AP108B Sigma-AldrichGoat Anti-Guinea Pig IgG Antibody, biotin conjugate

Several studies showed that the up-regulation of glial glutamate transporter-1 (GLT-1) participates in the acquisition of brain ischemic tolerance induced by cerebral ischemic preconditioning or ceftriaxone pretreatment in rats. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance induced by intermittent hypobaric hypoxia (IH) preconditioning (mimicking 5,000 m high-altitude, 6 h per day, once daily for 28 days), immunohistochemistry and western blot were used to observe the changes in the expression of GLT-1 protein in hippocampal CA1 subfield during the induction of brain ischemic tolerance by IH preconditioning, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of brain ischemic tolerance in rats. The basal expression of GLT-1 protein in hippocampal CA1 subfield was significantly up-regulated by IH preconditioning, and at the same time astrocytes were activated by IH preconditioning, which appeared normal soma and aplenty slender processes. The GLT-1 expression was decreased at 7 days after 8-min global brain ischemia. When the rats were pretreated with the IH preconditioning before the global brain ischemia, the down-regulation of GLT-1 protein was prevented clearly. Neuropathological evaluation by thionin staining showed that 200 nmol DHK blocked the protective role of IH preconditioning against delayed neuronal death induced normally by 8-min global brain ischemia. Taken together, the up-regulation of GLT-1 protein participates in the acquisition of brain ischemic tolerance induced by IH preconditioning in rats.

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (GLAST [glutamate aspartate transporter], GLT1 [glutamate transporter 1], EAAC1 [excitatory aminoacid transporter 1]) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Here we describe, through immunoblotting, immunofluorescence assays and β-counter analysis of [(3)H] l-glutamate uptake, the expression, distribution and activity of the glutamate transporters in in vitro cultures of embryonic dorsal root ganglia sensory neurons, sensory neurons+satellite cells and satellite cells. In this work we demonstrated that glutamate transporters are expressed in all cultures with a peculiar pattern of distribution. Even if GLAST is strongly detected in satellite cells, it is slightly expressed also in sensory neurons. GLT1 immunostaining is very weak in DRG neurons, but it was evident in the satellite cells. Finally, EAAC1 is localized in the soma and in the neuritis of sensory neurons, while it is not detectable in satellite cells. Moreover, all the cell cultures showed a strong sodium-energy-dependent glutamate uptake activity and it is more marked in neurons alone or in co-culture with satellite cells compared to satellite cells alone. Finally, we show that the complete or partial pharmacological inhibition of glutamate transporters virtually completely or partially abolish glutamate uptake in all cell culture. These results, that demonstrate that functionally active glutamate transporters can be studied in dorsal root ganglia cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any changes occur in in vitro models of peripheral nervous system damage.Copyright © 2011. Published by Elsevier Ltd.