Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K252a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.

Calcium-mediated mechanisms of ischemic injury and protection.

Our understanding of calcium's role in cerebral ischemia continues to evolve from the initial recognition that it may be harmful to the ischemic cell. A multitude of experiments have supported the hypothesis that excessive influx of calcium into the cell under ischemic conditions is a major mechanism of cell injury and death. Pharmacological intervention to restore cellular calcium homeostasis is protective in many models of cell anoxia. Principle routes of calcium entry are the voltage-sensitive (VSCC) and N-methyl-D-aspartate linked receptor operated (ROCC) calcium channels. Regional variations in channel densities have been described and it is now known that these classes of channels are located in different regions of the neurons. Activation of both channel types has been identified in in vivo models of cerebral ischemia. Although the ROCC is predominant in number, the VSCC appears to activate at higher cerebral blood flow values suggesting that it is an earlier conduit for calcium than the glutamate-driven ROCC. Intracellular calcium is well recognized as a second messenger system and there is increasing appreciation that it induces immediate early genes (IEG). Since IEGs function as transcriptional regulating factors, the differential expression of specific target genes may be of importance for determining death or survival of the ischemic tissue.

Cerebral acidosis in focal ischemia: II. Nimodipine and verapamil normalize cerebral pH following middle cerebral artery occlusion in the rat.

The effects of prostacyclin, nimodipine, and verapamil on local cerebral pH (LCpH) and CBF (LCBF) in middle cerebral artery (MCA)-occluded rats were compared with those in controls and others receiving nimodipine carrier. LCpH and LCBF were determined simultaneously by a double-label autoradiographic technique. The infusions were intravenous, started 15 min following the occlusion, and ended at decapitation 4 h postocclusion. The dosages were 0.5 micrograms/kg/min for nimodipine, 40 micrograms/kg/min for verapamil, and 5 ng/kg/min for prostacyclin. Cortical LCpH in the MCA territory of control and carrier-infused rats varied between 6.72 +/- 0.05 and 6.76 +/- 0.05 (means +/- SEM). These values were significantly lower than the LCpH in the same structures in the contralateral hemisphere (7.09 +/- 0.06; p less than 0.05). LCBF on the side of occlusion varied between 54 +/- 5 ml/100 g/min for the parietal and 57 +/- 7 ml/100 g/min for the sensorimotor cortex, while on the contralateral side, LCBF in these same structures was 190 +/- 18 and 191 +/- 4 ml/100 g/min, respectively. LCpH was not modified by prostacyclin treatment following MCA occlusion, but the pH in the structures that were acidotic in the controls became indistinguishable from contralateral values in nimodipine- and verapamil-treated animals. In contrast, LCBF was statistically higher than controls in many structures only in rats treated with prostacyclin. This suggested that the correction of LCpH produced by calcium blockers was not related to an effect they had on blood flow. Animals receiving calcium blockers tended to have smaller areas of infarction. These results may have therapeutic implications in cerebral ischemia.

Effect of thiamine deficiency and its reversal on cerebral blood flow in the rat. Observations on the phenomena of hyperperfusion, "no reflow," and delayed hypoperfusion.

Local CBF (LCBF) was determined in the same rat model and at the same intervals of thiamine deficiency and reversal as in previous studies of local cerebral glucose utilization (LCGU) and pH (LCpH). The results showed that prior to the appearance of the clinical sequelae of thiamine deficiency (opisthotonus, which usually occurs on day 18 of deficiency) cerebral structures such as the mammillary body, vestibular nucleus, inferior colliculus, and thalamus showed significant hyperperfusion, reaching greater than 200% of control values. At opisthotonus, there was a general decline in LCBF, but, in addition, the larger of these structures developed inhomogeneous perfusion with patches of hyperperfusion adjacent to others of low flow. Seven days of thiamine replenishment at opisthotonus resulted in delayed hypoperfusion notably in the mammillary body, inferior colliculus, and thalamic nuclei. Superimposition of the LCBF, LCGU, and LCpH data reveals that structures known to be vulnerable to the development of histological lesions in this model showed an early phase of hyperperfusion uncoupled from declining LCGU and normal LCpH. Then, following a significant but only focal rise in LCGU between days 11 and 14 of deficiency, hyperperfusion persisted while the pH was dropping and LCGU was rapidly declining. The phase of patchy perfusion occurred only in the histologically vulnerable structures when LCGU was very low and acidosis was at its peak, suggesting that it may have resulted from these opposing influences on LCBF. Following replenishment with thiamine, the vulnerable structures showed delayed hypoperfusion coupled to LCGU.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo binding of nimodipine in the brain: I. The effect of focal cerebral ischemia.

