Recurrent mild cerebral ischemia: enhanced brain injury following acute compared to subacute recurrence in the rat.

BACKGROUND: In the current study, a transient cerebral ischemia producing selective cell death was designated a mild ischemic insult. A comparable insult in humans is a transient ischemic attack (TIA) that is associated with functional recovery but can have imaging evidence of minor ischemic damage including cerebral atrophy. A TIA also predicts a high risk for early recurrence of a stroke or TIA and thus multiple ischemic insults are not uncommon. Not well understood is what the effect of differing recovery times between mild ischemic insults has on their pathophysiology. We investigated whether cumulative brain damage would differ if recurrence of a mild ischemic insult occurred at 1 or 3 days after a first insult. RESULTS: A transient episode of middle cerebral artery occlusion via microclip was produced to elicit mild ischemic changes-predominantly scattered necrosis. This was followed 1 or 3 days later by a repeat of the same insult. Brain damage assessed histologically 7 days later was substantially greater in the 1 day recurrent group than the 3 days recurrent group, with areas of damage consisting predominantly of regions of incomplete infarction and pannecrosis in the 1 day group but predominantly regions of selective necrosis and smaller areas of incomplete infarction in the 3 days group (P < 0.05). Enhanced injury was reflected by greater number of cells staining for macrophages/microglia with ED1 and greater alterations in GFAP staining of reactive astrocytes in the 1 day than 3 days recurrent groups. The differential susceptibility to injury did not correspond to higher levels of injurious factors present at the time of the second insult such as BBB disruption or increased cytokines (tumor necrosis factor). Microglial activation, with potential for some beneficial effects, appeared greater at 3 days than 1 day. Also blood analysis demonstrated changes that included an acute increase in granulocytes and decrease in platelets at 1 day compared to 3 days post transient ischemia. CONCLUSIONS: Dynamic changes in multiple inflammatory responses likely contribute to the time dependence of the extent of damage produced by recurrent mild ischemic insults. The time of mild stroke recurrence is crucial with early recurrence producing greater damage than subacute recurrence and this supports urgency for determining and implementing optimal stroke management directly after a TIA.

Recombinant hirudin treatment modulates aquaporin-4 and aquaporin-9 expression after intracerebral hemorrhage in vivo.

Edema formation has been linked to thrombin toxicity induced by blood clot at the acute stage of intracerebral hemorrhage. Thrombin induces cell toxicity in neuron, microglia and astrocyte. Aquaporin (AQP) 4 and 9 are proteins expressed on astrocyte in rat brain and involved in the brain water accumulation in brain edema. Recombinant hirudin (r-Hirudin) is a direct inhibitor of thrombin that can block the toxicitic effect of thrombin. In this study, we demonstrated that autologous whole blood infusion in caudate nucleus up-regulates the expression of AQP4 and AQP9 mRNAs and proteins. AQP4 and AQP9 mRNAs expression peaked at about 6 h after blood infusion. The AQP4 protein peaked at about 48 h while AQP9 at about 24 h after blood infusion. Thrombin induced up-regulation of AQP4 and AQP9 were inhibited by r-Hirudin administration and significantly decreased the expression of both AQPs. We further investigated the relationship between edema formation and expression of AQP4 and AQP9. The data presented here may be helpful in optimizing r-Hirudin as an anti-thrombin drug in the treatment of edema at the acute stage of ICH.

Transient hypertension concurrent with forepaw stimulation enhances functional MRI responsiveness in infarct and peri-infarct regions.

Although functional magnetic resonance imaging (fMRI) is gaining use as a tool to assess cerebral recovery following various insults, the effects of potential confounders such as hypertension are poorly defined. We hypothesized that after stroke, transient hypertension during an fMRI study could produce a detected activation unrelated to neuronal activity within the infarct. Thus, the effect of norepinephrine induced increases in blood pressure (BP) on the fMRI response to forepaw stimulation were investigated in controls or 1 week after transient middle cerebral artery occlusion in rats. Images were smoothed spatially and voxels correlating to either forepaw stimulation or the change in BP time courses were analyzed. Transient hypertension increased the signal intensity and numbers of voxels correlating to the BP time courses within and adjacent to the ischemic infarct and these exceeded the response in the contralateral hemisphere or in controls. With left paw stimulation at normotension, there was a loss of activation in right sensory-motor cortex -- a region with necrosis and disruption of cerebral vessels. As BP increased left paw stimulation also resulted in the detection of activation in the infarcted sensory-motor cortex and peri-infarct regions. Thus, BP changes synchronous with tasks in fMRI studies can result in MR signal changes consistent with a loss of cerebral blood flow (CBF) autoregulation rather than neuronal activation in necrotic brain. After stroke, the use of stressful tasks associated with BP changes in fMRI studies should be limited or the BP change should be considered as a potential source of MR signal changes.

