Drug-free patients with major depression show an increased electrophysiological response to valid and invalid feedback.

BACKGROUND: Depressed patients are biased in their response to negative information. They have been found to show a maladaptive behavioral and aberrant electrophysiological response to negative feedback. The aim of this study was to investigate the behavioral and electrophysiological response to feedback validity in drug-free depressed patients. METHOD: Fifteen drug-free in-patients with unipolar major depression disorder (MDD) and 30 demographically matched controls performed a time-estimation task in which they received valid and invalid (i.e. related and unrelated to performance) positive and negative feedback. The number of behavioral adjustments to the feedback and the feedback-related negativity (FRN) were measured. RESULTS: Patients made fewer correct adjustments after valid negative feedback than controls, and their FRNs were larger. Neither patients nor controls adjusted their time estimates following invalid negative feedback. CONCLUSIONS: The FRN results suggest that depressed drug-free in-patients have an atypical rostral anterior cingulate response to feedback that is independent of feedback validity. Their behavioral response to invalid negative feedback, however, is not impaired. This study confirms the notion that the behavioral responses of depressed individuals to negative feedback are context dependent.

Microglial reaction in the rat cerebral cortex induced by cortical spreading depression.

The response of microglial cells to cortical spreading depression (CSD) was studied in rat brain by immunocytochemistry. CSD was elicited for one hour by the topical application of 4M potassium chloride solution and the microglial reaction examined immunocytochemically after 4, 16, 24 and 72 hours. CSD was sufficient to induce a microglial reaction throughout the cortex at 24 hours. Activated microglial cells furthermore showed a striking de-novo expression of major histocompatibility complex class II antigens. In contrast, no microglial reaction was observed in the cortex of sham-operated animals. This microglial reaction in response to CSD was not associated with histologically detectable neuronal damage. These results support the view that microglial cells are extremely sensitive to changes of the brain microenvironment. Their activation may be related to changes of ion homeostasis in the brain which are not sufficient to trigger neuronal injury.

Gene expressions after thrombolytic treatment of middle cerebral artery clot embolism in mice.

BACKGROUND AND PURPOSE: Thrombolytic treatment of stroke may result in reperfusion injury. To investigate the role of selective gene expressions, C57Bl/6J mice were subjected to middle cerebral artery (MCA) clot embolism, followed after 1 hour by intracarotid infusion of 10 mg/kg recombinant tissue plasminogen activator (rtPA) or vehicle. METHODS: Before the onset of treatment and at 1, 3, 6, and 24 hours of recirculation, animals were frozen in situ and hsp70, c-fos, junB, and NSE mRNAs were imaged on cryostat sections using in situ hybridization autoradiography. Cerebral protein synthesis (CPS) and ATP content were measured on adjacent brain sections. RESULTS: hsp70 mRNA was upregulated in the penumbral cortex of untreated animals and in the MCA core region of animals receiving rtPA (ie, regions characterized by a mismatch between high ATP levels and suppressed CPS). c-fos and junB mRNAs were transiently expressed mainly in the peri-infarct intact cortex for up to 3 to 6 hours in the treated and up to 24 hours in the untreated animals. In both groups, NSE mRNA declined in the central parts of the MCA territory together with a loss of silver impregnation, but this decline was more pronounced in the untreated animals. CONCLUSIONS: The genomic expression pattern after thrombolytic recanalization of clot embolism resembles that of other types of transient ischemia such as reversible thread occlusion, although the outcome is markedly different. The investigated gene expressions, notably hsp70 mRNA, reflect the kind and severity of the ischemic stress, but they do not predict reversibility of the ischemic injury.

Model of kinetic behavior of deoxyglucose in heterogeneous tissues in brain: a reinterpretation of the significance of parameters fitted to homogeneous tissue models.

