Helmut Schröck

Mechanisms of stroke protection by physical activity

Regular physical activity is associated with a decrease of cerebrovascular and cardiovascular events, which may relate to enhanced endothelium‐dependent vasodilation. Here, we provide evidence that physical activity protects against ischemic stroke via mechanisms related to the upregulation of endothelial nitric oxide synthase (eNOS) in the vasculature. Voluntary training on running wheels or exercise on a treadmill apparatus for 3 weeks, respectively, reduced cerebral infarct size and functional deficits, improved endothelium‐dependent vasorelaxation, and augmented cerebral blood flow in wild‐type mice. The neuroprotective effects of physical training were completely absent in eNOS‐deficient mice, indicating that the enhanced eNOS activity by physical training was the predominant mechanism by which this modality protects against cerebral injury. Our results suggest that physical activity not only decreases stroke risk, but also provides a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

Physical Activity Improves Long-Term Stroke Outcome via Endothelial Nitric Oxide Synthase–Dependent Augmentation of Neovascularization and Cerebral Blood Flow

Physical activity upregulates endothelial nitric oxide synthase (eNOS), improves endothelium function, and protects from vascular disease. Here, we tested whether voluntary running would enhance neovascularization and long-term recovery following mild brain ischemia. Wild-type mice were exposed to 30 minutes of middle-cerebral artery occlusion (MCAo) and reperfusion. Continuous voluntary running on wheels conferred long-term upregulation of eNOS in the vasculature and of endothelial progenitor cells (EPCs) in the spleen and bone marrow (BM). This was associated with higher numbers of circulating EPCs in the blood and enhanced neovascularization. Moreover, engraftment of TIE2/LacZ-positive BM-derived cells was increased in the ischemic brain. Four weeks after the insult, trained animals showed higher numbers of newly generated cells in vascular sites, increased density of perfused microvessels and sustained augmentation of cerebral blood flow within the ischemic striatum. Moreover, running conferred tissue sparing and improved functional outcome at 4 weeks. The protective effects of running on angiogenesis and outcome were completely abolished when animals were treated with a NOS inhibitor or the antiangiogenic compound endostatin after brain ischemia, and in animals lacking eNOS expression. Voluntary physical activity improves long-term stroke outcome by eNOS-dependent mechanisms related to improved angiogenesis and cerebral blood flow.

Intracerebroventricular injection of streptozotocin induces discrete local changes in cerebral glucose utilization in rats

The purpose of the present study was to investigate whether or not cerebral glucose utilization is changed locally after damage of the neuronal insulin receptor by means of intracerebroventricular (icv) streptozotocin (STZ) administered in a subdiabetogenic dosage (1.5 mg/kg bw.). STZ was administered at the start of the study, and 2 and 21 days later bilaterally into the cerebral ventricles in rats of a mean age of 18 months. The local distribution of cerebral glucose utilization was analyzed in conscious rats on the 42nd day after the first STZ injection using the quantitative (14C)‐2‐deoxyglucose method. Of the 35 brain structures investigated from autoradiograms of brain sections, 17 showed a reduction in glucose utilization. Decreases in glucose utilization were observed in the frontal, parietal, sensory motor, auditory and entorhinal cortex and in all hippocampal subfields. In contrast, glucose utilization was increased in two white matter structures. The decrease in cerebral glucose utilization observed in cortical and hippocampal areas in the present study may correspond to changes in morphobiological parameters which have been found in patients with Alzheimers disease. The present data are in accordance with the hypothesis that an impairment in the control of neuronal glucose metabolism at the insulin receptor site may exist in sporadic dementia of Alzheimer type (DAT), and can be studied by the icv STZ animal model.

Lack of Capillary Recruitment in the Brains of Awake Rats during Hypercapnia

The present study investigates the question of whether increases in CBF induced by hypercapnia in awake rats are accompanied by increases in the number of perfused capillaries. For the detection of perfused capillaries, gamma-globulin-coupled fluorescein isothiocyanate was injected intravenously. In 10 brain structures the density of perfused capillaries per square millimeter was determined from coronal sections using a highly sensitive fluorescent microscopical method that, in contrast to others, avoided air drying of the frozen brain sections. The results showed an inhomogeneous local distribution of the density of perfused capillaries during normo- and hypercapnia. The density of perfused capillaries was unchanged during hypercapnia compared with normocapnia, although blood flow was markedly increased. It is concluded that a capillary recruitment does not exist in the brain during the high-flow situation of hypercapnia.

Gross persistence of capillary plasma perfusion after middle cerebral artery occlusion in the rat brain

The densities of perfused and existing capillaries were measured in different cortical regions of rat brains after subtemporal occlusion of the middle cerebral artery (MCA). Capillary perfusion patterns (perfused capillaries versus nonperfused capillaries) were verified immediately after MCA occlusion in one group of rats and 1 h later in a second group using fluorescent double staining of capillary morphology and plasma perfusion in identical brain sections. In addition, local cerebral blood flow (CBF) was measured in another group of rats 1 h after MCA occlusion using the autoradiographic iodo-[14C]-antipyrine method. Although cortical CBF was decreased by up to 75% 1 h after MCA occlusion, plasma perfusion was not completely stopped in most capillaries (circulation time of Evans blue, 10 s). Only small patchy perfusion deficits (<0.1 mm2 of brain section) were detected in the capillaries immediately and 1 h after MCA occlusion in all brains except for one that exhibited a more extensive lack of capillary perfusion. The data show that a drastic reduction of cortical CBF after MCA occlusion is not accompanied by a corresponding amount of nonperfused capillaries.

