Free Communications: Experiment 2–Kainic Acid

Abstract

Abstract *KatsunariYoshida, *KiyotakaHashizume, and *TatsuyaTanaka *Department of Neurosurgery, Asahikawa Medical College, Asahikawa, Japan Purpose: It is thought that neurons are generated during the embryonic and postnatal periods but not after adolescence. We have found that pyramidal cell loss and pyknosis occur in a kainic acid–induced limbic seizure rat model. In this study, bromodeoxyuridine (BrdU), a marker of cell proliferation, was used to determine the relation between seizure-induced hippocampal degeneration and neurogenesis. Methods: Male Wistar rats (250–300 g) were stereotaxically injected with kainic acid (0.5 (g) into the unilateral amygdala. All of the rats received a single injection of BrdU (100 mg/kg) intraperitoneally (i.p.) before killing. Under deep sodium pentobarbital anesthesia (60 mg/kg i.p.), each rat was transcardially perfused with saline and then with 4% paraformaldehyde. Animals were killed on days 3, 7, 14, and 28 (three to six animals each day) after seizure induction. The brains were embedded in paraffin and cut in 4-μm sections. For preliminary examination, newborn cells positive for BrdU were counted in the dentate gyrus and pyramidal cell layers by diaminobenzidine (DAB) staining. Then double-labeled immunofluorescence staining was performed by using primary antibodies to BrdU and (-tubulin III (TuJ1) as immature neuronal markers and conjugated fluorescein isothiocyanate (FITC) and rhodamine as secondary antibodies. Results: In kainic acid–induced limbic seizure models, the number of newborn neuronal cells increased significantly in the dentate gyrus from days 3 to 7 compared with those in controls. Several newborn cells were observed in the pyramidal cell layer (CA3), where cell loss and pyknosis were evident. In another investigation using cryostat sections, mossy fiber reorganization in the dentate inner molecular layer was observed by Timm staining 2 weeks after kainic acid injection. Conclusions: The rate of neurogenesis in the dentate granule cell layer during the early postnatal period and adulthood appears to be influenced by factors such as aging, environmental stimulation, glucocorticoid hormone levels, and glutamatergic input to the dentate gyrus. Recent investigations using a variety of experimentally induced pathologies, including seizures, cerebral ischemia, and toxin injection, showed alterations in dentate neurogenesis after brain injury. Repetitive seizure activities induce not only neuronal cell loss but also seizure-induced neurogenesis in the dentate gyrus and CA3. The dentate gyrus is positioned to be able to control hippocampal throughput, and it has been suggested that the dentate gyrus functions as a gate for controlling the propagation of seizures. Increase of hippocampal neurogenesis appeared in association with neuronal damage. Generation of new neurons and mossy fiber sprouting in the hippocampus may be an important neurobiologic element underlying the clinical effects of kainic acid–induced limbic seizure.