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The susceptibility of the brain to ischaemic injury dramatically limits its viability following interruptions in blood flow. However, data from studies of dissociated cells, tissue specimens, isolated organs and whole bodies have brought into question the temporal limits within which the brain is capable of tolerating prolonged circulatory arrest. This Review assesses cell type-specific mechanisms of global cerebral ischaemia, and examines the circumstances in which the brain exhibits heightened resilience to injury. We suggest strategies for expanding such discoveries to fuel translational research into novel cytoprotective therapies, and describe emerging technologies and experimental concepts. By doing so, we propose a new multimodal framework to investigate brain resuscitation following extended periods of circulatory arrest.

In the history of stroke research the term letzte Wiese (English last meadow or last field) was introduced in the early fifties of the past century to describe the risk of low blood perfusion in the periphery of a vascular territory. The term appears 1952 in the discussion of a colloquium held by the German Society of Physiological Chemistry in the small city Mosbach, located close to the scenic river Neckar in the north of the federal state Baden-Wurttemberg.1 The symposium entitled Chemie und Stoffwechsel des Nervengewebes (chemistry and metabolism of nervous tissue) comprised 5 presentations on various aspects of brain function and metabolism. One of these was given by Erich Opitz, Chair of the Physiological Institute of the University Kiel. In his presentation Energieumsatz des Gehirns in situ unter aeroben und anaeroben Bedingungen (energy metabolism of the brain under aerobic and anaerobic conditions), he addressed the magnitude of metabolic activity required to maintain the structural integrity of the brain (Strukturumsatz). In the discussion after his presentation, Max Schneider, Chair of the Physiological Institute of the University of Cologne, remarked that a differentiation between structural and functional activity of the brain can be obtained by gradually reducing the blood perfusion of an isolated head preparation and recording the suppression and recovery of the electroencephalogram. The lowest flow rate down to which spontaneous electroencephalogram activity persists is the amount required to maintain functional activity, and the lowest flow rate which is tolerated for some time without causing irreversible suppression of the electroencephalogram corresponds to the amount required to preserve structural integrity. This approach anticipated the latter differentiation between core and penumbra of evolving brain infarction introduced by Astrup et al2 after occlusion of the middle cerebral artery. However, as pointed out by Schneider,3 global measurements of blood flow …

Mutations in the X-chromosomal L1CAM gene lead to severe neurological deficits. In this study, we analyzed brains of female mice heterozygous for L1 (L1+/-) to gain insights into the brain structure of human females carrying one mutated L1 allele. From postnatal day 7 onward into adulthood, L1+/- female mice show an increased density of neurons in the neocortex and basal ganglia in comparison to wild-type (L1+/+) mice, correlating with enhanced metabolic parameters as measured in vivo. The densities of astrocytes and parvalbumin immunoreactive interneurons were not altered. No significant differences between L1+/- and L1+/+ mice were seen for cell proliferation in the cortex during embryonic days 11.5-15.5. Neuronal differentiation as estimated by analysis of doublecortin-immunoreactive cortical cells of embryonic brains was similar in L1+/- and L1+/+ mice. Interestingly, at postnatal days 3 and 5, apoptosis was reduced in L1+/- compared to L1+/+ mice. We suggest that reduced apoptosis leads to increased neuronal density in adult L1+/- mice. In conclusion, L1+/- mice display an unexpected phenotype that is not an intermediate between L1+/+ mice and mice deficient in L1 (L1-/y), but a novel phenotype which is challenging to understand regarding its underlying molecular and cellular mechanisms.

Experimental research into brain ischemia contributes substantially to the understanding of ischemic injury mechanisms but suffers from its limited relevance for clinical treatment strategies. One of the reasons is the use of experimental models and methods that do not or only partially replicate the pathophysiology of naturally occurring brain ischemia. To facilitate the understanding and interpretation of experimental data, the most widely used experimental models and analytical methods of brain ischemia are reviewed. Particular emphasis is given to the pathophysiological particularities of the various ischemia models, the application of imaging methods for the reliable differentiation between infarct core, penumbra, benign oligemia and normal tissue, as well as to the final outcome of experimental interventions. Based on this analysis, recommendations are given to improve the translational power of brain ischemia-related experimental research.

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68+ macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis.

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.

science, stroke research and industry. The primary objective of the workshop was to exchange views on the topic of stroke and to identify research activities that could potentially result in major advances in the areas of stroke prevention, treatment and recovery. These research priorities should address the most pressing scientific, clinical and industrial needs in the field of stroke. Participants agreed that answers to the questions posed by these research priorities must significantly help in reducing the stroke burden to the European Union. The following is a summary of the ideas that were presented, discussed and agreed upon during the Stroke Workshop. Several of the key topics, particularly those in basic science stroke research and imaging, span prevention, therapy and recovery, thus conceivably making contributions to multiple areas of stroke management.

