[Therapeutically induced arteriogenesis in the brain. A new approach for the prevention of cerebral ischemia with vascular stenosis]. / Therapeutisch induzierte Arteriogenese im Gehirn. Ein neuer Ansatz zur Prävention der zerebralen Ischämie bei stenosierenden Gefässerkrankungen.

Stroke is the leading cause of disability and a major cause of death in Germany and the western world. Ischemic stroke involves different pathophysiologic mechanisms such as thromboembolic vascular occlusion, cerebral micro- or macroangiopathy, extracranial arterial stenosis, and cardiac embolism. Experimental and clinical studies have shown that arteriogenesis, the adaptive growth of pre-existing collateral arteries, can be therapeutically enhanced in peripheral circulation and the heart. We examined the consequences to time course and hemodynamics of brain arteriogenesis in a chronic hypoperfusion model following systemic administration of the hemopoietic growth factor called granulocyte macrophage colony stimulating factor (GM-CSF). Treatment with GM-CSF led to the growth of intracranial collateral arteries, which improved the cerebral hemodynamic reserve and significantly reduced energy failure when brains were additionally challenged by hypotension. Therapeutically induced arteriogenesis may be of considerable interest for preventing infarction in patients with uncompensated cerebrovascular disease.

Expression of c-jun, mitogen-activated protein kinase phosphatase-1, caspase-3 and glial fibrillary acidic protein following cortical cold injury in rats: relationship to metabolic disturbances and delayed cell death.

The expression of c-jun, mitogen-activated protein kinase phosphatase-1 (mkp-1), caspase-3 and glial fibrillary acidic protein (gfap) was examined at 1, 3 and 7 days after cortical cold injury in rats by in situ hybridisation and immunocytochemistry. Alterations of gene expression were related to metabolic disturbances and delayed cell death, as revealed by cerebral protein synthesis autoradiography, ATP bioluminescence, pH fluorescence and terminal transferase biotinylated dUTP nick end labelling (TUNEL). Protein synthesis autoradiographies depicted sharply demarcated cortex lesions, which were almost congruent with areas exhibiting ATP depletion (lesion volume: 16.9+/-11.8 mm(3) after 7 days). Lesions were surrounded by a region of tissue alkalosis, which was most prominent 1 day after trauma. Delayed cell injury, as revealed by TUNEL, was noticed in a thin rim around the lesion border on day 1 (tissue volume: 1.7+/-0.8 mm(3)) and, to lesser extent, days 3 and 7 post-lesioning. However, only a small percentage of cells in this area were positive for activated caspase-3 protein. TUNEL(+) cells were further seen in the ventrobasal thalamus after 7 days. In the thalamus, the appearance of DNA-fragmented cells was closely accompanied by activated caspase-3 expression. In situ hybridisations revealed that cell injury both in the peri-lesion rim and ventrobasal thalamus was associated with increased c-jun and gfap, but not mkp-1 and caspase-3 mRNA levels. Gene responses were not confined to areas revealing irreversible cell death: mkp-1 mRNA was bilaterally upregulated in the lesion-remote entorhinal cortex, cingulate cortex and reticular thalamus at 7 days after trauma, and caspase-3 mRNA was slightly, but significantly downregulated in the entorhinal cortex after 3 and 7 days. Gfap mRNA was elevated in all regions exhibiting tissue alkalosis. Our data suggest that delayed cell injury after cortex trauma may be apoptotic in the ventrobasal thalamus, but not the peri-lesion rim. The dissociated responses of c-jun, mkp-1 and caspase-3 mRNAs may represent important factors influencing tissue viability.

A control system approach for evaluating somatosensory activation by laser-Doppler flowmetry in the rat cortex.

