Candesartan, an angiotensin II AT₁-receptor blocker and PPAR-γ agonist, reduces lesion volume and improves motor and memory function after traumatic brain injury in mice

Abstract

Traumatic brain injury (TBI) results in complex pathological reactions, the initial lesion worsened by secondary inflammation and edema. Angiotensin II (Ang II) is produced in the brain and Ang II receptor type 1 (AT₁R) overstimulation produces vasoconstriction and inflammation. Ang II receptor blockers (ARBs) are neuroprotective in models of stroke but little is known of their effect when administered in TBI models. We therefore performed controlled cortical impact (CCI) injury on mice to investigate whether the ARB candesartan would mitigate any effects of TBI. We administered candesartan or vehicle to mice 5 h before CCI injury. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%, decreased the number of dying neurons, lessened the number of activated microglial cells, protected cerebral blood flow (CBF), and reduced the expression of the cytokine TGFβ1 while increasing expression of TGFβ3. Candesartan-treated mice also showed better motor skills on the rotarod 3 days after injury, and improved performance in the Morris water maze 4 weeks after injury. These results indicate that candesartan is neuroprotective, reducing neuronal injury, decreasing lesion volume and microglial activation, protecting CBF and improving functional behavior in a mouse model of TBI. Co-treatment with a peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist significantly reduced some of the beneficial effects of candesartan after CCI, suggesting that PPARγ activation may contribute to part or to all of the neuroprotective effect of candesartan. Overall, our data suggest that ARBs with dual AT₁R-blocking and PPARγ activation properties may have therapeutic value in treating TBI.

Figure 1

Neuroprotective effect of candesartan treatment on lesion volume following CCI injury in mice. (a) Experimental design. Osmotic pumps containing candesartan (CD, 1 mg/kg/day) or vehicle (VH) were implanted subcutaneously 5 h before craniotomy (sham) or CCI injury. Mice were pre-trained in the rotarod task (R), at 1 and 2 days before CCI and tested at 1 and 3 days post-injury (dpi). MWM testing began at 24 dpi, and continued daily until 28 dpi, when the mice were killed. (b) Representative sections of injured brains at 3 dpi stained with cresyl-violet (top). The dotted line indicates the lesion area composed of the cavity and edematous area. Candesartan treatment significantly reduced the mean lesion volume by 43% compared with vehicle-treated mice at 3 dpi (mean±SD n=7–8, ***p<0.001) and by 31% at 28 dpi (mean±SD, n=11–12, *p<0.05).

Figure 2

Candesartan treatment reduces the number of dying cells and activated microglial cells after CCI injury. Sections of brains from cortex (a, b) or hippocampus (d, e) taken from mice treated with candesartan (CD) or vehicle (VH) and killed at 3 dpi, showing dying cells, labeled by TUNEL (green) and the neuronal marker NeuN (red) (c, f). Graphs show the number of TUNEL-positive cells at 3 dpi was significantly reduced after injury in CD-treated mice (CCI-CD) compared with those treated with VH (CCI-VH) in the cortex and in the CA1, CA2/3, and dentate gyrus (DG) of the hippocampus (mean±SEM, n=4, *p<0.05). (g, h) At 3 dpi, increased numbers of Iba-1-positive microglia (red) were found in brain sections taken from VH-treated mice in comparison with CD-treated mice. In the VH-treated mice, the microglia had the ameboid and hypertrophy morphology characteristic of activated microglia (g′), in comparison with the more ramified morphology in CD-treated animals (h′). (i, j) Quantitative analysis of Iba-1-positive cells in injured cortex showed a reduction after CD treatment in the number of Iba-1-positive cells and in the fluorescence intensity of Iba-1 staining in the perilesional area (mean±SEM, n=4, *p<0.05). Scale bars represent 50 μm (a–e, g, and h) and 25 μm (g′, h′).

Figure 3

Changes in the CBF and blood pressure after CCI injury in mice treated with vehicle or candesartan. Mice were administered candesartan or vehicle by daily injection for 3 days, starting 5 h before injury. (a) Regional CBF (rCBF) was measured before injury, during, 2 and 18 h after injury, and was expressed as % baseline values (arbitrary units) in the ipsilateral hemisphere. At 2 and 18 h post-injury, candesartan treatment increased CBF values compared with vehicle. (mean±SEM, n=12–17, *p<0.05, ***p<0.001). (b) Blood pressure (BP) in CCI mice with vehicle (CCI-VH) or candesartan-treated (CCI-CD) measured before, and 2 and 24 h after injury, (mean±SEM, n=6).

