Hyperlipidemia attenuates vascular endothelial growth factor-induced angiogenesis, impairs cerebral blood flow, and disturbs stroke recovery via decreased pericyte coverage of brain endothelial cells.

OBJECTIVE: Therapeutic angiogenesis aims at the promotion of vascular growth, usually under conditions of atherosclerosis. It was unknown how hyperlipidemia, a risk factor that is closely associated with atherosclerosis of brain vessels in humans, influences vascular endothelial growth factor-induced angiogenesis and stroke recovery. APPROACH AND RESULTS: Wild-type and apolipoprotein-E (ApoE)(-/-) mice were kept on regular or cholesterol-rich diet for mimicking different severities of hyperlipidemia. Mice were treated intracerebroventricularly with recombinant human vascular endothelial growth factor for 21 days (0.02 µg/d) and subsequently subjected to 90-minute middle cerebral artery occlusion followed by 1 or 24 hours of reperfusion. Histochemical, autoradiographic, and regional bioluminescence techniques were used to evaluate effects of blood lipids on postischemic angiogenesis, histopathologic brain injury, cerebral blood flow, protein synthesis and energy state, and pericyte coverage of brain endothelial cells. Hyperlipidemia dose-dependently attenuated vascular endothelial growth factor-induced capillary formation and pericyte coverage of brain endothelial cells, abolishing the improvement of cerebral blood flow during subsequent stroke, resulting in the loss of the metabolic penumbra and increased brain infarction. The enhanced angiogenesis after vascular endothelial growth factor treatment was accompanied by increased expression of the adhesion protein N-cadherin, which mediates endothelial-pericytic interactions, in ischemic brain microvessels of wild-type mice on regular diet that was blunted in wild-type mice on Western diet and ApoE(-/-) mice on either diet. CONCLUSIONS: The compromised vessel formation and hemodynamics question the concept of therapeutic angiogenesis in ischemic stroke where hyperlipidemia is highly prevalent.

Vascular endothelial growth factor promotes pericyte coverage of brain capillaries, improves cerebral blood flow during subsequent focal cerebral ischemia, and preserves the metabolic penumbra.

BACKGROUND AND PURPOSE: Therapeutic angiogenesis aims at improving cerebral blood flow by amplification of vascular sprouting, thus promoting tissue survival under conditions of subsequent ischemia. It remains unknown whether induced angiogenesis leads to the formation of functional vessels that indeed result in hemodynamic improvements. Observations of hemodynamic steal phenomena and disturbed neurovascular integrity after vascular endothelial growth factor delivery questioned the concept of therapeutic angiogenesis. METHODS: Mice were treated with recombinant human vascular endothelial growth factor (0.02 µg/d; intracerebroventricular) for 3 to 21 days and subsequently exposed to 90-minute middle cerebral artery occlusion. Angiogenesis, histological brain injury, IgG extravasation, cerebral blood flow, protein synthesis and energy state, and pericyte coverage on brain capillaries were evaluated in a multiparametric approach combining histochemical, autoradiographic, and regional bioluminescence techniques. RESULTS: Vascular endothelial growth factor increased brain capillary density within 10 days and reduced infarct volume and inflammation after subsequent middle cerebral artery occlusion, and, when delivered for prolonged periods of 21 days, enhanced postischemic blood-brain barrier integrity. Increased cerebral blood flow was noted in ischemic brain areas exhibiting enhanced angiogenesis and was associated with preservation of the metabolic penumbra, defined as brain tissue in which protein synthesis has been suppressed but ATP preserved. Vascular endothelial growth factor enhanced pericyte coverage of brain endothelial cells via mechanisms involving increased N-cadherin expression on cerebral microvessels. CONCLUSIONS: That cerebral blood flow is increased during subsequent ischemic episodes, leading to the stabilization of cerebral energy state, fosters hope that by promoting new vessel formation brain tissue survival may be improved.

Changes of cerebral blood flow during the secondary expansion of a cortical contusion assessed by 14C-iodoantipyrine autoradiography in mice using a non-invasive protocol.