Coupling between functional cortical activity and blood flow is a regulatory principle that adjusts the supply of substrates to the metabolic needs of the tissue. The flow response is usually expressed as the maximum increase over baseline; control system analysis allows the description of the entire time course and the main dynamic features of the regulative principle. In chloralose-anesthetized rats, forepaws were stimulated by trains of electric pulses of 0.3 or 5 ms duration. Blood flow was recorded in the contralateral somatosensory cortex by laser-Doppler flowmetry and correlated with the amplitude of primary somatosensory evoked potentials (SEP). Changes were analyzed by a control system approach. Pulses of 0.3 or 5 ms evoked SEPs of similar amplitude, whereas flow responses differed: 0.3 ms pulses led to a peak and plateau characteristic, 5 ms pulses evoked a plateau characteristic. The flow response evoked by 0.3 ms pulses can be modeled mathematically by an initial feedforward regulative principle followed after some delay by feedback controlled flow stabilization, whereas 5 ms pulses lack the feedforward component. The absence of an electrophysiological difference points to a dissociation between electrophysiological and hemodynamic responses and may be of importance for the understanding of flow coupling.

[Glutamate hypothesis of stroke]. / Die Glutamathypothese des Schlaganfalls.

Neuronal injury following focal cerebral ischemia is widely attributed to the excitatory effects of glutamate. However, critical analysis of published data on glutamate toxicity in vitro and the comparison of this data with in vivo release of glutamate and the therapeutic effect of glutamate antagonists raises doubts about a neurotoxic mechanism. An alternative explanation for glutamate-mediated injury is energy failure due to peri-infarct spreading depression-like depolarizations. These depolarizations cause a sharp increase in metabolic activity and therefore produce a mismatch between blood flow and the oxygen requirements of the tissue. The generation of peri-infarct spreading depressions and the associated metabolic workload can be suppressed by glutamate antagonists. As a result, energy failure is also prevented, and the volume of ischemic infarct decreases. Interventions to improve ischemic resistance should therefore aim at improving the oxygen supply or reducing the metabolic workload, rather than interfering with the consequences of a putative excitotoxic injury cascade.

Effects of electromagnetic radiation of mobile phones on the central nervous system.

With the increasing use of mobile communication, concerns have been expressed about the possible interactions of electromagnetic radiation with the human organism and, in particular, the brain. The effects on neuronal electrical activity, energy metabolism, genomic responses, neurotransmitter balance, blood-brain barrier permeability, cognitive function, sleep, and various brain diseases including brain tumors are reviewed. Most of the reported effects are small as long as the radiation intensity remains in the nonthermal range, and none of the research reviewed gives an indication of the mechanisms involved at this range. However, health risks may evolve from indirect consequences of mobile telephony, such as the sharply increased incidence rate of traffic accidents caused by telephony during driving, and possibly also by stress reactions which annoyed bystanders may experience when cellular phones are used in public places. These indirect health effects presumably outweigh the direct biological perturbations and should be investigated in more detail in the future.

Non-invasive imaging methods for the characterization of the pathophysiology of brain ischemia.

Non-invasive imaging methods are increasingly used to study the evolution and therapy of brain diseases under both clinical and experimental conditions. In the animal experiment, these methods can be supplemented by invasive tissue assays to allow precise characterization of the underlying pathophysiology. Based on such an approach, this review evaluates the importance of in vivo nuclear magnetic resonance (NMR) and positron emission tomography (PET) for the understanding of the pathophysiology of brain ischemia.

Prediction of hemorrhagic transformation after thrombolytic therapy of clot embolism: an MRI investigation in rat brain.

