STIMULATION OF BETA(2)-ADRENOCEPTORS INHIBITS APOPTOSIS IN RAT-BRAIN AFTER TRANSIENT FOREBRAIN ISCHEMIA

We have previously demonstrated that the neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo was mos t likely mediated by an increased nerve growth factor (NGF) expression . In the present study, we examined whether clenbuterol was capable of inhibiting apoptosis caused by ischemia. Transient forebrain ischemia was performed in male Wistar rats (300 to 350 g) by clamping both com mon carotid arteries and reducing the blood pressure to 40 mm Hg for 1 0 minutes. Clenbuterol (0.1, 0.5, and 1.0 mg/kg intraperitoneally) was administered 3 hours before ischemia or immediately after ischemia. T he brains were removed for histologic evaluation 7 days after ischemia . The time course of DNA fragmentation was determined 1, 2, 3 and 4 da ys after ischemia. Staining with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was used for further an alysis of DNA fragments in situ 3 days after ischemia. The NGF protein was assayed by enzyme-linked immunosorbent assay. Ten-minute forebrai n ischemia damaged 80% to 90% of the neurons in the hippocampal CAl re gion evaluated 7 days after ischemia. Pretreatment with clenbuterol (0 .5 and 1.0 mg/kg) reduced the neuronal damage by 18.1% (P < 0.01) and 13.1% (P < 0.05), respectively. The neuroprotective effect also was fo und when clenbuterol (0.5 mg/kg) was administered immediately after is chemia (P < 0.05). The DNA laddering appeared in striatum 1 day and in hippocampus 2 days after ischemia and peaked on the third day in both regions. The DNA laddering was nearly abolished in the hippocampus an d partially blocked in striatum and cortex by 0.5 mg/kg clenbuterol. T hese results were confirmed by TUNEL staining. Clenbuterol (0.5 mg/kg intraperitoneally) elevated the NGF protein level by 33% (P < 0.05) in the hippocampus and 41% (P < 0.05) in the cortex 6 hours after ischem ia. Three days after ischemia, the NGF levels in these regions were no longer different between the clenbuterol-treated and control groups. This study clearly demonstrates that clenbuterol possesses a neuroprot ective activity and a marked capacity to inhibit DNA degradation after global ischemia. The results suggest that clenbuterol increases NGF e xpression during the first hours after global ischemia and thereby pro tects neurons against apoptotic;damage.