Prior deafferentation confers long term protection to CA1 against transient forebrain ischemia and sustains GluR2 expression.

Hippocampal CA1 pyramidal neurons undergo delayed neurodegeneration after transient forebrain ischemia, and the phenomenon is dependent upon hyperactivation of l-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of glutamate receptors, resulting in aberrant intracellular calcium influx. The GluR2 subunit of AMPA receptors is critical in limiting the influx of calcium. The CA1 pyramidal neurons are very sensitive to ischemic damage and attempts to achieve neuroprotection, mediated by drugs, have been unsuccessful. Moreover, receptor antagonism strategies in the past have failed to provide long-term protection against ischemic injury. Long-term protection against severe forebrain ischemia can be conferred by fimbria-fornix (FF) deafferentation, which interrupts the afferent input to CA1. Our study evaluated the long-term protective effect of FF deafferentation, 12 days prior to induction of ischemia, on vulnerable CA1 neurons. Our results indicate that at 7 and 28 days post-ischemia, prior FF deafferentation protected 60% of neurons against ischemic cell death. Furthermore, we sought to evaluate whether FF deafferentation also sustained GluR2 levels in these neurons. GluR2 protein and mRNA expression were sustained by deafferentation at 70% of control following ischemia. Correlation studies revealed a positive correlation between GluR2 protein and mRNA level. These results demonstrate that protection conferred by FF deafferentation was long-term and related to sustained GluR2 expression.

The neurotoxicity of tissue plasminogen activator?

Tissue plasminogen activator (tPA), a fibrin specific activator for the conversion of plasminogen to plasmin, stimulates thrombolysis and rescues ischemic brain by restoring blood flow. However, emerging data suggests that under some conditions, both tPA and plasmin, which are broad spectrum protease enzymes, are potentially neurotoxic if they reach the extracellular space. Animal models suggest that in severe ischemia with injury to the blood brain barrier (BBB) there is injury attributed to the protease effects of this exogenous tPA. Besides clot lysis per se, tPA may have pleiotropic actions in the brain, including direct vasoactivity, cleaveage of the N-methyl-D-aspartate (NMDA) NR1 subunit, amplification of intracellular Ca++ conductance, and activation of other extracellular proteases from the matrix metalloproteinase (MMP) family, e.g. MMP-9. These effects may increase excitotoxicity, further damage the BBB, and worsen edema and cerebral hemorrhage. If tPA is effective and reverses ischemia promptly, the BBB remains intact and exogenous tPA remains within the vascular space. If tPA is ineffective and ischemia is prolonged, there is the risk that exogenous tPA will injure both the neurovascular unit and the brain. Methods of neuroprotection, which prevent tPA toxicity or additional mechanical means to open cerebral vessels, are now needed.

Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy.

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Training the brain to survive stroke.

BACKGROUND: Presently, little can be done to repair brain tissue after stroke damage. We hypothesized that the mammalian brain has an intrinsic capacity to adapt to low oxygen which would improve outcome from a reversible hypoxic/ischemic episode. Acclimation to chronic hypoxia causes increased capillarity and tissue oxygen levels which may improve the capacity to survive ischemia. Identification of these adaptations will lead to protocols which high risk groups could use to improve recovery and reduce costs. METHODS AND FINDINGS: Rats were exposed to hypoxia (3 weeks living at ½ an atmosphere). After acclimation, capillary density was measured morphometrically and was increased by 30% in the cortex. Novel implantable oxygen sensors showed that partial pressure of oxygen in the brain was increased by 40% in the normal cortex. Infarcts were induced in brain with 1 h reversible middle cerebral artery occlusions. After ischemia (48 h) behavioural scores were improved and T2 weighted MRI lesion volumes were reduced by 52% in acclimated groups. There was a reduction in inflammation indicated by reduced lymphocytes (by 27-33%), and ED1 positive cells (by 35-45%). CONCLUSIONS: It is possible to stimulate a natural adaptive mechanism in the brain which will reduce damage and improve outcome for a given ischemic event. Since these adaptations occur after factors such as HIF-1α have returned to baseline, protection is likely related more to morphological changes such as angiogenesis. Such pre-conditioning, perhaps with exercise or pharmaceuticals, would not necessarily reduce the incidence of stroke, but the severity of damage could be reduced by 50%.

