Published online before print November 6, 2008, doi: 10.1161/STROKEAHA.108.526673
(Stroke. 2009;40:285.)
© 2009 American Heart Association, Inc.
Original Contributions |
Blockade of Bradykinin Receptor B1 but Not Bradykinin Receptor B2 Provides Protection From Cerebral Infarction and Brain Edema
From Department of Neurology (M.A., S.B., G.S., C.K.), Department of Anesthesiology (M.Brede), and Institute for Clinical Biochemistry and Pathobiochemistry (T.R.), University of Würzburg, Würzburg, Germany; Departamento de Biofisica (J.B.P.), Universidade Federal de São Paulo, São Paulo, Brazil; Max-Delbrück-Center for Molecular Medicine (M.Bader), Berlin-Buch, Germany.
Correspondence to Christoph Kleinschnitz, MD, Department of Neurology, Julius-Maximilians-University of Würzburg, Josef-Schneider Strasse 11, D-97080 Würzburg, Germany. E-mail christoph.kleinschnitz{at}mail.uni-wuerzburg.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Background and Purpose— Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by 2 different receptors (B1 and B2 receptor [B1R and B2R]) and comprise induction of edema formation and release of proinflammatory mediators.
Methods— Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan’s blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected.
Results— B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8±4.7 mm3 vs 50.1±9.1 mm3, respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0±9.5 mm3 vs 50.1±9.1 mm3, respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema.
Conclusions— These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.
Key Words: bradykinin • edema • endothelin-1 • inflammation • stroke
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Brain edema is a frequent cause of secondary infarct growth and subsequent deterioration of neurological symptoms.1,2 Recently, the life-saving effect of decompressive surgery has been established in patients with malignant middle cerebral artery (MCA) infarction leading to large brain edema.3 However, craniectomy is very invasive and so far no medication, eg, steroids or hyperosmolaric solutions (eg, mannitol, sorbitol) has proven to effectively reduce brain edema.4–6 The molecular mechanisms underlying edema formation in ischemic stroke are largely unknown. The kallikrein–kinin system plays an important role in the regulation of vascular permeability and has been invoked in edema formation.7–9 Kinins (eg, bradykinin, kallidin) are biologically highly active proinflammatory peptide hormones that are released by kallikreins from their precursors, kininogens, during various kinds of tissue injury, including focal and global brain ischemia.10–13 All components of the so-called kallikrein–kinin system have been identified in the brain.14–16 The cellular effects of kinins are mediated by 2 different bradykinin receptors. B1R is expressed at low levels under normal conditions but is induced selectively during inflammation by soluble mediators, eg, IL-1β or tumor necrosis factor-
. In contrast, B2R is constitutively expressed and mediates the majority of bradykinin physiological effects.8,17–20 Activation of B1R and B2R triggers inflammatory processes in the target organ such as the release of proinflammatory cytokines or the attraction of immune cells, as well as increased vascular permeability.8,21,22 In the present study, we analyzed in parallel the effect of B1R and B2R deficiency or blockade on infarct size, edema formation, and inflammatory processes in a mouse model of focal ischemic stroke. We could show that inhibition of B1R but not B2R protects from ischemic brain damage and is associated with less edema formation and attenuation of the local postischemic inflammatory response.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Induction of Cerebral Ischemia
A total of 276 mice were used in this study. Animal experiments were approved by the Regierung von Unterfranken and conducted according to the recently published recommendations for research in mechanism-driven basic stroke studies.23 B1R and B2R knockout (KO) mice used in this study were described previously.24,25 B1R KO mice and animals deficient in both kinin receptors are normotensive.24–26 C57BL/6 wild-type (WT) mice (Charles River; Sulzfeld, Germany) served as controls. Focal cerebral ischemia was induced in 6- to 8-week-old mice by 60 minutes of transient middle cerebral artery occlusion (tMCAO), as described (Expanded Materials and Methods, available online at http://stroke.ahajournals.org).27,28 For polymerase chain reaction (PCR) studies, sham-treated C57BL/6 mice (n=4) were used as controls. To determine the effect of pharmacological B1R inhibition in WT mice, the selective B1R inhibitor R-715 (Ac-Lys-[D-βNal7, Ile8]desArg9-BK; Biomatik Corporation) was administered intravenously 1 hour after tMCAO at a dosage of 500 µg/kg body weight (n=10) or 1 mg/kg body weight (n=10), respectively.29 For the selective blockade of B2R, Hoe-140 (D-Arg0-Hyp3-Thi5-D-Tic7-Oci8-BK; 0.2 mg/kg body weight; Sigma Aldrich) was injected (n=10).30
Laser Doppler flowmetry (Moor Instruments) was used in some animals (n=6/group) to monitor regional cerebral blood flow in the MCA territory (supplemental Figure IA, available online at http://stroke.ahajournals.org) and the cerebral vasculature was assessed by perfusion with black ink (n=3/group; supplemental Figure IB). Long-term survival of WT, B1R KO, and B2R KO mice after tMCAO was investigated over a period of 5 days (n=10/group).
|
Assessment of Functional Outcome
Twenty-four hours after tMCAO, the modified Bederson score31 was used to determine global neurological function according to the following scoring system: 0, no deficit; 1, forelimb flexion; 2, decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning; and 5, no movement. Motor function and coordination were evaluated by the grip test.32 For this test, the mouse was placed midway on a string between 2 supports and rated as follows: 0, falls off; 1, hangs onto string by 1 or both forepaws; 2, same as for 1, and attempts to climb onto string; 3, hangs onto string by 1 or both forepaws plus 1 or both hind paws; 4, hangs onto string by forepaws and hind paws plus tail wrapped around string; and 5, escape (to the supports).
Determination of Infarct Size
Infarct volumes were quantified by planimetry from 2,3,5-triphenyltetrazolium chloride-stained (TTC; Sigma Aldrich) mouse brains, as described (online).27,28
Determination of Brain Edema
Brain edema was determined by comparing the wet and dry weight of the infarcted hemispheres. The permeability of the cerebral vasculature was assessed by extravasation of Evan’s blue tracer (Sigma Aldrich; n=4/group; online).