Effects of tissue heterogeneity on regional CMRglc (rCMRglc) calculated by use of the deoxyglucose (DG) method at 45 min following the pulse of DG were evaluated in simulation studies. A theoretical model was developed to describe the kinetics of DG uptake and metabolism in heterogeneous brain tissues. Rate constants were fitted to simulation data for mixed tissue and rCMRglc computed on the basis of this tissue heterogeneity model. The results were compared with those obtained by use of the original model of the DG method for homogeneous tissue, both without (3K model) and with (4K model) a term to describe an apparent loss of deoxyglucose-6-phosphate (DG-6-P). As a direct consequence of tissue heterogeneity, the effective rate constant for phosphorylation of DG, k3*, declined with time. To compensate for the time-changing k3*, estimates of the dephosphorylation rate constant, k4*, were artifactually high when the 4K model was used, even though no dephosphorylation of DG-6-P actually occurred. The present study demonstrates that the finding of a significant k4*, at least within 45 min following a pulse of DG, may not represent dephosphorylation at all, but rather the consequence of measuring radioactivity in a heterogeneous tissue and applying a model designed for a homogeneous tissue. Furthermore, the high estimates of k4* resulted in significant overestimation of rCMRglc. When rCMRglc was computed with the conventional single-scan or autoradiographic method at 45 min after a pulse of DG, the 3K and tissue heterogeneity models yielded values that were within 5% of the true weighted average value for the heterogeneous tissue as a whole. We conclude that the effects of tissue heterogeneity alone can give the appearance of product loss, even when none occurs, and that the use of the 4K model with the assumption of product loss in the 45-min experimental period recommended for the DG method may lead to overestimation of the rates of glucose utilization.

Prevention of periinfarct direct current shifts with glutamate antagonist NBQX following occlusion of the middle cerebral artery in the rat.

The effect of the glutamate (AMPA subtype) receptor antagonist NBQX on periinfarct direct current (DC) shifts and cortical ATP depletion volume was examined in rats subjected to 3 h of occlusion of the middle cerebral artery (MCA). MCA occlusion produced an immediate DC shift in the periphery of the ischemic territory. Vehicle-treated (untreated) animals developed one to five additional DC shifts (median, 2) during the 3-h occlusion time. NBQX treatment (2 x 30 mg/kg i.v. immediately after MCA occlusion and 1 h later) significantly reduced the number of DC deflections (median, 0; range, 0-2; p < 0.05) without changing blood flow in the border zone of the infarct (untreated, 50.6 +/- 10.6%; NBQX-treated: 51.9 +/- 7.7% of control; mean +/- SD). NBQX treatment significantly decreased the cortical volume of ATP depletion (untreated, 75.3 +/- 11.4 mm3; NBQX-treated, 47.9 +/- 10.1 mm3; p < 0.05). Moreover, a significant linear relationship between the number of periinfarct DC shifts and the volume of cortical ATP depletion was obtained (y = 38.3 + 9.4x; r = 0.866; p < 0.001). The reduction of brain infarct volume by NBQX treatment is explained by the suppression of DC shifts and the decrease of metabolic workload in hemodynamically compromised cortex.

Cerebral blood flow, glucose utilization, regional glucose, and ATP content during the maturation period of delayed ischemic injury in gerbil brain.

Coupling between local perfusion and metabolism was examined in Mongolian gerbils during the development of delayed neuronal death using a combination of double-tracer autoradiography and imaging of local energy state. Animals were anesthetized with 1.5% halothane and forebrain ischemia was produced by occluding both common carotid arteries. After 5 min of ischemia, brains were recirculated for 6 h and 1, 2, or 4 days. At the end of the experiment, regional cerebral blood flow (CBF) and glucose utilization (CMRglc) were determined in identical brain section with [131I]iodoantipyrine and [14C]deoxyglucose, respectively. Adjacent sections were taken for imaging of ATP and glucose using substrate-specific bioluminescence reactions. In the CA1 subfield of control animals, CBF and CMRglc amounted to 81 +/- 8 ml 100 g-1min-1 and 69 +/- 2 mumol 100 g-1min-1, respectively, and the calculated CBF/CMRglc ratio was 1.18 +/- 0.12 ml/mumol (mean +/- SD). After ischemia, the CBF/CMRglc ratio increased to 1.31 +/- 0.14, 1.43 +/- 0.16, 1.45 +/- 0.16, and 1.56 +/- 0.18 ml/mumol following 6 h and 1, 2, or 4 days recirculation, respectively. Glucose levels did not change during the 6 h to 4 day recirculation period in the hippocampal CA1 subfield. In the same region, ATP levels were unchanged during 6 h to 2 day postischemic recovery but reduced to about 70% after 4 days of recirculation. The results indicate that a mismatch of the flow--metabolism couple following transient ischemia does not appear to contribute to the postischemic maturation of delayed neuronal death in selectively vulnerable brain regions.

