Riitta Miettinen

Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat model of temporal lobe epilepsy.

The present study was designed to address the question of whether recurrent spontaneous seizures cause progressive neuronal damage in the brain. Epileptogenesis was triggered by status epilepticus (SE) induced by electrically stimulating the amygdala in rat. Spontaneous seizures were continuously monitored by video-EEG for up to 6 months. The progression of damage in individual rats was assessed with serial magnetic resonance imaging (MRI) by quantifying the markers of neuronal damage (T2, T1 rho, and Dav) in the amygdala and hippocampus. The data indicate that SE induces structural alterations in the amygdala and the septal hippocampus that progressively increased for approximately 3 weeks after SE. T2, T1 rho, and Dav did not normalize during the 50 days of follow-up after SE, suggesting ongoing neuronal death due to spontaneous seizures. Consistent with these observations, Fluoro-Jade B-stained preparations revealed damaged neurons in the hippocampus of spontaneously seizing animals that were sacrificed up to 62 days after SE. The presence of Fluoro-Jade B-positive neurons did not, however, correlate with the number of spontaneous seizures, but rather with the time interval from SE to perfusion. Further, there were no Fluoro-Jade B-positive neurons in frequently seizing rats that were perfused for histology 6 months after SE. Also, the number of lifetime seizures did not correlate with the severity of neuronal loss in the hilus of the dentate gyrus assessed by stereologic cell counting. The methodology used in the present experiments did not demonstrate a clear association between the number or occurrence of spontaneous seizures and the severity of hilar cell death. The ongoing hippocampal damage in these epileptic animals detected even 2 month after SE was associated with epileptogenic insult, that is, SE rather than spontaneous seizures.

Pattern of neuronal death in the rat hippocampus after status epilepticus. Relationship to calcium binding protein content and ischemic vulnerability

The pattern of hippocampal cell death has been studied following hippocampal seizure activity and status epilepticus induced by 110-min stimulation of the perforant pathway in awake rats. The order of vulnerability of principal cells in the different hippocampal subfields--as determined by silver impregnation--was found to be very similar to the pattern found in ischemia; i.e., dentate hilus greater than CA1, subiculum greater than CA3c greater than CA3a,b greater than dentate granule cells. The hilar somatostatin-containing cells were the most vulnerable cell type, whereas all other subpopulations of nonprincipal neurons--visualized by immunocytochemistry for the calcium binding proteins parvalbumin and calbindin--were remarkably resistant. Pyramidal cells in the CA3 region containing neither of the examined calcium binding proteins were more resistant to overexcitation than CA1 pyramidal cells, most of which do contain calbindin. This indicates that no simple relationship exists between vulnerability in status epilepticus and neuronal calcium binding protein content, and that local and/or systemic hypoxia during status epilepticus may be responsible for the ischemic pattern of cell death.

Neuroprotective Effects of Dexmedetomidine in the Gerbil Hippocampus after Transient Global Ischemia

Background: Cerebral ischemia induces a massive release of norepinephrine associated with neuronal death in the brain. It has been demonstrated that alpha2 ‐adrenoceptor agonists decrease the release and turnover of noradrenaline, and this might prove advantageous in counteracting the neurodegeneration in ischemic brain. Therefore, in the present study, the authors tested whether dexmedetomidine, a selective alpha2 ‐receptor agonist, has neuroprotective effects in a gerbil transient global ischemia model. Methods: Ischemia was induced by bilateral carotid occlusion for 5 min in diethylether‐anesthetized normothermic gerbils. Dexmedetomidine was administered subcutaneously in four different treatment paradigms (6–8 animals/group): 3 or 30 micro gram/kg 30 min before and thereafter at 3, 12, 24, and 48 h after the occlusion, or 3 or 30 micro gram/kg at 3, 12, 24, and 48 h after the occlusion. Control animals were subjected to forebrain ischemia but received only saline injections. One week after occlusion, animals were transcardially perfused for histochemistry. Neuronal death in the CA1 and CA3 regions of the hippocampus and in the hilus of the dentate gyrus was evaluated in silver‐stained 60‐micro meter coronal sections. Results: Compared with saline‐treated ischemic animals, dexmedetomidine at a dose of 3 micro gram/kg given before and continued after the induction of ischemia reduced the number of damaged neurons in the CA3 area (2 +/‐ 3 vs. 17 +/‐ 20 degenerated neurons/mm2; P <0.05). Also in the dentate hilus, the number of damaged neurons was reduced by dexmedetomidine (3 micro gram/kg) given before and continued after ischemia (5 +/‐ 7 vs. 56 +/‐ 42 degenerated neurons/mm2; P <0.01). Conclusions: The present data demonstrate that dexmedetomidine effectively prevents delayed neuronal death in CA3 area and in the dentate hilus in gerbil hippocampus when the management is started before the onset of ischemia and continued for 48 h after reperfusion. Inhibition of ischemia‐induced norepinephrine release may be associated with neuroprotection by dexmedetomidine.

