Peter Andiné

The Effect of MK-801 on Cortical Spreading Depression in the Penumbral Zone following Focal Ischaemia in the Rat

Cortical spreading depression (CSD) is a transient depression of neuronal activity that spreads across the cortical surface. In the present studies, we have investigated CSD activity in the penumbral zone following permanent middle cerebral artery (MCA) occlusion in the rat (n = 16/group), using double-barreled Ca2+ -sensitive microelectrodes. Measurements of CSD activity were made for 3 h in each animal. During this time, a varying number of spontaneous CSDs were seen in the control group (total was 30, with a range of 0–7/rat). These CSDs were of varying duration: “small” (∼1 min) and “big” (5–45 min) CSDs. During a CSD, the extracellular [Ca2+] decreased to 0.11 ± 0.07 mM (mean ± SD). After 3 h, the extracellular [Ca2+] in the cortex (penumbral zone) was either normal (10/16 rats) or lowered to 0.5 mM (2/16 rats) or to 0.1 mM (4/16 rats). In the caudate nucleus (ischaemic core area), all rats had an extracellular [Ca2+] of ∼0.1 mM when measured after the 3 h recording period. Neuropathological evaluation of the brains of the animals, which had been allowed to survive for 24 h after MCA occlusion, revealed ischaemic damage in the dorsolateral cortex and caudate nucleus. Administration of the noncompetitive NMDA antagonist, MK-801 (3 mg/kg i.p.), 30 min after MCA occlusion resulted in 24 and 29% reductions in the volume of hemispheric and cortical damage, respectively, which was highly significant (p < 0.0001); no protection was seen against caudate damage. MK-801 also significantly (p < 0.05) reduced the number of CSDs (total was 10 with a range of 0–2/rat) and the degree of decrease in extracellular [Ca2+] during CSDs (low level was 0.19 ± 0.15 mM, mean ± SD). Both the number of “small CSDs” and significantly the “big CSDs” were reduced by MK-801. The “small CSDs” were present in the border zone and also deeper within the infarct, whereas “big CSDs” and anoxic depolarisations only occurred closer to the infarct core. CSDs were not seen outside the border zone of the infarct. In conclusion, there was a good correlation between extracellular [Ca2+] changes and ischaemic damage in the infarcted region following MCA occlusion. MK-801 reduced the ischaemic damage in the penumbral zone in parallel with attentuation of the number and amplitude of CSDs and thereby a reduced intracellular Ca2+ overload.

The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection

The neuroprotective effect of kynurenic acid, an unspecific antagonist of excitatory amino acid receptors, was evaluated in a model of hypoxic-ischemia in neonatal rats. One-week-old rats were subjected to ligation of the left carotid artery and exposure to 7.7% O2/92.3% N2 for 2 h. Kynurenic acid (300 mg/kg) was administered i.p. immediately after the period of hypoxic-ischemia in one group (n = 32) and compared with saline-treated (n = 27). After 2 weeks the rats were sacrificed and the brain damage evaluated by comparing the weight of the lesioned and unlesioned hemispheres. In rats receiving kynurenic acid the reduction in weight of the lesioned hemisphere was 25.4 +/- 3.3% as compared to 37.8 +/- 3.6% in saline-treated controls (P less than 0.001). The results suggest that excitatory amino acids are involved in the development of postischemic damage in the immature brain.

Hypoxia-ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists

In a model of perinatal hypoxic-ischemic brain damage, we examined the neuroprotective efficacy of posttreatment with the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist NBQX. Unilateral brain damage developed in 95% of rat pups subjected to hypoxia-ischemia with a 27.8 +/- 1.2% weight deficit of the damaged hemisphere. MK-801 in doses of 0.3 and 0.5 mg/kg i.p. reduced the brain damage by 61% (p < 0.001) and 43% (p < 0.001), respectively. A higher dose of MK-801 (0.75 mg/kg) did not offer neuroprotection. Treatment with NBQX (40 mg/kg) reduced the hemispheric lesion by 28% (p < 0.05). In conclusion, posttreatment with both NBQX and low doses of MK-801 reduced perinatal brain damage. The NMDA receptor antagonist offered stronger neuroprotection which is in agreement with a proposed NMDA receptor hyperactivity around postnatal day 7 in rats.

