Bradykinin is known for its pathophysiological role as mediator of inflammation. Following cerebral ischemia, bradykinin promotes the secondary brain damage through an increase of vascular permeability and brain edema formation, again hallmarks of inflammation. It is; not clear, whether bradykinin also activates inflammatory cells and regulates microcirculatory blood flow in the brain. The purpose of the study is to investigate the reaction of bradykinin upon cerebral leukocyte- and thrombocyte-endothelium interactions as well as microvascular perfusion.Intravital fluorescence microscopy of pial blood vessels was performed in gerbils. Intracarotid injection of bradykinin resulted in increased numbers of rolling and adherent leukocytes as well as rolling platelets at the venular endothelium. This was reversed by administration of a bradykinin B2 receptor antagonist. In contrast, after additional administration of a B1 receptor antagonist, microvascular blood-flow and capillary density was decreased.We conclude that bradykinin initiates leukocyte- and platelet-endothelium interactions in the cerebral microcirculation via activation of B2 receptors. Activation of B1 receptors ensures regular cerebral perfusion. Thus, to attenuate secondary brain damage, inhibition of B2 but not B1 receptors might be of therapeutical benefit. (C) 2012 Elsevier B.V. All rights reserved.

Nitrous oxide is a widely used anesthetic gas. The aim of this study was to investigate the effect of this agent on inflammatory side effects in the brain. The cerebral microcirculation of Mongolian gerbils was investigated by fluorescent intravital microscopy for up to 7 h after induction of anesthesia. Anesthesia was induced and maintained with isoflurane or halothane alone or in combination with nitrous oxide (70%). The number of leukocytes that were rolling along and firmly adherent to the endothelial wall of cerebral venules was significantly elevated in animals anesthetized with nitrous oxide in combination with isoflurane and halothane compared to isoflurane and halothane alone. A significantly increased number of neutrophil granulocytes invading the brain parenchyma in histological slices from animals treated with the combination of isoflurane or halothane and nitrous oxide compared to controls treated with isoflurane or halothane alone was observed. Our data show that prolonged anesthesia with nitrous oxide induces inflammation of the cerebral microcirculation and brain.

OBJECTIVE:The aim of this study was to measure microvascular perfusion (MVP) on the brain surface in global ischemia and reperfusion by means of intravital fluorescence microscopy. METHODS:Global ischemia was induced in gerbils for 15 minutes with 3 hours of reperfusion. The passage of a rhodamine bolus (25 mul intravenously) from an arteriole to a venule was analyzed by intravital fluorescence microscopy through a cranial window. After the changes of fluorescence intensities in an arteriole and venule, the arteriovenous transit time and the MVP were calculated using the integral difference method. Additionally, regional cerebral blood flow was assessed by laser Doppler flowmetry and vessel diameters and blood pressure were recorded. RESULTS:The baseline mean MVP was 2.21 +/- 0.89 sec(-1) in the control group, remaining stable throughout observation in sham operated animals. In ischemic animals, the MVP was 2.11 +/- 0.47 sec(-1) at baseline, showing a significant decrease during ischemia to 0.07 +/- 0.16 sec(-1) (3%; P < 0.01). There was postischemic maximum hyperperfusion of 2.72 +/- 0.40 sec(-1) (134 +/- 11%; P < 0.05) at 15.4 +/- 6.9 minutes and hypoperfusion of 1.63 +/- 0.57 sec(-1) (77 +/- 13%; P = 0.19) at 36.6 +/- 16.4 minutes. There was a strong, significant correlation between MVP and regional cerebral blood flow (R = 0.82; P < 0.0001). CONCLUSION:MVP on the brain surface can be calculated from the transit time of a dye bolus from an arteriole to a venule. MVP shows a high correlation to regional cerebral blood flow. The assessment of MVP allows one to easily and repeatedly quantify perfusion changes of the microvascular network on the brain surface.

