Nils Henrik Diemer

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Abstract: Rats were implanted with 0.3‐mm‐diameter dialysis tubing through the hippocampus and subsequently perfused with Ringers solution at a flow rate of 2 (μ1/min. Samples of the perfusate representing the extracellular fluid were collected over 5‐min periods and subsequently analyzed for contents of the amino acids glutamate, as‐partate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10‐min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight‐and threefold during the ischemic period; the taurine concentration also was increased 2.6‐fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of γ‐aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.

Cellular Origin of Ischemia-Induced Glutamate Release from Brain Tissue In Vivo and In Vitro

Abstract: The uptake and release of d‐[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) were studied in cultured glutamatergic neurons (cere‐bellar granule cells) and astrocytes at normal (5 mM) or high (55 mM) potassium and under conditions of hypo‐glycemia, anoxia or “ischemia” (combined hypoglycemia and anoxia). In glutamatergic neurons it was found that “ischemic” conditions led to a 2.4‐fold increase in the potassium‐induced release of d‐[3H]aspartate as compared to normal conditions. Hypoglycemia or anoxia alone affected the release only marginally. The ischemia‐induced increase in the evoked d‐[3H]aspartate release was shown to be calcium‐dependent. In astrocytes no difference was found in the potassium‐induced release between the four conditions and the K+‐induced release was not calcium‐dependent. The uptake of d‐[3H]‐aspartate was found to be stimulated at high potassium in both glutamatergic neurons (98%) and in astrocytes (70%). This stimulation of d‐aspartate uptake, however, was significantly reduced under conditions of anoxia or “ischemia” in both cell types. In glutamatergic neurons (but not in astrocytes) hypoglycemia also decreased the potassium stimulation of d‐aspartate uptake. In a previous report it was shown, using the microdialysis technique, that during transient cerebral ischemia in vivo the extracellular glutamate content in hippocampus was increased eightfold. In the present paper it is shown that essentially no increase in extracellular glutamate is seen under ischemia when the perfusion is performed using calcium‐free, cobalt‐containing perfusion media. The results from the in vitro and in vivo experiments indicate that the glutamate accumulated extracellularly under ischemia in vivo originates from transmitter pools in glutamatergic neurons. Moreover, the released glutamate cannot be efficiently disposed of due to a lack of activation by potassium of the high‐affinity glutamate uptake system in neurons and astrocytes under ischemic conditions.

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.

Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat

The fluorescent dye 6-methoxy-8-p-toluene sulfonamide quinoline (TSQ) was used to monitor the distribution of zinc in the hippocampus and fascia dentata of adult rats subjected to 20 min of cerebral ischemia. In normal brains TSQ stains only neuropil, in particular the mossy fiber layers in the dentate hilus (CA4) and CA3, but within 2 h after ischemia, TSQ-fluorescent cells were observed in the dentate hilus. At longer survival times TSQ-positive cells stained positively with acid fuchsin, a sign of cellular degeneration. At the same time a decrease in the TSQ fluorescence of the mossy fiber terminals in the dentate hilus (CA4) and the CA3 mossy fiber layer was noted. The observations suggest that zinc many play a role in the selective death of dentate hilar neurons after cerebral ischemia.

Regional Cerebral Glucose Phosphorylation and Blood Flow After Insertion of a Microdialysis Fiber Through the Dorsal Hippocampus in the Rat

Abstract: Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.

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.

Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-1 pyramidal cell loss.

SummarySomatostatin (SS)- and cholecystokinin (CCK)-immunopositive cell somata in the rat hippocampus were quantitated at day 1, 2, 3 and 4 after cerebral ischemia. A significant (P<0.01) 60%–80% loss of hilar and CA-3c SS neurons took place. No CCK neurons were lost. Damage to SS neurons was significant on the second postischemic day and preceded the delayed loss of CA-1 neurons. We speculate that loss of SS neurons, which presumably innervate the inhibitory GABAergic (γ-aminobutyric acid) interneurons, may induce hyperactivity stimulating the Ca-1 neurons to death.

Cellular reactions to implantation of a microdialysis tube in the rat hippocampus

SummaryMicrodialysis tubes, used for measurements of extracellular neurotransmitter concentrations, were implanted in rat dorsal hippocampus to study the adjacent tissue reaction. The brain was examined 1–60 days after the implantation. Within the first 2 days, normal neuropil and only occasional hemorrhage surrounded the microdialysis tube. Three days following the implantation astrocytes close to the dialysis tube, hypertrophied. Hypertrophic astrocyte processes invaded the spongy fiber wall. There was no increase in the number of astrocytes. Fourteen days after the fiber insertion layers of reticulin-positive fibers separated astrocytes and the remaining neuropil from the fiber wall. Late tissue changes (1 and 2 months) consisted of collagen deposits and occasional granuloma formation. These results can be used to predict the optimal time for commencing microdialysis after the fiber implantation.

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.

Experimental subarachnoid hemorrhage: subarachnoid blood volume, mortality rate, neuronal death, cerebral blood flow, and perfusion pressure in three different rat models.

Objective To investigate which of three subarachnoid hemorrhage (SAH) models is the most suitable for studies of pathological and pathophysiological processes after SAH. Methods SAH was induced in rats via intracranial endovascular perforation (perforation model), blood injection into the cisterna magna (300 microl), or blood injection into the prechiasmatic cistern (200 microl). The subarachnoid blood volume was quantitatively measured. Cerebral blood flow (CBF) (as assessed with laser Doppler flowmetry), intracranial pressure, and mean arterial blood pressure were recorded for 90 minutes after SAH. Mortality was recorded, and neuronal death was assessed in animals that survived 7 days after SAH. Results The subarachnoid blood volume was close to the injected amount after prechiasmatic SAH. In the other models, the volume varied between 40 and 480 microl. The mortality rates were 44% in the perforation SAH group, 25% in the prechiasmatic SAH group, and 0% in the cisterna magna SAH group; the corresponding values for neuronal death were 11, 44, and 28%. Cerebral perfusion pressure approached baseline values within 5 minutes after SAH in all three models. CBF decreased to approximately 35% of baseline values immediately after SAH in all groups; it gradually increased to normal values 15 minutes after SAH in the cisterna magna SAH group and to 60 and 89% of baseline values 90 minutes post-SAH in the perforation and prechiasmatic SAH groups. CBF was significantly correlated with the subarachnoid blood volume. Conclusion The prechiasmatic SAH model seems to be the most suitable for study of the sequelae after SAH; it produces a significant decrease in CBF, an acceptable mortality rate, and substantial pathological lesions, with high reproducibility. The CBF reduction is predominantly dependent on the amount of subarachnoid blood.