Invasive Hemodynamics
For invasive hemodynamics mice were anesthetized with 2.5% isoflurane and catheterized via the right carotid artery with a high-fidelity 1.4-F Millar micro-tip catheter as described.33 Systolic and diastolic blood pressure and heart rate were measured 10 minutes after intravenous application of 0.9% NaCl (controls), R-715 (1 mg/kg body weight), and Hoe-140 (0.2 mg/kg body weight; n=5/group). Hemodynamic data were digitized via a MacLab system (AD Instruments) connected to an Apple G4 PowerPC computer (Apple Computer, Inc) and analyzed.
PCR Studies
Ischemic infarctions mostly spared the neocortex of B1R KO mice (Figures 2, 3
). Therefore, basal ganglia (instead of total hemispheres), which were regularly included in the ischemic infarctions also in B1R KO mice (Figures 2,3), were dissected from the brains in WT and B1R KO mice at 4 hours (n=8 per group) and 24 hours (n=8 per group) after tMCAO. This mode of sampling excluded biased PCR results because of different infarct sizes between the groups. Real-time reverse-transcription polymerase chain reaction against murine B1R and B2R, IL-1β, transforming growth factor β-1, tumor necrosis factor-, and endothelin-1 was performed using routine procedures (online).
Histology and Immunohistochemistry
Formalin-fixed brains embedded in paraffin from WT and B1R and B2R KO mice at day 1 after tMCAO (n=6/group) were cut into 4-µm-thick sections 0.5 mm anterior from bregma and stained with hematoxylin and eosin. Invading immune cells (T cells, macrophages, neutrophilic granulocytes) within the infarcted basal ganglia were detected by routine immunohistochemistry and quantified (online).
Statistical Analysis
Data are expressed as mean±SD. For statistical analysis PrismGraph 4.0 (GraphPad Software) software package was used. Brain edema formation in WT and B1R KO animals as measured by extravasation of Evan’s blue (Figure 4B) was compared using the nonparametric Mann-Whitney test. For comparison of survival curves (Figure 2B), the log-rank test was used. All other data were tested for Gaussian distribution with the D'Agostino and Pearson omnibus normality test and then analyzed by Bonferroni-corrected 1-way ANOVA or 2-way ANOVA (reverse-transcription polymerase chain reaction data). P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
B1R Blockade Protects From Focal Cerebral Ischemia
In a first set of experiments, we analyzed the mRNA expression pattern of B1R and B2R in ischemic brains from C57BL/6 mice over time (Figure 1). Both receptors were constitutively expressed at low levels in sham-treated animals. B1R and B2R mRNA expression significantly increased
9-fold after 4 hours (P<0.0001), and in the case of B1R further increased until 24 hours (16.4±2.9-fold induction; P<0.01; Figure 1). Taken together, these data indicate that both kinin receptors are expressed in the murine brain and, just like their ligand bradykinin,12 undergo induction after focal cerebral ischemia, suggesting a functional role of the kallikrein–kinin system in ischemic stroke.
|
To investigate the functional role of B1R and B2R in experimental cerebral ischemia 60 minutes of tMCAO was induced in WT and B1R and B2R KO mice. Twenty-four hours after reperfusion, the infarct volumes in B1R KO animals assessed by TTC staining were dramatically reduced to 30% of the infarct volumes in WT mice (Figure 2A; 16.8±4.7 mm3 vs 50.1±9.1 mm3, respectively; P<0.0001). Importantly, infarct volumes in B1R-null mice did not further increase between day 1 and day 3 (16.8±4.7 mm3 vs 21.3±4.3 mm3, respectively; P>0.05) and remained significantly smaller compared to WT controls (21.3±4.3 mm3 vs 54.2±7.1 mm3, respectively; P<0.01; Figure 2A). The reduction in infarct size was functionally relevant, because the Bederson score assessing global neurological function (0.6±0.9 vs 2.9±0.8, respectively; P<0.0001) and the grip test that specifically measures motor function and coordination (2.7±0.9 vs 4.1±0.7, respectively; P<0.0001) were significantly better in B1R-deficient mice than in WT mice at day 1 after tMCAO (Figure 2B). In contrast, lack of B2R did not confer neuroprotection after focal cerebral ischemia because the infarct volumes and neurological deficits in B2R KO mice 24 hours after reperfusion were comparable to those in WT animals (Figure 2A,B). Moreover, B1R blockade significantly increased long-term survival in mice after tMCAO. Whereas 6 of 10 WT mice and 4 of 10 B2R KO died 5 days after infarct induction, 100% (10/10) of B1R-deficient mice survived (P<0.05; Figure 2B). Consistent with the TTC stains, histological analysis revealed massive ischemic infarction of the basal ganglia and neocortex in WT animals, but only limited infarctions that were mostly restricted to the basal ganglia in B1R KO mice (Figure 2C).
|
As congenital B1R deficiency protects mice from ischemic stroke, pharmacological targeting of B1R should provide similar protection. To test the protective potential of B1R inhibition, WT mice received 2 different doses of R-715 1 hour after the induction of tMCAO. Whereas 500 µg R-715/kg body weight had no significant impact on infarct size and neurological status, the higher dose of 1 mg R-715/kg body weight markedly reduced the infarcted brain volume at 24 hours compared to untreated control mice (15.0±9.5 mm3 vs 50.1±9.1 mm3, respectively; P<0.01; Figure 3A). At this concentration, R-715–treated mice showed a significantly better Bederson (1.3±0.7 vs 2.9±0.8, respectively; P<0.01) and grip test score (2.7±0.9 vs 4.3±0.7, respectively; P<0.01). In contrast, the selective B2R inhibitor Hoe-140 did not confer neuroprotection when applied in the acute phase after ischemic stroke (Figure 3A and 3B).
|
It is known that the kallikrein–kinin system plays an important role in blood pressure regulation8 that could influence edema formation and infarct volume after ischemic stroke. We therefore analyzed the effect of the pharmacological B1R and B2R inhibitors R-715 and Hoe-140 on blood pressure and heart rate in C57BL/6 mice. Ten minutes after a single injection of R-715 (1 mg/kg body weight), a dosage that was neuroprotective when applied 1 hour after tMCAO (Figure 3A,B), or Hoe-140 (0.2 mg/kg body weight), no significant differences in systolic (86.4± 8.0 mm Hg vs 83.8±12.3 mm Hg or 85.7±6.5 mm Hg; P>0.05) or diastolic blood pressure (55.6±5.6 mm Hg vs 55.2±14.6 mm Hg or 56.4±6.7 mm Hg; P>0.05) and heart rate (491.4±70.2 min–1 vs 487.4±23.3 min–1 or 469.4±62.7 min–1; P>0.05) were found compared to vehicle-treated controls (Supplemental Figure II). This is in line with our previously published findings showing that B1R KO mice and animals deficient in both B1R and B2R are normotensive.24–26 These results exclude that blood pressure alterations caused the different infarct volumes and edema formation after B1R or B2R blockade in experimental stroke.