Although changes of cerebral blood flow (CBF) in and around traumatic contusions are well documented, the role of CBF for the delayed death of neuronal cells in the traumatic penumbra ultimately resulting in secondary contusion expansion remains unclear. The aim of the current study was therefore to investigate the relationship between changes of CBF and progressive peri-contusional cell death following traumatic brain injury (TBI). CBF and contusion size were measured in C57Bl6 mice under continuous on-line monitoring of (ETp)CO2 before, and at 15 min and 24 h following controlled cortical impact by 14C-iodoantipyrine autoradiography (IAP-AR; n = 5-6 per group) and by Nissl staining, respectively. Contused and ischemic (CBF < 10%) tissue volumes were calculated and compared over time. Cortical CBF in not injured mice varied between 69 and 93 mL/100mg/min depending on the anatomical location. Fifteen minutes after trauma, CBF decreased in the whole brain by approximately 50% (39 +/- 18 mL/100mg/min; p < 0.05), except in contused tissue where it fell by more than 90% (3 +/- 2 mL/100mg/min; p < 0.001). Within 24 h after TBI, CBF recovered to normal values in all brain areas except the contusion where it remained reduced by more than 90% (p < 0.001). Contusion volume expanded from 24.9 to 35.5 mm3 (p < 0.01) from 15 min to 24 h after trauma (+43%), whereas the area of severe ischemia (CBF < 10%) showed only a minimal (+13%) and not significant increase (22.3 to 25.1 mm3). The current data therefore suggest that the delayed secondary expansion of a cortical contusion following traumatic brain injury may not be caused by a reduction of CBF alone.

Establishing a photothrombotic 'ring' stroke model in adult mice with late spontaneous reperfusion: quantitative measurements of cerebral blood flow and cerebral protein synthesis.

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 (lCBF) measured by laser-Doppler flowmetry declined promptly after irradiation to 43% of the baseline value at 30 mins poststroke. Using double tracer autoradiography, quantitative lCBF 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.

Ischemia induces a translocation of the splicing factor tra2-beta 1 and changes alternative splicing patterns in the brain.

Alternative splice-site selection is regulated by the relative concentration of individual members of the serine-arginine family of proteins and heterogeneous nuclear ribonucleoproteins. Most of these proteins accumulate predominantly in the nucleus, and a subset of them shuttles continuously between nucleus and cytosol. We demonstrate that in primary neuronal cultures, a rise in intracellular calcium concentration induced by thapsigargin leads to a translocation of the splicing regulatory protein tra2-beta1 and a consequent change in splice-site selection. To investigate this phenomenon under physiological conditions, we used an ischemia model. Ischemia induced in the brain causes a cytoplasmic accumulation and hyperphosphorylation of tra2-beta1. In addition, several of the proteins binding to tra2-beta1, such as src associated in mitosis 68 and serine/arginine-rich proteins, accumulate in the cytosol. Concomitant with this subcellular relocalization, we observed a change in alternative splice-site usage of the ICH-1 gene. The increased usage of its alternative exons is in agreement with previous studies demonstrating its repression by a high concentration of proteins with serine/arginine-rich domains. Our findings suggest that a change in the calcium concentration associated with ischemia is part of a signaling event, which changes pre-mRNA splicing pathways by causing relocalization of proteins that regulate splice-site selection.

Therapeutic induction of arteriogenesis in hypoperfused rat brain via granulocyte-macrophage colony-stimulating factor.

BACKGROUND: Colony-stimulating factors (CSFs) have been shown to effectively induce arteriogenesis in the hindlimb. Moreover, clinical trials demonstrated positive effects of CSFs on arteriogenesis in patients with coronary artery disease. However, patients with cerebrovascular disease have not yet profited from treatments aimed at the growth of brain vessels. Thus far, angiogenesis studies have failed to demonstrate improvement of stroke outcome. Arteriogenesis differs from angiogenesis in that it substitutes arterial collaterals for the occluded artery. METHODS AND RESULTS: We tested in a novel brain arteriogenesis rat model (occlusion of vertebral plus left carotid artery [3-VO]) the application of CSFs or saline over 7 or 21 days. On 3-VO postmortem, latex perfusion demonstrated a time- and treatment-dependent arteriogenesis of the posterior cerebral artery (PCA). In saline-treated animals, the PCA diameter increased by 39%; in granulocyte-macrophage (GM)-CSF-treated animals, this increase was significantly faster (72% after 1 week). Functionally, saline-treated animals exhibited a decline of CO2 reactivity (mm Hg) from 1.48% to 0.1% compared with GM-CSF-treated animals (1.43% arterial pCo2 change after 1 week). This difference remained significant after 3 weeks. This functional improvement correlated with increased numbers of CD68-positive macrophages in histological sections of the PCA in GM-CSF--treated animals and only a few macrophages in saline-treated animals. CONCLUSIONS: To the best of our knowledge, this is the first report of stimulation of arteriogenesis in the brain. The subcutaneous application of GM-CSF led to functional improvement of brain hemodynamic parameters.