We report the regional variation in [3H]nimodipine binding in vivo during focal cerebral ischemia. After intravenous injection, 30 min of circulation of [3H]nimodipine was sufficient to establish a secular equilibrium of distribution in the brain. Rats sustained left middle cerebral and common carotid artery occlusions for 5 min, and 4, 24, and 48 h (n greater than or equal to 6 epr group). They were decapitated 30 min after injection of 250 microCi of [3H]nimodipine and their brains were submitted to autoradiography. The concentrations of [3H]nimodipine in plasma and brain structures, corrected for metabolism of nimodipine, were used to calculate the regional volumes of distribution (V) in the ischemic left (L) and control right (R) hemispheres. Log (VL/VR) was then defined as the group mean of the logarithms of the left-to-right ratio of V of [3H]nimodipine. In the lateral caudate, binding of [3H]nimodipine on the ischemic side was highest within 5 min of occlusion. Log (VL/VR) in this region for the combined sham-operated and normal control rats and those after 5 min and 4 and 24 h of ischemia were -0.014 +/- 0.025, 0.137 +/- 0.056*, -0.201 +/- 0.367, and -0.049 +/- 0.370 (mean +/- SD, *represents p less than 0.01 compared with controls). By contrast, in the superior frontal cortex, values for log (VL/VR) in the same sequence were -0.016 +/- 0.025, 0.028 +/- 0.056, 0.284 +/- 0.228*, and 0.224 +/- 0.069*, thus showing a significant rise in [3H]nimodipine binding only at 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral acidosis in focal ischemia: I. A method for the simultaneous measurement of local cerebral pH with cerebral glucose utilization or cerebral blood flow in the rat.

We developed a technique for the simultaneous autoradiographic determination of local cerebral pH (LCpH) using [14C]dimethyloxazolidinedione ([14C]DMO) with either local cerebral glucose utilization (LCGU) using [14C]deoxyglucose ([14C]DG) or local CBF (LCBF) using [14C]iodoantipyrine ([14C]IAP). The method evolved from the observation that [14C]DMO is "unstable," which is shown here to be due to sublimation of the compound. Thus, the technique consists of administering to rats [14C]DMO followed by either [14C]DG or [14C]IAP. The first autoradiographic film that results contains both labeled compounds. The brain sections are then exposed in a fume hood until the complete disappearance of [14C]DMO, which usually required 10 days. The second film thus contains only the non-DMO isotope administered. Blood levels of the two isotopes are calculated from the total concentrations of isotope and the rate of decline of [14C]DMO in blood. This information is then used to determine cerebral pH and the other cerebral function of interest. Using this double-label technique in normal rats, LCpH, LCGU, and LCBF can be determined with the same accuracy as with single-label techniques. In rats with middle cerebral artery occlusion, this technique yielded LCpH and LCBF values 4 h after occlusion that were not statistically different from those obtained by separate determinations. Thus, interactions between LCpH and either LCBF or LCGU can be studied by this technique in normal and pathological conditions.

Relationship between extracellular glutamate concentration and voltage-sensitive calcium channel function in focal cerebral ischemia in the rat.