Activation of platelets induces interactions with platelets, endothelial cells, and leukocytes. In vivo observation of these interactions in the cerebral microcirculation is rare. The purpose of the present study was to develop a model enabling the in vivo observation of platelet kinetics in the cerebral microcirculation. Intravital fluorescence microscopy was performed in the Mongolian gerbil. Platelets of a donor were labeled ex vivo with carboxyfluorescein diacetat-succinimidylester (CFDA-SE), providing long-term fluorescence. Platelet function was tested ex vivo by flow cytometric analysis and in vivo by analyzing platelet-endothelium interactions. Labeled platelets stimulated with adenosine diphosphate ADP (200 μmol/L) or thrombin (1000 U/L) showed aggregation in flow cytrometric analysis, whereas unstimulated platelets were not aggregated. Irradiation of the brain surface after intravenous injection of the photosensitizing dye Photosan first induced rolling and firm adherence of platelets on arteriolar and venular endothelium, followed by the formation of a thrombus obstructing the vessel. Quantitative analysis (nx100 μm −1 min −1 ) before and after 6 mins of irradiation showed 2.6±3.2 versus 29.0±28.9 rolling, and 0.0±0.0 versus 1.7±2.3 firm adherent platelets in arterioles, and 3.9±3.3 versus 36.6±20.9 rolling and 0.0±0.0 versus 13.6±8.9 firm adherent platelets in venules. Thus, we conclude that ex vivo labeling of platelets with CFDA-SE does not activate platelets. Platelet aggregation and adhesion was achieved by platelet-specific stimulation such as ADP, thrombin or irradiation. In vivo assessment of physiologic and pathophysiologic mechanisms of platelets in the cerebral microcirculation can be achieved in this model.

Cerebral ischemia induces activation of leukocyteendothelium interactions requiring upregulation of specific adhesion molecules including the selectins. The aim of the current study was to elucidate the therapeutic potency of P–selectin blockade on microcirculatory disturbances and secondary brain damage after global cerebral ischemia. Global cerebral ischemia for 15 minutes was induced in Mongolian gerbils. Functional blockade of P–selectin was achieved by pretreatment with the antibody RB 40.34 (2 mg/kg, n = 7). In vivo observation of brain microcirculation was performed by epifluorescence microscopy of a cranial window. Survival was assessed daily up to 4 days after ischemia. In the control group leukocyte rolling increased during reperfusion with a maximum at 3 h (28 ± 14 · 100μm –1 · min –1 ) and was significantly reduced by the P–selectin antibody (13 ± 9 ·100μm –1 ·min –1 ,p <0.05). No effect on firm leukocyte adhesion was observed (4 ± 3 vs. 2 ± 1 · 100μm –1 · min –1 ). The survival of animals that received the Pselectin antibody (28 %) was significantly reduced compared with controls (71 %). Anti–P–selectin antibody reduces leukocyte rolling but has no positive effect on survival. Our data question the role of the inflammatory response in the development of secondary brain damage and do not support this kind of therapeutical approach in global cerebral ischemia.

Cerebral tissue acidosis following ischemia or traumatic brain injury contributes to cytotoxic brain edema formation. In vitro lactacidosis induces swelling of glial cells by intracellular Na+- and Cl−-accumulation by the Na+/H+-antiporter, Cl−/HCO3−-antiporters and the Na+–K+–2Cl−-cotransport. The present study aimed to elucidate whether mechanisms of lactacidosis-induced glial swelling are dependent on intra- or extracellular Ca2+-ions. Therefore, C6 glioma cells were exposed to a lactacidosis of pH 6.2 in standard or calcium-free medium and following intracellular calcium chelation. Cell volume and intracellular pH were assessed by flow cytometry. Lactacidosis of pH 6.2 induced a prompt and sustained swelling of suspended C6 glioma cells reaching a maximum of 128% within 60min. Omission of Ca2+ from the suspension medium strongly attenuated cell swelling while chelation of intracellular Ca2+ had no effects. Intracellular acidosis was not affected by either treatment. The present data show a strong dependency of lactacidosis-induced glial swelling upon extracellular Ca2+ while intracellular acidosis is not affected by omission of [Ca2+]e. Therefore, our data suggest that the Na+–K+–2Cl−-cotransporter, the only so far known transporter involved in cell volume regulation but not in pHi regulation during lactacidosis, is activated in a [Ca2+]e-dependent manner.

