Increased brain injury and worsened neurological outcome in interleukin-4 knockout mice after transient focal cerebral ischemia

Abstract

Background and purpose: Stroke causes brain injury with activation of an inflammatory response that can contribute to injury. We tested the hypothesis that the anti-inflammatory cytokine interleukin-4 (IL-4) reduces injury after stroke using IL-4 knockout (KO) adult male mice.

Methods: IL-4 KO and wild-type mice were subjected to transient middle cerebral artery occlusion. Outcome was assessed by triphenyltetrazolium chloride staining for infarct volume, neuroscore and spontaneous activity for behavioral outcome, and immunostaining and stereological counting for cellular response.

Results: Infarction volume at 24 hours was significantly larger in IL-4 KO mice, neurological score was significantly worse, and spontaneous activity was reduced compared with wild-type mice. Increased macrophage/microglial infiltration, increased numbers of myeloperoxidase-positive cells, and increased Th1/Th2 ratio were observed in the infarct core in IL-4 KO mice. Reduced astrocyte activation was observed in the cortical penumbra in IL-4 KO mice. Recombinant IL-4 administered intracerebroventricularly before middle cerebral artery occlusion significantly reduced infarct volume, improved neurological score, reduced macrophages/microglia, and lowered the Th1/Th2 ratio in IL-4 KO mice, but not in wild-type.

Conclusions: Loss of IL-4 signaling in KO mice was associated with worse outcome, and this was reversed by giving exogenous IL-4. Worsened outcome was associated with increased inflammation in the core, which was reversed in IL-4 KO but not significantly changed in wild-type mice by exogenous IL-4. This is consistent with IL-4 signaling leading to reduced inflammation in the core and a possible beneficial role for activated astrocytes in the penumbra.

Figure 1

Ischemic infarct volume and neurological score were increased in IL-4 KO mice. (A) Representative TTC-stained coronal sections. (B) Quantification of infarct volume expressed as a percent of hemispheric volume (P=0.032, n=10). (C) The neurological deficit assessed at 24 hr was greater in IL-4 KO (P=0.039, n=10).

Figure 2

Spontaneous activity is reduced more in IL-4 KO mice following MCAO assessed for 24 hr starting 2 hr after MCAO. (A) Total travel distance was calculated based on x, y position and accumulated path length. Total distance was significantly reduced after MCAO compared to naïve or sham surgery. Total travel distance for IL-4 KO MCAO was significant shorter than WT MCAO (n=12, p<0.05). (B) Average velocity did not differ between groups. (C) Beam breaks in IL-4 KO MCAO were significantly fewer than WT (n=12, p<0.05). (D) Active time is the total time spent moving/24 hr. (E–G) Individual representative travel patterns demonstrate decreased spontaneous activity after MCAO. For all panels * P<0.05 vs sham and naive of the same genotype, # P <0.05 vs WT MCAO.

Figure 3

GFAP, CD68 and MPO immunoreactive cells in the cortical penumbra and ischemic core were assessed at 24 hr reperfusion. The upper picture shows a whole brain section in which the cortical penumbra (CP) and ischemic core (IC) areas are indicated. Panel (A) shows glial activation in the CP and IC from WT and IL-4 KO animals (20x). (B) Images and quantitation of GFAP⁺ astrocytes or CD68⁺ microglia/macrophages at 40x magnification in CP. Increase in GFAP⁺ cells is greater in WT, with no statistical difference in CD68⁺ between genotypes. (C) CD68⁺ cells and MPO⁺ leukocytes (20x) were counted in the IC after MCAO. CD68+ and MPO⁺ cells were greater in the IL-4 KO group. Scale bars, 50 μm. * P <0.05 vs sham of the same genotype, # P<0.05 vs WT MCAO. n=4 for MCAO IL-4 KO, n=5 MCAO WT, n=3 both sham groups.

Figure 4

CD68 (A) and GFAP (B) expression in the ischemic hemisphere assessed by Western. (A) CD68 protein level increases after MCAO are greater in IL-4 KO, p=0.037. n=3 sham, n=6 WT MCAO, n=7 IL-4 KO MCAO. (B) GFAP protein levels increased similarly in IL-4 KO and WT after MCAO. * P <0.05 vs sham same genotype, # P<0.05 vs WT MCAO, n=3 all groups.

Figure 5

Administration of RhIL-4 protects only in IL-4 KO mice. (A) Infarct volume in WT was unchanged with RhIL-4 (n=7) but significantly decreased in IL-4 KO-RhIL-4 (n=9) compared to IL-4KO-vehicle (p=0.0006, n=8). (B) Neurological score was unchanged in WT-RhIL-4 (p=0.28, n=4) but significantly decreased in IL-4 KO-RhIL-4 (n=9) compared to vehicle treated (n=5, p=0.01); (C) RhIL-4 did not change CD68⁺ cells in the ischemia core in WT but significantly decreased CD68⁺ cells in IL-4 KO-RhIL-4 compared to IL-4KO-vehicle (p=0.002, n=4/group). (D) IL-4 treatment did not change CD68⁺ cells in penumbra of WT (n=4) or IL-4 KO (p=0.068, n=4/each group). (E) Images of T-bet⁺ cells (Th1) or GATA-3⁺ (Th2) at 20x magnification in IC. Scale bar, 50 μm (F) Th1/Th2 ratio was greater in IL-4 KO-vehicle compared to WT-vehicle (p=0.008) or IL-4KO-RhIL-4 (p=0.008, n=4/group). Th1/Th2 ratio was unchanged in WT with/without RhIL-4 (n=4/group). (G) RhIL-4 significantly decreased T-bet⁺ cells in the core of IL-4 KO compared to IL-4KO-vehicle, p=0.029 (H) Fewer GATA3⁺ cells were in the ischemic core in IL-4 KO-vehicle compared to WT-vehicle (p=0.007, n= 4/group). * P <0.05 vs vehicle injected same genotype, # P<0.05 vs WT-vehicle.