Ischemic injury in experimental stroke depends on angiotensin II.

Since pharmacological interactions of the renin-angiotensin system appear to alter the neurological outcome of stroke patients significantly, we examined the effect of elevated levels of angiotensin II and the role of its receptor subtype AT1 in brain infarction in transgenic mice after focal cerebral ischemia. Angiotensinogen-overexpressing and angiotensin receptor AT1 knockout mice underwent 1 h or 24 h permanent middle cerebral artery occlusion (MCAO). The current study revealed a much smaller penumbra size, i.e., brain tissue at risk, in angiotensinogen-overexpressing animals compared with their wild-type subgroup after 1 h MCAO, but an enlarged infarct size after 24 h. In contrast, a smaller lesion area of energy failure and a much larger penumbral area were found in AT1 knockout mice compared with wild-type littermates. Lower perfusion thresholds for ATP depletion and protein synthesis inhibition after MCAO in AT1-deficient mice and reduced cell damage in an in vitro model using embryonic neurons of AT1 knockout mice suggest injury mechanisms independent of arterial blood pressure. Our data, therefore, demonstrate a direct correlation between brain angiotensin II and the severity of ischemic injury in experimental stroke.

Longitudinal assessment of infarct progression, brain metabolism and behavior following anterior cerebral artery occlusion in rats.

BACKGROUND: Stroke patients suffering from occlusion of the anterior cerebral artery (ACAo) develop cognitive and executive deficits. Experimental models to investigate such functional impairments and recovery are rare and not satisfyingly validated. NEW METHOD: We stereotactically injected the vasoconstrictor endothelin-1 (ET-1) close to the ACA of rats and assessed magnitude and course of CBF reduction using [(14)C]iodoantipyrine autoradiography and [(15)O]H2O-PET. [(18)F]FDG-PET and T2-weighted MRI determined regional metabolic and structural alterations. To test cognitive and executive functions, we analyzed decision-making in a food-carrying task, spatial working memory in a spontaneous alternation task and anxiety in an elevated plus maze test before and 1 month after ACAo. RESULTS: CBF decreased immediately after ET-1 injection, started to recover 1-2h and returned to control 4h thereafter. Metabolic and structural lesions developed permanently in the ACA territory. Hypometabolism occurring bilaterally in the piriform region may reflect diaschisis. Behavioral testing after ACAo revealed context-dependent changes in decision making, exploratory activity and walking speed, as well as decreased anxiety and spatial working memory. COMPARISON WITH EXISTING METHOD(S): Aside from modeling a known entity of stroke patients, ACAo in rats allows to longitudinally study deterioration of cognitive and executive function without major interference by disturbed primary motor function. It complements therefore stroke research since common models using middle cerebral artery occlusion (MCAo) all affect motor function severely. CONCLUSION: The established ACAo model in rats effectively reflects deficits characteristic for ACA stroke in humans. It is furthermore highly suitable for longitudinal assessment of cognitive and executive functions.

Characterization of a novel chronic photothrombotic ring stroke model in rats by magnetic resonance imaging, biochemical imaging, and histology.

A novel photothrombotic ring stroke model was characterized by multiparametric magnetic resonance imaging, imaging of cerebral blood flow (CBF), adenosine triphosphate (ATP), pH, and histology. Ischemia was initiated by transosseous irradiation of a predefined brain area intravenously perfused by the photosensitive dye erythrosin B in male Wistar rats. In the region of the primary ring-lesion, the phototoxic reaction caused necrosis reflected by low relative ATP levels (28 +/- 15%), alkalosis (pH: 7.35 +/- 0.50), and histologic evidence at 14 days after lesion induction. In the ring-encircled interior region (region-at-risk), spontaneous tissue reperfusion (relative CBF: 93 +/- 3%) enabled partial tissue preservation. This was demonstrated by a less impaired energy metabolism (ATP: 65 +/- 23%), normal pH (7.01 +/- 0.50), and still normal cellular structures shown by histologic staining. Analysis of the temporal characteristics within the region-at-risk revealed a slow continuous increase of the apparent diffusion coefficient of water (ADC) to 144 +/- 16% of control (14d) and an early vasogenic edema, reflected by an increase of the T2 relaxation time to 143 +/- 17% of control (2d). Both final ADC and T2 correlated well with the tissue pH within the region-at-risk, thus emphasizing the usefulness of this multiparametric noninvasive imaging approach.