Effect of propentofylline (HWA 285) on extracellular purines and excitatory amino acids in CA1 of rat hippocampus during transient ischaemia

1 The adenosine uptake blocker propentofylline (HWA 285) has previously been shown to protect hippocampal CA1 pyramidal cells from ischaemia‐induced delayed neuronal death. The influence of propentofylline, on the extracellular concentrations of purines, aspartate and glutamate in the CA1 of the rat hippocampus during transient forebrain ischaemia was investigated. 2 Twenty min of ischaemia was induced by four‐vessel occlusion in Wistar rats, extracellular compounds were sampled by use of microdialysis and EEG was recorded by a tungsten electrode attached to the dialysis probe. 3 Propentofylline (10 mgkg−1 i.p.) did not influence the basal levels of any of the compounds in the hippocampal dialysates. 4 The EEG became isoelectric within 20 s after induction of ischaemia. 5 Extracellular adenosine, inosine, hypoxanthine, aspartate and glutamate increased several fold during ischaemia and remained elevated during early reflow. Within 2 h of reperfusion the concentration of all compounds was normalized. Xanthine increased upon reperfusion and remained elevated after 2 h. 6 Propentofylline (10 mg kg−1 i.p.) administered 15 min before ischaemia significantly enhanced the ischaemia‐evoked increase of adenosine but attenuated the increases of the other purine catabolites and of glutamate. 7 In separate in vitro experiments, propentofylline did not inhibit adenosine deaminase activity. 8 The present data show that propentofylline enhances extracellular adenosine and lowers extracellular glutamate in vivo during ischaemia. These findings may be important in relation to the neuroprotective properties of propentofylline.

Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia.

Excitatory amino acids (EAAs) have been implicated to play a part in the development of hypoxic-ischemic brain injury in the neonate. The aim of the present study was to follow changes of intra- and extracellular (microdialysis) amino acids in the cerebral cortex in a model where cortical hypoxic-ischemic damage is produced consistently. Hypoxic-ischemia (unilateral ligation of the carotid artery + 2 h of exposure to 7.8% oxygen) caused a depletion of tissue ATP, phosphocreatine and glucose with a concomittant accumulation of AMP and lactic acid in cortical tissue. These changes were accompanied by a decrease of tissue aspartate and glutamine whereas the contents of gamma-aminobutyric acid (GABA), phenylalanine, leucine, isoleucine, valine and alanine increased. In the extracellular fluid GABA, glutamate, aspartate, taurine, glycine and alanine all increased multi-fold during hypoxic-ischemia. Aspartate and glutamate returned to near initial levels 2 h after the end of the insult, whereas the elevation of glycine persisted during recovery. In conclusion, the high extracellular levels of EAAs and glycine may exert injurious effects during and after hypoxic-ischemia.

Changes in Extracellular Amino Acids and Spontaneous Neuronal Activity During Ischemia and Extended Reflow in the CA1 of the Rat Hippocampus

Abstract: This study addresses the possible involvement of an agonist‐induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four‐vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple‐unit activity. Multiple‐unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and ‐γ‐aminobutyric acid increased in both acute and chronic experiments (seven‐to 26‐fold). Multiple‐unit activity recovered to preischemic levels following 4–6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple‐unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple‐unit activity recovered faster and reached 140% of control at 8h, paralleled by an increase in extracellular aspartate (5.5‐fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.

Calcium Uptake Evoked by Electrical Stimulation is Enhanced Postischemically and Precedes Delayed Neuronal Death in CA1 of Rat Hippocampus: Involvement of N-Methyl-D-Aspartate Receptors

Extracellular calcium concentration changes in the CA1 of the hippocampus during burst activity were measured during postischemic reflow, and the involvement of N-methyl-D-aspartate (NMDA) receptors was evaluated, In adult Wistar rats global ischemia was induced by four-vessel occlusion for 20 min, After 6 h of postischemic reflow, the animals were halothane-anesthetized and reintubated, A double-barrelled calcium-sensitive microelectrode was advanced through stratum oriens, pyramidale, and radiatum in 50-μm steps. At each step the perforant pathway was stimulated (15 Hz, 30 s), and changes in extracellular calcium concentration were recorded. High-frequency stimulation elicited burst firing and transient decreases in extracellular calcium concentration, which are interpreted as neuronal calcium uptake. In control hippocampus, the extracellular calcium decreases were maximal in the stratum pyramidale. Six to eight hours after ischemia, a threefold enhancement of extracellular calcium decreases was found in the dendritic layers of the CA1. The NMDA-receptor antagonist ketamine (15–30 mg/kg intraperitoneally) reduced these electrically evoked calcium decreases. Seven days after ischemia, there was a 60–90% loss of pyramidal cells in the CA1. In conclusion, the cellular calcium uptake, possibly through NMDA receptors evoked by neuronal activity, is enhanced during early postischemia and precedes delayed neuronal death.