Figure 4

Candesartan treatment modulates TGFβ1 and TGFβ3 expression after CCI. TGFβ1 immunoreactivity (red) was decreased by candesartan treatment in the ipsilateral cortex (b) and hippocampus (d) in comparison with that in vehicle-treated mice (a, c, respectively). (f) Quantitative analysis of TGFβ1 immunoreactivity showed that candesartan treatment led to a reduction of TGFβ1 immunoreactivity of 47% in the cortex and 49% in the hippocampus compared with vehicle-treated groups (mean±SEM, n=4, *p<0.05). Conversely, candesartan treatment increased TGFβ3 immunoreactivity in the cortex (h) and hippocampus (j) in comparison with immunoreactivity in vehicle mice (g, i, respectively). (l) Quantitative analysis of TGFβ3 immunoreactivity showed that candesartan treatment led to an increase in TGFβ3 immunoreactivity of 50% in the cortex and 36% in the hippocampus compared with vehicle-treated groups (mean±SEM, n=4, *p<0.05). Higher magnification images show TGFβ1 and TGFβ3 colocalization with the astroglial marker, GFAP (e, k, respectively). Scale bars represent 50 μm (a–d and g–j) and 25 μm (e, k).

Figure 5

Candesartan treatment improves motor and cognitive function in mice after CCI. (a) Time (seconds) that mice were able to remain on the rotarod in pre-training and at 1 and 3 days post-injury (dpi). Candesartan treatment (CD) enhanced the ability of mice to stay on the rotarod after either sham (SH) surgery or CCI mice compared with vehicle (VH)-treated mice but did not alter the ability of naive (NAI) mice to perform this test (mean±SEM, n=8–12, ***p<0.008, **p<0.005, *p<0.05) at 1 and 3 dpi. (b) MWM testing showed that after CCI injury mice treated with CD spent more time in the northwest (NW) quadrant from where the platform was removed in the probe trial, compared with mice receiving VH at 28 dpi. Thus, CD treatment led to a greater ability to learn and remember the location of a hidden platform (mean±SEM, n=5 SH-VH, n=3 SH-CD, n=7 CCI-VH, n=8 CCI-CD, *p<0.05).

Figure 6

The influence of PPARγ antagonist on the neuroprotective effects of candesartan following brain injury. Mice were administered vehicle, candesartan (CD) and/or the PPARγ antagonist, T0070907 by daily injection for 3 days, starting 5 h before injury. (a) Effects on PPARγ mRNA expression. PPARγ mRNA expression was not significantly altered after injury (CCI) and/or after CD treatment in the perilesional cortex as compared with vehicle-treated naive (NAI) mice at 3 dpi (mean±SEM, n=4). (b) Effects on lesion volume. At 3 dpi, CD significantly reduced the lesion volume (*p<0.05, CCI-VH vs CCI-CD). T0070907 administration alone (CCI-T0) or together with CD (CCI-CD+T0) did not alter the lesion volume compared with the vehicle group (NS, p>0.05), nor was it significantly different than in mice treated with CD alone (NS, p>0.05, CCI-VH vs CCI-CD+T0) (mean±SEM, n=7–8). (c) Effects on Iba-1-positive microglial cells. CD significantly reduced the number of Iba-1-positive cells in the injured cortex (***p<0.001, **p<0.005, CCI-VH vs CCI-CD). This effect was abolished by co-treatment with T0070907 (*p<0.05, CCI-CD vs CCI-CD+T0). Treatment with T0070907 alone (CCI-T0) did not change the number of Iba-1-positive cells (NS, p>0.05, CCIVH vs CCI-T0) (mean±SEM, n=4–7). (d) Effects on the ability of mice to remain on the rotarod. CCI significantly reduced the ability of mice to remain on the rotarod (⁺⁺p<0.01, SHVH vs CCI-VH). CD treatment alone significantly enhanced the ability of CCI injured mice to remain on the rotarod at 1 dpi (*p<0.05, CCI-VH vs CCI-CD). The protective effect of CD at 1 dpi was no longer significant after co-administration of T0070907 (NS, p>0.05, CCI-VH vs CCI-CD-T0) (mean±SEM, n=7; data were analyzed by two-way ANOVA with Bonferroni post-tests). VH, vehicle; CD, candesartan; T0, T0070907; NS, not significant.