Ornithine decarboxylase activity and putrescine levels in reversible cerebral ischemia of Mongolian gerbils: effect of barbiturate.

Reversible cerebral ischemia was produced in anesthetized Mongolian gerbils by occluding both common carotid arteries. After 5 min of ischemia, brains were recirculated for 8 or 24 h. Treated animals received a single intraperitoneal injection of pentobarbital (50 mg/kg) immediately after the aneurysm clips were removed. At the end of the experiments, animals were reanesthetized and their brains frozen in situ. Tissue samples were taken from the cerebral cortex, lateral striatum, CA1 subfield of the hippocampus, thalamus, and cerebellum for measuring ornithine decarboxylase (ODC) activity and putrescine levels. In addition, 20-microns-thick coronal tissue sections were taken from the level of the striatum and stained with hematoxylin/eosin for evaluating the extent of ischemic neuronal necrosis in the lateral striatum. In control animals ODC activity and putrescine levels amounted, respectively, to 0.32 +/- 0.03 nmol/g/h and 10.2 +/- 0.5 nmol/g in the cerebral cortex; 0.34 +/- 0.02 nmol/g/h and 12.8 +/- 0.5 nmol/g in the lateral striatum; 0.58 +/- 0.05 nmol/g/h and 10.5 +/- 0.7 nmol/g in the hippocampal CA1 subfield; 0.35 +/- 0.01 nmol/g/h and 9.8 +/- 0.4 nmol/g in the thalamus; and 0.25 +/- 0.01 nmol/g/h and 8.3 +/- 0.6 nmol/g in the cerebellum. After 5 min cerebral ischemia and 8 h recirculation, a significant 7- to 16-fold increase in ODC activity was observed in all forebrain structures studied. Following 24 h recirculation, ODC activity normalized in the cortex, striatum, and thalamus but was still significantly above control values in the hippocampal CA1 subfield.(ABSTRACT TRUNCATED AT 250 WORDS)

Repeated negative DC deflections in rat cortex following middle cerebral artery occlusion are abolished by MK-801: effect on volume of ischemic injury.

Following permanent occlusion of the left middle cerebral artery (MCA) in rats, electrophysiological and hemodynamic characteristics of the periinfarct border zone were investigated in sham-operated (n = 6), untreated (n = 6), and MK-801-treated (3.0 mg/kg; n = 6) animals. For this purpose, direct current potential (DC), EEG, and blood flow (laser-Doppler flowmetry) were recorded from the cortex in the periphery of the MCA territory. In sham-operated rats, a single negative cortical DC deflection was observed after electrocoagulation of the cortex, whereas in untreated MCA-occluded animals, three to eight transient DC deflections were monitored during the initial 3 h of ischemia. The duration of these cortical DC shifts gradually increased from 1.2 +/- 0.3 to 3.7 +/- 2.7 min (mean +/- SD; p less than 0.05) during this time. In animals treated intraperitoneally with MK-801 (3.0 mg/kg) immediately after MCA occlusion, the number of cortical DC shifts significantly declined to one to three deflections (p less than 0.005). The EEG of the treated animals revealed low-amplitude burst-suppression activity. In the untreated and treated experimental group, the reduction of cortical blood flow amounted to 69 +/- 25 and 49 +/- 13% of control, respectively. Despite the more pronounced cortical oligemia, MK-801 treatment resulted in a significant decrease of the volume of the ischemically injured tissue from 108 +/- 38.5 (untreated group) to 58 +/- 11.5 (p less than 0.05) mm3. Our results suggest that repetitive cortical DC deflections in the periinfarct border zone contribute to the expansion of ischemic brain infarcts.

Effect of thrombolysis on the dynamics of infarct evolution after clot embolism of middle cerebral artery in mice.

