Wolfgang Kuschinsky

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.

Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA fragmentation and ischemic cell damage following middle cerebral artery occlusion in rats

Permanent occlusion of the middle cerebral artery in rats was used to assess the effects of focal ischemia on the expression of members of the bcl-2 family which have been implicated in the regulation of programmed cell death. Intraluminal occlusion of one middle cerebral artery for 6 h resulted in histologically detectable brain damage within the ipsilateral caudate putamen, basolateral cortex and parts of the thalamus. In the infarcted basolateral cortex and thalamus fragmentation of DNA was detected in many nuclei using in-situ end-labeling of DNA breaks by terminal transferase, whereas only scattered labeled nuclei were visible in the infarcted caudate putamen. Immunohistochemical analysis revealed activation of c-Fos in the infarcted cortex and thalamus and in the non-infarcted cingulate cortex as has been shown by others. A decrease in immunoreactivity for Bcl-2, and Bcl-X and an increase in immunostaining for Bax was observed exclusively in neurons within the ischemic cortex and thalamus. Within the infarcted caudate putamen, however, protein levels of all bcl-2 family members declined and c-Fos remained absent. By reverse transcription and polymerase chain reaction it was demonstrated that levels of bcl-2 mRNA markedly decreased in the ipsilateral hemisphere, whereas the amount of bax mRNA was elevated. These findings suggest that a shift in the ratio of cell death repressor Bcl-2 to cell death effector Bax and a concomitant activation of c-Fos may contribute to neuronal apoptosis in the infarcted thalamus and cortex.

Expression of vascular endothelial growth factor and its receptors in rat neural stem cells.

Neural stem cells serve neurogenesis in several areas of the adult mammalian brain. The present study investigates an additional feature, i.e. the expression of vascular endothelial growth factor (VEGF) and its receptors in cultured neurospheres from hippocampus, subventricular zone, and olfactory bulb in adult rats. RT-PCR showed that all three lines expressed VEGF mRNA, but different sets of VEGF receptor mRNAs. Stimulation by either 50 ng/ml VEGF-A165 or by 24 h of anoxia resulted in an altered pattern of receptor expression for VEGF-R2 (Flk-1), VEGF-R3 (Flt-4), neuropilin-1, and neuropilin-2, whereas VEGF-R1 (Flt-1) remained unexpressed. The basal expression of VEGF and of several of its receptor mRNAs indicates a hitherto unknown angiogenic potential of neural stem cells.

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.

Alterations in Rat Brain Proteins after Desflurane Anesthesia

BackgroundVolatile anesthetics disappear from an organism after the end of anesthesia. Whether changes of protein expression persist in the brain for a longer period is not known. This study investigates the question of whether the expression of proteins is altered in the rat brain after the end of desflurane anesthesia. MethodsThree groups (n = 12 each) of rats were anesthetized with 5.7% desflurane in air for 3 h. Brains were removed directly after anesthesia, 24 h after anesthesia, or 72 h after anesthesia. Two additional groups (n = 12 each) served as naive conscious controls, in which the brains were removed without previous anesthesia 3 or 72 h after the start of the experiment. Cytosolic proteins were isolated. A proteome-wide study was performed, based on two-dimensional gel electrophoresis and mass spectrometry. ResultsCompared with conscious controls, significant (P < 0.05) increase/decrease was found: 3 h of anesthesia, 5/2 proteins; 24 h after anesthesia, 13/1 proteins; 72 h after anesthesia, 6/4 proteins. The overall changes in protein expression as quantified by the induction factor ranged from −1.67 (decrease to 60%) to 1.79 (increase by 79%) compared with the controls (100%). Some of these regulated proteins play a role in vesicle transport and metabolism. ConclusionDesflurane anesthesia produces changes in cytosolic protein expression up to 72 h after anesthesia in the rat brain, indicating yet unknown persisting effects.

Expression of Hemoglobin in Rodent Neurons

Hemoglobin is the major protein in red blood cells and transports oxygen from the lungs to oxygen-demanding tissues, like the brain. Mechanisms that facilitate the uptake of oxygen in the vertebrate brain are unknown. In invertebrates, neuronal hemoglobin serves as intracellular storage molecule for oxygen. Here, we show by immunohistochemistry that hemoglobin is specifically expressed in neurons of the cortex, hippocampus, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal hemoglobin distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels. Probing for low oxygen levels in the tissue, we provide evidence that hemoglobin α-positive cells in direct neighborhood with hemoglobin α-negative cells display a better oxygenation than their neighbors and can be sharply distinguished from those. Neuronal hemoglobin expression is upregulated by injection or transgenic overexpression of erythropoietin and is accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions. Thus we provide a novel mechanism for the neuroprotective actions of erythropoietin under ischemic—hypoxic conditions. We propose that neuronal hemoglobin expression is connected to facilitated oxygen uptake in neurons, and hemoglobin might serve as oxygen capacitator molecule.

