Blocking of bradykinin receptor B1 protects from focal closed head injury in mice by reducing axonal damage and astroglia activation

Abstract

The two bradykinin receptors B1R and B2R are central components of the kallikrein-kinin system with different expression kinetics and binding characteristics. Activation of these receptors by kinins triggers inflammatory responses in the target organ and in most situations enhances tissue damage. We could recently show that blocking of B1R, but not B2R, protects from cortical cryolesion by reducing inflammation and edema formation. In the present study, we investigated the role of B1R and B2R in a closed head model of focal traumatic brain injury (TBI; weight drop). Increased expression of B1R in the injured hemispheres of wild-type mice was restricted to the later stages after brain trauma, i.e. day 7 (P<0.05), whereas no significant induction could be observed for the B2R (P>0.05). Mice lacking the B1R, but not the B2R, showed less functional deficits on day 3 (P<0.001) and day 7 (P<0.001) compared with controls. Pharmacological blocking of B1R in wild-type mice had similar effects. Reduced axonal injury and astroglia activation could be identified as underlying mechanisms, while inhibition of B1R had only little influence on the local inflammatory response in this model. Inhibition of B1R may become a novel strategy to counteract trauma-induced neurodegeneration.

Figure 1

Induction of B1R and B2R is restricted to the later phase after focal closed head injury. Relative (Rel.) gene expression of B1R (left) and B2R (right) in the lesioned hemispheres of wild-type mice on day 1 (d1) or day 7 (d7) after weight-drop injury or sham operation (n=4 per group and time point). ^*P<0.05, 1-way analysis of variance (ANOVA), Bonferroni's post hoc test compared with sham-operated mice. ns, not significant.

Figure 2

Blocking of B1R, but not B2R, improves functional outcome after weight-drop injury. (A) B1r^−/− mice had a significantly lower neurological severity score (NSS) on day 3 (d3) and day 7 (d7) after focal closed head trauma compared with wild-type (WT) mice while the early functional deficits after 1 hour were similar. No protection was observed in B2r^−/− mice (n=18 per group). (B) WT mice treated with the specific B1R blocker R-715 (1 mg/kg body weight) starting from 1 hour after trauma also developed less severe neurological deficits on day 3 (d3) and day 7 (d7) compared with vehicle-treated mice (Ctrl), thereby confirming our results in transgenic mice (n=7 per group). ^*P<0.05, ^**P<0.001, Kruskal–Wallis test with post hoc Dunn's multiple comparison test compared with WT mice or untreated mice, respectively. ns, not significant.

Figure 3

B1R deficiency reduces delayed axonal damage and neuronal apoptosis after weight-drop injury. (A) The injury-induced rise of β-amyloid precursor protein (β-APP) mRNA expression as an indicator of diffuse axonal injury seen in wild-type (WT) mice on day 7 (d7) after head trauma was significantly attenuated in B1r^−/− mice (n=4 per group and time point). ^###P<0.0001, 2-way analysis of variance (ANOVA), Bonferroni post hoc test compared with sham-operated mice; ^**P<0.001, 2-way ANOVA, Bonferroni post hoc test compared with WT mice. (B) (left) Representative brain sections from WT and B1r^−/− mice 7 days after weight-drop injury. Apparently more axons stained positive for non-phosphorylated neurofilament (anti-SMI32, arrows), a marker for axonal damage, in the cortices of WT mice compared with B1r^−/− mice on day 7 (d7) after weight-drop injury and increased traumatic axonal damage could be confirmed by counting the numbers of neurofilament-positive retraction bulbs (right; n=4 per group). ^***P<0.0001, unpaired, two-tailed Student's t-test compared with WT mice. Bar=10 μm. (C) Left panels show representative brain sections from WT and B1r^−/− mice 7 days after weight-drop injury, immunolabeled for the neuronal marker NeuN and subjected to TUNEL assay to detect apoptosis. Right panels show the number of TUNEL-positive neurons per brain slice in the injured hemispheres of both groups on day 7 (d7) (n=4 per group). ^*P<0.05, unpaired, two-tailed Student's t-test compared with WT mice. Bar=100 μm. ns, not significant; Rel., relative.

Figure 4

B1R deficiency reduces delayed astrogliosis after weight drop injury. (A) Relative (Rel.) gene expression of the astrocyte activation markers glial fibrillary acidic protein (GFAP), nestin, and vimentin in the lesioned hemispheres of wild-type (WT) mice or B1r^−/− mice on day 1 (d1) or day 7 (d7) after weight-drop injury or sham operation. Note that all astrocyte markers were significantly lower expressed in the brains of B1r^−/− mice on day 7 (n=4 per group and time point). (B) In line with our mRNA expression data, significantly less cells stained positive for GFAP in the injured brains of B1r^−/− mice compared with WT on day 7 (d7) after focal closed head trauma (n=6 per group and time point). ^#P<0.05, ^###P<0.0001, 2-way analysis of variance (ANOVA), Bonferroni post hoc test compared with sham-operated mice; ^*P<0.05, 2-way ANOVA, Bonferroni post hoc test compared with WT mice. n.d., not detectable; ns, not significant.

Figure 5

B1R deficiency has only little effects on the local inflammatory response after weight-drop injury. (A) Relative (Rel.) gene expression of several prototypic pro- and antiinflammatory cytokines in the lesioned hemispheres of wild-type (WT) mice or B1r^−/− mice on day 1 (d1) or day 7 (d7) after weight-drop injury or sham operation. Note that only tumor necrosis factor (TNF)α levels were significantly lower in B1r^−/− mice at the later phase after brain trauma (n=4 per group and time point). (B) In line with these findings, the numbers of CD11b+ microglia/macrophages invading the injured brain were similar between both groups (n=6 per group and time point). ^##P<0.001, 2-way analysis of variance (ANOVA), Bonferroni post hoc test compared with sham-operated mice; ^**P<0.001, 2-way ANOVA, Bonferroni post hoc test compared with WT mice. IL, interleukin; ns, not significant; TGF, transforming growth factor.