|
B1R Deficiency Reduces Brain Edema Formation After Focal Cerebral Ischemia
To further investigate the mechanism by which B1R deficiency protects from cerebral ischemia, the extent of brain edema formation as measured by the free water content of the ischemic hemisphere was assessed (Figure 4A). At day 1 after tMCAO, brain water content in the ischemic hemisphere of B1R KO mice was significantly reduced compared to WT littermates (77.8±0.7% vs 80.5±1.6%, respectively; P<0.01). Again, no differences were observed between WT and B2R KO mice (Figure 4A; P>0.05). In line with these findings, permeability of the cerebral vasculature as quantified by the volume of Evan’s blue extravasation within the ischemic brain parenchyma was dramatically lower in mice lacking the B1R compared to controls (6.7±1.6 mm3 vs 81.7±17.8 mm3, respectively; P<0.0001; Figure 4B). Most importantly, brain edema was even nearly absent in brain regions (basal ganglia), where infarction was present in B1R KO mice (Figure 4B, dotted line), indicating that the reduction of edema formation was not just an unspecific epiphenomenon attributable to smaller infarct volumes. To further analyze the underlying mechanism that prevented edema formation in B1R-deficient mice, we analyzed the expression profile of endothelin-1, which has been shown to be critically involved in regulating vascular integrity and edema formation under various pathophysiological conditions, including ischemic stroke, in the ischemic basal ganglia of WT and B1R KO mice over time (Figure 4C). Endothelin-1 mRNA levels were elevated early at 4 hours after tMCAO and massively increased until 24 hours in WT animals. In contrast, no significant endothelin-1 induction was observed in B1R KO mice at any time point after tMCAO, and endothelin-1 expression was significantly lower after 24 hours compared to WT controls (2.6±0.6-fold induction vs 25.2±6.3 induction, respectively; P<0.001).
|
MCAO. **P<0.01. 1-way ANOVA compared to WT mice, Bonferroni post hoc test. B, Representative corresponding coronal brain sections (left) of WT and B1R KO mice at day 1 after tMCAO after injection of Evan’s blue. Note that Evan’s blue extravasation was even absent in areas where infarction was present in B1R KO mice (basal ganglia; dotted line). B, Volume of Evan’s blue extravasation (right) determined by planimetry in the ischemic hemisphere of WT and B1R KO mice 24 hours after 60 minutes of
B1R Deficiency Reduces Postischemic Inflammation in the Brain
Several studies have demonstrated that B1R deficiency modulates the inflammatory response under various pathophysiological conditions.34,35 We therefore analyzed the mRNA expression levels of several prototypic proinflammatory and antiinflammatory cytokines in the brains of WT and B1R KO mice after tMCAO over time (Figure 5). The amount of IL-1β mRNA in the infarcted basal ganglia of WT mice was elevated already 4 hours after tMCAO and further increased until 24 hours compared to sham-treated controls (54-fold induction). In contrast, IL-1β mRNA expression was significantly lower in B1R KO mice after 4 hours (4.3±3.8-fold induction vs 25.8±8.3-fold induction, respectively; P<0.05) and 24 hours after ischemia (13.1±4.7-fold induction vs 54.2±18.6 induction, respectively; P<0.01). Four hours after tMCAO, transforming growth factor β-1 transcripts were already markedly elevated in the ischemic brains of B1R KO mice compared to sham-treated controls, but not in WT animals (2.4±0.41-fold induction vs 0.9±0.2-fold induction, respectively; P<0.05) and transforming growth factor β-1 expression sustained until day 1. Tumor necrosis factor- levels did not differ between WT and B1R KO mice at any time point (Figure 5).
|
To further analyze the extent of the inflammatory response, we quantified the amount of immune cells invading the ischemic basal ganglia by immunohistochemistry (Figure 6A,B). Twenty-four hours after the induction of tMCAO, the majority of neutrophilic granulocytes were detected in the brain parenchyma of WT mice (Figure 6A, arrows), whereas some of them were still in the process of evading cerebral blood vessels (Figure 6A, arrowheads). In contrast, significantly less neutrophilic granulocytes (14.0±4.6 vs 29.0±11.3; P<0.05), macrophages (14.0±5.3 vs 32.6±11.1; P<0.001), and T cells (1.0±0.6 vs 4.0±1.0; P<0.0001) invaded the ischemic basal ganglia of B1R KO mice compared to WT controls (Figure 6A,B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
The main finding of the current study is that B1R blockade profoundly reduces infarct volumes and neurological deficits after experimental ischemic stroke in mice by salvaging the neocortex. Within the basal ganglia that were similarly affected by infarctions in WT and B1R KO mice, brain edema formation was less in B1R KO mice and the local inflammatory response was attenuated.
Although B1R and B2R have been detected in the rodent central nervous system, little is known about the expression of kinin receptors in the mouse brain after experimental stroke.8,18 Real-time reverse-transcription polymerase chain reaction revealed that both receptors are profoundly induced at very early stages after tMCAO. The time course of B1R and B2R induction in our study thus paralleled the formation of their ligand bradykinin after tMCAO in mice,12 suggesting a functional relevance for the kallikrein–kinin system in the acute phase of ischemic stroke. The cells expressing B1R and B2R in the (infarcted) brain remain to be further characterized but might be neurons, microglia, and endothelial cells.8
Previous studies could show that the blockade of the B1R protects from ischemic tissue damage, eg, in models of myocardial or renal infarction.34–37 This protective effect was accompanied by reduced ischemia-related inflammation. We further extend these findings by demonstrating that B1R is also critically involved in infarct development in the brain. Importantly, decreased stroke volumes in B1R-deficient mice were accompanied by a significant reduction in neurological deficits and mortality, and the pharmacological B1R blockade was still effective when performed 1 hour after the induction of stroke. This underlines the functional significance of this novel approach and indicates a potential suitability of selective B1R inhibitors13 for clinical application during the acute phase of ischemic stroke in humans.
