Thus, an impairment of the fluid absorption from the retinal parenchyma shoud be considered as a major pathogenic mechanism of edema formation[2].Under normal and healthy conditions the inner retina is continuously dehydrated by Muller cells, whereas the subretinal space and outer retina are kept dry by the pumping mechanism of the retinal pigment epithelium[3].Miiller cells are the principal glial cells of the vertebrate retina which span the entire thickness of the retina. They are believed to play an important role in the maintenance and regulation of the BRB[9], as they constitute an anatomical link between the retinal neurons, retinal blood vessels and the vitreous [7,8]. It has been suggested that Muller cells mediate the retinal water homeostasis via transcellular water fluxes that are facilitated by the co-expression of Aquaporin4(AQP4) and distinct potassium channels in perivascular membrane domains.[10].Aquaporins are transmembrane water channels. AQP1, AQP4, and AQP9 have been identified in the brain, but it is AQP4 with a postulated role in brain edema that has become the focus of increased medical interest [11]. It has been suggested that AQP4 expression on brain astrocytes mediates fluid uptake from the extracellular space around synapses[11,24]. Increased AQP4 expression has been reported in a wide spectrum of human neuropathological conditions, including ischemia, trauma, brain tumours and multiple sclerosis[12,13].Our previous study has also shown that AQP4 level was increased in the retina of hypoxia exposure rat [14]. Kir4.1 channels are located in membranes which are in close contact with spaces outside of the neural retina, i.e., in Muller cell processes enwrapping blood vessels and at both limiting membranes of the retina [15-17]. The co-expression of Kir4.1 and AQP4 in distinct membrane domains of Muller cells has led to the suggestion that the water transport through Muller cells is tightly coupled to the spatial buffering Kir currents mediated by the cells[10].Diabetic retinopathy is now considered to be a state of inflammation. In the early stage of diabetes, microglia activation and many inflammatory cytokines are involved in the pathophysiology of diabetic retinopathy. It is possible that expression of AQP4 and Kir4.1 are related with the inflammatory cytokine since previous study has found that vascular endothelial growth factor(VEGF) and Interleukin-1β(IL-1β) may involve in the expression of AQP4[18] and altered Kir currents in cultured astrocytes by inflammation cytokines[19]. Minocycline is a second-generation, semisynthetic tetracycline that exerts anti-inflammatory effects that are completely separate from its antimicrobial actions. It has been used as a inhibitor of microglia in many researchs[20-23]. Many rescent studies has found that minocycline could reduce the expression of inflammatory mediator, inhibit neuron apotosis, delay the progress of Alzheimer disease, Huntingtor's disease and protect local ischemic neuron. Whether minocycline could be beneficial to reduce retina edema has not been reported. In the present study, we investigate the mechanism of retina edema and minocycline's effect on retina edema using diabetic rat as reserach subject.PartⅠChanges of expression of AQP4,Kir4.1,VEGF and Occludin in the early diabetic rats SectionⅠPurpose:This study was undertaken to examine the expression of AQP4 and Kir4.1 in the retina of early diabetic rats. Methods:Diabetic rats were induced with streptozotocin injection and divided into four groups at random(control, diabetic 1week, diabetic 2weeks, diabetic 4weeks). The expression of AQP4 and Kir4.1 in retinas in experimental and control groups were investigated by double immunofluorescence, Western blotting and Real-time RT PCR. Results:The level of AQP4 and Kir4.1 in the retina of diabetic 1 week and diabetic 2weeks group were comparable to that in the retina of control goup. In the diabetic 4weeks group, AQP4 expression was increased and Kir4.1 expression was decreased when compared with control group.Conclusion:AQP4 and Kir4.1 expression were differential in the retina of early diabetic rats, which the former was up-regulated and the latter was down-regulated.SectionⅡExpression of VEGF and Occludin and assessment of Blood retinal barrier leakage in the early diabetic ratsPurpose:This study was undertaken to examine VEGF and Occludin expressin and assess Blood retinal barrier leakage and retina edema in the retina of early diabetic rats. Methods:Diabetic