Changes Of Glutamate Metabolism In The Rat Retina Following Chronic Experimental Ocular Hypertension

Background and objectiveGlutamate is the predominant excitatory neurotransmitter throughout the vertebrate central nervous system, including the retina, but can be toxic to retinal ganglion cells (RGCs) at hight concentrations. It is therefore essential that glutamate levels be tightly regulated within the retina. The termination of glutamatergic transmission requires the removal of glutamate from the extracellular space by means of excitatory amino acid transporter (EAAT) molecules, present in both glial cells and neurons, thus amino acid transporter play a key role in regulating the balance between physiological signalling and pathological overactivation.. Under normal conditions, the accumulation of synaptically released glutamate is mainly prevented by a powerful glial uptake system Glial cells, mainly Muller glia in retina, which express the glutamine synthetase. glutamine synthetase confers upon glial cells an almost unlimited capacity to remove extracellular glutamate. Malfunction of glutamate transport and GS will leads high concentrations of glutamate in the extracellular space, many researchers have reported elevated vitreous glutamate in human and experimental monkey glaucoma, indicatinge high concentration of glutamate play a key role in this disease. But the exact cause of high extracellular glutamate remains uncertain. This study was conducted to investigate whether changes of glutamate transporter and relevant enzyme occurs in the model of intraocular hypertensionMaterials and methods78 male Wistar rats (250-300g) were used for the experiments, the left eyes of all the rats were manipulated with episcleral veins ligation to elevatethe intraocular pressure as the test group. While the contralateral eyes were kept untouched as the control. All the rat intraocular pressure was measured and recorded regularly. 12 rats of the rats were sacrificed and the eyes were enucleated after 2 months and paraffin-embedded sections through the optic disc of each eye were prepared in a standard manner and stained with hematoxylin and eosin. The thickness of the retina were measured .66 rats were divided into 3 groups and sacrificed after 1 week ,1 month ,2 months. Total RNA was prepared from the 8 rats retinas of each group and the reverse transcriptase -polymerase chain reaction (RT-PCR) analysis were used to detect the glutamate transporter GLAST and glutamine synthetase( GS) expression. The glutamine synthetase activity of 6 rats in each group were evaluated with spectrophotometric assay. The retina of 8 rats in each group were prepared into synaptosomes and the L-[3H] -glutamate transport assay was performed with liquid scintillation counting. Results1. ligation of there episcleral veins can cause intraocular pressure elevation for at least two months without re-treatment, approximately 2.8-fold increase in IOP right after the surgery, 2.2-fold increase at 1 week after surgery . Although the IOP decrease partly afterwards, the difference of the IOP between two group is more than lOmmHg. After the hematoxylin and eosin stain the thickness of the retina after 2 months' high IOP become thinner than the control (P<0.05).2. The level of GLAST mRNA and glutamine synthetase mRNA in the all test group decreased compared with those in the control (PO.05).3. Under the high IOP the activity of the glutamine synthetase decreased compared with those in control (PO.05).4. 1 week after the surgery there was obvious decrease in the activity of the L-[3H] glutamate uptake in the test group compared with the control. The uptake activity decreased gradually as the time of elevation in IOP prolonged .5. When the kinetic parameters determined for the uptake system in control and test group were compared, a 13% decrease in Vmax and an increase of 37% in Km values was observed.