Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Increased CD8(+) T cells associated with Chlamydia pneumoniae in symptomatic carotid plaque.

BACKGROUND AND PURPOSE: The presence of Chlamydia pneumoniae has been reported in carotid atheroma, but its causative effect in the activation of an atherosclerotic plaque to a prothrombotic state remains unproved. Antigen- mediated activation of T lymphocytes within plaque may represent a mechanism by which infection can result in plaque conversion. The goal of the present study was to characterize the T-cell subtype profile related to the presence of C pneumoniae in patients with symptomatic versus asymptomatic carotid atherosclerosis. METHODS: We studied 14 plaques (5 symptomatic and 9 asymptomatic) positive for C pneumoniae confirmed by polymerase chain reaction and 14 plaques (6 symptomatic and 8 asymptomatic) from age- and stenosis-matched patients negative for C pneumoniae by polymerase chain reaction. T-cell subpopulations of T-helper, T-cytotoxic, and T-memory lymphocytes were identified through indirect enzyme immunohistochemistry with anti-CD3+, anti-CD4+, anti-CD8+, and anti-CD45RO+ monoclonal antibodies, respectively. Results are expressed as the number of positive cells per millimeter squared. RESULTS: In the absence of C pneumoniae, symptomatic plaques had a modest but significant increase of CD3+ (89.6 versus 55.3, P=0.013), CD4+ (57.3 versus 32.7, P=0.01), and CD45RO+ (82.8 versus 43.7, P=0.007), but not CD8+ T cells (28.5 versus 25.5, P=0.245) compared with asymptomatic. However, in the presence of C pneumoniae, there was significant increase of all T-lymphocyte subtypes in symptomatic plaques, including CD8+ (76.8 versus 30.3, P=0.03), CD3+ (192.1 versus 80.4, P=0.004), CD4+ (111.9 versus 37.9, P=0.003), and CD45RO+ (120.2 versus 72.9, P=0.003) cells compared with asymptomatic plaques. With use of 2-way ANOVA, both the presence of chlamydia and symptoms were associated with significantly higher T-cell counts (P<0.005 for all subtypes). CONCLUSIONS: Although all patients with symptomatic disease show a modest elevation in the concentration of intraplaque lymphocytes, a preferential increase in CD8+ class I-restricted T cells is observed in symptomatic carotid plaque positive for C pneumoniae. These data provide incentive to further explore the role of Chlamydia in the modification of immune-mediated mechanisms in active atherosclerotic plaque.

Proinflammatory cytokine expression contributes to brain injury provoked by chronic monocyte activation.

BACKGROUND: We have proposed that an increased interaction between monocyte/macrophages and blood vessel endothelium predisposes subjects to strokes. The effect of chronic monocyte activation on the development of cerebral infarcts was thus studied in rats after provocation of a modified local Swartzman reaction, in brain vasculature. MATERIALS AND METHODS: Two weeks after an IV bolus of bacillus Calmette-Guérin (BCG), we studied spontaneous superoxide production, integrin expression, endothelial adhesion of monocytes and the neurological symptoms, brain histology, and cytokine immunoreactivity after a provocative dose of LPS (30-300 microg/rat i.c.v.). RESULTS: Monocyte migration into the brain was stimulated by BCG priming. The incidence of paralysis and death in response to LPS was markedly increased in BCG-primed rats. Histological evaluation of the brains of neurologically impaired and moribund animals revealed intravascular thrombosis and pale and hemorrhagic infarcts. Infiltrates of leukocytes expressing immunoreactive IL-1:, IL-6, and TNF-alpha were found around blood vessels, cerebral ventricles, and meninges, and were accompanied by a profound microglial expression of IL1P, endothelial expression of IL-6, and expression of TNF-alpha and TNF-R 1 in glia and neurons of cortex and hippocampus. Treatment (2 x 100 microg/10 ,I, i.c.v.) with recombinant human (rh-)TNF 55kDa receptor completely prevented, and treatment with rh-IL- I receptor antagonist significantly decreased the incidence of paralysis and death in response to BCG + LPS. The improvement of neurological symptoms was accompanied by reduced histological damage and supppression of IL-1P/ expression in the brain tissue. CONCLUSIONS: The data demonstrate that chronic monocyte activation predisposes subjects to thrombosis and hemorrhage via an exaggerated release of proinflammatory cytokines.

Brain vessels normally undergo cyclic activation and inactivation: evidence from tumor necrosis factor-alpha, heme oxygenase-1, and manganese superoxide dismutase immunostaining of vessels and perivascular brain cells.