rats were induced with streptozotocin injection and divided into four groups at random(control, diabetic lweek, diabetic 2weeks, diabetic 4weeks). The expression of VEGF and Occludin in retinas in experimental and control groups were investigated by double immunofluorescence, Western blotting and Real-time RT PCR. Blood-retinal barrier and retina edema were assessed by rhodamine isothiocyanate(RhIC).Results:The level of VEGF in the retina of diabetic 1 week, diabetic 2weeks and diabetic 4weeks group were increased compared with control goup. The level of Occludin in the retina of diabetic 1 week, diabetic 2weeks and diabetic 4weeks group were decreased compared with control goup. Blood retinal barrier disruption and retinal edema was also observed in the retina of diabetic rats as evidenced by the accumulation of RhIC. RHIC leakage was located from internal limiting membrane to outer plexiform layer in the retina of diabetic rats. Enchanced RHIC leakage was observed in outer plexiform layer.ConcIusion: Increased VEGF level and decrease Occludin level were observed in the retina of early diabetic rats. VEGF expression was up-regulated and Occludin expression was down-regulated with time. Inner blood retinal barrier was disrupted in the retina of early diabetic rats.PartⅡSectionⅠEffect of minocycline on the expression of VEGF,Occludin and Blood-retinal barrier assessment in the retina of early diabetic rat.:Purpose:This study was undertaken to examine the expression of VEGF and Occludin in the retina of normal and 4 weeks diabetic rats and minocycline'effect on their expressions. Methods:Adult SD rats were randomly divided into 4 groups:Normal SD rats were used as control group; Diabetic 4weeks group; Diabetic 4weeks plus minocycline treated group; Minocycline group:normal rats treated with minocycline alone. Expression of VEGF and Occludin in the retina was investigated by double immunofluorescence, Western blotting and Real-time RT PCR. Blood-retinal barrier and retina edema were assessed by rhodamine isothiocyanate(RhIC). RESULTS:The results showed increased expression of VEGF, and decreased expression of Occludin in the retina of diabetic 4weeks rats. Compared with diabetic 4weeks group, VEGF expression was decreased and Occludin expression was increased in the diabetic 4weeks plus minocycline treated group(P< 0.05). VEGF and Occludin levels in the minocycline group were comparable to that in the control group (P>0.05. Blood retinal barrier disruption and retinal edema was also observed in the retina of diabetic rats as evidenced by the accumulation of RhIC. Conclusion:Minocycline treatment could efficiently reduce the expression of VEGF and increase the expression of Occludin.. Minocycline has no effect on the expression of VEGF and Occludin in the retina of normal rats.SectionⅡEffect of minocycline on the expression of AQP4, Iba-1, IL-1βand Kir4.1 in the retina of early diabetic rat.Purpose:This study was undertaken to examine the expression of AQP4, Iba-1, IL-1βand Kir4.1 in the retina of normal and 4 weeks diabetic rats and minocycline'effect on their expressions. Methods:Adult SD rats were randomly divided into 4 groups:Normal SD rats were used as control group; Diabetic 4w group; Diabetic 4w plus minocycline treated group; Minocycline group:normal rats treated with minocycline alone. Expression of AQP4 and Kir4.1 in the retina was investigated by double immunofluorescence, Western blotting and Real-time RT PCR. Expression of Iba-1 and IL-1βin the retina was investigated by Western blotting and Real-time RT PCR. RESULTS:The results showed increased expression of AQP4, Iba-1, IL-1β, and decreased expression of Kir4.1 in the retina of diabetic rats. Compared with diabetic 4w group, AQP4, Iba-1, IL-1βexpression was decreased and Kir4.1 expression was increased in the diabetic 4w plus minocycline treated group(P< 0.05). AQP4, Iba-1, IL-1βand Kir4.1 levels in the minocycline group were comparable to that in the control group (P>0.05) Conclusion:AQP4 and Kir4.1 expression were differential in the retina of early diabetic rats, which the former was up-regulated and the latter was down-regulated. Therefore, fluid absorption from the retinal parenchyma by Muller cells may be impaired. While minocycline treatment could efficiently reverse the changes, it may be beneficial to the clearance of retinal fluid and reduced the retinal edema. Minocycline has no effect on the expression of AQP4, Iba-1, IL-1βand Kir4.1 in the retina of normal rats.