The role of leukocyte–endothelial interactions (LEI) as part of the inflammatory response after global cerebral ischemia (GCI) is hardly understood and may be detrimental as well as beneficial. Objective of the current study was to investigate the cause–effect relationship of activated leukocytes for the development of ischemic brain damage. Mongolian gerbils were subjected to 15min of global cerebral ischemia. A cranial window was implanted for quantitative analysis of the pial microcirculation focusing on leukocyte–endothelium interactions by intravital fluorescence microscopy up to 3h of reperfusion. Subsequently the animals were daily screened for neurological deficits and the evolving brain damage was assessed histologically after 4 days. After global cerebral ischemia the number of rolling and adherent leukocytes increased 20- and >23-fold, respectively upon 3h of reperfusion as compared to controls (P<0.05). Ischemic animals developed neurological deficits and showed a significant loss of neurons in selective vulnerable areas of the brain. The extent of leukocyte activation, i.e. the maximum number of rollers and stickers directly correlated to the number of viable neurons on day 4 in hippocampus, cortex, and striatum. We conclude that there is a relationship between activation of leukocyte–endothelium interactions and the reduction of ischemic brain damage after global cerebral ischemia. Activation of leukocytes may have neuroprotective potential or indicate regenerative processes.

Cytotoxic brain edema is a major contributor of tissue damage following cerebral ischemia and traumatic brain injury. The pathophysiology of cytotoxic edema formation is still not well understood. Although it is widely believed that oxidative stress causes cytotoxic brain edema, experimental proof is lacking. The aim of the present study was therefore to examine the effect of oxidative stress on cell volume of glial cells. C6 glial cells were exposed to hydrogen peroxide and the superoxide forming complex hypoxanthine/xanthine oxidase (HX/XO). Exposure to hydrogen peroxide (0.5-5 mM) resulted in initial cell shrinkage by 5.7 +/- 1.5% (mean +/- SEM; p < 0.05) and was followed by a dose-dependent recovery to baseline. Exposure to superoxide anions generated by HX/XO provoked a delayed, but sustained decrease of cell volume by 11.8 +/- 0.9% (p < 0.05). Cell volume showed no tendency to recover upon sustained exposure to superoxide. Neither hydrogen peroxide nor HX/XO exposure was associated with a decrease of cell viability. Thereby, the present study demonstrates that oxidative stress by hydrogen peroxide and superoxide anions does not induce cytotoxic cell swelling and suggests that free radicals are not directly involved in the formation of cytotoxic brain edema.

Perifocal depolarizations (PFD) have been observed after traumatic brain injury, are known to disturb cerebrovascular reactivity and thus may contribute to the morphological consequences of brain injury. In this investigation, the role of PFD was studied in focal brain lesions with/without induction of delayed hypotension. Cerebral freeze lesions were induced in anesthetized normotensive rats that underwent perfusion fixation of brains 5 min, 4 h or 24 h after lesioning, respectively, to obtain quantitative histopathology. In additional groups, a 45-min period of moderate hypobaric hypotension was applied 15 min post-trauma and brains were perfusion fixed after 4 h or 24 h. In a second series, the direct current (DC) potential and cortical laser-Doppler flow (LDF) were measured adjacent to lesions under normotensive or hypotensive conditions. Sham procedures were carried out in rats that underwent hypotension alone. Lesioning resulted in a significant LDF decrease to 50% of baseline, further decreased during hypotension to less than 40% of control (P < 0.05). Sham animals had LDF values between 60 and 70% of control when subjected to hypotension. Focal brain injury always induced a negative DC shift shortly after lesioning. In 6 of 8 rats that underwent cold lesion plus hypotension, a second PFD was observed approximately 2.5 min after onset of hypotension accompanied by a relative LDF increase by 25 +/- 12%. Lesion expansion was significantly worsened by hypotension (8.19 +/- 0.56 mm(3) at 24 h) compared with normotensive rats (7.01 +/- 0.3 mm(3) at 24 h, P < 0.01). We conclude that hypotension triggers depolarizations by an ischemic mechanism that contributes to final tissue damage.