Reversible focal ischemia may lead to delayed tissue injury despite primary restoration of blood flow and metabolism. The authors investigated whether such delayed changes also occur after thrombolytic treatment of thromboembolic stroke. Clot embolism of the middle cerebral artery (MCA) was produced in C57/B16J mice by intracarotid injection of heterologous clots. One hour after embolism, one group was treated with intracarotid infusion of rt-PA (10 mg/kg). The untreated control group received an equal amount of vehicle. Just before onset of treatment and after 1, 3. 6, and 24 hours, animals were frozen in situ and cerebral blood flow (CBF), cerebral protein synthesis (CPS), ATP content, and DNA fragmentations (TUNEL) were imaged on cryostat sections using double tracer autoradiography. bioluminescence, and immunohistochemical techniques, respectively. In untreated animals (n = 20), CPS was suppressed in approximately 68% of hemispheric transsection at 1 hour after embolization. The ATP depleted area was smaller (approximately 58%), but between 6 and 24 hours it merged with that of CPS suppression. TUNEL-positive neurons became visible between 6 and 24 hours exclusively in regions with ATP depletion. rt-PA-induced thrombolysis (n = 20) led to the gradual improvement of blood flow. At 24 hours. ATP depletion was fully reversed and the CPS suppression area declined to approximately 16% of hemispheric transsection. Despite progressive metabolic recovery, large numbers of neurons became TUNEL-positive and animals died between 24 and 48 hours. Thrombolysis after clot embolism restores metabolic activity including protein synthesis, but the therapeutic benefit is limited by secondary injury that requires additional treatment to improve final outcome.

Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat.

The ischemic threshold of protein synthesis and energy state was determined 1, 6, and 12 h after middle cerebral artery (MCA) occlusion in rats. Local blood flow and amino acid incorporation were measured by double tracer autoradiography, and local ATP content by substrate-induced bioluminescence. The various images were evaluated at the striatal level in cerebral cortex by scanning with a microdensitometer with 75 microns resolution. Each 75 x 75 microns digitized image pixel was then converted into the appropriate units of either protein synthesis, ATP content, or blood flow. The ischemic threshold was defined as the flow rate at which 50% of pixels exhibited complete metabolic suppression. One hour after MCA occlusion, the threshold of protein synthesis was 55.3 +/- 12.0 ml 100 g-1 min-1 and that of energy failure was 18.5 +/- 9.8 ml 100 g-1 min-1. After 6 and 12 h of MCA occlusion, the threshold of protein synthesis did not change (52.0 +/- 9.6 and 56.0 +/- 6.5 ml 100 g-1 min-1, respectively) but the threshold of energy failure increased significantly at 12 h following MCA occlusion to 31.9 +/- 9.7 ml 100 g-1 min-1 (p less than 0.05 compared to 1 h ATP threshold value; all values are mean +/- SD). In focal cerebral ischemia, therefore, the threshold of energy failure gradually approached that of protein synthesis. Our results suggest that with increasing duration of ischemia, survival of brain tissue is determined by the high threshold of persisting inhibition of protein synthesis and not by the much lower one of acute energy failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuated c-fos mRNA induction after middle cerebral artery occlusion in CREB knockout mice does not modulate focal ischemic injury.

To elucidate the mechanism of ischemia-induced signal transduction in vivo, we investigated the effect of the targeted disruption of the alpha and delta isoforms of the cAMP-responsive element-binding protein (CREB) on c-fos and heatshock protein (hsp) 72 gene induction. Permanent focal ischemia was induced by occlusion of the middle cerebral artery of the CREB mutant mice (CREB(-/-), n = 5) and the wild-type mice (n = 6). Three hours after onset of ischemia, the neurologic score was assessed and pictorial measurements of ATP and cerebral protein synthesis (CPS) were carried out to differentiate between the ischemic core (where ATP is depleted), the ischemic penumbra (where ATP is preserved but CPS is inhibited), and the intact tissue (where both ATP and CPS are preserved). There were no significant differences in neurologic score or in ATP, pH, and CPS between the two groups, suggesting that the sensitivity of both strains to ischemia is the same. Targeted disruption of the CREB gene significantly attenuated c-fos gene induction in the periischemic ipsilateral hemisphere but had no effect on either c-fos or hsp72 mRNA expression in the penumbra. The observations demonstrate that CREB expression, despite its differential effect on c-fos, does not modulate acute focal ischemic injury.