BACKGROUND AND PURPOSE: Thrombolytic treatment of stroke carries the risk of hemorrhagic transformation. Therefore, the potential of MRI for prediction of recombinant tissue plasminogen activator (rtPA)-induced bleeding is explored to identify patients in whom rtPA treatment may provoke such complications. METHODS: Spontaneously hypertensive rats (SHR) (n=9) were submitted to middle cerebral artery (MCA) clot embolism, followed 3 hours later by intra-arterial infusion of 10 mg/kg rtPA. Untreated SHR (n=9) were infused with saline. MRI imaging was performed before treatment and included apparent diffusion coefficient (ADC), T2, and perfusion mapping and contrast enhancement with gadolinium-DTPA. The distribution of intracerebral hemorrhages was studied 3 days later by histological staining. RESULTS: Clot embolism led to the rapid decline of ADC in the territory of the occluded artery. Tissue lesion volume derived from ADC imaging increased by 155+/-69% in the untreated animals and by 168+/-87% in the treated animals (P=NS), determined on the histological sections after 3 days. This same lesion growth in both groups indicated absence of therapeutic effect after 3-hour treatment delay. Hemorrhagic transformations were significantly more frequent in treated SHR (P<0.05). In untreated rats, hemorrhages were found in the border zone of the ischemic territory; in treated animals, hemorrhagic transformations occurred in the ischemic core region. rtPA-induced hemorrhages were predicted by a disturbance of the blood-brain barrier in 3 of 4 animals before treatment by Gd-DTPA contrast enhancement but not by ADC, T2, or perfusion imaging. The region of contrast enhancement colocalized with subsequent bleeding in these animals. CONCLUSIONS: The disturbance of blood-brain barrier but not of other MR parameters allows risk assessment for hemorrhagic transformation induced by subsequent thrombolytic treatment.

Dynamic changes of ADC, perfusion, and NMR relaxation parameters in transient focal ischemia of rat brain.

The potential of multiparametric MRI parameters for differentiating between reversibly and irreversibly damaged brain tissue was investigated in an experimental model of focal brain ischemia in the rat. The middle cerebral artery (MCA) was occluded by intraluminal suture insertion for 60 or 90 min, followed by 4.5 h of reperfusion. The apparent diffusion coefficient (ADC) of brain water, T(1) and T(2) relaxation times, and CBF(i), an MR-derived index of cerebral perfusion, were repeatedly measured and correlated with the outcome from the ischemic impact. A novel user-independent approach for segmentation of ADC maps into classes of increasing injury was introduced to define regions of interest (ROIs) in which these parameters were evaluated. MCA occlusion led to a graded decline of ADC, which corresponded with both the severity of flow reduction and an increase in T(1) and T(2) relaxation times. Removal of the suture led to a triphasic restitution of blood flow consisting of a fast initial rise, a secondary decline, and final normalization. Postischemic reperfusion led to a rise of ADC irrespective of the duration of ischemia. However, the quality of recovery declined with increasing severity of the ischemic impact. Throughout the observation time, T(1) and T(2) showed a continuous increase, the intensity of which correlated with the severity of ADC decline during ischemia. Particularly with longer ischemia time, elevated T(2) in combination with reduced ADC yielded a lower probability of recovery during recirculation, while intraischemic perfusion information contributed less to the prediction of outcome. In conclusion, the combination of MR parameters at the end of ischemia correlated with the probability of tissue recovery but did not permit reliable differentiation between reversibly and irreversibly damaged tissue.

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.

Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.

GTPase RhoB: an early predictor of neuronal death after transient focal ischemia in mice.

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.

Aggravation of brain injury after transient focal ischemia in p53-deficient mice.

The transcriptional factor p53 is a regulatory protein which contributes to the preservation of tissue integrity by promoting either DNA repair or apoptosis. To establish the pathophysiological role of this protein in ischemia, we produced 1 h transient middle cerebral artery (MCA) occlusion in normal and in p53-deficient mice and investigated the resulting tissue damage by multiparametric imaging. Possible genetic influences on the angioarchitecture of the MCA territory and blood flow were examined by intravascular latex infusion and laser-Doppler flowmetry. Wild-type (p53(+/+)), heterozygous (p53(+/-)) and homozygous (p53(-/-)) mice deficient for the p53 gene did not differ in respect to angioarchitecture or the effect of vascular occlusion on blood flow and general physiological parameters. Twenty-four hours after 1 h MCA occlusion, mice revealed a gene dose-dependent decline in the size of metabolic disturbances (ATP depletion and inhibition of protein synthesis) and histological injury (Cresyl Violet staining). DNA fragmentations detected by terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) did not differ in the three groups and were only present in ATP-depleted tissue. Our findings suggest that after transient focal brain ischemia p53 prevents rather than aggravates brain injury, and that this effect is brought about by mechanisms that are unrelated to the pro-apoptotic properties of this gene.