BACKGROUND:During many neurovascular procedures temporary occlusion of cerebral arteries is inevitable. Neuroprotective drugs may reduce the risk of cerebral infarction in this situation. Increasing evidence indicates neuroprotective properties of magnesium in cerebral ischemia. Previous experimental studies on the neuroprotective efficacy of magnesium-treatment in transient focal ischemia provide widely differing results using different magnesium doses and treatment-regimens. The present study was conducted to find the maximum protective dose of intravenous magnesium sulphate in a rat model of transient focal ischemia.METHODS:45 male Sprague-Dawley rats were subjected to 90 minutes of middle cerebral artery occlusion (MCAO) by an intraluminal thread. Animals were randomly assigned to one of 4 treatment arms: (1) vehicle (2) MgSO(4) 1x0.75 mmol/kg (3) MgSO(4) 2x1 mmol/kg (4) MgSO(4) 1 mmol/kg+0.5 mmol/kg/h. Local cortical blood flow (LCBF) was continuously measured by laser-Doppler flowmetry. Functional deficits were quantified daily, infarct volumes were assessed histologically after 7 days.RESULTS:Magnesium serum levels below 3 mmol/l were well tolerated by the animals. Above 3 mmol/l cardiodepressive effects limited neuroprotection. Total infarct volumes in groups 3 and 4 were significantly reduced by 32% and 42%, respectively, compared to controls. Postoperative neurological recovery was significantly improved in magnesium-treated groups.CONCLUSION:Continuous magnesium-administration with stable serum concentrations between 2 and 3 mmol/l offered the best protection and was well tolerated. Serum concentrations above 3 mmol/l should not be exceeded. An elevation of magnesium serum levels could be useful for brain tissue protection during procedures which are prone to the risk of temporary vessel occlusion.

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein-kinin system, promotes neuronal tissue damage as well as disturbances in blood-brain barrier function through activation of B-2 receptors. In a rat model of focal cerebral ischemia, blockade of B-2 receptors before initiation of ischemia with the B-2 receptor antagonist, LF 16-0687 Ms, afforded substantial neuroprotection. in order to assess the potential clinical value of this approach, we evaluated the effect of LF 16-0687 Ms given at reperfusion following focal cerebral ischemia on local cerebral blood flow (LCBF), neurological outcome, and infarct size. Sprague-Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Animals were assigned to one of four treatment arms (n = 7 each): (1) vehicle, (2) LF 16-0687 Ms (1.0 mg/kg/day), (3) LF 16-0687 Ms (3.0 mg/kg/day), or (4) LF 16-0687 Ms (10.0 mg/kg/day) given at reperfusion and repetitively over 2 days. Neurological recovery was examined daily, and infarct volume was assessed histologically on day 7 after ischemia. Physiological parameters and local CBF were not influenced by the treatment. Significant improvement of neurological outcome was observed on postischemic day 3 in animals receiving 1.0 and 3.0 mg/kg/day of LF 16-0687 Ms (P < 0.05). Inhibition of B-2 receptors significantly reduced infarct volume in all treated animals predominantly in the cortex. B-2 receptor blockade with LF 16-0687 Ms showed neuroprotective effectiveness even when therapy was initiated upon reperfusion, i.e. 90 min after induction of ischemia. Therefore, blockade of B-2 receptors seems to be a promising therapeutic approach after focal cerebral ischemia, which deserves further experimental and clinical evaluation. (c) 2005 Elsevier B.V. All rights reserved.

