Martin Balslev Jørgensen

Selective neuron loss after cerebral ischemia in the rat: Possible role of transmitter glutamate

Male Wistar rats were subjected to 20 min of cerebral ischemia by means of 4‐vessel occlusion. The topography of regional, selective neuron loss in this model corresponded to areas with pronounced glutamate high affinity uptake (presynaptic receptors), suggesting that transmitter glutamate is involved in the mechanism of neuron damage. One group of animals was injected with the glutamate antagonist, glutamic acid diethyl ester (GDEE) before ischemia. The regional neuron loss was rated using a semiquantitative scale. No statistically significant difference was found between the groups. The results do not exclude a possible role of transmitter glutamate in the pathogenesis of ischemic brain damage. More specific and potent glutamate antagonists are needed in order to clarify such a mechanism.

Ischemic damage in hippocampal CA1 is dependent on glutamate release and intact innervation from CA3.

The removal of glutamatergic afferents to CA1 by destruction of the CA3 region is known to protect CA1 pyramidal cells against 10 min of transient global ischemia. To investigate further the pathogenetic significance of glutamate, we measured the release of glutamate in intact and CA3-lesioned CA1 hippocampal tissue. In intact CA1 hippocampal tissue, glutamate increased sixfold during ischemia; in the CA3-lesioned CA1 region, however, glutamate only increased 1.4-fold during ischemia. To assess the neurotoxic potential of the ischemia-induced release of glutamate, we injected the same concentration of glutamate into the CA1 region as is released during ischemia in normal, CA3-lesioned, and ischemic CA1 tissue. We found that this particular concentration of glutamate was sufficient to destroy CA1 pyramids in the vicinity of the injection site in intact and CA3-lesioned CA1 tissue when administered during control (non-ischemic) conditions. In contrast, the same amount injected during ischemia in the CA3-lesioned CA1 region destroyed pyramidal cells in a widely distributed zone around the injection site in the CA1 region. It is concluded that the ischemia-induced damage of pyramidal cells in CA1 is dependent on glutamate release and intact innervation from CA3.

Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia

ABSTRACT‐ Rats exposed to 10 min of complete cerebral ischemia develop necrosis of the CA‐1 region of the hippocampus after 2–3 days. We studied the involvement of synaptic transmission for this process by ablation of the afferent input (which is mainly glutamatergic) to CA1 by bilateral destruction of CA‐3 neurons (Schafferotomi). The deafferentiation completely prevented the ischemic nerve cell destruction as revealed by histological studies after 6 days. The role of intracellular Ca++ overload was assessed by measurement of the interstitial Ca++ concentration. In control animals the interstitial Ca++ concentration decreases abruptly to 10% of the initial value 1.6 min after the onset of ischemia. The denervated hippocampi, however, showed no decrease during the 10 min of ischemia and hippocampi injected with 2‐amino‐5‐phosphovalerate (APV), a competitive antagonist of the glutamate N‐methyl‐D‐aspartate (NMDA) receptors, displayed a significantly reduced decrease (45% of the initial value) during ischemia. It is concluded that calcium influx via the glutamate‐operated channels during the ischemic period is an important link in the development of ischemic brain cell damage.

Delayed c-fos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study

The relative levels of c-fos mRNA in individual neurons of the hippocampal formation of rats is dramatically increased following 20 min of cerebral ischemia induced by 4-vessel occlusion. After 24 h of recirculation, a number of scattered neurons in the dentate hilus became hybridization positive. This effect appeared to peak between 24 and 48 h. A few neurons in the pyramidal cell layer of CA1 expressed c-fos as early as 24 h, but the most intense labeling in this region was seen at 72 h of recirculation. These results correlate well with the known distribution of delayed ischemic necrosis in the brain.

Leao's spreading depression in the hippocampus explains transient global amnesia. A hypothesis.

Transient global amnesia has been explained by epileptic mechanisms or transient ischemic attacks affecting the hippocampus. None of these two mechanisms appear likely. The animal experimental phenomenon entitled spreading depression of cortical electrical activity (SD) or spreading depression of Leao has been implicated in migraine pathogenesis and may be relevant to transient global amnesia. In experimental animals, SD in the hippocampus causes a temporary functional ablation lasting minutes to hours with full functional recovery. Glutamate, which is present in large amounts in the hippocampus, may experimentally elicit spreading depression, and strong emotional events may possibly liberate glutamate and bring about this reaction in human patients.

Removal of the entorhinal cortex protects hippocampal CA-1 neurons from ischemic damage

SummaryThe excitatory (glutamatergic) innervation seems to determine a nerve cells vulnerability to complete, transient ischemia. Interruption of the excitatory afferents to the hippocampus by removal of the entorhinal cortex prior to ischemia allows examination of this hypothesis. Groups of adult male Wistar rats were subjected to 20 min of ischemia (fourvessel occlusion) 4 days following a sham procedure, unilateral or bilateral entorhinotomy. CA-1 pyramidal cell survival following ischemia was assessed by light microscopic examination (cell counts) 4 days after ischemia. Compared to control animals unilateral entorhinotomy protected 50% of the CA-1 pyramidal neurons ipsilateral to the lesion, whereas bilateral entorhinotomy resulted in 84% protection. The pathophysiology of ischemic brain damage is discussed, and it is suggested that the protection of CA-1 pyramidal neurons after entorhinotomy is due to interruption of the input to the dentate granule cells, which forms a link in the trisynaptic pathway from the entorhinal cortex to the CA-1.