This study sought to establish the photothrombotic ‘ring’ stroke model with late spontaneous reperfusion in adult mice. By applying a 3.0-mm diameter laser ring-beam (514 nm, 0.21 mm thick, 0.65 W/cm2) onto the exposed skull for 60 secs with concurrent erythrosin B (4.25 mg/kg) intravenous infusion for 15 secs, the centrally located cortical region within the ring locus was progressively encroached by an annular ring-shaped perfusion deficit. In this region, local cerebral blood flow (ICBF) measured by 1aser-Doppler flowmetry declined promptly after irradiation to 43% of the baseline value at 30 mins poststroke. Using double tracer autoradiography, quantitative ICBF measured with [14C]iodoantipyrine was 46 to 17 to 58 ml/100 g/mins at 4 h to 48 h to 7 days postischemia in this area. Cerebral protein synthesis (CPS), as detected by [3H]leucine incorporation into protein, transiently decreased to 57% to 38% to 112% at 4 h to 48 h to 7 days postischemia in the center region. Morphologically, some neurons in the center region appeared swollen at 4 h. At 48 h, the majority of neurons were severely swollen with eosinophilia and pyknosis, whereas at 7 days poststroke’ the tissue morphology became partly restored. The center within the mouse photothrombotic ring lesion thus exhibits reversible alterations of local CBF, CPS and tissue morphology that are reminiscent of the cortical penumbra in other models of focal cerebral ischemia.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a powerful arteriogenic factor in the hypoperfused rat brain. To test the pathophysiological relevance of this response, the influence of GM-CSF on brain energy state was investigated in a model of hemodynamic stroke. Sprague-Dawley rats were submitted to three-vessel (bilateral vertebral and unilateral common carotid artery) occlusion (3-VO) to induce unilaterally accentuated brain hypoperfusion. One week later, hemodynamic stroke was induced by additional lowering of arterial blood pressure. Experiments were terminated by in situ freezing of the brain. ATP was measured in cryostat sections by using a bioluminescence method. The use of 3-VO, in combination with 15 min of hypotension of 50, 40, or 30 mmHg, did not produce disturbances of energy metabolism, however, focal areas of ATP depletion were unilaterally detected after 3-VO, in combination with 15 min of hypotension of 20 mmHg. Treating such animals with GM-CSF (40 μg·kg -1 ·d -1 ) during the 1-week interval between 3-VO and induced hypotension significantly reduced the hemispheric volume of energy depletion from 48.8 ± 44.2% (untreated group, n = 10) to 15.8 ± 17.4% (treated group, n = 8, P = 0.033). GM-CSF-induced arteriogenesis is another approach to protect the brain against ischemic injury.

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.

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.

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.

Experimental and clinical studies have provided evidence for spontaneous and therapeutically induced arteriogenesis after occlusion of major peripheral or cardiac vessels. Such evidence is lacking for the cerebrovascular system. In halothane-anesthetized rats, different degrees of brain hypoperfusion were induced by one- to four-vessel occlusion, that is, one or both common carotid arteries in combination with or without bilateral vertebral artery occlusion. The flow decline was monitored by laser Doppler flowmetry, the residual hemodynamic reserve by testing flow reactivity to ventilation with 6% CO2 and arteriogenesis by intravascular latex infusion and immunohistochemistry of vascular proliferation and monocyte adhesion. The optimum condition for induction of arteriogenesis was three-vessel (one carotid plus both vertebral arteries) occlusion, which led to reduction of blood flow to about 50% and complete suppression of CO2 reactivity, but no histologic injury. One week after three-vessel occlusion, the ipsilateral posterior cerebral artery significantly enlarged by 39%, and after 3 weeks by 72%, paralleled by the partial return of CO2 reactivity and the appearance of immunohistochemical markers of arteriogenesis. Three-vessel occlusion is a reliable model for the induction of arteriogenesis in the adult brain and is a new approach for exploring the potentials of arteriogenesis for the prevention of progressing brain ischemia.

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 Conclusions — The disturbance of blood-brain barrier but not of other MR parameters allows risk assessment for hemorrhagic transformation induced by subsequent thrombolytic treatment.

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.

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.

Treatment of clinical stroke with recombinant tissue plasminogen activator (rt-PA) carries the risk of hemorrhagic complications. Hence, predictors of therapeutic outcome with respect to (a) reperfusion and (b) tissue recovery would be very useful to identify potentially salvageable brain tissue. Magnetic resonance (MR) parameters, especially the apparent diffusion coefficient of water (ADC), perfusion-weighted imaging (PWI) and T(2) relaxometry are thought to provide this information. We evaluated the prognostic implications of ADC, PWI and T(2) relaxometry immediately before initiation of thrombolytic treatment in a model of clot embolism in rats. Animals (n = 14) were treated with intraarterial rt-PA (10 mg/kg) at 90 min after embolism. MR imaging was repeatedly performed at 4.7 T before and up to 5.5 h after embolism. ADC was calculated from diffusion-weighted images (b-values: 30, 765, 1500 s/mm(2)), arterial spin tagging was used for PWI, and quantitative T(2) relaxometry was performed with a Carr-Purcell-Meiboom-Gill (CPMG) sequence. A reperfusion index was calculated to assess the quality of thrombolytic recanalization. The decline of ADC at the end of the experiment to below 80% of control was defined as unfavorable outcome. The probability of tissue injury at the end of the experiments increased with the severity of ADC changes before the initiation of treatment (probability of unfavorable outcome: 21%, 44%, 65% for ADC values of 80-90%, 70-80% and70% of control, respectively). Pretreatment PWI or T(2) relaxometry also correlated with outcome but-alone or in combination with pretreatment ADC maps-did not improve injury prediction over that obtained by ADC alone. Outcome was influenced positively by successful reperfusion the quality of which, however, could not be predicted by pre-treatment MR characteristics. The data demonstrate that ADC mapping performed before the initiation of thrombolytic treatment provides reliable risk assessment of impeding brain injury but due to uncertainties of postischemic reperfusion does not allow precise outcome prediction in individual experiments.

The temporospatial relationship between microglial and astrocytic reactions and delayed thalamic cell death was examined 1-7 days following a traumatic cold lesion of the rat sensorimotor cortex using immunocytochemistry in combination with terminal deoxynucleotidyltransferase-mediated biotinylated dUTP nick end labeling (TUNEL) of nuclear DNA fragmentation. No or only occasional TUNEL-positive cells were found in the thalamic relay nuclei up to 3 days after trauma. After 7 days, on the other hand, a considerable number of TUNEL-positive cells were seen in the ventrobasal, the ventrolateral and posterior thalamic nuclei. Already 3 days after trauma, i.e., before cell injury was detectable, many protoplasmic astrocytes, which were reactive for glial fibrillary acidic protein, and ramified microglia, which were positive for complement receptor type 3b (CR3b) but negative for major histocompatibility complex (MHC) class II antigen, were noticed in the thalamus. The number of labeled astro- and microglia further increased after 7 days, when DNA fragmentation became evident. At this time, the morphology of microglia shifted towards bushy and rod-like cells, and microglia became also reactive for MHC class II antigen. Clusters of CR3b- and MHC class II-positive microglia were found in the ventrobasal thalamus. The present findings demonstrate that trauma-induced microglial and astrocytic reactions appear in the thalamus prior the onset of cell damage.