HIPPOCAMPAL PLASTICITY IN ALZHEIMER'S DISEASE : CHANGES IN HIGHLY POLYSIALYLATED NCAM IMMUNOREACTIVITY IN THE HIPPOCAMPAL FORMATION

The highly polysialylated neural cell adhesion molecule (PSA‐NCAM) is one of the most promising molecules that contributes to plasticity in the central nervous system. We evaluated PSA‐NCAM immunoreactivity in the hippocampal formation of Alzheimers disease (AD) patients. We found significant increases over control levels in the optical density ratios of PSA‐NCAM immunoreactivity in the outer molecular layer/granule cell layer (ODoml/grl) and in the inner molecular layer/granule cell layer (ODiml/grl) in the dentate gyrus of AD patients. The optical density of the granule cell layer in the dentate gyrus did not differ significantly between AD patients and control subjects. However, the number of PSA‐NCAM‐immunopositive infragranule cells was higher in the AD group compared with control subjects. The major finding in the CA1, subiculum and entorhinal cortex of AD patients was the disorganization of PSA‐NCAM‐immunoreactive fibres. These results indicate that neuronal remodelling occurs, especially in the dentate gyrus of patients with AD.

Pharmacological consequences of cholinergic plus serotonergic manipulations.

The present study investigated pharmacological consequences of combined cholinergic and serotonergic blockade. Raphe medianus (RM) lesions (5,7-DHT) had no effect on spatial learning, but augmented scopolamine 0.8 mg/kg induced learning deficit. Pilocarpine (4 mg/kg) could reverse scopolamine (0.8 mg/kg), but not scopolamine (0.8 mg/kg) + RM lesion induced spatial learning impairment. However, a higher dose of pilocarpine could restore spatial learning deficit induced by scopolamine (0.8 mg/kg) and RM lesions. These findings support the important role of cholinergic-serotonergic interaction in the regulation of spatial learning and suggests that the combined cholinergic-serotonergic deficit in patients with Alzheimers disease may have an impact on therapeutic approaches which seek to normalize AD related cognitive impairments.

Characterization of Target Cells for Aberrant Mossy Fiber Collaterals in the Dentate Gyrus of Epileptic Rat

Previous studies have demonstrated formation of recurrent excitatory circuits between sprouted mossy fibers and granule cell dendrites in the inner molecular layer of the dentate gyrus (9, 28, 30). In addition, there is evidence that inhibitory nonprincipal cells also receive an input from sprouted mossy fibers (39). This study was undertaken to further characterize possible target cells for sprouted mossy fibers, using immunofluorescent staining for different calcium-binding proteins in combination with Timm histochemical staining for mossy fibers. Rats were injected intraperitoneally with kainic acid in order to induce epileptic convulsions and mossy fiber sprouting. After 2 months survival, hippocampal sections were immunostained for parvalbumin, calbindin D28k, or calretinin followed by Timm-staining. Under a fluorescent microscope, zinc-positive mossy fibers in epileptic rats were found to surround parvalbumin-containing neurons in the granule cell layer and to follow their dendrites, which extended toward the molecular layer. In addition, dendrites of calbindin D28k-containing cells were covered by multiple mossy fiber terminals in the inner molecular layer. However, the calretinin-containing cell bodies in the granule cell layer did not receive any contacts from the sprouted fibers. Electron microscopic analysis revealed that typical Timm-positive mossy fiber terminals established several asymmetrical synapses with the soma and dendrites of nonpyramidal cells within the granule cell layer. These results provide direct evidence that, in addition to recurrent excitatory connections, inhibitory circuitries, especially those responsible for the perisomatic feedback inhibition, are formed as a result of mossy fiber sprouting in experimental epilepsy.

Estrogen treatment improves spatial learning in APP + PS1 mice but does not affect beta amyloid accumulation and plaque formation.