Laser-Doppler (LD) fluxmetry (LDF) is a widely used method for the measurement of relative tissue perfusion. Assessing LD-flux at multiple locations using a scanning technique greatly reduces movement artefacts and makes repetitive measurements at the same location possible. However, measurements in brain are often confounded by superficial cortical vessels. Commonly applied strategies to circumvent this problem, such as defining a cut-off point to exclude the high flux data of vessels or calculating the median from multiple locations to estimate regional cerebral blood flow (rCBF) all have specific shortcomings. The aim of this study was to analyse LD-data by mathematically discriminating between parenchymal and vessel data based on the distribution of flux data. Data was obtained by scanning the cortex of 15 male Sprague-Dawley rats using a matrix of 6x10 equidistant (500 microm) points. Standard statistical analysis as well as cluster analysis using the complete linkage algorithm was performed. The LD-data showed a bimodal frequency distribution with low values representing parenchymal and high values representing vessel flux. Parenchyma and vessels were reliably discriminated by cluster analysis. This was shown by mapping the vessel clusters on the scan matrix with the location of the superficial cortical vessels using Chi-square testing (p<0.0001). The parenchymal data followed a Gaussian normal distribution (p<0.851), whereas the vessel data did not (p<0.0001). Thus, cluster analysis is useful to discriminate parenchymal from vessel flux, thereby significantly improving the accuracy of LD-scanning data.

The volume of an experimental necrotic lesion of the cortex expands up to 400% of its initial size within the first 24 h after the insult. Lesion expansion, a clinically well known phenomenon, is often accompanied by perifocal brain edema and consequently difficult to image and to analyze by magnetic resonance imaging (MRI). Therefore we aimed to validate a T(2)-weighted spin echo sequence upon its ability to distinguish necrotic from edematous brain tissue. Male Sprague-Dawley rats (n = 5 per group) were subjected to a cortical freezing lesion leading to immediate tissue necrosis with subsequent perifocal vasogenic brain edema. Immediately and 4, 12, and 24 h after the lesion the maximal area of necrosis was quantified longitudinally by coronal T(2)-weighted spin echo MRI-scans. After the last scan, animals were sacrificed for direct comparison of the lesion area obtained by MRI and histomorphometry. In parallel groups of animals, lesion expansion was quantified by histology. The acquired T(2)-maps clearly distinguish the cortical necrosis from perifocal edema and healthy brain. Focal freezing led to a cortical lesion of 5.24 +/- 0.36 mm(2) immediately after trauma (0 h; 100%) which expanded progressively to a maximum of 6.82 +/- 0.34 mm(2) after 24 h (131%; *p < 0.01 vs. 0 h). Lesion expansion quantified by histology was almost identical (132% within 24 h). Histological assessment resulted in smaller absolute lesion areas compared to MRI, most likely due to shrinking during tissue processing (4.72 +/- 0.26 mm(2) vs. 6.82 +/- 0.34 mm(2), p < 0.01). The current study shows that necrotic brain tissue can be distinguished from surrounding brain edema by T(2)-mapping. The technique is sensitive enough to detect small changes in necrosis expansion in vivo as validated by histology. The presented technique may be a useful future tool for the non-invasive identification of necrotic brain tissue following brain injury (e. g., from trauma or ischemia).

Notwithstanding that the control of a normal cell volume seems to assume a high cell biological priority, as demonstrated by the spontaneous normalisation of the cell volume following anosmotic exposure, the cellular swelling response may also have important functions to support the homeostasis. Prominent examples are the clearance of excessive K+or glutamate concentrations from the interstitial compartment in order to defend a normal neuronal function. This requires not only physiological transmembrane Nat and K+-concentration gradients, but also an absence, or at least very low levels of excitatory transmitter compounds in the perisynaptic (interstitial) compartment. Yet, cell swelling may also reflect sequelae of cell injury, may be even an early indication of impending cell death. For example, cell swelling from arachidonic acid or higher levels of acidosis may eventually merge into an irreversible insult of a viable cell.