Abnormalities in the homeostasis of the renin-angiotensin system have been implicated in the pathogenesis of vascular disorders, including stroke. The authors investigated whether angiotensinogen (AGN) knockout mice exhibit differences in brain susceptibility to focal ischemia, and whether such differences can be related to special features of the collateral circulation. Wild-type and AGN-knockout mice were submitted to permanent suture occlusion of the middle cerebral artery (MCA). The collateral vascular system was visualized by systemic latex infusion, and the ischemic lesions were identified by cresyl-violet staining. The core and penumbra of the evolving infarct were differentiated by bioluminescence and autoradiographic imaging of A TP and protein biosynthesis, respectively. In wild-type mice, mean arterial blood pressure was 95.0 ± 8.6 mm Hg, and the diameter of fully relaxed anastomotic vessels between the peripheral branches of the anterior and middle cerebral arteries 26.6 ± 4.0 μm In AGN knockouts, mean arterial blood pressure was significantly lower, 71.5 ± 8.5 mm Hg ( P

Intracerebral hemorrhage is a major complicating factor of thrombolytic therapy of stroke. To investigate the incidence of bleeding in animals with a diseased vascular system, thrombolysis was carried out in spontaneously hypertensive rats (SHR) after clot embolism of the right middle cerebral artery (MCA). Three hours after embolism SHR were treated with either recombinant tissue-plasminogen activator (rt-PA) or saline, and neurological deficits and intracerebral hemorrhages were evaluated after 3 days survival. Rt-PA-treated SHR exhibited a significantly higher incidence of hemorrhages than untreated rats but neurological deficits and survival rates showed a non-significant trend for improvement. This model offers the possibility of investigating the pathophysiology of post-thrombolytic complications in a clinically relevant small animal model.

Lesion-remote metabolic changes were examined 1-7 days after neocortical cold injury using tissue ATP, glucose and lactate bioluminescent imaging, pH-dependent fluoroscopy and cerebral protein synthesis (CPS) autoradiography. One day after lesioning an alkaline pH shift (0.35 +/- 0.19 units above contralateral) was noticed in the lesion-remote cortex, the underlying white matter, the striatum, hippocampus and thalamus, which slowly resolved within 7 days and probably reflected the spread of vasogenic edema. Closely associated with the pH shift, elevations in tissue glucose and lactate levels were found, which reached maximum levels after 3 days (7.4 +/- 2.4 vs 4.2 +/- 1.2 micromol/g glucose, 6.6 +/- 2.3 vs 2.1 +/- 0.6 micromol/g lactate) but, in contrast to the alkalosis, remained elevated after 1 week. Thus, neocortical trauma is associated with long-lasting metabolic changes, which are intimately linked with the distribution of post-traumatic alkalosis.

Neuronal death after brain ischemia is mainly due to necrosis but there is also evidence for involvement of apoptosis. To test the importance of apoptosis, we investigated the effect of targeted disruption of the apoptosis-suppressive gene bcl-2 on the severity of ischemic brain injury. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery in homozygous (n=7) and heterozygous (n = 6) bcl-2 knockout mice as well as in their wildtype littermates (n=5). Bcl-2 ablation did not influence cerebral blood flow but it significantly increased infarct size and neurological deficit score at 1 day after reperfusion in a gene-dose dependent manner. The exacerbation of tissue damage in the absence of Bcl-2 underscores the importance of apoptotic pathways for the manifestation of ischemic injury after transient vascular occlusion.

The Zac1 gene encodes a zinc finger protein that regulates both apoptosis and cell cycle arrest in vitro. Furthermore, Zac1 protein seems to trans-activate the gene encoding the type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP). Northern blot analysis revealed high levels of Zac1 mRNA in the rodent brain. In the present study, we demonstrate by in situ hybridization histochemistry a progressive increase in Zac1 transcripts in the mouse brain from day 1 to day 3 following transient focal cerebral ischemia. Moreover, we observed an up-regulation of PACAP type I receptor mRNA expression showing a similar temporospatial distribution. Late induction of cell death promoting Zac1 in the post-ischemic brain may be attributed to delayed or secondary cell death. Co-induction of the type I receptor for neurotrophic PACAP however, points to a role in restorative processes.

Time for primary review 28 days. More than any other organ of the body, brain integrity depends on the continuous blood supply of oxygen and glucose for covering the energy demands of the tissue. Cessation or severe reduction of blood flow results in almost instantaneous biochemical and functional deficits which become rapidly irreversible unless blood flow is promptly restored. The ischemia time that is tolerated by the brain depends, among others, on the density of ischemia, the tissue concentration of primary and secondary energy stores and the rate of energy consumption which, in turn, depends on the temperature, the degree of functional activity and the absence or presence of anesthetics and other drugs. There is also a major difference between the ischemic vulnerability of different regions of the brain. As an example, the CA1 sector of hippocampus may suffer irreversible injury after ischemia of as short as 5 min [94]whereas other nerve cell populations survive normothermic cerebrocirculatory arrest of as long as 1 h [77]. The pathophysiology of cerebral ischemia is further complicated by the fact that the severity of cell injury is modulated by numerous indirect or secondary consequences of the primary ischemic impact. Recirculation disturbances, stress responses, peroxidative changes, or the activation of genomic responses are only few examples of the large number of hemodynamic and molecular responses which determine the final outcome [153, 97]. The interpretation of experimental data of cerebral ischemia, therefore, requires intimate knowledge of the models and conditions under which the data have been collected. The number and diversity of such models, has become very large over the years because researchers have sought in vain for the ideal approach to study ischemic injury and its therapeutic reversal. Such an ideal model should be relevant to the clinical situation, it should be … * Corresponding author. Tel.: +49 (221) 4726 211; Fax: +49 (221) 4726 325; E-mail: hossmann@mpin-koeln.mpg.de

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 μm) 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.

