Benzodiazepines protect hippocampal neurons from degeneration after transient cerebral ischemia: An ultrastructural study

The ability of full and partial benzodiazepine receptor agonists to prevent DNA fragmentation and neuronal death after transient cerebral ischemia was investigated in the Mongolian gerbil. Diazepam (10 mg/kg, i.p.) or the par tial agonist imidazenil (3 mg/kg, i.p.) was administered 30 and 90 min afte r transient forebrain ischemia produced by occlusion of the carotid arterie s for 5 min. Treatment with diazepam completely protected CA1b hippocampal pyramidal neurons in 94% of the animals and partially protected pyramidal n eurons in 6% of the animals, as assessed with a standard Nissl stain three and four days after ischemia. DNA fragmentation was examined by the termina l dUTP nick-end labeling (TUNEL) reaction. Prior to cell death, there were no TUNEL-positive neurons in area CA1b. By three days after ischemia, when neuronal degeneration was nearly complete, 14 out of 16 gerbils exhibited a positive TUNEL reaction throughout area CA1b stratum pyramidale. In 13 out of 14 gerbils treated with diazepam, no TUNEL-positive neurons were observ ed in this region. Imidazenil was less effective than diazepam with respect to both neuroprotection and prevention of DNA fragmentation. Three days af ter ischemia, six out of eight gerbils treated with imidazenil showed parti al to complete neuroprotection. Imidazenil completely prevented DNA fragmen tation in only one of the animals; varying degrees of TUNEL reaction persis ted in the remainder. To determine whether the neurons protected by diazepa m had a normal ultrastructure, gerbils were killed two to 30 days after isc hemia and the hippocampal neurons in area CA1b were examined by electron mi croscopy. Within the first 48 h after ischemia, early cytoplasmic changes o f varying degrees (e.g., vacuolation, rough endoplasmic reticulum stacking, swollen mitochondria) and electron-dense dendrites were observed in gerbil s not treated with diazepam. Degeneration was nearly complete by three days after ischemia. In contrast, pyramidal neuron ultrastructure appeared norm al in gerbils that exhibited complete area CA1b neuroprotection (defined at the light microscope level) by diazepam when studied two. seven or 30 days after ischemia. In gerbils with partial protection of area CA1b, most of t he remaining neurons exhibited varying degrees of necrosis when studied 30 days after ischemia. No apoptotic bodies were observed. We conclude that: (i) diazepam can fully protect CA1 pyramidal cells from t he toxic effects of transient cerebral ischemia; (ii) when diazepam affords only partial neuroprotection, the residual CA1 pyramidal cells exhibit ult rastructural abnormalities consistent with necrotic damage; and (iii) diaze pam is a more efficacious neuroprotectant than the partial benzodiazepine r eceptor agonist, imidazenil. (C) 2000 IBRO. Published by Elsevier Science L td. All rights reserved.