Ischemic cell death occurs when extracellular glutamate levels increase, causing tissue depolarization and an excessive rise in intracellular calcium concentrations. The relative occurrence of the depolarization events and the changes in glutamate concentration in ischemia have not been studied. In a model of focal cerebral ischemia in the rat, three measurements were made simultaneously in vivo: cerebral blood flow (CBF) by the H2-clearance method, extracellular glutamate concentration by microdialysis, and activation of the voltage-sensitive calcium channel (VSCC) by its binding to [3H]nimodipine. Effects of probe implantation on these measurements were accounted for. The CBF to control ratio obtained during the experiments spanned the range of 1.08 to 0.07. Binding to [3H]nimodipine became significantly activated when CBF fell to approximately 0.49 of its control value while extracellular glutamate concentrations increased significantly only at a CBF ratio of < 0.33. Activation of the VSCC at this high CBF ratio may be due to ischemic depolarization, which has been shown to activate the binding to [3H]nimodipine. It may be useful to define a CBF threshold of 50% of normal in focal ischemia for opening of the VSCC. The same threshold has been linked to an overall depression of protein synthesis and to activation of a number of molecular responses.

Relevance of interstitial glutamate to selective vulnerability in focal cerebral ischemia.

Glutamate concentrations in striatum and cortex were measured by means of in vivo cerebral microdialysis before and for 4 h after middle cerebral and ipsilateral common carotid artery occlusion in rats. The peak glutamate concentration reached 7.28 +/- 3.60 microM in dialysate from striatum and 5.64 +/- 2.24 microM in that from cortex. An index of exposure of each region to glutamate was calculated by integrating glutamate concentrations after occlusion. During ischemia the striatum was exposed to statistically higher cumulative concentrations of glutamate than the cortex (p < 0.01). The difference in vulnerability between striatum and cortex may arise from the additional time needed for the cortex to be exposed to cumulative threshold levels of glutamate.

Nimodipine prevents hyperglycemia-induced cerebral acidosis in middle cerebral artery occluded rats.

The effects of acute moderate hyperglycemia on local cerebral pH (LCpH) and local cerebral blood flow (LCBF) were studied in rats infused with glucose before middle cerebral artery (MCA) occlusion, and compared with findings in MCA occlusion alone. The effects of nimodipine infusion on LCBF and LCpH in MCA-occluded hyperglycemic rats were also studied. LCpH and LCBF were determined simultaneously by a double-label autoradiographic technique. Hyperglycemia was induced by an intraperitoneal injection of 2 g/kg D-glucose before MCA occlusion. Nimodipine-treated rats received the drug as an intravenous infusion of 0.5 micrograms/kg/min starting 15 min after occlusion, and ending at decapitation 4 h postocclusion. Cortical LCpH of five structures in the MCA territory of hyperglycemic rats varied between 6.64 +/- 0.04 and 6.72 +/- 0.02 (mean +/- SEM). These values were significantly lower than LCpH in the same ischemic structures in the control rats, which varied between 6.76 +/- 0.04 and 6.82 +/- 0.03 (p less than 0.05 for four of five structures). Cortical LCpH of hyperglycemic nimodipine-treated rats ranged between 6.94 +/- 0.02 and 7.05 +/- 0.02, indicating significant elevations in LCpH (p less than 0.001) compared with the untreated ischemic hyperglycemic animals. LCBF in the ischemic structures was not modified by hyperglycemia or nimodipine treatment. This suggests that nimodipine, by mechanisms other than improvement in blood flow, can prevent the enhanced cerebral tissue acidosis produced by hyperglycemia before incomplete focal ischemia.

The effects of a competitive NMDA receptor antagonist (CGS-19755) on cerebral blood flow and pH in focal ischemia.

We report the effects of intravenous infusion of CGS-19755, a potent competitive N-methyl-D-aspartate (NMDA) antagonist, on local cerebral pH (LCpH) and local CBF (LCBF) in rats with occluded left middle cerebral and common carotid arteries. LCpH and LCBF were determined simultaneously by a double-label autoradiographic technique 4 h after vascular occlusion in three groups: no treatment, carrier infused, and a group receiving CGS-19755 at 10 mg/kg bolus immediately after occlusion followed by infusion at 5 mg kg-1 h-1 for 4 h. Compared with rats receiving carrier, several cortical structures on the side of occlusions showed significantly higher CBF in rats receiving CGS-19755. This drug also corrected the pH in several left cortical structures to values significantly higher than in the rats receiving carrier. The correction in LCpH was not limited to those regions showing significant elevations in LCBF. In the nonoccluded hemisphere, CGS-19755 significantly increased the hemispheric mean blood flow from 122 +/- 17 to 221 +/- 64 ml 100 g-1 min-1 (mean +/- SD of all structures, p less than 0.01) without any changes in LCpH. Cortical but not basal ganglia infarct volume was significantly smaller in rats receiving CGS-19755 than in the carrier-treated group. These results suggest that, at least partially, the neuroprotective effect of CGS-19755 in ischemia may be related to changes in CBF and pH in addition to its antagonist effect on the NMDA receptor.