The effects of a traumatic neocortical lesions, induced by transcranial cold injury, on brain metabolism and gene expression were examined. The surrounding of the lesions was characterized by increased glucose and lactate levels without major disturbances of protein synthesis or energy state. A transient pH decrease by 0.4 units was noticed 1 h post-injury, which shifted towards alkaline values by 3 h. The metabolic disturbances did not differ between injured animals with spontaneous spreading depressions (SD, n = 14) and those without SD (n = 7). In SD animals, c-fos mRNA was strongly elevated in the injury-remote cortex, but hsp72 mRNA was not enhanced. Thus, in contrast to focal ischemia, the metabolic dysfunction around traumatic cortical lesions is not aggravated by SD.

In the present study the neuroprotective effect of mild hypothermia (decrease of temperature from 37 degrees C to 33 degrees C) during and after transient ischemia in brain tissue at different stages of development was tested in vitro by measuring energy metabolism, glutamate release and protein biosynthesis rate (PSR) in hippocampal slices. Slices were taken from immature (E40) and mature (E60) guinea pig fetuses and adult guinea pigs. The slices were exposed to ischemia-like conditions (oxygen/glucose deprivation, OGD) for periods of between 10 to 40 min followed by a 2-h or 12-h recovery phase. During OGD, mild hypothermia slowed down the depletion of energy stores only in slices from immature fetuses, but had no effect on slices prepared from mature fetuses and adult animals. Hypothermia also reduced glutamate release significantly during oxygen/glucose deprivation. Lowering temperature to 33 degrees C had no effect on energy metabolism and only a minor effect on PSR of slices from mature fetuses and adult animals subjected to 2 h of recovery. However, 12 h after OGD PSR was markedly improved by mild hypothermia in slices from mature animals and in slices from adults that had been exposed to OGD for only 20 or 30 min. The inhibition of PSR was more severe in the slices from adults than in those from mature fetuses subjected to the same duration of OGD. Age- and temperature-related differences in glutamate release during OGD did not fully agree with corresponding disparities in the values for PSR obtained 12 h after OGD. These results indicate that the neuroprotective effect of mild hypothermia was not mediated by a temperature-dependent retardation of the depletion of energy stores during OGD. Age-related disparities in the vulnerability of the brain to ischemia and the neuroprotective efficiency of mild hypothermia appear to be only partially reflected by the varying levels of glutamate release during ischemia but best reflected by the extent of PSR inhibition. It is concluded that mild hypothermia may be a suitable therapeutical intervention for the suppression of hypoxic-ischemic cell damage during birth.

The Ref-1 protein is a bifunctional nuclear enzyme involved in repair of DNA lesions and in redox regulation of DNA-binding activity of AP-1 family members, such as Fos and Jun transcription factors. In the present study, we demonstrate by in situ hybridization that transient global ischemia induced by cardiac arrest activates ref-1 mRNA expression in the granular cells of the rat dentate gyrus after 6 h and in CA1 pyramidal neurons of the hippocampus proper after 24 h, respectively. Immunohistochemical analysis revealed nuclear accumulation of Ref-1 protein in granular cells of the ischemia-resistant dentate gyrus, whereas Ref-1 protein expression progressively decreased in vulnerable CA1 neurons of the post-ischemic hippocampus from 24 h onwards. At the same time point, intense nuclear c-Jun immunoreactivity was observed in both neuronal cell populations. Our data suggest that oxidative stress induced by ischemia-reperfusion may increase neuronal ref-1 expression. However, inability of ref-1 mRNA translation and nuclear translocation of encoded protein in CA1 pyramidal neurons may inhibit repair of oxidative DNA damage or cellular adaptive responses leading to delayed neuronal cell death.

Results from experiments performed with permanent non-neuronal cell lines suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concluded that disturbances in ER calcium homeostasis may contribute to the suppression of PS triggered by a severe metabolic stress (W. Paschen, Med. Hypoth ., 47 (1996) 283–288). To elucidate how an emptying of ER calcium stores of these cells would effect PS and ribosomal aggregation of non-transformed fully differentiated cells, experiments were run on primary neuronal cell cultures. ER calcium stores were depleted by treating cells with thapsigargin (TG, a selective, irreversible inhibitor of ER Ca 2+ -ATPase), cyclopiazonic acid (CPA, a reversible inhibitor of ER Ca 2+ -ATPase), or caffeine (an agonist of ER ryanodine receptor). Changes in intracellular calcium activity were evaluated by fluorescence microscopy using fura-2-loaded cells. Protein synthesis was determined by measuring the incorporation of [ 3 H]leucine into proteins. The degree of aggregation of ribosomes was evaluated by electron microscopy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused about 70% inhibition of PS, and PS recovered completely 60 min after treatment. Caffeine produced an inhibition of PS to about 50% of control. Loading cells with the calcium chelator BAPTA-AM (33.3 μM) alone suppressed PS without reversing TG- or caffeine-induced inhibition of PS, indicating that the suppression of PS was caused by a depletion of ER calcium stores and not by an increase in cytosolic calcium activity. TG-treatment of cells induced a complete disaggregation of polysomes which was not reversed within the 4 h recovery period following TG-treatment. After caffeine treatment of cells, we observed a heterogeneous pattern of ribosomal aggregation: in some neurons ribosomes were almost completely aggregated while in other cells a significant portion of polyribosomes were disaggregated. The results indicate that a depletion of neuronal ER calcium stores disturbs protein synthesis in a similar way to the effects of transient forms of metabolic stress (ischemia, hypoglycemia or status epilepticus), thus implying that a disturbance in ER calcium homeostasis may contribute to the pathological process of stress-induced cell injury.