Arteriogenesis in hypoperfused rat brain.

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% CO(2) 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 CO(2) 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 CO(2) 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.

Spreading depression activates unfolded protein response.

Preconditioning is a process where a preceding non-lethal form of stress activates a stress response that protects cells against an otherwise lethal form of stress. Preconditioning can be induced in various ways including short-term ischemia or spreading depression. Here we investigated the effect of 1 h repetitive spreading depression on the unfolded protein response (UPR), a stress response activated under conditions associated with endoplasmic reticulum (ER) dysfunction. Spreading depression induced processing of xbp1 mRNA, indicative of an activation of UPR. Processing of xbp1 was paralleled by a rise in grp78 mRNA levels resulting from an activation of a signal transduction pathway that depends on protein synthesis. Preconditioning-induced activation of UPR may preserve ER functioning under pathological conditions interfering with ER functions.

Heterozygosity for the mutated X-chromosome-linked L1 cell adhesion molecule gene leads to increased numbers of neurons and enhanced metabolism in the forebrain of female carrier mice.

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.

Conflict Processing in the Rat Brain: Behavioral Analysis and Functional µPET Imaging Using [F]Fluorodeoxyglucose.

Conflicts in spatial stimulus-response tasks occur when the task-relevant feature of a stimulus implies a response toward a certain location which does not match the location of stimulus presentation. This conflict leads to increased error rates and longer reaction times, which has been termed Simon effect. A model of dual route processing (automatic and intentional) of stimulus features has been proposed, predicting response conflicts if the two routes are incongruent. Although there is evidence that the prefrontal cortex, notably the anterior cingulate cortex (ACC), plays a crucial role in conflict processing, the neuronal basis of dual route architecture is still unknown. In this study, we pursue a novel approach using positron emission tomography (PET) to identify relevant brain areas in a rat model of an auditory Simon task, a neuropsychological interference task, which is commonly used to study conflict processing in humans. For combination with PET we used the metabolic tracer [(18)F]fluorodeoxyglucose, which accumulates in metabolically active brain cells during the behavioral task. Brain areas involved in conflict processing are supposed to be activated when automatic and intentional route processing lead to different responses (dual route model). Analysis of PET data revealed specific activation patterns for different task settings applicable to the dual route model as established for response conflict processing. The rat motor cortex (M1) may be part of the automatic route or involved in its facilitation, while premotor (M2), prelimbic, and ACC seemed to be essential for inhibiting the incorrect, automatic response, indicating conflict monitoring functions. Our findings and the remarkable similarities to the pattern of activated regions reported during conflict processing in humans demonstrate that our rodent model opens novel opportunities to investigate the anatomical basis of conflict processing and dual route architecture.

Oxygen therapy improves energy metabolism in focal cerebral ischemia.

Oxygen therapy (OT) with hyperbaric oxygen (HBO) or normobaric hyperoxia (NBO) improves the oxygenation of penumbral tissue in experimental ischemic stroke. However, whether this results in the improvement of energy metabolism is unclear. We investigated the effect of both OTs on tissue acidosis and on ATP production. Beginning 25 min after filament middle cerebral artery occlusion (MCAO), mice breathed either air, 100% O2 (NBO), or 100% O2 at 3 ata (HBO) for 60 min. Regional tissue pH was measured using the umbelliferone fluorescence. Regional ATP concentration was depicted by substrate-specific bioluminescence. Severity of ischemia did not differ among groups in laser-Doppler flowmetry. Both NBO (70.1±14.0 mm³) and, more effectively, HBO (57.2±11.9 mm³) significantly reduced volume of tissue acidosis compared to air (89.4±4.0 mm³), p<0.05). Topographically, acidosis was less pronounced in the medial striatum and in the cortical ischemic border areas. This resulted in significantly smaller volumes of ATP depletion (77.8±7.7 mm³ in air, 61.4±15.2 mm³ in NBO and 51.2±14.4 mm³ in HBO; p<0.05). In conclusion, OT significantly improves energy metabolism in the border zones of focal cerebral ischemia which are the areas protected by OT in this model.

Whiskers area as extracerebral reference tissue for quantification of rat brain metabolism using (18)F-FDG PET: application to focal cerebral ischemia.