Studies of vascular biology during the past decade have identified an expanding list of agonists and antagonists that regulate local hemostasis, inflammation, and reactivity in blood vessels. Interactions at the blood-endothelial interface are intricate and complex and have been postulated to play a role in the initiation of stroke and the progression of brain injury during early hours of ischemia, particularly in conjunction with reperfusion injury (Hallenbeck, 1996). In the current study of normal and activated vessels in rat brain, immunoreactive tumor necrosis factor-alpha (TNF-alpha), heme oxygenase-1 (HO-1), and manganese superoxide dismutase (MnSOD) exhibit concentric perivascular rings involving vessel wall and surrounding parenchyma that appear to coincide with one another in serial sections. The ring patterns suggest periodic radial expansion of these molecules released through a process of cyclic activation and inactivation of brain vessel segments. In this process, the rings appear randomly scattered instead of affecting all vessels within a high power field (HPF) synchronously. The average number of vessels per HPF (mean +/- SD) with perivascular cuffs of immunoreactive MnSOD increased from 51 +/- 28 in Wistar, 72 +/- 46 in Wistar-Kyoto, and 84 +/- 30 in Sprague Dawley rats (no spontaneous strokes) to 184 +/- 72 in spontaneously hypertensive stroke-prone rats (spontaneous strokes). Perivascular immunoreactive cuffs are also increased in spontaneously hypertensive rats by induction of cytokine expression by lipopolysaccharide (64 +/- 15 vs. 131 +/- 32 /HPF). The patterns of TNF-alpha, HO-1, and MnSOD in naïve animals are interpreted to indicate that focal hemostatic balance normally fluctuates in brain vessels and influences surrounding parenchymal cells. Perivascular immunoreactive cuffs representing this process are more frequent in animals with lipopolysaccharide-induced endothelial activation or genetic stroke proneness.

Lipopolysaccharide-induced ischemic tolerance is associated with increased levels of ceramide in brain and in plasma.

Intravenous administration of lipopolysaccharide (LPS) (0.9 mg/kg) has been shown to induce ischemic tolerance in spontaneously hypertensive rats (SHR). TNF-alpha is believed to play a crucial role in preconditioning as its inhibition with TNF-alpha-binding protein abolished tolerance. Our recent studies (Liu et al., Am. J. Physiol. 278 C144, 2000) have demonstrated that ceramide, a downstream messenger in TNF-alpha signaling, is a mediator of hypoxia-induced tolerance in neuronal cells. To test the hypothesis that ceramide contributes to LPS-induced tolerance in vivo, SHR were injected intravenously with either LPS or saline and the levels of ceramide in brain and in plasma were determined by reversed phase HPLC. LPS injection resulted in a significant increase of ceramide in plasma with a maximum at 24 h (8.32+/-1.14 pmol/microl (LPS) vs. 2.65+/-0.62 pmol/microl (saline)). LPS also induced ceramide upregulation in brain cortex, which started between 6 and 12 h and remained elevated up to 48 h after LPS injection. Fluorescent NBD-C6 ceramide was able to cross blood-brain barrier and was found in brain vessels, perivascular cells and in brain parenchyma 30 min after intravenous injection. These findings demonstrate that LPS preconditioning leads to elevation of ceramide in brain and plasma and, in conjunction with previous work, suggests that ceramide plays a role in LPS-induced protection against brain ischemic injury in vivo.

Cortical spreading depression increases protein synthesis and upregulates basic fibroblast growth factor.

Protective effects of cortical spreading depression (CSD) against ischemic damage have been demonstrated in cortex when elicited at either 24 h or 3 days prior to ischemia. The present study was carried out to investigate possible mechanisms of neuroprotection following CSD. In Sprague-Dawley rats, 5 M KCl, 5 M NaCl, or physiological saline was applied to the cortex for 1 h. Repetitive CSD waves were elicited only in the KCl group. Measurements of cerebral glucose utilization demonstrated a marked reduction in affected cortex and subcortical regions in both the NaCl and the KCl groups, whereas cortical and hippocampal protein synthesis was discretely increased only in the KCl group. Immunohistochemistry of GFAP demonstrated a rapid activation in reactive astrocytes at 3 days in the KCl group whereas only a discrete activation was observed in the NaCl group. Similar changes were observed for basic fibroblast growth factor. Our results suggest that CSD-induced ischemic tolerance is not due to a reduction in energy metabolism but rather is associated with an upregulation of trophic factors and glial cell activation which might provide a mechanism for a long-lasting neuroprotection.