Regional quantitative determination of brain glucose in tissue sections: a bioluminescent approach.

A bioluminescent technique is described for the regional quantitative determination of brain glucose. A close linear interrelationship was obtained between the optical density of the bioluminescent images and the glucose content in tissue samples. The regression coefficients of this correlation permit the quantification of glucose bioluminescent pictures using an image-processing system. Regional distribution of glucose was correlated to regional tissue pH under both physiological and pathophysiological conditions.

Triple-tracer autoradiography of cerebral blood flow, glucose utilization, and protein synthesis in rat brain.

A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyglucose ([14C]DG), and 3H-amino acids as radioactive tracers. Autoradiographic differentiation between isotopes was performed by taking advantage of different half-lives, solubility of labeled tracers in a wash solution, and sensitivity of the photographic material to disintegrations of the radionuclides. Blood flow autoradiograms using 131I-IAP were obtained by immediate exposure of brain sections to Kodak NMB film for 24 h. During 131I autoradiography contamination by 3H was absent and by 14C was negligible at tissue concentrations of less than 0.45 microCi/g brain tissue. After complete decay of 131I, reexposure of brain sections to Kodak NMB film for 2 weeks provided autoradiograms that stemmed exclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultrofilm for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.

Quantitative measurement of local cerebral blood flow in the anesthetized mouse using intraperitoneal [14C]iodoantipyrine injection and final arterial heart blood sampling.

Autoradiographic measurement of local cerebral blood flow (CBF) with [14C]iodoantipyrine (IAP) is limited in mice by the difficulty in cannulating vessels and the blood loss for repeated blood sampling. The authors modified and validated the method to measure local CBF with [14C]IAP in mice by combining intraperitoneal tracer application with a single blood sampling from the heart at the end of the experiment. Experiments were carried out in male SV129 mice under halothane anesthesia. After intraperitoneal administration of 15 microCi [14C]IAP, arterial blood samples were collected repeatedly and anesthetized animals were immersed in liquid nitrogen. In addition, frozen blood from the heart was sampled to obtain the final blood [14C]radioactivity. Correlation analysis between the sampling time and [14C]radioactivity of the arterial blood revealed a highly significant linear relationship (P < 0.001, r = 0.978) and a lag time of the [14C]tracer in arterial blood of 3.3 +/- 0.6 seconds. [14C]radioactivity of the final arterial blood sample (444 +/- 264 nCi/mL) was almost equal to that of the heart blood (454 +/- 242 nCi/mL), and the absolute difference in each animal was 3.3 +/- 4.2% (mean +/- SD). The convolution integrals for the CBF calculation were determined either by integrating the radioactivity of individual arterial blood samples or by assuming a linear rise from [14C]tracer lag time after intraperitoneal [14C]IAP injection to the value measured in the blood sample from the frozen heart. Regional flow values calculated by the two methods differed by less than 11% (not significant). This method allows the quantitative measurement of local CBF in anesthetized mice without any vessel catheterization and will make mutant mice a more powerful tool to elucidate the molecular mechanisms of brain injuries by combining flow studies with molecular-biological methods.

Dynamics of regional brain metabolism and gene expression after middle cerebral artery occlusion in mice.

The evolution of brain infarcts during permanent occlusion of the middle cerebral artery (MCA) was studied in mice using multiparametric imaging techniques. Regional protein synthesis and the regional tissue content of ATP were measured on adjacent cryostat sections at increasing intervals after vascular occlusion ranging from 1 hour to 3 days. The observed changes were correlated with the expression of the mRNA of hsp70, c-fos, c-jun, and junB, as well as the distribution of DNA double-strand breaks visualized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL). One hour after MCA occlusion, the tissue volume with suppressed protein synthesis was distinctly larger than that in which ATP was depleted. With ongoing ischemia time, the ATP-depleted area gradually expanded and, within 1 day, merged with the region of suppressed protein synthesis. Expression of hsp70 mRNA occurred mainly in the penumbra (defined as the region of suppressed protein synthesis but preserved ATP), peaking at 3 hours after vascular occlusion. Expression of the immediate-early genes c-jun, c-fos, and junB increased both in the penumbra and the periinfarct normal tissue already at 1 hour after vascular occlusion, with slightly different regional and temporal patterns for each of these genes. DNA fragmentations were clearly confined to neurons; they appeared after 1 day in the infarct core (defined as the region of suppressed ATP) and never were detected in the penumbra. The late appearance of TUNEL after infarcts had reached their final size and the absence in the penumbra points against a major pathogenetic role of apoptosis. Permanent MCA occlusion in mice thus produces a gradually expanding infarct, the final size of which is heralded by the early inhibition of protein synthesis.