The functional genome of CA1 and CA3 neurons under native conditions and in response to ischemia

BackgroundThe different physiological repertoire of CA3 and CA1 neurons in the hippocampus, as well as their differing behaviour after noxious stimuli are ultimately based upon differences in the expressed genome. We have compared CA3 and CA1 gene expression in the uninjured brain, and after cerebral ischemia using laser microdissection (LMD), RNA amplification, and array hybridization.ResultsProfiling in CA1 vs. CA3 under normoxic conditions detected more than 1000 differentially expressed genes that belong to different, physiologically relevant gene ontology groups in both cell types. The comparison of each region under normoxic and ischemic conditions revealed more than 5000 ischemia-regulated genes for each individual cell type. Surprisingly, there was a high co-regulation in both regions. In the ischemic state, only about 100 genes were found to be differentially expressed in CA3 and CA1. The majority of these genes were also different in the native state. A minority of interesting genes (e.g. inhibinbetaA) displayed divergent expression preference under native and ischemic conditions with partially opposing directions of regulation in both cell types.ConclusionThe differences found in two morphologically very similar cell types situated next to each other in the CNS are large providing a rational basis for physiological differences. Unexpectedly, the genomic response to ischemia is highly similar in these two neuron types, leading to a substantial attenuation of functional genomic differences in these two cell types. Also, the majority of changes that exist in the ischemic state are not generated de novo by the ischemic stimulus, but are preexistant from the genomic repertoire in the native situation. This unexpected influence of a strong noxious stimulus on cell-specific gene expression differences can be explained by the activation of a cell-type independent conserved gene-expression program. Our data generate both novel insights into the relation of the quiescent and stimulus-induced transcriptome in different cells, and provide a large dataset to the research community, both for mapping purposes, as well as for physiological and pathophysiological research.

Autoradiographic analysis of the regional distribution of Glut3 glucose transporters in the rat brain

Glut3 is a glucose transporter protein which facilitates the transport of glucose across the neuronal membranes. The local distribution of Glut3 in the brain is not well known. The present study had the aim to verify the local distribution of Glut3 in the brain and to compare it with the local glucose utilization. A polyclonal antibody directed against the C-terminal peptide sequence of Glut3 was applied to cryosections of rat brains. A secondary antibody was added which had been coupled to 35S. Using autoradiography and radioactive standards, 17 cerebral structures were investigated. The results show moderate differences of Glut3 density in the structures investigated ranging from -23% to +41% of the mean density. The pineal gland was an exception with a density 66% lower than mean. Local cerebral glucose utilization (LCGU) was analyzed in identical brain structures by application of the quantitative autoradiographic 2-deoxyglucose method to conscious rats. The range of LCGU was from -59% to +55% of the mean. No correlation was found between the moderately heterogeneous Glut3 transporter density and the strongly heterogeneous local cerebral glucose utilization. The results show that the local density of Glut3 glucose transporter protein does not reflect the local level of glucose utilization in the brain.

Impact of Actin Filament Stabilization on Adult Hippocampal and Olfactory Bulb Neurogenesis

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca2+-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn−/−) mice as a model system for actin filament stabilization. In Gsn−/− mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca2+]i increases and exocytotic neurotransmitter release were enhanced in Gsn−/− synaptosomes. Importantly, treatment of Gsn−/− synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca2+ influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn−/− brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn−/− mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca2+ influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn−/− hippocampus is associated with a special vascular niche for neurogenesis.

The influence of nicotine on local cerebral blood flow in rats.

Local cerebral blood flow (LCBF) was measured in conscious rats during an acute nicotine infusion. LCBF was measured using the autoradiographic iodoantipyrine method. LCBF was unchanged in most brain structures during nicotine infusion compared to controls. Significant (P less than 0.05) increases were found in 3 structures (lateral geniculate body, superior colliculus, anteroventral nucleus of the thalamus). These structures have already shown increases in local glucose utilization in a previous study [2]. The observed increases in LCBF are interpreted to be secondary to metabolic activation by nicotine indicating the lack of a direct action of nicotine on cerebral blood vessels.

A flow sensitive alternating inversion recovery (FAIR)-MRI protocol to measure hemispheric cerebral blood flow in a mouse stroke model.

Blood flow imaging is an important tool in cerebrovascular research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides three-dimensional noninvasive quantitative methods of cerebral blood flow (CBF) imaging, but these MRI techniques have not yet been validated for mice. The authors compared CBF imaging using flow sensitive alternating inversion recovery (FAIR)-MRI and (14)C-Iodoantipyrine (IAP)-autoradiography in a mouse model of acute stroke. Twenty-nine male 129S6/SvEv mice were subjected to filamentous left middle cerebral artery occlusion (MCAo). CBF imaging was performed with (14)C-IAP autoradiography and FAIR-MRI using two different anesthesia protocols, namely intravenous infusion of etomidate or inhalation of isoflurane, which differentially affect perfusion. Using (14)C-IAP autoradiography, the average CBF in ml/(100 g*min) was 160+/-34 (isoflurane, n=5) vs. and 59+/-21 (etomidate, n=7) in the intact hemisphere and 43+/-12 (isoflurane, n=5) vs. 36+/-12 (etomidate, n=7) in the MCAo hemisphere. Using FAIR-MRI, the corresponding average CBFs were 208+/-56 (isoflurane, intact hemisphere, n=7), 84+/-9 (etomidate, intact hemisphere, n=7), 72+/-22 (isoflurane, MCAo hemisphere, n=7) and 48+/-13 (etomidate, MCAo hemisphere, n=7). Regression analysis showed a strong linear correlation between CBF measured with FAIR-MRI and (14)C-IAP autoradiography, and FAIR-MRI overestimated CBF compared to autoradiography. FAIR-MRI provides repetitive quantitative measurements of hemispheric CBF in a mouse model of stroke.