Recovery of the brain after a period of cerebral ischemia depends greatly on the restoration of nutritive blood flow, which, however, may be severely disturbed. Early post-ischemic deficits (no-reflow) multiply with increasing duration of ischemia. The pathophysiology is multifactorial and includes vascular factors (endothelial blebs, compression by swollen glial cells), blood factors (viscosity changes due to erythrocyte sludging, platelet aggregation, blood dehydration), and general cardiovascular factors (post-ischemic hypotension, venous congestion). Treatment of no-reflow requires a combination therapy (e.g., hypertensive flush, small volume hypertonic solutions, fibrinolysis) for interfering with as many of these factors as possible. Delayed post-ischemic hypoperfusion develops after a preceding phase of post-ischemic hyperemia and is characterized by increased vasotonus. Hypoperfusion is associated with a disturbed coupling between brain function, metabolism, and blood flow, and may lead to secondary stimulation of anaerobic metabolism. Causal factors include disturbed blood/vessel wall interactions (expression of adhesion molecules, generation of free radicals) and possibly down-regulation of endothelial nitric oxide synthase. Treatment of post-ischemic hypoperfusion includes neutrophil elimination and free radical scavengers but is still unsatisfactory. Improvement of reperfusion deficits is a challenging task that must be solved before proceeding to specific molecular interventions for the treatment of ischemic cell injury.

Bo K Siesjo was an outstanding brain researcher and will be remembered as such. He was born in Sweden on 8 January 1930 and died in his home country on the 27 June 2013. He graduated from medical school at the Lund's University in 1958 and defended his PhD thesis at the same university in 1962. His postgraduate training took place in Sweden and Cambridge, and in the late 1980s he was a visiting scientist in Nice. After his graduation from medical school, he had appointments at the Lund's University and University Hospital until his retirement in 1995. In Lund he created the Laboratory for Experimental Brain Research, and the Swedish Research Council established a special professorship for him in 1974. He, subsequently, became the chief of the research department at Lund's University Hospital. After his retirement he went to Hawaii to build up a neuroscience research department at Queens Medical Center in Honolulu. Siesjo's research focus was on cerebral metabolism in the normal and injured brain, and an early important topic of his interest was the regulation of pH in the extra- and intracellular fluids in the brain. He published a textbook on the brain's energy metabolism and authored or co-authored more than 500 publications listed on PubMed. In 1970s his research focused on the ischemic metabolic cascade, the mechanisms of ischemic cell death, and the energy reserves in the brain, work that bore rich fruit. Two classic papers from this period stand out for their influence on the field, ie, his 1981 paper with Astrup and Symon on the concept of the ischemic penumbra,1 and his synthesis paper on the mechanisms of ischemic cell death.2 In his later years of research he also investigated the pathophysiology of chronic brain diseases, such as dementia of the Alzheimer's type. In his research carrier, Siesjo became one of the most highly cited scientists in his field. Bo Siesjo was the main catalyst for the founding in 1981 of the Journal of Cerebral Blood Flow and Metabolism and the organization of its parent professional society, the International Society of Cerebral Blood Flow and Metabolism. He was also the recipient of a number of awards that include the Mihara Award in 1989, the J Allan Taylor International Prize in Medicine in 1992, the KJ Zulch Prize in 1992, and the Lifetime Achievement Award by the International Society for Cerebral Blood Flow and Metabolism in 2003. Siesjo's research in Lund was renowned worldwide, and many young scientists from all over the world came to work with him. Many of them, who got their scientific start in his laboratory, later became well-established researchers in their home countries; more than 20 of them achieved full professorships in institutions worldwide. In addition to his scientific interests, Bo Siesjo had an intense interest in art that led him to establish an art collection concentrated mainly on modern national art. Bo was married with Bodil, and they had three children, Peter, Viveka, and Bjorn. All honor to his name.

Successful resuscitation of the brain requires complete microcirculatory reperfusion, which, however, may be impaired by activation of blood coagulation after cardiac arrest. The study addresses the question of whether postischemic thrombolysis is effective in reducing cerebral no-reflow phenomenon.14 adult normothermic cats were submitted to 15-min cardiac arrest, followed by cardiopulmonary resuscitation (CPR) and 30 min of spontaneous recirculation. The CPR protocol included closed-chest cardiac massage, administration of epinephrine 0.2 mg/kg, bicarbonate 2 mEq/kg per 30 min, and electrical defibrillation shocks.During CPR, animals in the treatment group (n = 6) received intravenous bolus injections of 100 U/kg heparin and 1 mg/kg recombinant tissue type plasminogen activator (rt-PA), followed by an infusion of rt-PA 1 mg/kg per 30 min.Microcirculatory reperfusion of the brain was visualized by labeling the circulating blood with 300 mg/kg of 15% fluorescein isothiocyanate albumin at the end of the recirculation period. Areas of cerebral no-reflow--defined as the absence of microvascular filling--were identified by fluorescence microscopy at eight standard coronal levels of forebrain, and expressed as the percentage of total sectional area. One animal in the treatment group was excluded from further analysis because of intracerebral hemorrhage due to brain injury during trepanation. Autopsy revealed the absence of intracranial, intrathoracic, or intra-abdominal bleeding in all the other animals. In untreated animals (n = 8), no-reflow affected 28 +/- 13% of total forebrain sectional areas, and only 1 out of 8 animals showed homogenous reperfusion (i.e., no-reflow15% of total forebrain sectional areas). Thrombolytic therapy (n = 5) significantly reduced no-reflow to 7 +/- 5% of total forebrain sectional areas and all treated animals showed homogenous reperfusion at the microcirculatory level.The present data demonstrate that thrombolytic therapy improves microcirculatory reperfusion of the cat brain when administered during reperfusion after cardiac arrest.