Nitric oxide modulates endothelin 1-induced Ca2+ mobilization and cytoskeletal F-actin filaments in human cerebromicrovascular endothelial cells.

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1-stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1-mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO-ET-1 interactions may contribute to regulation of microvascular function.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia.

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice.

Cytokines are recognized to play an important role in acute stroke. Tumor necrosis factor-alpha (TNF) is one of the pro-inflammatory cytokines and is expressed in ischemic brain. We hypothesized that TNF might play a role in the regulation of tolerance to ischemia when administered prior to the ischemic episode. We studied the effects of pretreatment of TNF administered intravenously, intraperitoneally, or intracisternally in mice that were subjected to middle cerebral artery occlusion (MCAO) 48 h later. MCAO was performed in BALB/C mice by direct cauterization of distal MCA, which resulted in pure cortical infarction. A significant reduction in infarct size was noted in mice pretreated by TNF at the dose of 0.5 microgram/mouse (p < 0.01) intracisternally. At the doses used in this study, administration of TNF by intravenous or intraperitoneal routes was not effective. Immunohistochemical analysis of brains subjected to 24 h of MCAO revealed a significant decrease in CD11b immunoreactivity after TNF pretreatment compared with control MCAO. Preconditioning with TNF affects infarct size in a time- and dose-dependent manner. TNF induces significant protection against ischemic brain injury and is likely to be involved in the signaling pathways that regulate ischemic tolerance.

Temporal impairment of microcirculatory perfusion following focal cerebral ischemia in the spontaneously hypertensive rat.

Microcirculatory impairments have theoretically been proposed as a potential factor in the development of ischemic injury, but few attempts have been made to directly assess microvascular patency following stroke. To address this issue we investigated the temporal changes in microvascular perfusion induced by permanent focal ischemia. Halothane-anesthetized spontaneously hypertensive rats were subjected to middle cerebral artery occlusion (MCAO) of 5 min to 4 h duration. Two fluorescent tracers (FITC-dextran and Evans blue) were then sequentially administered i.v. and allowed to circulate for 10 and 5 s respectively. Tissue sections were examined by fluorescent microscopy, and the mean number of perfused microvessels/mm2 calculated for cortical areas representing non-ischemic (Region A), perifocal/penumbral (Region B) and core ischemic (Region C) regions. For sham-operated controls, virtually all microvessels perfused with tracer within 5 s. In contrast MCAO induced significant reductions in the number of perfused microvessels in Regions B and C. The most marked impairments in perfusion were observed in core MCA territory (e.g. 2-10% of control values for 5 s circulation period) while, initially, the deficit was less severe in penumbral cortex. However, a secondary perfusion impairment developed over time in the perifocal/penumbral region, so that the deficit was greater 4 h after MCAO than at earlier time points (e.g. 72%, 71% and 22% of control value for 0.5, 1 and 4 h MCAO respectively; 10 s circulation period). In conclusion, MCAO induced severe impairments in microcirculatory perfusion within the core ischemic region, and to a lesser extent in the penumbra. However, the development of a more severe perfusion deficit in the penumbra within 4 h of MCAO supports the hypothesis that microcirculatory failure in this region contributes to its recruitment to the ischemic infarct.

Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats.

Ischemic tolerance was induced in spontaneously hypertensive rats (SHR) by injection of a single dose of lipopolysaccharide (LPS) (0.9 mg/kg, i.v.) 1-7 days prior to permanent middle cerebral artery occlusion (MCAO). Infarct volume, evaluated 24 h after MCAO, was significantly reduced by LPS administration 2, 3 or 4 days prior to MCAO (22.8, 25.9 and 20.5%, respectively). The beneficial effect of LPS pre-treatment was completely nullified by concurrent administration of TNFbp. On this basis, the tolerance to ischemia induced by LPS is likely to be mediated by TNF-alpha.

Interleukin-1 mediates induction of tolerance to global ischemia in gerbil hippocampal CA1 neurons.