The kallikrein–kinin system plays an important role in regulating blood pressure,8 which in turn could influence infarct volume after ischemic stroke. The pharmacological blockade of B1R and B2R had no effect on blood pressure and heart rate in our study. This is in line with our previous findings demonstrating that B1R KO mice and animals deficient in both kinin receptors (B1R/B2R double KO mice) are normotensive.24–26 Moreover, B1R antagonists did not change blood pressure in normotensive rats.38,39 These results indicate that blood pressure effects cannot account for the profound stroke protection in B1R-deficient mice or after pharmacological blockade of B1R.
Besides preventing infarct development within the neocortex, brain edema formation within areas of infarction in the basal ganglia was also reduced after blockade of B1R, as indicated by lack of Evan’s blue extravasation regularly seen in WT mice. Brain edema is considered an important secondary step in lesion development after stroke and reaches its maximum between day 1 and day 3.1,2 Interestingly, induction of endothelin-1 transcripts was nearly absent in the ischemic basal ganglia of B1R-null mice. In contrast, high levels of endothelin-1 were found at 4 hours and 24 hours after experimental stroke in WT animals, thus confirming previous observations.40,41 Endothelin-1 has been shown to be critically involved in regulating vascular integrity and edema formation under various pathophysiological conditions, including ischemic stroke.42,43 Mice overexpressing endothelin-1 developed more brain edema and larger cerebral infarctions after tMCAO.44 Moreover, pharmacological blockade of the endothelin type A receptor attenuated ischemic brain injury, edema formation, and blood–brain barrier disruption in rats.40,45 Most interestingly, high serum levels of endothelin-1 have very recently been shown to predict severe cerebral edema in patients with acute ischemic stroke after recombinant tissue plasminogen activator treatment.46 The molecular pathways that link the kallikrein–kinin system and endothelin-1–driven mechanism in ischemic stroke need to be further established.
The second mechanism by which B1R deficiency probably conveyed neuroprotection after focal cerebral ischemia was attenuation of the local inflammatory response.47 Twenty-four hours after tMCAO, the majority of invading neutrophilic granulocytes was already detected in the brain parenchyma of WT mice, whereas some cells were still evading the cerebral blood vessels, which confirms previous observations.47 In contrast, the number of neutrophils in the ischemic basal ganglia of B1R KO mice was significantly reduced. Leukocyte invasion has been shown to contribute to stroke development, probably by impairing reperfusion of the cerebral microvasculature.48,49 In addition, less T cells infiltrated the infarcted brain of B1R-deficient animals. Interestingly, several recent studies could demonstrate that 24 hours after 60 minutes of tMCAO, brain infarct volumes were significantly reduced in mice lacking T cells.50,51 The mechanism by which T-cell deficiency mediates neuroprotection in experimental stroke seems to be at least partly mediated by reduced interactions between lymphocytes and platelets in the cerebral microvasculature, leading to improved tissue reperfusion. Finally, the expression of various soluble immune mediators after tMCAO was altered. B1R mutant mice expressed less IL-1β within the ischemic brain, whereas the amount of transforming growth factor β-1 was increased compared to WT controls. IL-1β is a prototypic proinflammatory cytokine that has been attributed to aggravate ischemic brain damage.47 In contrast, transforming growth factor β-1 exerts pleiotropic immune functions and has been shown to mediate neuroprotection in different stroke models, eg, by augmenting antiapoptotic mechanisms.52 The cells producing these cytokines after tMCAO need to be further characterized but might include neurons, microglia, or invading immune cells.47
Previous investigations using B2R-deficient mice in experimental stroke produced contradictory results. Gröger et al 12 reported that B2R KO mice are protected from cerebral ischemia after tMCAO. The reasons for this discrepancy to our results are not clear at present but may be explained by different study designs, eg, the relatively small sample size.12 Differences in the time of brain ischemia (45 min vs 60 min) obviously did not account for the divergent results, because we also observed identical infarct volumes after 45 minutes of tMCAO at day 1 in WT and B2R KO mice (not shown). In obvious contrast to Gröger et al,12 Xia et al53 postulated that postischemic brain injury is exacerbated in mice lacking the B2R. Numerous studies did not find a detrimental effect of B2R deficiency or blockade on stroke outcome.12,54,55 Infarct size in WT mice after 90 minutes of MCAO was surprisingly small in the study by Xia et al,53 whereas the B2R KO mice had developed infarcts that were the size that one would usually expect in WT mice after 90 minutes of tMCAO. We and others27,28,55,56 could demonstrate that 60 minutes of occlusion of the MCA already causes infarct volumes between 50 and 80 mm3 at day 1 after MCAO, eg, in C57BL/6, SV129, or Swiss mice. In the article by Xia et al,53 90 minutes of occlusion only led to infarct volumes of 12.8±7.3 mm3 in the WT group. These very small infarctions in the control group are highly suggestive for insufficient vessel occlusion and additional reasons for those discrepant findings have been addressed elsewhere.57,58
Taken together, our present study provides evidence that blocking of the B1R can diminish brain edema and cerebral infarction in mice and may open new avenues for acute stroke treatment in humans in the future. Whether this novel strategy is also applicable in severe central nervous system pathologies other than ischemic stroke, such as intracranial bleeding or head trauma, needs to be further established.
|
Acknowledgments |
|---|
The authors thank Melanie Glaser for excellent technical assistance.
Sources of Funding
This work was supported by grants from the Interdisciplinary Clinical Research Center (IZKF) Würzburg (to C.K. and T.R.: IZKF E-35).
Disclosures
None.
Received May 22, 2008; accepted June 4, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
1. Ayata C, Ropper AH. Ischaemic brain oedema. J Clin Neurosci. 2002; 9: 113–124.[CrossRef][Medline] [Order article via Infotrieve]
2. Bardutzky J, Schwab S. Antiedema therapy in ischemic stroke. Stroke. 2007; 38: 3084–3094.
3. Vahedi K, Hofmeijer J, Juettler E, Vicaut E, George B, Algra A, Amelink GJ, Schmiedeck P, Schwab S, Rothwell PM, Bousser MG, van der Worp HB, Hacke W; DECIMAL, DESTINY, and HAMLET Investigators. Early decompressive surgery in malignant infarction of the middle cerebral artery: a pooled analysis of three randomised controlled trials. Lancet Neurol. 2007; 6: 215–222.[CrossRef][Medline] [Order article via Infotrieve]
4. Qizilbash N, Lewington SL, Lopez-Arrieta JM. Corticosteroids for acute ischaemic stroke. Cochrane Database Syst Rev. 2002; 2: CD000064.[Medline] [Order article via Infotrieve]
5. Righetti E, Celani MG, Cantisani T, Sterzi R, Boysen G, Ricci S. Glycerol for acute stroke. Cochrane Database Syst Rev. 2004; 2: CD000096.[Medline] [Order article via Infotrieve]
6. Bereczki D, Fekete I, Prado GF, Liu M. Mannitol for acute stroke. Cochrane Database Syst Rev. 2007; 183: CD001153.
7. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev. 1992; 441: 1–80.
