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 (10mg/kg, i.p.) or the partial agonist
imidazenil (3mg/kg, i.p.) was administered 30 and 90min after transient forebrain
ischemia produced by occlusion of the carotid arteries for 5min. Treatment with
diazepam completely protected CA1b hippocampal pyramidal neurons in 94% of the animals and partially protected pyramidal neurons in 6% of the animals, as assessed with a standard Nissl
stain three and four days after
ischemia. DNA fragmentation was examined by the terminal 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 observed in this region.
Imidazenil was less effective than
diazepam with respect to both neuroprotection and prevention of DNA fragmentation. Three days after
ischemia, six out of eight gerbils treated with
imidazenil showed partial to complete neuroprotection.
Imidazenil completely prevented DNA fragmentation in only one of the animals; varying degrees of TUNEL reaction persisted in the remainder. To determine whether the neurons protected by
diazepam had a normal ultrastructure, gerbils were killed two to 30 days after
ischemia and the hippocampal neurons in area CA1b were examined by electron microscopy. Within the first 48h after
ischemia, early cytoplasmic changes of varying degrees (e.g., vacuolation, rough endoplasmic reticulum stacking, swollen mitochondria) and electron-dense dendrites were observed in gerbils not treated with
diazepam. Degeneration was nearly complete by three days after
ischemia. In contrast, pyramidal neuron ultrastructure appeared normal 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 the 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 the toxic effects of
transient cerebral ischemia; (ii) when
diazepam affords only partial neuroprotection, the residual CA1 pyramidal cells exhibit ultrastructural abnormalities consistent with necrotic damage; and (iii)
diazepam is a more efficacious
neuroprotectant than the partial
benzodiazepine receptor agonist,
imidazenil.