We have recently demonstrated marked neuroprotective efficacy of a combination therapy with magnesium (calcium- and glutamate-antagonist), tirilazad (antioxidant) and mild hypothermia (MTH) in a rat model of transient focal cerebral ischemia. In the present study, we investigated MTH under conditions of permanent focal cerebral ischemia. In part I, 20 Sprague–Dawley rats were subjected to 6 h of permanent, laser-Doppler flowmetry (LDF) controlled middle cerebral artery occlusion (MCAO). Drugs were administered 30 min before and 1 h after MCAO. Hypothermia (33 °C) was maintained for 2 h. Infarct size was planimetrically determined after 6 h. In part II, 29 rats were assigned to the same treatment arms and subjected to 7 days of permanent MCAO. Neurological deficits and body weight were assessed daily. Infarct size was determined on day 7. In part I, MTH significantly reduced infarct formation by 52% after 6 h. In part II, high mortality within the first 3 days was observed in both groups. Treated animals showed a significantly better postoperative weight gain on day 7 and neurological recovery on days 6 and 7 compared to controls without significant differences in infarct volume. MTH seems to exert its neuroprotective properties even in a setting of permanent cerebral ischemia. High mortality and absence of infarct reduction after 7 days might be due to model limitations. Neurological recovery, the most important clinical outcome parameter, is significantly improved in 7-day survivors. Significant neuroprotection under conditions of permanent ischemia and former promising results in transient ischemia justify further investigations of MTH.

Treatment of patients suffering from severe head injury is so far restricted to general procedures, whereas specific pharmacological agents of neuroprotection including hypothermia have not been found to improve the outcome in clinical trials. Albeit effective, symptomatic measures of the preclinical rescue of patients (i.e. stabilization or reestablishment of the circulatory and respiratory system) or of the early clinical care (e.g. prompt diagnosis and treatment of an intracranial space occupying mass, maintenance of a competent circulatory and respiratory system, and others) by and large constitute the current treatment based on considerable organizational and logistical efforts. These and other components of the head injury treatment are certainly worthwhile of a systematic analysis as to their efficacy or remaining deficiencies, respectively. Deficits could be associated with delays of providing preclinical rescue procedures (e.g. until intubation of the patient or administration of fluid). Delays could also be associated in the hospital with the diagnostic establishment of intracranial lesions requiring prompt neurosurgical intervention.

Brain edema and secondary growth of a traumatic brain tissue necrosis are important manifestations of secondary brain damage and of prognostic significance in severe head injury. Aim of the current study was to analyze the interdependency of the resulting brain swelling from the size of the focal traumatic lesion. Male Sprague-Dawley rats were intubated and mechanically ventilated. A trephination was made over the left parietal cortex for induction of a cold lesion. Different injury severities were achieved by varying the contact time of the cooled copper-cylinder and the exposed cortex. Animals were randomized into 12 experimental groups. Hemispheric brain swelling was measured in groups A1-A6 (n = 4-8) by gravimetry 24 hrs after lesions of six increasing severity levels. Correspondingly, in animals of groups B1-B6 (n = 5-7) the volume of necrosis was planimetrically assessed in histological serial sections of the brain obtained 24 hrs after trauma of different severity. In groups A1-A6. hemispheric brain swelling (increase in weight) was growing with increasing contact duration of the cold probe with the exposed cerebral cortex, i.e. from 7.7 +/- 0.4% (5 s) to a maximum of 9.9 +/- 0.5% (25 s). Longer contact periodes (30 s) were not further effective to increase hemispheric brain swelling. The contact times and extent of swelling were linearly correlated between 5 s and 25 s (r = 0.47; p < 0.01). The volume of necrosis in groups B1-B6 increased from 35.7 +/- 3.7 mm3 (5 s) to 106.3 +/- 10.3 mm3 (30 s). There was again a linear correlation between the duration of contact of the cold probe (i.e. injury severity) with the brain cortex and the volume of necrosis (r = 0.77; p < 0.01). Accordingly. the lesion volume could be increased in a reproducible manner from 35.7 up to 106.3 mm3 by extending the contact times of the cooling device and cerebral cortex. Hemispheric swelling, predominantly due to vasogenic brain edema, was expanding in relationship with the volume of necrosis.