In vivo binding of nimodipine in the brain: II. Binding kinetics in focal cerebral ischemia.

We report the binding characteristics of [3H]nimodipine to normal and ischemic brain in vivo. We used the 1,4-dihydropyridine, nimodipine, to label the L-type voltage-sensitive calcium channel in focal cerebral ischemia after occlusion of both the middle cerebral and ipsilateral common carotid arteries in rats. Varying concentrations of [3H]nimodipine were infused 3.5 h after the onset of ischemia and circulated for 30 min before the brain was obtained for autoradiography and determination of regional nimodipine content. In separate sets of experiments, the metabolites of nimodipine were determined and the conditions for equilibrium of nimodipine distribution were established. Increased nimodipine uptake was observed in ischemic regions. This increased binding was saturable and specific with an affinity constant, KD, of 0.45 nM and a maximal regional binding capacity, Bmax, ranging from 3.1 to 10.9 pmol/g. Only binding to ischemic tissue was specific and saturable whereas that in nonischemic tissue was nonspecific. In vivo binding of nimodipine may be used to identify cell membrane depolarization and calcium channel activation in focal cerebral ischemia.

Metabolic and histological reversibility of thiamine deficiency.

The rapid improvement in the clinical manifestations of thiamine deficiency with thiamine supplementation is well known. To study this process in more detail, we rendered rats thiamine deficient either by dietary deprivation alone (DD) or, in addition, by daily pyrithiamine administration (DD + PT). We observed the cerebral metabolic and histological responses of these rats after 1 or 7 days of thiamine supplementation both prior to and at the onset of clinical sequelae. The cerebral metabolic response to thiamine deficiency and replenishment was determined with the [14C]deoxyglucose technique for measurement of local cerebral glucose utilization (LCGU). Our results indicate that thiamine replenishment reverses the LCGU changes resulting from thiamine deprivation of short duration. However, prolonged thiamine deprivation may result in LCGU changes that are not completely reversible by thiamine replenishment, before the appearance of the clinical or histological consequences of thiamine deficiency.

Cortical spreading depression protects against subsequent focal cerebral ischemia in rats.

The possibility that cortical spreading depression (CSD) may have neuroprotective action during subsequent focal cerebral ischemia was examined in rats. Three days before the imposition of focal cerebral ischemia CSDs were elicited by applying potassium chloride (KC1) for 2 h through a microdialysis probe implanted in the occipital cortex. Control animals were handled identically except that saline was infused instead of KC1. Focal ischemia was produced by the intraluminal suture method and cortical and subcortical infarct volumes were measured 7 days later. Neocortical infarct volume was reduced from 124.8 +/- 49.5 mm(3) in the controls to 62.9 +/- 59.5 mm(3) in the animals preconditioned with CSD (p = 0.012). There was no difference between the two groups in the subcortical infarct volume or in CBF, measured by the hydrogen clearance method, during or immediately after the ischemic interval. Our data indicate that preconditioning CSD applied 3 days before middle cerebral artery occlusion may increase the brain's resistance to focal ischemic damage and may be used as a model to explore the neuroprotective molecular responses of neuronal and glial cells.

Reduction of functional capillary density in human brain after stroke.

