Brain Disorders & Therapy

Cellular damage and changes in glutamate transporters in a murine model of ischemic stroke

Global Summit on Stroke

August 03-05, 2015 Birmingham, UK

Sabah Khan

Scientific Tracks Abstracts: Brain Disord Ther

Abstract:

Ischemic stroke or cerebral ischemia is a leading cause of death and adult disability but there is no effective treatment for
this devastating disease. It is, therefore, important to understand stroke induced cellular damage and biochemical changes
to find new potential targets for treatment of this disease. Glutamate plays a key role in the pathophysiology of stroke1 and
glutamate levels in the brain are regulated by both plasma and vesicular glutamate transporters2. We have previously shown
that the levels of specific plasma glutamate transporters are reduced following middle cerebral artery occlusion3. Recently
we have optimised a murine model of cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) in
our laboratory. In the present study we have examined cellular damage and the changes in glutamate transporters (EAAT2,
VGluT1 & VGluT2) following cerebral ischemia using western blotting and immunohistochemistry.
All experiments were performed on 10- 12 week old (26–30 g) C57BL/6 mice (Harlan-Olac, Bicester, UK) under UK
Home Office License. Cerebral ischemia was induced in mice using arterial clips under inhalation anaesthesia with isoflurane
for 15 minutes and sham-operated mice underwent the same procedure except that the carotid arteries were not occluded.
Following 3-7days reperfusion time the mice were killed by scheduled 1 and the brains removed. The brain samples taken
from both sides the cortex or the hippocampus were homogenised and protein was extracted using SDS-PAGE sample buffer.
Proteins were transferred to polyvinylidene difluoride membrane (Amersham, GE Healthcare, UK) and probed with primary
antibodies (GLTt-1, GLAST, VGluT1- & VGluT2,) followed by horse- radish peroxidase-conjugated anti-rabbit or antimouse
IgG . The bound antibodies were visualised by using a Laser 3000 chemiluminescence detection system (Amersham
Biosciences, Amersham, UK). Densitometry analysis in relation to total protein was performed using Image quantification
software. The result showed consistent reduction in western blot of GLT-1, VGluT-1 and VGluT-2 as compared to β-actin in
BCCAO animals. For immunohistochemistry the mice were re-anesthetised with isoflurane by inhalation and perfused with
4% paraformaldehyde. Brain sections (40-50um) were cut and processed for immunohistochemistry using specific antibodies
(Neun, GFAP, GS, Glt-1, VGluT1- & VGluT2,) and analysed using an AxioImager Z.1 epifluorescence microscope (Carl
Zeiss, Welwyn Garden City, UK).
The results suggest that vesicular glutamate transporters VGLUT1 and 2 along with specific plasma glutamate transporters
may be potential targets for the prevention of excitotoxicity induced by ischemic stroke.