We investigated the effects of ovariectomy (OVX) and 17 beta-estradiol (0.18 mg per pellet) treatment on spatial learning and memory, hippocampal beta amyloid (A beta) levels, and amyloid plaque counts in double transgenic mice (A/P) carrying mutated amyloid precursor protein (APPswe) and presenilin-1 (PS1-A246E). After OVX at 3 months of age, the mice received estrogen treatment for the last 3 months of their lifetime before they were killed at 6, 9, or 12 months of age. Estrogen treatment in A/P OVX mice increased the number of correct choices in a position discrimination task in the T-maze, and slightly improved their performance in a win-stay task (1/8 arms baited) in the radial arm maze (RAM). However, estrogen treatment did not reverse the A beta-dependent cognitive deficits of A/P mice in the water maze (WM) spatial navigation task. Furthermore, ovariectomy or estrogen treatment in OVX and sham-operated A/P mice had no effect on hippocampal amyloid accumulation. These results show that the estrogen treatment in a transgenic mouse model of Alzheimers disease (AD) improves performance in the same learning and memory tasks as in the normal C57BL/6J mice. However, the estrogen effects in these mice appeared to be unrelated to A beta-induced cognitive deficits. Our results do not support the idea that estrogen treatment decreases the risk or alleviates the symptoms of Alzheimers disease by inhibiting the accumulation of A beta or formation of amyloid plaques.

Effects of quisqualic acid nucleus basalis lesioning on cortical EEG, passive avoidance and water maze performance.

The study examines the effects of unilateral quisqualic acid nucleus basalis (NB) lesioning on cortical EEG and learning behavior. Lesions produced both gliosis in the ventral pallidum and a marked reduction in the cortical ChAT activity. Normal cortical EEG activity was abolished on the side of NB lesion, i.e., slow wave activity and the incidence of high voltage spindles was higher on the side of lesion compared with the control side. NB lesioning impaired passive avoidance retention, but not spatial learning ability. These results suggest that EEG and passive avoidance deficits induced by NB quisqualic acid lesion may result from the damage specifically to cholinergic neurons. Thus, the restoration of EEG and passive avoidance performance defects in quisqualic-lesioned rats may be used as an index of the efficacy of the cholinergic replacement therapies.

Estimation of the Total Number of Cholinergic Neurons Containing Estrogen Receptor-α in the Rat Basal Forebrain

This study was undertaken to estimate the total number of cholinergic cells and the percentage of cholinergic cells that contain estrogen receptor-α (ERα) in the rat basal forebrain. Double immunostaining for choline acetyltransferase (ChAT) and ERα was carried out on 50-μm-thick free-floating sections. Because routine mounting method causes considerable flattening of the sections, we embedded immunostained sections in Durcupan, an epoxy resin known to cause virtually no shrinkage. When this procedure was used the section thickness was well preserved, individual cells could be clearly identified, and subcellular localization of ERα immunoreactivity was easy to verify. Cell counting in these sections revealed that the rat basal forebrain contains 26,390 ± 1097 (mean ± SEM) cholinergic neurons. This comprises 9674 ± 504 in the medial septum-vertical diagonal band of Broca, 9403 ± 484 in the horizontal diagonal band of Broca, and 7312 ± 281 in the nucleus basalis. In these nuclei, 60%, 46%, and 14% of the cholinergic neurons were colocalized with ERα, respectively. We believe that our results are an improvement on existing data because of the better distinction of individual neurons that the Durcupan embedding method brings.

α2-Adrenoceptor agonist, dexmedetomidine, protects against kainic acid-induced convulsions and neuronal damage

Kainic acid (KA)-induced convulsions are accompanied by histopathological changes that are most prominent in the temporal lobe structures. In the present study, we investigated whether a selective alpha2-adrenoceptor agonist, dexmedetomidine could attenuate KA-induced epileptic convulsions and subsequent neuronal damage in the rat hippocampus. Rats were pretreated 30 min before KA injection (9 mg/kg, i.p.) with dexmedetomidine (3 micrograms/kg, s.c.). The behavior of animals was observed for at least 3 h. Dexmedetomidine suppressed the development (p < 0.001), generalization (p < 0.05) and severity (p < 0.01) of convulsions. In addition, histological analysis revealed that dexmedetomidine-treated animals without convulsions or with only partial convulsions had no neuronal damage in the principal cell layers of the hippocampus. A selective alpha2-antagonist, atipamezole (1 mg/kg, s.c.) potentiated KA-induced convulsions and increased the mortality in status epilepticus. In conclusion, the present study demonstrated that dexmedetomidine, in addition to possessing anticonvulsant properties, has a neuroprotective effect in the KA model of status epilepticus.