Electron microscopic investigation of rat brain after brief cardiac arrest.

Rats were submitted to 10-min cardiac arrest, followed by resuscitation and survival for 1 day, 3 days or 1 week. Five regions of interest (CA1 and CA3 sector of hippocampus, dentate gyrus, reticular nucleus of thalamus and parietal cortex) where studied by light and electron microscopy at each of the survival times, and compared with non-ischemic control rats. Cell counts revealed delayed neuronal loss of about 30% after 3 days in both CA1 and CA3 sectors. Ischemic cell changes consisting of cytoplasmic condensation and nuclear pyknosis appeared in these regions on day 7 and --to a lesser degree-- also affected dentate gyrus, the reticular nucleus of thalamus and cerebral cortex. Ultrastructural alterations were evaluated using an ultrastructural injury catalogue. In all brain regions similar, although quantitatively differently expressed, changes occurred except ribosomal disaggregation, which was restricted to neurons of hippocampal CA1 sector on the first day after cardiac arrest. Progressive alterations included swelling of mitochondria and endoplasmic reticulum, which was most pronounced in CA1 and CA3 sectors of hippocampus, as well as chromatin aggregation and alterations of neuronal volume, which affected mainly the granule cells of dentate gyrus. Other alterations, such as osmiophilic inclusions or the formation of nuclear pore complexes, were transient with a maximum on the first day after cardiac arrest. Treatment with the free-radical scavenger alpha-phenyl-N-tert-butyl nitrone (PBN) suppressed the formation of nuclear pores but otherwise did not markedly change the morphological outcome. In comparison to previous studies of global brain ischemia induced by arterial inflow occlusion of the same duration, the present data demonstrate remarkable preservation of tissue integrity in CA1 sector but also distinct changes in brain regions considered to be resistant to ischemic injury. Morphological alterations of brain after cardiac arrest do not follow the established pattern of selective vulnerability.

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.

Treatment with an endothelin type A receptor-antagonist after cardiac arrest and resuscitation improves cerebral hemodynamic and functional recovery in rats.

OBJECTIVE: Successful resuscitation of the brain after cardiac arrest requires unimpaired microcirculatory reperfusion. Postischemic cerebral hypoperfusion presumably is mediated through activation of endothelin type A receptors (ET(A)). The effect of the selective ET(A) antagonist BQ123 on cerebral blood flow and function was studied in a rat model of cardiac arrest. DESIGN: Prospective, randomized trial. SETTING: Experimental animal laboratory. SUBJECTS: Twelve male Sprague-Dawley rats (290-350 g). INTERVENTIONS: Cardiac arrest for 12 mins was induced by electrical fibrillation of the heart, followed by standardized cardiopulmonary resuscitation. BQ123 (0.8 mg/kg; n = 6) or its vehicle (saline; n = 6) was injected intravenously at 15 mins after the return of spontaneous circulation. MEASUREMENTS: Cortical blood flow was measured by laser-Doppler flowmetry, electrophysiological function by recording the amplitude of somatosensory evoked potentials, vascular reactivity by ventilation with 6% CO2, and the functional coupling of blood flow by recording the laser-Doppler flow (LDF) changes during somatosensory stimulation. Hemodynamic and functional cerebral recovery was monitored for 3 hrs after the return of spontaneous circulation. MAIN RESULTS: Forty-five minutes after the return of spontaneous circulation, postischemic hypoperfusion developed in both groups, as reflected by a decrease of the LDF signal to about 60% of the preischemic level. In untreated animals, hypoperfusion persisted throughout the observation time, but in animals receiving BQ123, LDF gradually returned to normal. CO2 reactivity in untreated animals was severely reduced for 2-3 hrs after the onset of recirculation, whereas after BQ123 treatment it returned to normal and after 2 hrs even above normal. The ET(A) antagonist also induced a more rapid recovery of the somatosensory evoked potentials amplitude and of the functional blood flow response to somatosensory stimulation, but these parameters did not recover completely within the observation period. CONCLUSIONS: Application of the ET(A) antagonist BQ123 during the early reperfusion period after cardiac arrest shortens postischemic cerebral hypoperfusion and accelerates the restoration of the cerebrovascular CO2 reactivity and the recovery of electrophysiologic function.

