Christoph Wiessner

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1−/−) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1−/− mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1−/− mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1−/− mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1−/− mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1−/− mice. However, these effects on macrophages and astrocytes in MCP-1−/− mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1−/− mice produced significantly less interleukin-1β in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.

A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.

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.

Cell Type Specific Upregulation of Vascular Endothelial Growth Factor in an MCA-occlusion Model of Cerebral Infarct

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

Anti–Nogo-A Antibody Infusion 24 Hours After Experimental Stroke Improved Behavioral Outcome and Corticospinal Plasticity in Normotensive and Spontaneously Hypertensive Rats

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti—Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 ± 1.5% after PCI (control group: 1.1 ± 0.5%) and 4.5 ± 2.2% after MCAO in SHR rats (control group: 1.8 ± 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti—Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.

Expression of Angiopoietin-1, Angiopoietin-2, and Tie Receptors after Middle Cerebral Artery Occlusion in the Rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.

Targeting monocyte chemoattractant protein-1 signalling in disease.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated in many inflammatory and autoimmune diseases. The G-protein-coupled receptor CCR-2B is probably the most important MCP-1 receptor in vivo, and loss of MCP-1 effector function alone is sufficient to impair monocytic trafficking in inflammation models. MCP-1 signalling appears to be a relevant target, especially in rheumatoid arthritis (RA). In RA patients, MCP-1 is produced by synovial cells and infiltrating monocytes, plasma MCP-1 concentrations correlate with swollen joint count, and elevated serum MCP-1 concentrations were found in juvenile RA in patients with active disease. Modulation of MCP-1 signalling in experimental RA showed beneficial effects on inflammation and joint destruction. With respect to chronic neuroinflammation, a critical role for MCP-1 has been established in animal models for multiple sclerosis. In acute neuroinflammation, experimental evidence for a detrimental role of MCP-1 in stroke and excitotoxic injury has been found. Several selective small molecular weight CCR-2B antagonists and MCP-1-blocking antibodies have been described. The proof for the validity of targeting MCP-1 signalling in disease, however, has yet to be established in clinical trials.

Effect of global system for mobile communication (GSM) microwave exposure on blood-brain barrier permeability in rat

Abstract We investigated the effects of global system for mobile communication (GSM) microwave exposure on the permeability of the blood-brain barrier using a calibrated microwave exposure system in the 900 MHz band. Rats were restrained in a carousel of circularly arranged plastic tubes and sham-exposed or microwave irradiated for a duration of 4 h at specific brain absorption rates (SAR) ranging from 0.3 to 7.5 W/kg. The extravasation of proteins was assessed either at the end of exposure or 7 days later in three to five coronal brain slices by immunohistochemical staining of serum albumin. As a positive control two rats were subjected to cold injury. In the brains of freely moving control rats (n = 20) only one spot of extravasated serum albumin could be detected in one animal. In the sham-exposed control group (n = 20) three animals exhibited a total of 4 extravasations. In animals irradiated for 4 h at SAR of 0.3, 1.5 and 7.5 W/kg (n = 20 in each group) five out of the ten animals of each group killed at the end of the exposure showed 7, 6 and 14 extravasations, respectively. In the ten animals of each group killed 7 days after exposure, the total number of extravasations was 2, 0 and 1, respectively. The increase in serum albumin extravasations after microwave exposure reached significance only in the group exposed to the highest SAR of 7.5 W/kg but not at the lower intensities. Histological injury was not observed in any of the examined brains. Compared to other pathological conditions with increased blood-brain barrier permeability such as cold injury, the here observed serum albumin extravasations are very modest and, moreover, reversible. Microwave exposure in the frequency and intensity range of mobile telephony is unlikely to produce pathologically significant changes of the blood-brain barrier permeability.