Both the period of total circulatory arrest to the brain and postischemic-anoxic encephalopathy (cerebral postresuscitation syndrome or disease), after normothermic cardiac arrests of between 5 and 20 mins (no-flow), contribute to complex physiologic and chemical derangements. The best documented derangements include the delayed protracted inhomogeneous cerebral hypoperfusion (despite controlled normotension), excitotoxicity as an explanation for selectively vulnerable brain regions and neurons, and free radical-triggered chemical cascades to lipid peroxidation of membranes. Protracted hypoxemia without cardiac arrest (e.g., very high altitude) can cause angiogenesis; the trigger of it, which lyses basement membranes, might be a factor in post-cardiac arrest encephalopathy. Questions to be explored include: What are the changes and effects on outcome of neurotransmitters (other than glutamate), of catecholamines, of vascular changes (microinfarcts seen after asphyxia), osmotic gradients, free-radical reactions, DNA cleavage, and transient extracerebral organ malfunction? For future mechanism-oriented studies of the brain after cardiac arrest and innovative cardiopulmonary-cerebral resuscitation, increasingly reproducible outcome models of temporary global brain ischemia in rats and dogs are now available. Disagreements exist between experienced investigative groups on the most informative method for quantitative evaluation of morphologic brain damage. There is agreement on the desirability of using not only functional deficit and chemical changes, but also morphologic damage as end points.

A fast version of the arterial spin tagging technique for the detection of cerebral perfusion is presented. Based on adiabatic spin inversion in combination with snapshot FLASH imaging our technique allows the recording of perfusion changes with a temporal resolution of about 3 s. Differences of cerebral perfusion dependent on the choice of anesthesia were observed in rat brain. Furthermore, with this arterial spin tagging method we demonstrated perfusion increases in the somatosensory cortex of anaesthetized rats during forepaw stimulation. Comparison of the activated areas in the T 2 * -weighted BOLD images and the perfusion-weighted images showed good spatial correspondence, but the sensitivity to the functional activation was more than ten times higher in the perfusion technique.

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 × 10−6 mm2/s and 659 ± 17 × 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 min−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.

Focal cerebral ischaemia was produced in 11 rats by permanent occlusion of the right middle cerebral artery (MCA) using a suture model modified to enable manipulation with the animals in situ in an NMR spectrometer. The development of the ischaemic insults and the resultant infarcts were observed for up to 6 h by localized 1H MRS and diffusion-weighted MRI while performing continuous monitoring of electroencephalogram and extracellular DC potential. The ischaemic areas were depicted as regions of hyperintensity in the diffusion-weighted images. Signals due to lactate became visible in the 1H spectra after MCA occlusion indicating the onset of anaerobic glycolysis. A depletion of N-acetylaspartate was seen in all animals post-occlusion. Transient or stepwise increases of lactate were observed to occur coincidentally with the events of spontaneous transient peri-infarct depolarization detected by the electrophysiological measurements. Expansion of the ischaemic area delineated in the diffusion-weighted images also accompanied peri-infarct depolarizations. These observations are consistent with transient peri-infarct depolarization playing a role in the growth of infarcts.

Diffusion-weighted magnetic resonance imaging was used for the description of experimental brain tumors in rat. To validate this approach, diffusion-weighted images (DWI) were compared with nativeT 1- andT 2-weighted images, and withT 1-weighted images following contrast enhancement with the tumor-specific contrast agent manganese (III) tetraphenylporphine sulfonate (MnTPPS). Three tumor types were studied: F98 glioma, RN6 Schwannoma, and E376 neuroblastoma. On heavily diffusion-weighted images, all three tumor types as well as the peritumoral edema were clearly hypointense with respect to the intact brain tissue.T 2-weighted images presented mainly peritumoral edema as hyperintense region. A clear demarcation of the tumor was possible only onT 1-weighted images after contrast enhancement with MnTPPS. The difference in signal intensity between tumor and homotopic regions in the contralateral hemisphere was comparable in DWIs and in contrast-enhancedT 1-weighted images. Spatial comparison of depicted lesion areas in all three imaging modalities indicated that hypointense region on DWI represents both tumor and edema but does not permit their spatial differentiation.

The apparent diffusion coefficient (ADC) of water is a sensitive indicator of water and ion homeostasis of brain. Resuscitation of the brain after cardiac arrest, the most frequent reason for global cerebral ischemia under clinical conditions, depends critically on the reversal of disturbances of water and ion homeostasis. We, therefore, investigated whether ADC imaging can be used to monitor the development and reversal of ischemic brain injury during and after cardiac arrest. Ten adult mongrel normothermic cats were anesthetized with alfentanil and midazolam, immobilized with pancuronium, and mechanically ventilated with O2/N2O. Arterial, left ventricular, central venous, and intracranial pressures were monitored throughout the experiment. Magnetic resonance imaging was performed in a 4.7T MR scanner with a shielded gradient system. Diffusion-weighted images (DWI) were obtained by pulsed gradient spin echoes (diffusion weighting factor b: 0, 500, 1000, 1500 s/mm2). Quantitative ADC images were calculated from DWIs by fitting signal intensities against b-factors. Fifteen minute cardiac arrest was induced in the magnet by electrical fibrillation. Resuscitation was also carried out in the magnet, using a pneumatic vest for remotely controlled closed chest cardiac massage. Seven of 10 animals were resuscitated successfully and subsequently monitored for 3 h. During cardiac arrest, ADC declined from 678 +/- 79 x 10(-6) to 430 +/- 128 x 10(-6) mm2/s (63% of baseline). In the successfully resuscitated animals ADC returned to 648 +/- 108 x 10(-6) mm2/s within 30 min and remained at this level throughout the 3 h of recirculation. Regional evaluations of ADC revealed a transient overshoot in brainstem and basal ganglia to 114% of control at 15 min before returning to baseline values after 40 min. Failure of cardiac resuscitation prevented ADC normalization and led to its further decline to below 50% of control. Postcardiac arrest normalization of ADC maps correlated with homogeneous return of ATP, glucose, and lactate to near normal, whereas failure of ADC normalization was associated with depletion of ATP and glucose and severe lactate accumulation. In conclusion, our data indicate, that normalization of ADC is a reliable indicator of cerebral recovery after resuscitation from cardiac arrest.