Neuropathological endpoints in experimental stroke pharmacotherapy: The importance of both early and late evaluation

SummaryThis study adresses the issue of endpoint selection in the evaluation of neuroprotective drugs in experimental focal ischaemia. Previous work with the permanent middle cerebral artery (MCA) occlusion model in the rat has demonstrated that the ischaemic lesion does not acquire its final appearance until at least 28 days after the ictus. Therefore, the effect of the NMDA receptor blocker MK-801 (dizocilpine maleate) was evaluated both early (3 days) and late (28 days) after MCA occlusion to determine if the previously reported protective effect of a single post-ischaemic dose of MK801 found in acute experiments remained after 28 days.Mk-801 (0.5mg/kg, i.v.) or isotonic saline was randomly given to rats 30 min after MCA occlusion. Infarct volume and volume of ipsilateral and contralateral hemispheres were estimated from camera lucida drawings of 8 defined coronal histological sections of the brain. As expected, a 40% (p<0.05) reduction of infarct size was found in MK-801 treated rats after 3 days. In animals evaluated 28 days after MCA occlusion, no significant difference in infarct size, total tissue loss (infarct volume + ipsilateral hemisphere atrophy) or remaining non-infarcted tissue (contralateral hemisphere — total tissue loss) was seen between the MK-801 and placebo treated rats.The results suggest that the single dose treatment with MK-801 postponed the evolution of the infarct, which at 3 days after MCA occlusion is still in progress, possibly by ameliorating oedema formation. It remains to be shown if a multiple dose treatment with NMDA receptor antagonists improves the final neuropathological outcome after experimental stroke. The study illustrates the importance of including a late endpoint when evaluating the efficacy of neuroprotective stroke therapy.

Evaluation of brain damage in a rat model of neonatal hypoxic-ischemia

In spite of improvements in obstetric and neonatal care, hypoxic-ischemic brain damage with severe neurologic disability is still a clinical reality. A model in 7-day-old rats has been introduced to study the pathophysiology of perinatal hypoxic-ischemic brain damage. Unilateral brain damage is produced in the cerebral cortex, striatum and hippocampus, i.e. a similar distribution as is often seen in human asphyxiated neonates. In the present investigation the model was evaluated further by comparing three different methods to assess the brain damage: weighing the hemispheres, morphometry and somatosensory evoked potentials. Seven-day-old rats were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (7.7% O2 at 36 degrees C). After 2 h of hypoxic-ischemia pCO2 and pO2 decreased in mixed arterial/venous blood. The evaluation of the damage 2 weeks after the insult, demonstrated close correlation between morphometry and weighing (r = 0.836, P less than 0.01). The amplitude of evoked potentials correlated to the other parameters (r = 0.814, P less than 0.01 and r = 0.824, P less than 0.01 respectively) and displayed a greater relative attenuation than the other methods but with a more pronounced variability. These results indicate that the degree of brain damage can be assessed by weighing for screening purposes.

Enhanced Calcium Uptake by CA1 Pyramidal Cell Dendrites in the Postischemic Phase despite Subnormal Evoked Field Potentials: Excitatory Amino Acid Receptor Dependency and Relationship to Neuronal Damage

After 6–12 h of recovery from transient cerebral ischemia, the pyramidal cells of the hipppocampal CA1 region take up excessive amounts of calcium upon electrical stimulation, which has been suggested to be important for the development of delayed neuronal death. The aim of this study was to further characterize this enhanced calcium uptake with respect to time-course of development, relationship to neuronal damage, and amplitude of evoked field potentials as well as the dependency on N-methyl-d-aspartate (NMDA) and non-NMDA receptors. Adult Wistar rats were used and calcium-sensitive microelectrodes were placed in the stratum radiatum of the CA1 hippocampus for recording of the extracellular calcium concentration ([Ca2+]ec) during 20 min of ischemia and for 6 h of reflow. High-frequency stimulation of the perforant pathway elicited burst firing in CA1 and a transient decrease in [Ca2+]ec which reflects neuronal uptake. Shifts in [Ca2+]ec could not be evoked 0–1 h after ischemia. However, from 1–2 h burst firing could be evoked and the accompanying shift in [Ca2+]ec increased thereafter in amplitude with prolonged reflow, exceeded preischemic levels after 4 h, and reached 250 ± 116% (mean ± SD) of control after 6 h of reflow (p < 0.05). The extracellular reference potential shift during electrical stimulation and the amplitude of evoked field potentials were still subnormal after 6 h [85 ± 25% and 83 ± 25%, respectively (mean ± SD)]. There was a significant correlation between the degree of stimulated calcium uptake at 6 h postischemia and the extent of CA1 damage evaluated 7 days after the ischemic insult (r = 0.849; p < 0.001). The shifts in [Ca2+]ec were reduced by the NMDA antagonist MK-801 (0.5–2 mg/kg, i.v.) to approximately 50% of the initial level during both control and postischemic conditions (p < 0.01). The non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[F]quinoxaline (NBQX) (42 ± 13 mg/kg, i.p.; mean ± SD) decreased the amplitude of the evoked field potentials (to 30 ± 28% of control, p < 0.05) and completely abolished the evoked shifts in [Ca2+]ec. In conclusion, the uptake of calcium into CA1 pyramidal cells during electrical stimulation was enhanced already 4 h after ischemia in spite of the fact that other measures of excitability were subnormal. This calcium uptake correlated to the extent of CA1 pyramidal cell damage and was dependent on both NMDA and non-NMDA receptor activation.