8. Leeb-Lundberg LM, Marceau F, Müller-Esterl W, Pettibone DJ, Zuraw BL. International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol Rev. 2005; 571: 27–77.
9. Renné T, Schuh K, Müller-Esterl W. Local bradykinin formation is controlled by glycosaminoglycans. J Immunol. 2005; 175: 3377–3385.
10. Kamiya T, Katayama Y, Kashiwagi F, Terashi A. The role of bradykinin in mediating ischemic brain edema in rats. Stroke. 1993; 244: 571–575.
11. Lehmberg J, Beck J, Baethmann A, Uhl E. Bradykinin antagonists reduce leukocyte-endothelium interactions after global cerebral ischemia. J Cereb Blood Flow Metab. 2003; 234: 441–448.
12. Gröger M, Lebesgue D, Pruneau D, Relton J, Kim SW, Nussberger J, Plesnila N. Release of bradykinin and expression of kinin B2 receptors in the brain: role for cell death and brain edema formation after focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 2005; 258: 978–989.
13. Rodi D, Couture R, Ongali B, Simonato M. Targeting kinin receptors for the treatment of neurological diseases. Curr Pharm Des. 2005; 1110: 1313–1326.
14. Camargo AC, Shapanka R, Greene LJ. Preparation, assay, and partial characterization of a neutral endopeptidase from rabbit brain. Biochemistry. 1973; 129: 1838–1844.
15. Kariya K, Yamauchi A, Sasaki T. Regional distribution and characterization of kinin in the CNS of the rat. J Neurochem. 1985; 446: 1892–1897.
16. Kizuki K, Suzuki T, Kudo M, Noguchi T. Immunohistochemical demonstration of tissue kallikrein in the neurons of rat brain. Brain Res. 1994; 6342: 305–309.
17. Hall JM. Bradykinin receptors: pharmacological properties and biological roles. Pharmacol Ther. 1992; 562: 131–190.
18. Walker K, Perkins M, Dray A. Kinins and kinin receptors in the nervous system. Neurochem Int. 1995; 261: 1–16.
19. Pizard A, Blaukat A, Müller-Esterl W, Alhenc-Gelas F, Rajerison RM. Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail. J Biol Chem. 1999; 274: 12738–12747.
20. Blaukat A, Micke P, Kalatskaya I, Faussner A, Müller-Esterl W. Downregulation of bradykinin B2 receptor in human fibroblasts during prolonged agonist exposure. Am J Physiol Heart Circ Physiol. 2003; 284: 1909–1916.
21. Sarker MH, Hu DE, Fraser PA. Acute effects of bradykinin on cerebral microvascular permeability in the anaesthetized rat. J Physiol. 2000; 528: 177–187.
22. Medeiros R, Cabrini DA, Ferreira J, Fernandes ES, Mori MA, Pesquero JB, Bader M, Avellar MC, Campos MM, Calixto JB. Bradykinin B1 receptor expression induced by tissue damage in the rat portal vein: a critical role for mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways. Circ Res. 2004; 94: 1375–1382.
23. Dirnagl U. Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab. 2006; 26: 1465–1478.[CrossRef][Medline] [Order article via Infotrieve]
24. Pesquero JB, Araujo RC, Heppenstall PA, Stucky CL, Silva JA Jr, Walther T, Oliveira SM, Pesquero JL, Paiva AC, Calixto JB, Lewin GR, Bader M. Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors. Proc Natl Acad Sci U S A. 2000; 97: 8140–8145.
25. Cayla C, Todiras M, Iliescu R, Saul VV, Gross V, Pilz B, Chai G, Merino VF, Pesquero JB, Baltatu OC, Bader M. Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension. FASEB J. 2007; 21: 1689–1698.
26. Xu J, Carretero OA, Sun Y, Shesely EG, Rhaleb NE, Liu YH, Liao TD, Yang JJ, Bader M, Yang XP. Role of the B1 kinin receptor in the regulation of cardiac function and remodeling after myocardial infarction. Hypertension. 2005; 45: 747–753.
27. Kleinschnitz C, Stoll G, Bendszus M, Schuh K, Pauer HU, Burfeind P, Renné C, Gailani D, Nieswandt B, Renné T. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med. 2006; 2033: 513–518.
28. Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, Stoll G. Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. Circulation. 2007; 11517: 2323–2330.
29. Gobeil F, Neugebauer W, Filteau C, Jukic D, Allogho SN, Pheng LH, Nguyen-Le XK, Blouin D, Regoli D. Structure-activity studies of B1 receptor-related peptides. Antagonists. Hypertension. 1996; 28: 833–839.
30. Wirth K, Hock FJ, Albus U, Linz W, Alpermann HG, Anagnostopoulos H, Henk S, Breipohl G, König W, Knolle J. Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies. Br J Pharmacol. 1991; 102: 774–777.[Medline] [Order article via Infotrieve]
31. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986; 173: 472–476.
32. Moran PM, Higgins LS, Cordell B, Moser PC. Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein. Proc Natl Acad Sci U S A. 1995; 9212: 5341–5345.
33. Brede M, Wiesmann F, Jahns R, Hadamek K, Arnolt C, Neubauer S, Lohse MJ, Hein L. Feedback inhibition of catecholamine release by two different alpha2-adrenoceptor subtypes prevents progression of heart failure. Circulation. 2002; 106: 2491–2496.
34. McLean PG, Ahluwalia A, Perretti M. Association between kinin B(1) receptor expression and leukocyte trafficking across mouse mesenteric postcapillary venules. J Exp Med. 2000; 192: 367–380.