A series of experiments was performed to determine the role of interleukin (IL)-1 in the induction of tolerance to global ischemia in Mongolian gerbils. In Group I, a 2-min "preconditioning" ischemia protected CA1 hippocampal neurons in gerbils subjected to 3.5 min ischemia 3 days later. CA1 neuronal density was: sham, 171 +/- 3/mm; 3.5 min ischemia, 30 +/- 30/mm; 2 and 3.5 min ischemia 162 +/- 6/mm. Experiments in Group II addressed the role of IL-1 in the induction of tolerance by sublethal ischemia. Arterial IL-1 alpha and IL-1 beta became elevated between 1 and 3 days after a 2-min ischemic exposure. IL-1 alpha was: sham, 6.4 +/- 0.6 ng/ml; and 2-day, 10.2 +/- 1.2 ng/ml. IL-1 beta was: sham, 6.4 +/- 0.5 ng/ml; and 2-day, 17.3 +/- 2 ng/ml. Recombinant human IL-1 receptor antagonist (IL-1ra) i.p. blocked ischemic tolerance induction by 2-min preconditioning ischemia: 2-min ischemia + vehicle, 162 +/- 6/mm; and 2-min ischemia + IL-1ra, 67 +/- 17/mm. Experiments in Group III assessed the capacity of IL-1 to induce tolerance to brain ischemia. IL-1 alpha i.p. (0, 10, 20 micrograms/kg) for 3 days prior to 3.5-min forebrain ischemia provided significant CA1 neuroprotection in a dose-dependent manner: 2 +/- 2, 68 +/- 83, and 129 +/- 42/mm, respectively. IL-1 beta (15 micrograms/kg) in combination with either IL-1ra (100 mg/kg) or IL-1ra vehicle i.p. on the same schedule demonstrated a significant CA1 neuroprotection that could be nullified by IL-1ra: IL-1 beta + IL-1ra vehicle, 153 +/- 16/mm; and IL-1 beta + IL-1ra, 67 +/- 36/mm. Recognition that tolerance arises from stimulation of a known receptor (IL-1RI) permits molecular analysis of the intracellular signaling that is critical for production of that state.

Polymorphonuclear leukocytes and microcirculatory perfusion in acute stroke in the SHR.

In order to determine the effect of depleting circulating polymorphonuclear neutrophils (PMN's) on brain microcirculation and lesion size in an acute stroke model, Spontaneously Hypertensive Rats (SHR) were injected intraperitoneally with either 2 ml RP-3 antineutrophil antibody followed in 4 hours by MCAO (n = 5), 2 ml saline followed in 4 hours by middle cerebral artery occlusion (MCAO) (n = 6), or 2 ml saline followed in 4 hours by sham operation (n = 3). After 4 hours of ischemia or a 4 hour interval (sham-operated animals), microvascular perfusion was assessed by means of an intravascular fluorescent tracer technique: FITC-dextran and Evans blue were injected intravenously 10 seconds and 5 seconds, respectively, before decapitation. Lesion volume was calculated by interpolation from histologic sections cut from 8 predefined stereotactic levels. MCAO with the normal complement of neutrophils led to significant impairment of perfusion in nutrient vessels and a maximal ischemic lesion volume. Depletion of circulating leukocytes by RP-3 significantly attenuated the microvessel perfusion impairment and reduced the volume of ischemic brain injury.

Development of susceptibility to audiogenic seizures following cardiac arrest cerebral ischemia in rats.

Susceptibility to audiogenic seizures (AGS) was investigated in Sprague-Dawley rats subjected to cardiac arrest cerebral ischemia (CACI), produced by compression of the major cardiac vessels. The onset of AGS was regularly observed 1 day after CACI of > 5 min duration. The duration of postischemic susceptibility to AGS was directly related to the density of cerebral ischemia, with 50% of more severely ischemic animals still showing AGS susceptibility 8 weeks after CACI. Lesioning of the inferior colliculi (IC) abolished the onset of AGS; no such effect was observed after lesioning the medial geniculate (MG). Glutamic acid decarboxylase (GAD) immunochemistry revealed approximately 50% loss of GAD-positive neurons in the IC, which was similar in animals with various durations of AGS susceptibility. Otherwise, there was a conspicuous sprouting of gamma-aminobutyric acid (GABA)-ergic terminals in the ventral thalamic nuclei, which peaked approximately 1 month after the CACI. Evaluation of GABA-A inhibitory function in the hippocampus by the paired pulse stimulation revealed changes indicating loss of GABA-A inhibition coinciding with the onset of AGS, and its return in animals tested 2 months after CACI. Our observations suggest a potential role of GABA-ergic dysfunction in the postischemic development of AGS.

Protective effect of spreading depression against neuronal damage following cardiac arrest cerebral ischaemia.