UNLABELLED: Diseases and dysfunction of the central nervous system are often associated with regional changes in cerebral glucose metabolism, which can be measured in vivo by PET using (18)F-FDG as the tracer. For quantification, the arterial tracer input function must be determined. For rodents in particular, direct measurement of blood radioactivity concentration is scarcely feasible for follow-up of individual animals because of the invasiveness of blood sampling. We show that the whiskers area of the rat's muzzle serves as an extracerebral reference region. The derived model also takes into account local variations of the lumped constant, which is crucial in pathologic tissue. METHODS: In 11 rats, the reference tissue kinetic parameters were determined from PET data and measured whole blood radioactivity concentration. Parametric images of cerebral kinetic rate constants were calculated using the directly measured input function, the reference tissue time-activity curve with individually fitted reference kinetic parameters, and the reference time-activity curve with fixed reference kinetic parameters calculated from the fitted parameters averaged over all animals. The need for kinetic modeling in disease models is demonstrated in 5 rats subjected to acute focal cerebral ischemia. (18)F-FDG metabolism and transport rate constants and local cerebral glucose metabolic rates were calculated. RESULTS: Cerebral kinetic constants derived from the 3 methods corresponded closely. The maximum difference in whole-brain kinetic parameters observed between the directly measured input function and the reference tissue time-activity curve with individually fitted reference kinetic parameters was less than 5%. Taking fixed reference parameters (the reference time-activity curve with fixed reference kinetic parameters calculated from the fitted parameters averaged over all animals) still provided whole-brain kinetic parameters with an accuracy of approximately 90%. In the rats subjected to focal cerebral ischemia, (18)F-FDG kinetic parameters in healthy tissue were not significantly different from whole-brain kinetic parameters in naive rats. The ischemic region was characterized by preserved glucose metabolism, although (18)F-FDG uptake was elevated significantly-that is, the lumped constant in the ischemic region was different from that of healthy brain tissue. CONCLUSION: The method presented here allows for the quantitative noninvasive determination of cerebral glucose consumption in rats, takes into account local variations of the lumped constant, and is suitable for follow-up measurements of individuals.

Distinct spatiotemporal patterns of spreading depolarizations during early infarct evolution: evidence from real-time imaging.

Experimental and clinical studies indicate that waves of cortical spreading depolarization (CSD) appearing in the ischemic penumbra contribute to secondary lesion growth. We used an embolic stroke model that enabled us to investigate inverse coupling of blood flow by laser speckle imaging (CBF(LSF)) to CSD as a contributing factor to lesion growth already in the early phase after arterial occlusion. Embolization by macrospheres injected into the left carotid artery of anesthetized rats reduced CBF(LSF) in the territories of the middle cerebral artery (MCA) (8/14 animals), the posterior cerebral artery (PCA) (2/14) or in less clearly defined regions (4/14). Analysis of MCA occlusions (MCAOs) revealed a first CSD wave starting off during ischemic decline at the emerging core region, propagating concentrically over large portions of left cortex. Subsequent recurrent waves of CSD did not propagate concentrically but preferentially circled around the ischemic core. In the vicinity of the core region, CSDs were coupled to waves of predominantly vasoconstrictive CBF(LSF) responses, resulting in further decline of CBF in the entire inner penumbra and in expansion of the ischemic core. We conclude that CSDs and corresponding CBF responses follow a defined spatiotemporal order, and contribute to early evolution of ischemic territories.

Shut-down of translation, a global neuronal stress response: mechanisms and pathological relevance.

Shut-down of translation is a global stress response required to block synthesis of proteins that cannot be correctly folded and thereby reduce the work load of the folding machinery, a primary target of the pathological process triggered by severe forms of stress. The short-term control of protein synthesis involves alterations in the activity of initiation factors mediated through changes in their phosphorylation states, the alpha subunit of eukaryotic initiation factor 2 being a key player in this process. While the stress-induced shut-down of translation is viewed as a protective response, the inability of vulnerable cells to restore protein synthesis after being exposed to a severe form of stress is a pathological process because it blocks the translation of messages coding for protective proteins required for restoration of function. In models of cerebral ischemia, prolonged suppression of protein synthesis is therefore always associated with extensive cell death. Endoplasmic reticulum (ER) dysfunction has been identified as the mechanism underlying ischemia-induced suppression of protein synthesis. GADD34 is a protein that plays a pivotal role in the recovery of cells from shut-down of translation induced by ER stress. After transient ischemia, a rise in GADD34 protein levels has been found in resistant but not in vulnerable cells. Knowledge of the mechanisms activated in resistant cells to restore protein synthesis after severe stress will help open up new avenues for therapeutic strategies to combat various disorders of the brain associated with impairment of the translational machinery.