Quantitative MRI reveals the elderly ischemic brain is susceptible to increased early blood-brain barrier permeability following tissue plasminogen activator related to claudin 5 and occludin disassembly.

Great uncertainty exists as to whether aging enhances the detrimental effects of tissue plasminogen activator (tPA) on vascular integrity of the ischemic brain. We hypothesized that tPA treatment would augment ischemic injury by causing increased blood-brain barrier (BBB) breakdown as determined by quantitative serial T(1) and T(2) magnetic resonance imaging (MRI), and the transfer constant for gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) from blood to brain in aged (18 to 20 months) compared with young (3 to 4 months) Wistar rats after middle cerebral artery occlusion, mediated through the acute disassembly of claudin 5 and occludin. Increased T(2) values over the first hour of postreperfusion were independently augmented following treatment with tPA (P<0.001) and aging (P<0.01), supporting a synergistic effect of tPA on the aged ischemic brain. Blood-brain barrier permeability for Gd-DTPA (K(Gd)) was substantial following reperfusion in all animal groups and was exacerbated by tPA treatment in the elderly rat (P<0.001). The frequency of hematoma formation was proportionately increased in the elderly ischemic brain (P<0.05). Both tPA and age independently increased claudin 5 and occludin phosphorylation during ischemia. Early BBB permeability detected by quantitative MRI following ischemic stroke is enhanced by increased age and tPA and is related to claudin 5 and occludin phosphorylation.

Effects of acute hypoxia and hyperthermia on the permeability of the blood-brain barrier in adult rats.

Acute mountain sickness (AMS) develops within a few hours after arrival at high altitude and includes headache, anorexia, nausea, vomiting, and malaise. This afflicts 15-25% of the general tourist population at moderate altitudes. High-altitude cerebral edema (HACE) is considered to be the end stage of severe AMS and has been suggested to be a vasogenic edema, raising the possibility that acute hypoxia may increase blood-brain barrier (BBB) permeability. At present, there are no good small-animal models to study this syndrome. We hypothesize 1) that acute hypoxia can damage the BBB and 2) that rat can be used as a model to study hypoxia-induced changes in BBB permeability, especially if hypoxia-induced hypothermia could be minimized with high ambient temperature (HAT). Male Wistar rats were exposed to 1, 2, and 7 days of hypobaric hypoxia (equivalent to 0.5 atm), and changes in the temperature and BBB permeability were studied. The extravasation of endogenous immunoglobulin G, a large molecule, did not increase during room temperature hypoxia but did increase when hypoxia was combined with HAT. Hypoxia caused a significant increase in the leakage of sodium fluorescein (mol wt 376 Da). The expression of endothelial barrier antigen (EBA), a protein associated with the BBB, was reduced to 50% between 24 and 48 h after exposure to hypoxia, and the loss was exacerbated by HAT. The values almost returned to control levels by 7 days, showing adaptation to hypoxia. Hypoxic rats exhibited sodium fluorescein leakage mainly in focal areas in the brain parenchyma. In conclusion, it is possible to have transient BBB damage through exposure to acute hypoxia, and this damage is exacerbated by increasing body temperature to more of a normothermic value.

Lessons in experimental ischemia for clinical stroke medicine.

PURPOSE OF REVIEW: This review covers experimental developments in the laboratory and their translation to clinical stroke trials over the year from 3 August 2001 to 2002. RECENT FINDINGS: Recent findings include novel observations in the areas of excitotoxicity, free radical injury, neuro-inflammation and apoptosis. A key clinical finding this year has been the translation of the effects of hypothermia in global ischemia to two successful trials in cardiac arrest with patients cooled within 4-8 h following resuscitation achieving good neurological outcomes, as compared with their normothermic controls. An era of molecular imaging in stroke research is presaged by the first reports of enhanced magnetic resonance or labeling with supramagnetic contrast agents. SUMMARY: Although none of the drugs in focal ischemia has translated from experimental models, for the first time there is evidence of cytoprotection for the brain that has been translated from the laboratory to man.