Local cerebral blood flow, Local cerebral glucose utilization, and flow-metabolism coupling during sevoflurane versus isoflurane anesthesia in rats

Background Compared to isoflurane, knowledge of local cerebral glucose utilization (LCGU) and local cerebral blood flow (LCBF) during sevoflurane anesthesia is limited. Methods LCGU, LCBF, and their overall means were measured in Sprague‐Dawley rats (8 groups, n = 6 each) during sevoflurane and isoflurane anesthesia, 1 and 2 MAC, and in conscious control animals (2 groups, n = 6 each) using the autoradiographic 2‐[(14) C]deoxy‐D‐glucose and 4‐iodo‐N‐methyl‐[(14) C]antipyrine methods. Results During anesthesia, mean cerebral glucose utilization was decreased: control, 56 +/− 5 [micro sign]mol [middle dot] 100 g‐1 [middle dot]‐1; 1 MAC isoflurane, 32 +/− 4 [micro sign]mol [middle dot] 100 g‐1 [middle dot] min‐1 (‐43%); 1 MAC sevoflurane, 37 +/− 5 [micro sign]mol [middle dot] 100 g‐1 [middle dot] min‐1 (‐34%); 2 MAC isoflurane, 23 +/− 3 [micro sign]mol [middle dot] 100 g‐1 [middle dot] min‐1 (‐58%); 2 MAC sevoflurane, 23 +/− 5 [micro sign]mol [middle dot] 100 g‐1 [middle dot] min‐1 (‐59%). Local analysis showed a reduction in LCGU in the majority of the 40 brain regions analyzed. Mean cerebral blood flow was increased as follows: control, 93 +/− 8 ml [middle dot] 100 g‐1 [middle dot] min‐1; 1 MAC isoflurane, 119 +/− 19 ml [middle dot] 100 g‐1 [middle dot] min‐1 (+28%); 1 MAC sevoflurane, 104 +/− 15 ml [middle dot] 100 g‐1 [middle dot] min‐1 (+12%); 2 MAC isoflurane, 149 +/− 17 ml [middle dot] 100 g‐1 [middle dot] min‐1 (+60%); 2 MAC sevoflurane, 118 +/− 21 ml [middle dot] 100 g‐1 [middle dot] min‐1 (+27%). LCBF was increased in most brain structures investigated. Correlation coefficients obtained for the relationship between LCGU and LCBF were as follows: control, 0.93; 1 MAC isoflurane, 0.89; 2 MAC isoflurane, 0.71; 1 MAC sevoflurane, 0.83; 2 MAC sevoflurane, 0.59). Conclusion Mean and local cerebral blood flows were lower during sevoflurane than during isoflurane anesthesia. This difference cannot be explained by differing changes in glucose utilization because glucose utilization was decreased to the same extent in both groups.

Changes in brain capillary diameter during hypocapnia and hypercapnia

Since changes in the surface area of capillaries may be relevant to capillary exchange, the distensibility of brain capillaries was investigated. Brain capillary diameters were measured after perfusion fixation of brain tissue at a constant perfusion pressure during hypo- or hypercapnia. Sections were embedded, stained, and analyzed by light microscopy. The results showed significant differences in mean capillary diameter between the hypocapnic and the hypercapnic group. In the eight brain structures analyzed, capillary diameters were always larger in the hypercapnic group. Mean capillary diameter was 4.93 ± 0.29 μm in the hypocapnic group and 5.91 ± 0.10 μm in the hypercapnic group (means ± SD). We conclude that brain capillaries exhibit a moderate degree of distensibility. Variations in the precapillary pressure of microvessels may therefore influence both capillary flow and capillary surface area.

Influence of the Endothelial Glycocalyx on Cerebral Blood Flow in Mice

The endothelial surface layer (glycocalyx) of cerebral capillaries may increase resistance to blood flow. This hypothesis was investigated in mice by intravenous administration of heparinase (2500 IU/kg body weight in saline), which cleaves proteoglycan junctions of the glycocalyx. Morphology was investigated by transmission electron microscopy. Cerebral perfusion velocity was recorded before and during heparinase or saline treatment using laser–Doppler flowmetry. In addition, cerebral blood flow (CBF) was measured 10 minutes after heparinase or saline treatment using the iodo[14C]antipyrine method. Laser–Doppler flowmetry and CBF measurements were performed during normocapnia and severe hypercapnia (Pco2: 120 mm Hg). After heparinase, morphology showed a reduced thickness of the glycocalyx in cortical microvessels by 43% (P < 0.05) compared with saline-treated controls. Under normocapnic conditions, a 15% (P < 0.05) transient increase of cerebral flow velocity occurred 2.5 to 5 minutes after heparinase injection. Laser–Doppler flow and CBF returned to control values ten minutes after the injection. However, during severe hypercapnia, heparinase treatment resulted in a persisting increase in laser–Doppler flow (6%, P < 0.05) and CBF (30%, P < 0.05). These observations indicate the existence of a flow resistance in cerebral capillaries exerted by the glycocalyx. The transient nature of the CBF increase during normocapnia may be explained by a vascular compensation that is exhausted during severe hypercapnia.