Immunohistochemical analysis of protein expression after middle cerebral artery occlusion in mice.

The effect of transient focal cerebral ischemia on protein regulation was studied in mice using multiparametric immunohistochemistry. Injury was characterized by measurements of blood flow, regional protein synthesis and terminal transferase biotinylated-dUTP nick end labeling (TUNEL). The proteins studied were selected from a previously established list of differentially regulated proteins and included the GTPases dynamin, RhoB, CAS and Ran BP-1, the transcription factors Nurr1 and p-Stat 6, the protein kinase MAPK p49, the splicing factors SRPK1 and hPrp16, the cell cycle control proteins cyclin B1 and Nek2, the inflammatory proteins FKBP12 and Rag2, the cell adhesion protein paxillin and the folding protein TCP-1. Regulation patterns were diverse and comprised ipsi- and/or contralateral up- and down-regulation with or without topical association to impeding cell death. Some proteins (SRPK1, TCP-1 and Nurr1) also exhibited post-ischemic translocation from the nucleus to the cytosol. Our observations stress the importance of regional analysis for the interpretation of proteomic data, and contribute to the identification of new pathways that may be involved in the evolution of post-ischemic brain injury.

Mechanisms underlying suppression of protein synthesis induced by transient focal cerebral ischemia in mouse brain.

Transient global cerebral ischemia triggers suppression of the initiation step of protein synthesis, a process which is controlled by endoplasmic reticulum (ER) function. ER function has been shown to be disturbed after transient cerebral ischemia, as indicated by an activation of the ER-resident eIF2alpha kinase PERK. In this study, we investigated ischemia-induced changes in protein levels and phosphorylation states of the initiation factors eIF2alpha, eIF2B epsilon, and eIF4G1 and of p70 S6 kinase, proteins playing a central role in the control of the initiation of translation. Transient focal cerebral ischemia was induced in mice by occlusion of the left middle cerebral artery. Transient ischemia caused a long-lasting suppression of global protein synthesis. eIF2alpha was transiently phosphorylated after ischemia, peaking at 1-3 h of recovery. eIF2B epsilon and p70 S6 kinase were completely dephosphorylated during ischemia and phosphorylation did not recover completely following reperfusion. In addition, eIF2B epsilon, eIF4G1, and p70 S6 kinase protein levels decreased progressively with increasing recirculation time. Thus, several different processes contributed to ischemia-induced suppression of the initiation of protein synthesis: a long-lasting dephosphorylation of eIF2B epsilon and p70 S6K starting during ischemia, a transient phosphorylation of eIF2alpha during early reperfusion, and a marked decrease of eIF2B epsilon, eIF4G1, and p70 S6K protein levels starting during vascular occlusion (eIF4G1). Study of the mechanisms underlying ischemia-induced suppression of the initiation step of translation will help to elucidate the role of protein synthesis inhibition in the development of neuronal cell injury triggered by transient cerebral ischemia.

Biochemical changes and gene expression following traumatic brain injury: Role of spreading depression.

The effects of spreading depression-like DC depolarizations on biochemical changes and gene expression were examined following trau-matic neocortical lesions, as induced by transcranial cold injury. The surrounding of traumatic cold lesions was characterized by increased glu-cose and lactate contents, 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. These changes were similar in animals with spontaneous spreading depression-like DC shifts (n = 14) and those without spreading depressions (n = 7), but there was a marked difference in the gene response. In injured animals without SD, only a short-lasting response of c-fos, junB, c-jun and MKP-1 mRNAs as well as c-Fos protein was bilaterally found in the piri-form cortex, and - with ipsilateral dominance - the dentate gyrus and hippocampal CA3/4 fields at 1 h after lesioning. In injure d animals with spreading depressions, on the contrary, a strong elevation was seen in layers II-IV and VI of the injury-remote ipsilateral cerebral cortex, which persisted over as long as 6 h. The expression of c-fos, junB and MKP-1 mRNAs was closely related to the time interval between the last spreading depression and the end of the experiments. Levels were highest shortly after transient DC shifts, and decreased thereafter mono-exponentially with half-lives of 48, 75 and 58 min for c-fos, junB and MKP-1 mRNAs, respectively. Thus, spreading depression is a prominent factor influencing the trauma-related gene response, but - in contrast to focal ischemia - it does not aggravate the metabolic dysfunction.