Hemodynamics and metabolism in stroke-prone spontaneously hypertensive rats before manifestation of brain infarcts.

Genomic screening of hybrids from stroke-prone (SHR-SP) and stroke-resistant spontaneously hypertensive rats (SHR) identified a STR1 locus on the rat chromosome 1, which correlates with the susceptibility to cerebral stroke but not with hypertension. The authors examined whether this genetic abnormality is associated with hemodynamic or metabolic alterations in the brain that can be detected before the manifestation of brain infarction. Starting at 6 weeks of age, SHR-SP were fed with a salt-rich diet to accelerate arterial hypertension. At the age of 12 weeks, animals developed functional symptoms and were age-matched with symptom-negative SHR-SP to differentiate between presymptomatic and postsymptomatic changes. Brains were investigated by multiparametric imaging comprising quantitative double-tracer autoradiography of CBF and cerebral protein synthesis (CPS); bioluminescence imaging of regional ATP, glucose, and lactate content; and umbelliferone fluoroscopic imaging of tissue pH. None of the animals exhibited focal hemodynamic or biochemical abnormalities. In symptom-negative SHR-SP, global CBF was 1.1+/-0.3 mL x g(-1) x min(-1), cortical CPS was 10.1+/-3.1 nmol x g(-1) x min(-1), and cortical ATP, glucose, lactate, and pH levels were in the normal range. In SHR-SP with functional symptoms, ATP, glucose, and lactate levels also were normal, but tissue pH exhibited periventricular alkalosis, CBF was significantly reduced to 0.7+/-0.2 mL x g(-1) x min(-1) (P < 0.001), and cortical CPS was significantly reduced to 6.7+/-2.1 nmol x g(-1) x min(-1) (P < 0.001). The decline in brain perfusion of SHR-SP correlated significantly with both the severity of functional deficits and the decline of protein synthesis. Our observations demonstrate that SHR-SP had already developed functional symptoms before the manifestation of overt brain infarcts and that the symptoms are initiated by a decline in global CBF and cortical CPS. Genetic abnormalities in SHR-SP are associated with a diffuse vascular process that results in global decompensation of blood flow well before the onset of focal brain infarction.

Evolution of brain infarction after transient focal cerebral ischemia in mice.

The evolution of brain infarction after transient focal cerebral ischemia was studied in mice using multiparametric imaging techniques. One-hour focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Cerebral protein synthesis (CPS) and the regional tissue content of adenosine triphosphate (ATP) were measured after recirculation times from 0 hours to 3 days. The observed changes were correlated with the expression of the mRNAs of hsp-70, c-fos, and junB, as well as the distribution of DNA double-strand breaks, visualized by TUNEL. At the end of 1 hour of ischemia, protein synthesis was suppressed in a larger tissue volume than ATP in accordance with the biochemical differentiation between core and penumbra. Hsp70 mRNA was selectively expressed in the cortical penumbra, whereas c-fos and junB mRNAs were increased both in the lateral part of the penumbra and in the ipsilateral cingulate cortex with normal metabolism. During reperfusion after withdrawal of the intraluminal filament, suppression of CPS persisted except in the most peripheral parts of the middle cerebral artery territory, in which it recovered between 6 hours and 3 days. ATP, in contrast, returned to normal levels within 1 hour but secondarily deteriorated from 3 hours on until, between 1 and 3 days, the ATP-depleted area merged with that of suppressed protein synthesis leading to delayed brain infarction. Hsp70 mRNA, but not c-fos and junB, was strongly expressed during reperfusion, peaking at 3 hours after reperfusion. TUNEL-positive cells were detected from 3 hours on, mainly in areas with secondary ATP depletion. These results stress the importance of an early recovery of CPS for the prevention of ischemic injury and suggest that TUNEL is an unspecific response of delayed brain infarction.