35. Souza DG, Lomez ES, Pinho V, Pesquero JB, Bader M, Pesquero JL, Teixeira MM. Role of bradykinin B2 and B1 receptors in the local, remote, and systemic inflammatory responses that follow intestinal ischemia and reperfusion injury. J Immunol. 2004; 172: 2542–2548.
36. Lagneux C, Bader M, Pesquero JB, Demenge P, Ribuot C. Detrimental implication of B1 receptors in myocardial ischemia: evidence from pharmacological blockade and gene knockout mice. Int Immunopharmacol. 2002; 2: 815–822.[CrossRef][Medline] [Order article via Infotrieve]
37. Yin H, Chao J, Bader M, Chao L. Differential role of kinin B1 and B2 receptors in ischemia-induced apoptosis and ventricular remodeling. Peptides. 2007; 28: 1383–1389.[CrossRef][Medline] [Order article via Infotrieve]
38. Emanueli C, Chao J, Regoli D, Chao L, Ni A, Madeddu P. The bradykinin B1 receptor and the central regulation of blood pressure in spontaneously hypertensive rats. Br J Pharmacol. 1999; 126: 1769–1776.[CrossRef][Medline] [Order article via Infotrieve]
39. Duka A, Duka I, Gao G, Shenouda S, Gavras I, Gavras H. Role of bradykinin B1 and B2 receptors in normal blood pressure regulation. Am J Physiol Endocrinol Metab. 2006; 291: 268–274.[CrossRef]
40. Matsuo Y, Mihara Si, Ninomiya M, Fujimoto M. Protective effect of endothelin type A receptor antagonist on brain edema and injury after transient middle cerebral artery occlusion in rats. Stroke. 2001; 32: 2143–2148.
41. Barone FC, Globus MY, Price WJ, White RF, Storer BL, Feuerstein GZ, Busto R, Ohlstein EH. Endothelin levels increase in rat focal and global ischemia. J Cereb Blood Flow Metab. 1994; 14: 337–342.[Medline] [Order article via Infotrieve]
42. Iglarz M, Clozel M. Mechanisms of ET-1-induced endothelial dysfunction. J Cardiovasc Pharmacol. 2007; 50: 621–628.[Medline] [Order article via Infotrieve]
43. Kirkby NS, Hadoke PW, Bagnall AJ, Webb DJ. The endothelin system as a therapeutic target in cardiovascular disease: great expectations or bleak house? Br J Pharmacol. 2008; 153: 1105–1119.[CrossRef][Medline] [Order article via Infotrieve]
44. Lo AC, Chen AY, Hung VK, Yaw LP, Fung MK, Ho MC, Tsang MC, Chung SS, Chung SK. Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet. J Cereb Blood Flow Metab. 2005; 25: 998–1011.[CrossRef][Medline] [Order article via Infotrieve]
45. Dawson DA, Sugano H, McCarron RM, Hallenbeck JM, Spatz M. Endothelin receptor antagonist preserves microvascular perfusion and reduces ischemic brain damage following permanent focal ischemia. Neurochem Res. 1999; 24: 1499–1505.[CrossRef][Medline] [Order article via Infotrieve]
46. Moldes O, Sobrino T, Millán M, Castellanos M, de la Ossa NP, Leira R, Serena J, Vivancos J, Dávalos A, Castillo J High Serum Levels of Endothelin-1 Predict Severe Cerebral Edema in Patients With Acute Ischemic Stroke Treated With t-PA. Stroke. 2008; doi:10.1161/STROKEAHA.107.495044.
47. Kleinschnitz C, Stoll G. Inflammation and the Role of Immune Mediators in the Pathophysiology of Ischemic Stroke. In: Romano GT, ed. Inflammation Research Perspectives. New York: Nova Science Publishers Inc; 2008; 81–112.
48. del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab. 2003; 23: 879–894.[CrossRef][Medline] [Order article via Infotrieve]
49. Arumugam TV, Salter JW, Chidlow JH, Ballantyne CM, Kevil CG, Granger DN. Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion. Am J Physiol Heart Circ Physiol. 2004; 287: 2555–2560.[CrossRef]
50. Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation. 2006; 113: 2105–2112.
51. Hurn PD, Subramanian S, Parker SM, Afentoulis ME, Kaler LJ, Vandenbark AA, Offner H. T- and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation. J Cereb Blood Flow Metab. 2007; 27: 1798–1805.[CrossRef][Medline] [Order article via Infotrieve]
52. Buisson A, Lesne S, Docagne F, Ali C, Nicole O, MacKenzie ET, Vivien D. Transforming growth factor-beta and ischemic brain injury. Cell Mol Neurobiol. 2003; 23: 539–550.[CrossRef][Medline] [Order article via Infotrieve]
53. Xia CF, Smith RS Jr, Shen B, Yang ZR, Borlongan CV, Chao L, Chao J. Postischemic brain injury is exacerbated in mice lacking the kinin B2 receptor. Hypertension. 2006; 47: 752–761.
54. Zausinger S, Lumenta DB, Pruneau D, Schmid-Elsaesser R, Plesnila N, Baethmann A. Effects of LF 16–0687 Ms, a bradykinin B(2) receptor antagonist, on brain edema formation and tissue damage in a rat model of temporary focal cerebral ischemia. Brain Res. 2002; 950: 268–278.[CrossRef][Medline] [Order article via Infotrieve]
55. Ding-Zhou L, Margaill I, Palmier B, Pruneau D, Plotkine M, Marchand-Verrecchia C. LF 16–0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia. Br J Pharmacol. 139: 153915–153947, 2003.
56. Prüss H, Prass K, Ghaeni L, Milosevic M, Muselmann C, Freyer D, Royl G, Reuter U, Baeva N, Dirnagl U, Meisel A, Priller J. Inducible nitric oxide synthase does not mediate brain damage after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 2008; 28: 526–539.[CrossRef][Medline] [Order article via Infotrieve]
57. Kleinschnitz C, Austinat M, Bader M, Renné T, Stoll G. Deficiency of bradykinin receptor B2 is not detrimental in experimental stroke (letter). Hypertension. 2008; 51: e41.
58. Xia CF, Smith RS, Shen B Jr, Yang ZR, Borlongan CV, Chao L, Chao J. Response to deficiency of bradykinin receptor B2 is not detrimental in experimental stroke. Hypertension. 2008; 51: e42–e43.