Effect of spreading depression (SD) on survival of CA1 hippocampal neurons was studied in Sprague-Dawley rats subjected to cardiac arrest cerebral ischaemia (CACI). SD was induced either by the topical application of KCI to the cerebral cortex (CSD) or by KCI perfusion of the hippocampus via a microdialysis cannula (HSD). CACI was carried out by intrathoracic compression of major cardiac vessels at 1, 3 and 7 days after CSD induction and 3 days after HSD, following which neuronal loss in the CA1 sector of the hippocampus was determined morphometrically. Our study revealed the significant protective effect of SD on survival of the CA1 pyramidal neurons in rats which were subjected to CACI 3 days later. No significant indication of a protective effect was observed in animals with SD induced 1 or 7 days prior to CACI nor in rats in which KCI was substituted by NaCl of equivalent concentration which did not induce any SD waves. Microdialysis assay during HSD showed a significant elevation of extracellular glutamate. Our studies, demonstrating an induction of a transitory period of neuronal resistance to ischaemia by preceding spreading depression, open an opportunity for elucidation of endogeneous factors responsible for the development of such resistance.

Cardiac arrest-induced complete cerebral ischaemia in the rat: dynamics of postischaemic in vivo calcium uptake and protein synthesis.

Dynamics of pathological changes following cardiac arrest induced cerebral ischaemia were related to the findings of double tracer autoradiography of 45Ca and 3H leucine uptake as respective indicators of ischaemic injury and metabolic disturbance. Abnormal calcium accumulation, determined by 45Ca uptake, was related to injured but still living neurons and to reactive glial elements. 45Ca autoradiography confirmed a high sensitivity to neuronal injury of the nucleus reticularis thalami (NRT), hippocampal CA1 pyramidal layer, inferior colliculus, ventral thalamic nucleus (VTN), caudate nucleus, and parietal cortex. 3H leucine incorporation revealed that an initially widespread inhibition of protein synthesis was followed by its considerable recovery. Observations concerning hippocampal CA1 sector and VTN suggested that a significant degree of protein synthesis, maintained at the late stage after postischaemic recovery, was related to survival and regeneration of neurons and not to the presence of glial elements.

Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes.

Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.

Putative neuroexcitation in cerebral ischemia and brain injury.

Involvement of neuroexcitatory mechanisms in cerebral ischemia and brain injury was explored in experimental models of repetitive forebrain ischemia by temporary occlusion of carotid arteries in gerbils and cryogenic injury to the cerebral cortex in rats and gerbils. Our observations in these models revealed a pattern of injury that involved some anatomic structures outside the areas of direct ischemic or traumatic insult. Such foci of injury revealed conspicuously abnormal uptake of 45Ca associated with slight or moderate neuronal alteration, whereas severely injured areas showed no 45Ca uptake. Electron microscopic observations revealed a characteristic presence of calcium in swollen dendrites, closely resembling pictures obtained in neuroexcitatory conditions such as epileptic seizures. Abnormal uptake of 45Ca was associated with apparent blood-brain barrier changes characterized by intracytoplasmic uptake of extravasated albumin into the neurons. Protein synthesis assayed by in vivo [3H]leucine incorporation was reduced in regions showing calcium accumulation. Our observations suggest that neuroexcitation may play an important role in development of secondary and chronic changes after ischemic or traumatic brain insults.

Role of neuroexcitation in development of blood-brain barrier and oedematous changes following cerebral ischaemia and traumatic brain injury.

Potential involvement of neuroexcitatory mechanisms was studied in: 1) repetitive forebrain ischaemia in gerbils, 2) global cerebral ischaemia in rats and 3) cryogenic injury to the cerebral cortex in rats and gerbils. Uptake of 45Ca was used as a marker of injury, whereas ultrastructural localization of calcium was assessed with an oxalate-pyroantimonate method. The blood-brain barrier was evaluated with immunostaining for serum albumin. Changes in extracellular glutamate were estimated by microdialysis and an enzymatic cycling assay. Changes in water content were assessed by specific gravity measurements. Repetitive ischaemia of 3 x 5 min carotid occlusions produced a cumulative effect with regard to development of oedema and neuronal injury. This was associated with several-fold increments in glutamate release after repeated insults, whereas there was no apparent correlation with energy metabolism disturbances. Other studies revealed in all models a development of secondary foci distant to the primary impact of ischaemia or cold lesions, which were characterized by calcium accumulation in swollen dendrites, chronic neuronal changes and intraneuronal uptake of serum proteins, all of these changes being potentially compatible with involvement of neuroexcitatory mechanisms.