The blood flow of brain tissue often returns to normal after an ischemic episode. As "luxury" rather than "reactive" reperfusion, this hyperemia is associated with low metabolism. It is not known to what extent the high blood flow accompanies a high, normal, or low density of capillaries. The resolution of this question may indicate whether the functional capillary density is variable and, if so, whether it is coupled to blood flow or metabolism. To answer these questions, we defined functional capillaries as capillaries that transport glucose. We then calculated the density of functional capillaries (Dcap) and the mean time of transit of blood through the capillaries (tcap) from hemodynamic variables obtained in vivo by positron tomography of five patients afflicted by cerebral ischemic stroke. Each patient was studied twice, within 36 h of the insult and 1 week later. We identified nominally "ischemic" regions in the first study as cortical gray matter regions, contiguous with the ischemic focus, in which the magnitude of blood flow did not exceed 20 ml 100 g-1 min-1. In these regions, values of metabolism and functional capillary density were proportionately low compared with normal values obtained in the contralateral hemisphere. The studies revealed a reduction of the functional density of exchange vessels in postischemic brain tissue as soon as 36 h after the insult. In "ischemic" regions, within 36 h of the insult, the net extraction of oxygen was inversely related to the capillary transit time and appeared to be limited mainly by the low functional density of the capillaries. Contrary to expectations, the reduced density persisted, even when more than adequate perfusion of the tissue returned. For these reasons, we concluded that changes of the capillary density were associated with changes of the metabolism of the tissue rather than with blood flow.

In vivo uptake of [3H]nimodipine into brain during cortical spreading depression.

We report autoradiographic measurements of the in vivo uptake of [3H]nimodipine during the nonischemic depolarization of cortical spreading depression (CSD) in rat brain. [3H]Nimodipine uptake in brain was determined regionally in rats undergoing CSD (n = 8) and was significantly increased in cortex (14 +/- 7%) and hippocampus (10 +/- 6%) on the stimulated side relative to the contralateral hemisphere when compared with the same measurements in a control group (n = 8). A similar measurement using the physiologically inert radiotracer [14C]iodoantipyrine to control for potential effects of CSD on radioligand distribution showed a minimal increase (2.4 +/- 0.7%) of radiotracer uptake in cortex after CSD. This increase was significantly less than that observed in the [3H]nimodipine uptake studies. We hypothesize that increased in vivo [3H]nimodipine uptake in CSD identifies regions of depolarization and thus infers activation of the L-type voltage sensitive calcium channels.

In vivo binding of [3H]nimodipine in rat brain after transient forebrain ischemia.

We report the regional variation in relative in vivo binding of the L-type voltage sensitive calcium channel (VSCC) antagonist [3H]nimodipine to brain following transient forebrain ischemia in the rat. At 30-min of reperfusion after 20 min of forebrain ischemia, [3H]nimodipine binding was significantly increased in striatum, CA3 and CA4, and dentate relative to binding in sham-operated rats, suggesting that VSCCs were responding to ischemic depolarization. Two h following ischemia, binding in all brain structures returned to normal levels indicating repolarization of cell membranes. At 24 h of recirculation, increased [3H]nimodipine binding was again observed in striatum and dentate. Binding remained elevated in the striatum and dentate, and increased binding became evident in the CA1 region of the hippocampus after 48 h of reperfusion. With the exception of the dentate gyrus, the second rise in [3H]nimodipine binding anticipated or coincided with the observed regional ischemic cell changes. These observations in global cerebral ischemia support previous work indicating that in vivo binding of [3H]nimodipine to the L-type VSCC may be an early and sensitive indicator of impending ischemic injury. Such measurements may be of use in identifying vulnerable brain regions and defining a therapeutic window of opportunity in models of cerebral ischemia.

Estimation of cerebral oxygen utilization rate by single-bolus 15O2 inhalation and dynamic positron emission tomography.