Expanded Materials and Methods
|
Induction of Cerebral Ischemia |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Focal cerebral ischemia was induced in 6- to 8-week-old mice by 60-min transient middle cerebral artery occlusion (tMCAO) as described.1–3 Mice were anesthetized with 2.5% isoflurane (Abbott; Wiesbaden, Germany) in a 70% N2O/30% O2 mixture. After a midline skin incision in the neck, the proximal common carotid artery and the external carotid artery were ligated, and a standardized silicon rubber-coated 6.0 nylon monofilament (6021; Doccol Corp; Redlands, Calif, USA) was inserted and advanced via the right internal carotid artery to occlude the origin of the right MCA. Operators (M.A., S.B. and C.K.) were blinded to the genotype, and operation time per animal did not exceed 15 minutes. The intraluminal suture was left in situ for 60 minutes. Then animals were reanesthetized and the occluding monofilament was withdrawn to allow reperfusion. Neurological function was assessed by 2 independent and blinded investigators 24 and 72 hours (B1R KO mice) after tMCAO. Global neurological status was scored according to Bederson et al.4 Motor function and coordination were graded using the grip test.5 For PCR studies sham-treated C57BL/6 mice (n=4) in which the filament was not advanced to the origin of the right MCA were used as controls. To determine the effect of pharmacological B1R inhibition in WT mice, the selective B1R inhibitor R-175 (Ac-Lys-[D-βNal7, Ile8]desArg9-BK)6 (Biomatik Corp; Cambridge, Canada) was administered intravenously 1 hour after tMCAO at a dosage of 500 µg/kg body weight (n=10) or 1 mg/kg body weight (n=10), respectively (0.9% NaCl carrier solution). For the selective blockade of B2R Hoe-140 (D-Arg0-Hyp3-Thi5-D-Tic7-Oci8-BK)7 (0.2 mg/kg body weight, 0.9% NaCl carrier solution; Sigma Aldrich, Steinheim, Germany) was injected 1 hour after tMCAO (n=10).
|
Laser-Doppler Flowmetry |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Laser-Doppler flowmetry (Moor Instruments, UK) was used in some animals (n=6/group) to monitor regional cerebral blood flow (rCBF) in the MCA territory (6 mm lateral and 2 mm posterior from bregma).8 After advancing the thread the decrease in rCBF was similar in all groups indicating sufficient occlusion of the MCA origin (16.3±1.5% of baseline level in WT mice versus 14.1±5.4% of baseline level in B2R KO mice and 16.6±3.8% of baseline level in B1R KO mice, respectively; P>0.05; supplemental Figure IA). Ten minutes after reperfusion rCBF was reconstituted to >60% of baseline levels and again did not significantly differ between WT and KO animals (72.1±4.2% of baseline level in WT mice and 70.2±5.5% of baseline level in B2R KO mice and 71.5±4.8% in B1R KO mice, respectively; P>0.05).
|
Cerebrovasculature Assessment |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
For cerebrovasculature assessment WT, B1R and B2R KO mice (n=3/group) were deeply anesthetized with CO2 and transcardially perfused with 4% paraformaldehyde (PFA), followed by 3 mL black ink diluted in 4% PFA (1:5 v/v). Brains were carefully removed, fixed in 4% PFA overnight at 4°C and the Circle of Willis and major arteries were examined under a microscope (supplemental Figure IB). A complete Circle of Willis was identified in all animals studied and the distribution of the MCA trunk and branch appeared to be anatomically identical among the genotypes. To further quantitatively examine the vascular structures, the development of the posterior communicating arteries (PComAs) which can affect brain sensitivity to ischemia9 was examined. The mean score of PComAs in both hemispheres showed no significant differences between the groups (3.0±0.0 in WT mice versus 3.7±0.6 in B2R KO mice and 3.7±0.6 in B1R KO mice, respectively; P>0.05).
|
Determination of Infarct Size |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Wild-type (n=10), B1R KO mice (n=10), B2R KO mice (n=10) as well as R-175- (n=10) and Hoe-140-treated (n=10) animals were sacrificed 24 hours after tMCAO. Additional groups of WT and B1R KO mice (n=5 each) were allowed to survive until day 3. Brains were quickly removed and cut in three 2-mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus; Holliston, Mass, USA). The slices were stained for 20 min at 37°C with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Aldrich; Germany) in PBS to visualize the infarctions.10 Planimetric measurements (ImageJ software; National Institutes of Health, USA) were performed blinded to the groups and were used to calculate infarct volumes using the following formula: Vinfarct (mm3)=(area section 1 (mm2)x2.0 mm)+(area section 2 (mm2)x2.0 mm)+(area section 3 (mm2)x2.0 mm). Correction for brain swelling (edema) was not performed since the infarct volumes were suspected to critically depend on the extent of edema formation in B1R KO, B2R KO and WT mice.
|
Determination of Brain Edema |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
C57BL/6 (n=8), B1R (n=8) and B2R KO mice (n=4) were killed 24 h after reperfusion. Brains were removed, hemispheres separated, and weighed to assess the wet weight (WW). Thereafter, the hemispheres were dried for 24 h at 110°C and the dry weight (DW) was determined. Hemispheric water content (%) was calculated using the following formula: ((WW–DW)/WW)x100.
To determine the permeability of the cerebral vasculature 2% Evan’s Blue tracer (Sigma Aldrich, Germany) diluted in 0.9% NaCl was IV injected 2 hours after the induction of tMCAO.11 After 24 hours WT and KO mice (n=4/group) were transcardially perfused with 4% PFA and brains were quickly removed and cut in 2 mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus; Holliston, Mass, USA). Planimetric measurements (ImageJ software; National Institutes of Health, USA) of the brain parenchyma stained by Evan’s Blue were performed to calculate edema volumes.