Endothelin type A-antagonist improves long-term neurological recovery after cardiac arrest in rats.

OBJECTIVE: Antagonists of endothelin (ET(A)) receptors improve postischemic hypoperfusion. In this study we investigated whether the selective ET(A)-antagonist BQ123 also improves postischemic functional recovery. STUDY DESIGN: Cardiac arrest of 12 mins duration was induced in rats by electrical fibrillation of the heart, followed by advanced cardiopulmonary resuscitation. BQ123 (0.8 mg/kg; n = 9) or its vehicle (saline; n = 9) was injected intravenously at 15 mins after the return of spontaneous circulation. The neurologic deficit was scored daily for 7 days after resuscitation by rating consciousness, various sensory and motor functions, and coordination tests. On day 7, we measured functional coupling of cerebral blood flow under halothane anesthesia by recording laser-Doppler flow during electrical forepaw stimulation, and we measured vascular reactivity to CO2 by measuring the laser-Doppler flow change during ventilation with 6% CO2. The brains were perfusion-fixated with 4% paraformaldehyde, and the histopathologic damage was evaluated in the CA1 sector of hippocampus, in the motor cortex, and in the cerebellum. RESULTS: Treatment with BQ123 had no effect on histopathologic damage, but it significantly improved neurologic recovery. In all nine treated rats, neurologic performance returned to near normal within 2 days whereas four of nine untreated animals developed spastic paralysis of the hind limbs and severe coordination deficits. BQ123 also normalized CO2 reactivity and improved the functional cerebral blood flow response to somatosensory stimulation. CONCLUSIONS: The ET(A)-antagonist BQ123 significantly improves neurologic outcome after 12 mins of cardiac arrest. The apparent restoration of vascular reactivity demonstrates a correlation between hemodynamic factors and functional recovery.

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.

Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential.

The effect of focal ischemia on tissue pH was studied at various times up to 6 hours after permanent middle cerebral artery occlusion in rats. Tissue pH was imaged by using umbelliferone fluorescence and correlated with cerebral blood flow, ATP content, and recordings of the steady potential. Circumscribed foci of allalosis (pH 7.32+/-0.11) were detected with increasing frequency in penumbral regions having near-to-normal ATP concentrations and cerebral blood flow values between 20% and 40% of control. Both the infarct core, defined by ATP loss and cerebral blood flow values of less than 20% of control, and the inner peri-infarct rim were consistently acidic (pH 6.03+/-0.36 and 6.53+/-0.24, respectively). Treatment with the glutamate antagonist dizocilpine (MK-801) suppressed negative shifts of the steady potential and reduced significantly the occurrence of alkalosis observed in 90% of untreated but only in 44% of treated animals. Penumbral alkalosis appeared to be a time-dependent event occurring 30 to 60 minutes after the passage of peri-infarct depolarizations. The diversity of penumbral pH changes reflects the local disturbance of pH regulation and, possibly, the differential fate of penumbral subareas.