Brain injury after focal ischemia evolves along two basically different pathophysiologies, depending on the severity of the primary flow reduction and the dynamics of postischemic recirculation. In permanent and gradually reversed focal ischemia as after thromboembolic occlusion, primary core injury is irreversible but the expansion of the core into the penumbra can be alleviated by hemodynamic and molecular interventions. Such alleviation can only be achieved within 3 hours after the onset of ischemia because untreated core injury expands to near maximum size during this interval. In promptly reversed transient ischemia as after mechanical vascular occlusion, primary core injury may recover but a secondary delayed injury evolves after a free interval of as long as 6 to 12 hours. This injury can be alleviated throughout the free interval but the longer window is without clinical relevance because transient mechanical vascular occlusion is not a model of naturally occurring stroke. As this difference is widely ignored in stroke research, most clinical trials have been designed with a far too long therapeutic window, which explains their failure. Transient mechanical vascular occlusion models should, therefore, be eliminated from the repertoire of preclinical stroke research.

Adult normothermic cats were submitted to 1-h complete cerebrocirculatory arrest by intrathoracic occlusion of the internal mammary, the innominate, and the subclavian arteries in combination with pharmacologically induced hypotension. After ischemia, recirculation was initiated at different blood pressure levels to manipulate the postischemia resuscitation conditions. The resulting spectrum of postischemic recovery was studied by combining nuclear magnetic resonance imaging of the apparent diffusion coefficient (ADC) with pictorial assays of brain tissue pH, ATP, glucose, and lactate. Before ischemia, the mean ADC (average of seven coronal slices of five cats) was 713 ± 40 × 10−6mm2/s. After 10-min ischemia, ADC declined to 68% of control and after 50 min slightly further to 63% of control. During recirculation after 1-h ischemia, recovery of ADC varied depending on the initial reperfusion pressure and other systemic variables. In two animals ADC only transiently increased followed by a secondary decline below the postischemic level. In three other animals ADC returned to near control within 1 h of recirculation. The comparison of ADC changes with previously reported changes in extracellular volume revealed a close relationship, supporting the notion that ADC is a function of the intra/extracellular water compartmentation. Recovery of ADC correlated closely with tissue pH and metabolic recovery, studied 3 h after the initiation of recirculation. Animals without recovery of ADC exhibited global depletion of ATP and glucose and severe lactacidosis, whereas animals with recovery of ADC showed replenishment of ATP and glucose to near control and a substantial reversal of lactacidosis. Our data demonstrate that imaging of ADC provides reliable information about the metabolic state of the brain and can be used to monitor, with high temporal and regional resolution, the manifestation and reversal of ischemic brain injury.

Transient cerebral ischemia causes long-lasting inhibition of protein synthesis despite recovery of energy metabolism. We investigated the question if this inhibition is due to the formation of a suppression factor which interferes with the function of the protein synthesizing machinery. For this purpose rats were submitted to 20 minutes four vessel-occlusion followed by recirculation times from 30 minutes to 7 days. Post-mitochondrial supernatant (PMS) from various brain regions was added to a self-contained, cell-free rabbit reticulocyte translational system, and the effect onin vitro protein synthesis was assessed by measuring14C-leucine incorporation over a duration of 45 minutes. PMS prepared at the end of ischemia from hippocampus, striatum and cerebellum inhibitedin vitro protein synthesis by 40%–60% but there was only a minor inhibition by PMS from cerebral cortex. During post-ischemic recirculation cortical PMS transiently induced inhibition ofin vitro protein synthesis by 30% but this effect gradually disappeared within one week. The inhibition caused by PMS from hippocampus, striatum and cerebellum was not reversed during recirculation and still amounted to about 40% after 7 days. Inhibition ofin vitro protein synthesis could be blocked by heating PMS to 100°C, indicating that the suppressor factor is a protein. The comparison of thein vitro effect of postischemic PMS with previously describedin vivo inhibition of protein synthesis demonstrates that the here observed suppressor factor is not able to explain the overall disturbance of protein synthesisin vivo. However, the inhibitory potency of this factor after as long as 7 days after recirculation points to an ongoing pathological process, the importance of which remains to be clarified.

Biosynthesis and accumulation of the polyamines putrescine, spermidine, and spermine are closely associated with cellular growth processes. We examined polyamine levels and the activity of their first rate-limiting enzyme, ornithine decarboxylase (ODC), in stereotactically induced experimental gliomas of the rat brain 1 and 2 weeks after implantation. Regional ODC activity and polyamine levels were determined in the tumor and in the ipsi- and contralateral striatum, white matter, and cerebral cortex. In the tumor, both ODC activity and polyamine levels markedly increased with progressive tumor growth, as compared to those in the white matter of the opposite hemisphere. In the peritumoral brain tissue, ODC activity did not change, but there was a marked increase of putrescine and, to a lesser degree, of spermidine and spermine almost throughout the whole ipsilateral hemisphere. ODC activity, therefore, seems to be a reliable marker of neoplastic growth in the brain, which may be of use for new clinical concepts of the diagnosis and therapy of brain tumors. The more diffuse distribution of polyamines, however, may be associated with the formation and spreading of edema, which would explain some of the biological effects of tumors on distant brain tissue.