Evolution of regional changes in apparent diffusion coefficient during focal ischemia of rat brain: the relationship of quantitative diffusion NMR imaging to reduction in cerebral blood flow and metabolic disturbances.

Middle cerebral artery occlusion was performed in rats while the animals were inside the nuclear magnetic resonance (NMR) tomograph. Successful occlusion was confirmed by the collapse of amplitude on an electrocorticogram. The ultrafast NMR imaging technique UFLARE was used to measure the apparent diffusion coefficient (ADC) immediately after the induction of cerebral ischemia. ADC values of normal cortex and caudate-putamen were 726 +/- 22 x 10(-6) mm2/s and 659 +/- 17 x 10(-6) mm2/s, respectively. Within minutes of occlusion, a large territory with reduced ADC became visible in the ipsilateral hemisphere. Over the 2 h observation period, this area grew continuously. Quantitative analysis of the ADC reduction in this region showed a gradual ADC decrease from the periphery to the core, the lowest ADC value amounting to about 60% of control. Two hours after the onset of occlusion, the regional distribution of ATP and tissue pH were determined with bioluminescence and fluorescence techniques, respectively. There was a depletion of ATP in the core of the ischemic territory (32 +/- 20% of the hemisphere) and an area of tissue acidosis (57 +/- 19% of the hemisphere) spreading beyond that of ATP depletion. Regional CBF (rCBF) was measured autoradiographically with the iodo[14C]antipyrine method. CBF gradually decreased from the periphery to the ischemic core, where it declined to values as low as 5 ml 100 g-1. When reductions in CBF and in ADC were matched to the corresponding areas of energy breakdown and of tissue acidosis, the region of energy depletion corresponded to a threshold in rCBF of 18 +/- 14 ml 100 g-1 min-1 and to an ADC reduction to 77 +/- 3% of control. Tissue acidosis corresponded to a flow value below 31 +/- 11 ml 100 g-1 min-1 and to an ADC value below 90 +/- 4% of control. Thus, the quantification of ADC in the ischemic territory allows the distinction between a core region with total breakdown of energy metabolism and a corona with normal energy balance but severe tissue acidosis.

Analysis of time courses of metabolic precursors and products in heterogeneous rat brain tissue: limitations of kinetic modeling for predictions of intracompartmental concentrations from total tissue activity.

The efficacy of various kinetic models to predict time courses of total radioactivity and levels of precursor and metabolic products was evaluated in heterogeneous samples of freeze-blown brain of rats administered [14C]deoxyglucose ([14C]DG). Two kinetic models designed for homogeneous tissues, i.e., a no-product-loss, three-rate-constant (3K) model and a first-order-product-loss, four-rate-constant (4K) model, and a third kinetic model designed for heterogeneous tissues without product loss [Tissue Heterogeneity (TH) Model] were examined. In the 45-min interval following a pulse of [14C]DG, the fit of the TH Model to total tissue radioactivity was not statistically significantly better than that of the 3K Model, yet the TH Model described the time courses of [14C]DG and its metabolites more accurately. The TH- and 4K-Model-predicted time courses of [14C]DG and its metabolites were similar. Whole-brain glucose utilization (CMRglc) calculated with the TH or 3K Model, approximately 75 mumol 100 g-1 min-1, was similar to values previously determined by model-independent techniques, whereas CMRglc calculated with the 4K Model was 44% higher. In a separate group of rats administered a programmed infusion to attain a constant arterial concentration of [14C]DG that minimizes effects of tissue heterogeneity as well as any product loss, CMRglc calculated with all three models was 79 mumol 100 g-1 min-1 at 45 min after initiation of the infusion. Statistical comparisons of goodness of fit of total tissue radioactivity were, therefore, not indicative of which models best describe the tissue precursor and product pools or which models provide the most accurate rates of glucose utilization.

A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 microns) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter (r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.