Selective dendrite damage in hippocampal CA1 stratum radiatum with unchanged axon ultrastructure and glutamate uptake after transient cerebral ischaemia in the rat

In this study of the rat hippocampal CA1 stratum radiatum 4 days after 20 min transient cerebral ischaemia, we demonstrated ultrastructural dendritic degeneration and loss, whereas most axons appeared undamaged. Autoradiographs 4 days after ischaemia showed unchanged Na+-dependent glutamate high affinity uptake. The role of glutamate and Ca2+ for this selective postsynaptic vulnerability is discussed.

Protection against ischemic hippocampal CAI damage in the rat with a new non-NMDA antagonist, NBQX

Two glutamate antagonists were tested in a rat model of complete, transient cerebral ischemia. Six days after 10 min ischemia the mean loss of hippocampal CAI pyramidal neurones was 73%. Administration of the AMPA (a‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionic acid) antagonist NBQX (2,3‐dihydro‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline) reduced the pyramidal neurone loss to 1%, 11% and 15%, when given before, immediately after or 1 h after ischemia, respectively. MK‐801 (dizocilpine), a competitive NMDA antagonist gave no protection in this model. We suggest that the AMPA receptor transduction mechanisms are sensitized by ischemia and that the postischemic blockade of the main glutamatergic input to the CA 1 cells with NBQX impairs the deleterious effect of “normal” postischemic excitatory transmission.

Neural grafting to ischemic lesions of the adult rat hippocampus

SummaryThe purpose of this study was to examine the structural and connective integration of developing hippocampal neurons grafted to ischemic lesions of the adult rat hippocampus. The 4-vessel occlusion model was used to cause transient cerebral ischemia which damages CA1 pyramidal cells in the dorsal hippocampus, but spares nonpyramidal neurons and afferents in the area. One week later, cell suspensions were made from the CA1 region of fetal (E18-20) rats and injected stereotaxically into the lesion. The recipient brains were examined 6 weeks to 6 months later for survival, morphology, and intrinsic and extrinsic connections of the grafts. The methods used included cell stains, histochemical staining for acetylcholinesterease (AChE), immunocytochemical staining for neuropeptides (cholelecystokinin (CCK), somatostatin (SS), enkephalin (Enk) and an astrocytic marker, glial fibrillary acidic protein (GFAP), as well as tracing by retrograde axonal transport of fluorochromes and light and electron microscopy of anterograde axonal degeneration. The grafts survived well (80%) and were often quite large. They were well integrated in the lesioned host brain area, contained both pyramidal cells and neuropeptidergic neurons and displayed a near normal GFAP immunoreactivity for astrocytes. The latter contrasted the dense gliosis of the host ischemic lesion. Judged by the AChE staining the grafts were innervated by cholinergic host septohippocampal fibers. Ingrowth of host hippocampal commissural fibers was demonstrated by Fink-Heimer staining for degenerating nerve terminals following acute lesions of the hippocampal commissures. At the ultrastructural level degenerating, electron dense terminals of host commissural origin were found even deep inside the graft neuropil in synaptic contact with mainly dendritic spines. A transplant efferent connection to the host brain was demonstrated by retrograde fluorochrome tracing and consisted of a homotypic projection to more posterior levels of the ipsilateral host CA1 and subiculum. Minor abnormal, efferent projections to the host dentate molecular layer were shown in Timm staining. We conclude that fetal CA1 neurons grafted to one week old ischemic lesions of the dorsal CA1 in adult rats become structurally well incorporated and can establish nerve connections with the host brain.

Nitric oxide does not act as a mediator coupling cerebral blood flow to neural activity following somatosensory stimuli in rats.

The possible role of nitric oxide (NO) on vibrissa-stimulated increase of regional cerebral cerebral blood flow (rCBF) and cerebral metabolic rate of glucose (rCMRglu) was investigated in conscious Wistar rats by using an inhibitor of NO synthase, NG-nitro-L-arginine (NOLAG) at a concentration of 30 mg/kg. In vivo autoradiography distribution with 14C-iodoantipyrine and 14C-deoxyglucose in two separate series showed CBF of 174% of control and CMRglu of 196% of control in the primary sensory cortex opposite the stimulated side in saline treated control animals. Similar increases were found in NOLAG-treated animals. Furthermore, NOLAG did not change either basal CMRglu or CMRO2. The findings suggest, that NO is not involved in coupling flow to the increased metabolism accompanying physiological sensory stimuli.