The applicability of the synthetic manganese porphyrin MnTPPS as tumor-selective MRI-contrast agent for brain tumors was investigated quantitatively by in vivo relaxometry. To exclude toxic effects of side products, the purification method for MnTPPS was improved. As a result, MnTPPS+Cl- (free acid) with a purity of more than 99.8% was obtained. For the in vivo quantification of the contrast effect the magnetization M(0) and the transversal relaxation time T2 were evaluated in different regions of rat brains with or without implanted gliomas and in temporal muscle. Measurements were performed before and after the application of MnTPPS. The ratio of the magnetization M(0) (TR = 3.5 sec) of the tissue under investigation and the contralateral striatum was defined as contrast ratio Rc(0). Without MnTPPS Rc(0) of edema (0.93 +/- 0.08) and tumor (0.91 +/- 0.07) did not differ from normal brain tissue (corpus callosum: 0.96 +/- 0.07; cortex: 0.98 +/- 0.05). T2 of edema (110 +/- 12 msec) and intracranial tumor (93 +/- 7 msec) were significantly longer than in normal tissue (corpus callosum: 73 +/- 8 msec; parietal cortex: 75 +/- 6 msec; striate nucleus: 78 +/- 7 msec, p < .01). Two hours after the injection of MnTPPS, magnetization of neoplastic tissue was selectively enhanced (TR = 3.5 sec), and T2 was reduced. The smallest dose of 0.06 mmol/kg body weight (bw) increased Rc(0) to 1.12 +/- 0.04, and 0.38 mmol/kg to 1.30 +/- 0.13 (p < .01). T2 of tumor decreased to 85 +/- 6 msec after 0.06 mmol/kg and to 65 +/- 6 msec after 0.38 mmol/kg bw.(ABSTRACT TRUNCATED AT 250 WORDS)

In a set of four interconnected papers DeGracia presents a novel network-based explanatory framework for conceptualizing the outcome of ischemic injury that deviates sharply from the currently prevailing approach of linear injury superposition. The thorough analysis of the increasing use of network theory in modern biology pro-vides the background for envisioning a new chapter of brain ischemia research which may be able to overcome the present dilemma of stroke treatment.

A brief period of cerebral ischemia is followed by severe inhibition of protein synthesis which is slowly reversed in the resistant but not in the selectively vulnerable regions of the brain. Inhibition occurs at the translational level, as evidenced by the disaggregation of ribosomes into monosomes. In order to evaluate the importance of this disturbance for the evolution of ischemic injury, the effect of the neuroprotective drug, pentobarbital, on ribosomal aggregation was studied in gerbils subjected to 5 min bilateral carotid artery occlusion. Pentobarbital (50 mg/kg, i.p.) was applied shortly after the ischemia, and the aggregational state of ribosomes was investigated by electron microscopy after recirculation times ranging from 15 min to 1 day. Pentobarbital treatment did not prevent the initial post-ischemic disaggregation but promoted the subsequent reaggregation in the selectively vulnerable neurons. This suggests that post-ischemic application of barbiturates exerts its beneficial effect by reversing the post-ischemic block of ribosomal reaggregation in vulnerable regions.

The ultrafast inversion recovery snapshot FLASH technique was used to determine the kinetics of the contrast agent manganese (III) tetraphenylporphine sulfonate (MnTPPS) in experimental brain tumors in rats. In the first part of the investigation this technique was validated with the conventional inversion recovery spin-echo method by comparing in vivo T1 data of a normal rat brain. Agreement between T1 values obtained from both techniques was complete, as tested for a large number of pixels in identical coronal slices. In the second part the fast IR snapshot FLASH method was applied to study the effect of the NMR contrast agent MnTPPS on the T1 relaxation time of experimental gliomas in rat brains. T1 of normal brain tissue (1024-1035 ms), tumor (1217 ms), and edema (1199 ms) was determined with the inversion recovery version of the snapshot FLASH imaging technique. After intraperitoneal injection of MnTPPS (0.25 mmol/kg body wt) T1 decreased exponentially to 56% of control in tumor and to 62% in muscle. In normal and edematous brain tissue no significant changes in T1 were observed up to 5 h after injection of the contrast agent. Once the T1 contrast between tumor and peritumoral brain tissue had reached a saturation, the enhancement persisted for several hours to days. Therefore application of this contrast agent resulted in a sharp demarcation between glioma and peri-tumoral edema.

The regional distribution of ATP, glucose, lactate and tissue pH was studied by bioluminescence and fluoroscopic imaging of intact cryostat sections of implantation tumors of cat. In tumors, marked heterogeneity of metabolites and pH was present: in solid parts ATP was similar to normal brain but glucose tended to decrease with an increase in lactate and pH to above normal; in necrobiotic regions ATP declined and pH became acidic. In peritumoral edema, ATP was consistently decreased whereas glucose, lactate and pH increased above normal. Correlation of ATP with water revealed an inverse relationship both in tumor and peritumoral edema but correlation of water with pH was direct in edema and inverse in tumors. The associations and dissociations of lactate, ATP and pH are interpreted in terms of aerobic and anaerobic glycolysis, as well as in respect to the extracellular localization of tumor edema. The findings are of relevance for the interpretation of volume selective NMR spectra and stress the importance of precise volume localization for differentiating between edema and tumor-mediated metabolic alterations.