This study shows that regional CMRO2 can be estimated by means of nonlinear regression using dynamic positron emission tomographic data acquired during 1 min following single-bolus inhalation of 15O2. The feasibility of simultaneous estimation of CBF, cerebral blood volume (CBV), oxygen extraction ratio (OER), and CMRO2 was assessed by simulations using the model of Mintun et al. Four oxygen metabolic measurements, each consisting of a CBF, CBV, and 15O2 bolus study, were carried out on three volunteers. Regional values for CBF, CBV, OER, and CMRO2 were derived in two ways: from the fits of the time-activity curves of the dynamic 15O2 bolus study alone [CMRO2(fit)] and from the three separate studies [CMRO2 (control)]. For the 56 regions of interest analyzed, using a fit interval of 60 s, CMRO2(fit) was 93.4 +/- 7.8% of CMRO2(control) (mean +/- SD) with a correlation coefficient of r = 0.95. CMRO2(control) ranged from 87 to 290 mumol/min/100 g. Individual simultaneous estimates of CBF, CBV, and OER were not reliable. Finally, we found that the validity of the model was limited in practice to the first minute after tracer inhalation.

The effect of nimodipine on the evolution of human cerebral infarction studied by PET.

Fourteen patients were studied by positron emission tomography (PET) within 48 h of onset of a hemispheric ischemic stroke and again 7 days later. After the first set of PET scans, the patients were randomized to receive either nimodipine (n = 7) or a carrier solution (n = 7) by intravenous infusion. The infusions were maintained until the end of the second PET studies. CBF, cerebral blood volume (CBV), oxygen extraction ratio (OER), CMRO2, and CMRglc were measured each time. These metabolic and perfusion measurements were performed by standard methods. A surface map of each metabolic and perfusion measurement in the cortical mantle was generated by interpolating between the available slices. The various surface maps representing the physiological characteristics determined in the same or subsequent studies were aligned so that all data sets could be analyzed identically using an array of square regions of interest (ROIs). The functional status of each ROI was recorded at the two intervals following the cerebrovascular accident to characterize the evolution of the infarct, penumbra, and normal brain regions. We presumed the ischemic penumbra to be cortical regions in the proximity of the infarct and perfused at CBF values between 12 and 18 ml/100 g/min on the first PET scan, while densely ischemic regions had CBF of less than 12 nl/100 g/min and normally perfused brain greater than 18 ml/100 g/min. In the densely ischemic zone, CBF increased more in the nimodipine-treated group than in the carrier group. As well, in this region nimodipine reversed the decline in CMRO2 noted in the carrier group, the difference in the changes being significant. In the penumbra zone, comparable trends were noted in OER and CMRO2 but the difference in the changes between the two groups did not reach statistical significance. Changes in CMRglc and CBV were comparable between the two groups in both cortical regions.

Ischemic penumbra: the therapeutic window.

The concept of ischemic penumbra has extended our understanding of the focal ischemic process and in many ways has revitalized our research. Simply stated, the penumbra is the part of the brain that is sandwiched between brain regions committed to die and those that receive enough blood to communicate. Therefore, it is ischemic brain tissue that has just enough energy to survive for a short time but not enough to communicate and function. The life expectancy of the penumbra is short. Although the penumbra is an elegant concept, in practice it has been a difficult one to exploit. For one thing, it is unstable in both time and space. Depending on the severity and the duration of the focal ischemia, it may be anywhere in the ischemic brain. We believe that nimodipine binding experiments have taught us a great deal about the ischemic penumbra. Second, cells in the penumbra may die not by necrosis but by apoptosis. If that is true, then our concepts about the benign transient ischemic attack may need revision. Third, the penumbra may regain its ability to survive not only through reperfusion but also by interrupting the process of commitment to apoptosis.

Dementia in Parkinson disease: a neuropathologic study.

Thirty-four autopsy cases conforming to the standard neuropathologic criteria of Parkinson disease were sex- and age-matched with controls who had died of infarct or trauma. All brains were reviewed for changes compatible with Alzheimer disease, and available clinical data were retrospectively reviewed. Nineteen (56 percent) of the Parkinson cases had shown some degree of dementia. The average parkinsonian brain weight was 1281 gm; it was 1365 gm for the controls (p less than 0.02). Plaques, neurofibrillary tangles, granulovacuolar degeneration, and cortical cell loss were present in all but one of the parkinsonian brains; these pathologic changes were present in fewer controls and to a lesser degree. The higher incidence of dementia in patients with Parkinson disease may be explained by the simultaneous presence of Alzheimer disease.