Introduction
In animal models of stroke there are two forms of ischemic insults; focal infarction (representative of thromboembolic stroke) and global cerebral ischemia with widespread but selective neuronal death (representative of cardiac arrest). Transient global ischemia causes delayed but specific CA1 pyramidal neuronal death. Fimbria-fornix pathway is an important modulator of hippocampal function and sends afferents to the CA1 pyramidal neurons and prior deafferentation (FF lesion) of this pathway confers neuroprotection after ischemic insult. Such vulnerability may in part be due to changes in expression of calcium impermeable AMPA (GluR2 containing) receptors in CA1 neurons. We hypothesized that maintenance of GluR2 receptors in neurons may be one of the underlying molecular mechanisms for this protection.
Methods
Adult male Wistar rats were exposed to 10 min of ischemia by 4 vessel occlusion (4VO) 13 days after deafferentation and sacrificed 7 days post ischemia. Coronal sections were taken for H&E staining. Immunofluorescent labeling for GluR2 and neuronal nuclear protein (NeuN) was done on adjacent sections. CA1 region of the dorsal hippocampus was divided into various sectors for cell counts. The optical intensity of GluR2 protein was quantified in these corresponding sectors using ImagePro Plus software. In another group, protein extracts of CA1 region were used to run SDS-PAGE. Subsequently, the protein was transferred onto nitrocellulose membrane and probed with anti-GluR2 and anti-actin immunoglobulins. Protein bands were visualized using enhanced chemiluminescence under FlourS-Max Imager (BioRad, Hercules, CA, USA). Densitometric data determined using QuantityOne software are presented as ratio of GluR2 normalized to densitometric values for actin. Quantitative RT-PCR experiments using the comparative Ct method for determining relative expression of GluR2 are underway. Student's t-test was used to determine the statistical significance between groups.
Results
On the ipsilateral lesioned ischemic side, the CA1 showed 70% surviving neurons compared to 27% on the contralateral. This protection was observed to be long term. Fluorescent intensity of GluR2 protein was higher on the ipsilateral than on contralateral CA1 (755±205 vs. 416±136; p<0.01). Densitometric measurements of GluR2 protein bands in western blots also showed higher values on the ipsilateral CA1 (0.233±0.07 vs. 0.119±0.01; p<0.02). Quantitative RT-PCR experiments using the comparative Ct method for determining relative expression of GluR2 gene to confirm immunofluorescent data are ongoing.
Conclusions
Profound neuroprotection following lesioning was observed in the ipsilateral medial CA1 region at 7 days following similar periods of ischemia, which would ordinarily induce complete cell death. There was no sparing of injury on the contralateral side. Regional GluR2 upregulation following FF deafferentation provides a protective effect on CA1 pyramidal neurons after transient forebrain ischemic insult.