Temporal profile of apoptosis at ipsilateral ventroposterior thalamic nucleus and substantia nigra in adult hypertensive rats after cortex infarction

BACKGROUND:Apoptosis was found at ipsilateral ventroposterior thalamic nucleus (VPN) and substantia nigra (SNR), which are distant from but connected to the ischemic cerebral cortex, in animals with normotension. However, the secondary brain damage has not been clearly investigated in hypertension yet. OBJECTIVE: The present study tested the hypothesis that in adult hypertensive rats, neuronal apoptosis is associated with neuron loss at VPN and SNR after cortical ischemia.DESIGN, TIME AND SETTING: All the experimental procedures of animal model, immunostaining and deoxynucleotidyl transferase nick end labeling (TUNEL) assay were completed in the Neurological Laboratory of the First Affiliated Hospital of Sun Yat-sen University between October 2006 and July 2008.MATERIALS: The primary antibodies of monoclonal mouse anti-rat microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) were purchased from Sigma Chemical, USA and BD Pharmingen, USA, respectively; The secondary antibodies of biotinylated goat anti-mouse IgG and Cy3-conjugated donkey anti-mouse IgG were purchased from DAKO, Denmark and Jackson Immunoresearch Lab, USA, respectively; TUNEL detection Kits were purchased from Roche Applied Science, Switzerland and Upstate, USA.METHODS:Right distal middle cerebral artery occlusion (MCAO) was performed by electrocoagulation in 32 renovascular hypertensive rats. The expressions of MAP2 (the neuron-specific marker) and GFAP (the glial cell-specific marker) were detected by immunostaining, and cell apoptosis were assessed by TUNEL assay at ipsilateral VPN and SNR 1, 2, 3 and 4 weeks after distal MCAO. Sham control was performed at the same time point.MAIN OUTCOME MEASURES: MAP2, GFAP positive cells and TUNEL-staining cells were respectively quantified by computing the positive unit (PU) value in 5 randomly selected visual fields of VPN and SNR (10×40) with Kontron IBAS 2.5 automated photo analysis system (Germany).RESULTS: In renovascular hypertensive rats, during the 4 weeks after distal MCAO, the expression of MAP2 was gradually but significantly decreased, and that of GFAP was increased at ipsilateral VPN and SNR. Meanwhile, apoptosis of neurons detected by TUNEL assay and double immunostaining was significantly increased at both areas compared to sham controls, with peak occurring at 2 weeks post MCAO, preceding the peak of neuron loss.CONCLUSION: Neuron loss occurring at VPN and SNR after distal MCAO in hypertensive rats could be a secondary event resulting from apoptosis. The temporal profile of apoptosis provides options for examining the time-window of anti-apoptotic therapy.