|
PCR Studies |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Ischemic infarctions mostly spared the neocortex of B1R KO mice (Figures 2 and 3
). Therefore, basal ganglia (instead of total hemispheres), which were regularly included in the ischemic infarctions also in B1R KO mice (Figures 2 and 3), were dissected from the brains in all groups at 4 hours (n=8 per group) and 24 hours (n=8 per group) after tMCAO. This mode of sampling excluded biased PCR results, eg due to different infarct sizes between the groups. Tissue homogenization, RNA isolation and Real-time RT-PCR were performed as described.12–14 Total RNA was prepared with a Miccra D-8 power homogenizer (ART, Germany) using the TRIzol reagent (Invitrogen, Germany) and was quantified spectrophotometrically. Then, 250 µg of total RNA were reversely transcribed with the TaqMan Reverse Transcription Reagents (Applied Biosystems, Germany) according to the manufacturer’s protocol using random hexamers. Relative levels of cytokine and B1R and B2R mRNA were quantified with the fluorescent TaqMan technology. PCR primers and probes specific for murine B1R (assay ID: Mm00432059_s1), B2R (assay ID: Mm00437788_s1), IL-1β (assay ID: Mm004344228_m1), Transforming growth factor (TGF)β-1 (assay ID: Mm00441724_m1), endothelin-1 (assay ID: Mm 00438656_m1) and TNF (assay ID: Mm00443258_m1) were obtained as TaqMan Gene Expression Arrays (Applied Biosystems, Germany). 18s rRNA (TaqMan Predeveloped Assay Reagents for gene expression, part number: 4319413E, Applied Biosystems, Germany) was used as an endogenous control to normalize the amount of sample RNA. The PCR was performed with equal amounts of cDNA in the GeneAmp 7700 sequence detection system (Applied Biosystems, Germany) using the TaqMan Universal PCR Master Mix (Applied Biosystems, Germany). Reactions (total volume 50 µl) were incubated at 50°C for 2 minutes, at 95°C for 10 minutes followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Water controls were included to ensure specificity. Each sample was measured in triplicate and data points were examined for integrity by analysis of the amplification plot. The comparative Ct method was used for relative quantification of gene expression as described.12–14 |
Histology and Immunohistochemistry |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
Formalin-fixed brains embedded in paraffin from WT, B1R and B2R KO mice at day 1 after tMCAO (n=6/group) were cut into 4-µm thick sections (0.5 mm anterior from bregma) at the level of the infarcted basal ganglia. After deparaffinization and rehydratation tissues were stained with hematoxylin and eosin (Sigma Aldrich, Germany). For immunohistochemistry antigen retrieval was achieved by pretreatment with proteinase (P8038; Sigma Aldrich, Germany). Thereafter, endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 minutes and unspecific binding was prevented by adding 10% BSA for 30 minutes. For staining of invading immune cells rat anti-mouse NIMP-R14 (neutrophilic granulocytes; SC-59338; Santa Cruz Biotechnology; Calif, USA), rat anti-human CD3 (T cells; MCA1477; Serotec, Germany) and rat anti-mouse F4/80 (macrophages; BM4008; Acris, Germany) were applied at a dilution of 1:100 in PBS containing 1% BSA overnight at 4°C. Subsequently the slides were incubated with a biotinylated anti-rat IgG (BA-4001, Vector Laboratories, USA) diluted 1:100 in PBS containing 1% BSA for 45 minutes at room temperature. The secondary antibody was linked via streptavidin to a biotinylated peroxidase (POD) according to the manufacturer’s instructions (StreptABComplex/HRP Duet, K 0492, DakoCytomation, Denmark). Following POD staining with 3,3'-Diaminobenziden (DAB) (Kem-En-Tec Diagnostics, Denmark) and counterstaining with aqueous hematoxylin was performed. Negative controls included omission of primary or secondary antibody; tissue sections from murine spleens served as positive controls. For quantification of immune cells identical brain sections (thickness 4-µm) of WT, B1R and B2R KO mice at the level of the basal ganglia (0.5 mm anterior from bregma) were selected and cell counting was performed under a Zeiss Axiophot microscope (Zeiss; Oberkochen, Germany).
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesInduction of Cerebral IschemiaLaser-Doppler FlowmetryCerebrovasculature AssessmentDetermination of Infarct SizeDetermination of Brain EdemaPCR StudiesHistology and...References |
|---|
1. Clark WM, Lessov NS, Dixon MP, Eckenstein F. Monofilament intraluminal middle cerebral artery occlusion in the mouse. Neurol Res. 1997; 196: 641–648.
2. Kleinschnitz C, Stoll G, Bendszus M, Schuh K, Pauer HU, Burfeind P, Renné C, Gailani D, Nieswandt B, Renné T. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med. 2006; 2033: 513–518.
3. Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, Stoll G. Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. Circulation. 2007; 11517: 2323–2330.
4. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986; 173: 472–476.
5. Moran PM, Higgins LS, Cordell B, Moser PC. Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein. Proc Natl Acad Sci U S A. 1995; 9212: 5341–5345.
6. Gobeil F, Neugebauer W, Filteau C, Jukic D, Allogho SN, Pheng LH, Nguyen-Le XK, Blouin D, Regoli D. Structure-activity studies of B1 receptor-related peptides. Antagonists. Hypertension. 1996; 28: 833–839.
7. Wirth K, Hock FJ, Albus U, Linz W, Alpermann HG, Anagnostopoulos H, Henk S, Breipohl G, König W, Knolle J. Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies. Br J Pharmacol. 1991; 102: 774–777.[Medline] [Order article via Infotrieve]
8. Connolly ES Jr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ. Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery. 1996; 383: 523–531.
9. Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab. 1993; 134: 683–692.
10. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke. 1986; 176: 1304–1308.
11. Belayev L, Busto R, Zhao W, Ginsberg MD. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 1996; 739: 88–96.[CrossRef][Medline] [Order article via Infotrieve]
12. Kleinschnitz C, Schroeter M, Jander S, Stoll G. Induction of granulocyte colony-stimulating factor mRNA by focal cerebral ischemia and cortical spreading depression. Brain Res Mol Brain Res. 2004; 131: 73–78.[Medline] [Order article via Infotrieve]
13. Kleinschnitz C, Brinkhoff J, Sommer C, Stoll G. Contralateral cytokine gene induction after peripheral nerve lesions: dependence on the mode of injury and NMDA receptor signaling. Brain Res Mol Brain Res. 2005; 136: 23–28.[CrossRef][Medline] [Order article via Infotrieve]
14. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25: 402–408.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  D. Westermann, T. Walther, K. Savvatis, F. Escher, M. Sobirey, A. Riad, M. Bader, H.-P. Schultheiss, and C. Tschope Gene Deletion of the Kinin Receptor B1 Attenuates Cardiac Inflammation and Fibrosis During the Development of Experimental Diabetic Cardiomyopathy Diabetes, June 1, 2009; 58(6): 1373 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tsuda Role of Bradykinin and Catecholamines in Cerebral Infarction and Brain Edema Stroke, April 1, 2009; 40(4): e103 - e103. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||