Post-occlusion treatment with BDNF reduces infarct size in a model of permanent occlusion of the middle cerebral artery in rat

In view of the protective effect of brain derived neurotrophic factor (BDNF) against metabolic/excitotoxic insults in vitro, we investigated whether BDNF could limit infarct size after permanent occlusion of the middle cerebral artery in rat (MCAO). BDNF was delivered into the territory of the middle cerebral artery via an osmotic mini-pump (1 μg/h). Infusion of BDNF was started shortly after MCAO, and 24 h later brains were removed for infarct volume determination. Intraoperative and postoperative measurements of physiological variables revealed no differences among vehicle-treated and BDNF-infused animals. However, we found a 33% reduction in total infarct volume in BDNF-treated animals (p<0.05), and a 37% reduction in cortical infarct volume (p<0.05).

Effect of global system for mobile communication microwave exposure on the genomic response of the rat brain

The acute effect of global system for mobile communication (GSM) microwave exposure on the genomic response of the central nervous system was studied in rats by measuring changes in the messenger RNAs of hsp70, the transcription factor genes c-fos and c-jun and the glial structural gene GFAP using in situ hybridization histochemistry. Protein products of transcription factors, stress proteins and marker proteins of astroglial and microglial activation were assessed by immunocytochemistry. Cell proliferation was evaluated by bromodeoxyuridine incorporation. A special GSM radiofrequency test set, connected to a commercial cellular phone operating in the discontinuous transmission mode, was used to simulate GSM exposure. The study was conducted at time averaged and brain averaged specific absorption rates of 0.3 W/kg (GSM exposure), 1.5 W/kg (GSM exposure) and 7.5 W/kg (continuous wave exposure), respectively. Immediately after exposure, in situ hybridization revealed slight induction of hsp70 messenger RNA in the cerebellum and hippocampus after 7.5 W/kg exposure, but not at lower intensities. A slightly increased expression of c-fos messenger RNA was observed in the cerebellum, neocortex and piriform cortex of all groups subjected to immobilization, but no differences were found amongst different exposure conditions. C-jun and GFAP messenger RNAs did not increase in any of the experimental groups. 24 h after exposure, immunocytochemical analysis of FOS and JUN proteins (c-FOS, FOS B, c-JUN JUN B, JUN D), of HSP70 or of KROX-20 and -24 did not reveal any alterations. Seven days after exposure, neither increased cell proliferation nor altered expression of astroglial and microglial marker proteins were observed. In conclusion, acute high intensity microwave exposure of immobilized rats may induce some minor stress response but does not result in lasting adaptive or reactive changes of the brain.

Time course of microglia activation and apoptosis in various brain regions after permanent focal cerebral ischemia in mice

Abstract We investigated the temporal course of microglia activation in different brain regions after permanent middle cerebral artery (MCA) occlusion in mice and compared this microglia response with the appearance of apoptotic cells, Microglia activation and morphological changes of microglial cells were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse CR3 complement receptor. Cells showing morphological and biochemical features of apoptosis were identified using the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and light microscopy. As early as 30 min after onset of MCA occlusion activated microglia with hypertrophic cell bodies and stout processes were detected in the periphery of the ischemic lesion as identified by diffusion-weighted magnetic resonance imaging. A wider distribution and a progressive increase in the number of activated microglia was found with increasing time. Only few TUNEL-positive cells with apoptotic features were observed within the lesion area at 6 h after onset of cerebral ischemia. From 12 h after MCA occlusion onward a tremendous increase in the number of TUNEL-positive cells was found. Within the thalamus from 24 h onward microglia cells with few processes, irregular morphology and fragmented appearance were detected. Microglia activation in the thalamus progressed up to 4 weeks after MCA occlusion, but had declined after 90 days. Neuronal degeneration in the thalamus as determined by anti-neuronal nuclei immunohistochemistry progressed from 6 days after MCA occlusion onward. Only a few TUNEL-positive cells were found in the thalamus. In summary, microglia activation both in the primary cortical lesion area and in the secondarily affected thalamus preceded the manifestation of tissue injury. These observations encourage further studies on the role of microglia in focal cerebral ischemia.