Protective Effects of Interleukin-1 Inhibition With Anakinra in Mouse Models of Ischemic Stroke With and Without Reperfusion

Abstract

Background: Severe brain ischemia is associated with life-threatening edema and inflammation. Interleukin-1 is a crucial mediator of inflammation, and its blockade showed benefits in experimental stroke. We studied anakinra, a modified recombinant human interleukin-1 receptor antagonist, in mouse models of moderate to severe ischemia/reperfusion and large hemispheric infarctions. Due to anakinra's short half-life, we used a novel subcutaneous infusion protocol and tested 2 drug doses.

Methods and results: We performed transient or permanent intraluminal middle cerebral artery occlusion (MCAo) in male C57BL/6J and Balb/c mice, the latter of which have poorer collaterals. Mice received a subcutaneous anakinra bolus (24 mg/kg), followed by continuous infusion of either 24 or 120 mg/kg per day, starting at reperfusion or 15 minutes after permanent MCAo. We evaluated acute (24 hours/48 hours) infarct volume and edema by magnetic resonance imaging, neurological function, and inflammatory responses. The mortality rate tended to be higher in Balb/c compared with C57BL/6J mice. In both strains, prolonged ischemia expanded the infarct size, with intraluminal permanent MCAo resulting in larger hemispheric infarctions and edema than transient MCAo. The high dose of anakinra reduced infarct volume and inflammation in C57BL/6 mice and improved the functional deficits in Balb/c mice following transient MCAo. It also showed a trend toward reducing infarction and edema after permanent MCAo in C57BL/6 mice.

Conclusions: The study demonstrates that a high dose of anakinra improves outcomes in mouse models of moderate infarction following ischemia/reperfusion, whereas its effect was less pronounced in a malignant hemispheric infarction model without reperfusion, where only a nonsignificant trend toward protection was observed.

Keywords: anakinra; cytokines; edema; inflammation; ischemic stroke; malignant infarction.

Figure 1. Beneficial effects of high‐dose anakinra in the transient ischemia model in C57BL/6 mice.

A, Experimental design of the study. The MCA was occluded for 45 minutes in C57BL/6J mice. B, Percentage of the blood flow drop during ischemia from basal flow and recovery at reperfusion were similar between groups. C, Representative T2w MRI images of the mouse brain 48 hours after ischemia for each treatment group and corresponding masks used to measure infarct volume. D, Infarct volume in the vehicle group (n=11 mice), low‐dose anakinra group (n=9 mice), and high‐dose anakinra group (n=10 mice). Permutation test comparing high‐dose anakinra vs vehicle showed significant differences in infarct volume (*P=0.046). E and F, Functional assessment of neurological deficits using the grip (E) and corner (F) tests. The corner test showed that ischemia increases laterality (% right turns) vs the corresponding basal value in the vehicle group (*P=0.028) and the low‐dose anakinra group (*P=0.017), but not in the high‐dose anakinra group (P=0.908). G, Assessment of general and focal neurological deficits, and a combination of them (composite neuroscore) showed no significant differences between groups. The graphs with boxes show the 25th to 75th percentiles, the median line, and whiskers from the minimum to the maximum value, and all points. Other graphs show the mean±SD. AK indicates anakinra; MCA, middle cerebral artery; MRI, magnetic resonance imaging; s.c., subcutaneous; and tMCAo, transient middle cerebral artery occlusion.

Figure 2. High‐dose anakinra reduced cortical proinflammatory gene expression 48 hours following 45‐minute tMCAo in C57BL/6J mice.

A, Experimental design of the study. B and C, mRNA expression of proinflammatory mediators in cortex (B) and striatum (C) of ischemic mice treated with the high dose of anakinra (n=10 mice) or the vehicle (n=11 mice), and sham‐operated mice (n=6 mice). Ischemia increased the mRNA expression of these molecules. The statistical significance between anakinra vs vehicle ischemic groups is indicated by *P<0.05 and **P<0.01, or the exact P value. Values are expressed as the mean±SD. AK indicates anakinra; s.c., subcutaneous; and tMCAo, transient middle cerebral artery occlusion.

Figure 3. High‐dose anakinra attenuated the glial reaction following 45‐minute tMCAo in C57BL/6J mice.

A, Experimental design of the study using ischemic mice treated with the high dose of anakinra (n=10 mice) or the vehicle (n=11 mice). B, Confocal images of coronal brain sections of each treatment group immunostained with glial fibrillary acidic protein (red), ionized calcium‐binding adapter molecule 1 (green), and DAPI (blue). C, Quantification of the immunoreaction per region shows that the high‐dose of anakinra reduced the astroglial immunoreactive area (glial fibrillary acidic protein positive) in the hippocampus (*P=0.049) of the ipsilateral hemisphere. Values are expressed as the mean±SD. AK indicates anakinra; GFAP, glial fibrillary acidic protein; Iba‐1, ionized calcium‐binding adapter molecule 1; IntDen, integrated density; and tMCAo, transient middle cerebral artery occlusion.

Figure 4. High‐dose anakinra improved the behavioral performance of Balb/c mice 48 hours after 45‐minute tMCAo.

A, Experimental design. B, Before treatment, the drop in blood flow after MCAo was slightly higher (*P=0.026) in the mice allocated to the high‐dose anakinra group (n=9) than in the vehicle group (n=12). Moreover, the recovery at reperfusion was better in the vehicle group (*P=0.029). C, Infarct volume and edema were not statistically different between groups (n=9 high‐dose anakinra, n=8 vehicle mice that completed the study). D, Representative T2w images for each group obtained 48 hours after ischemia and corresponding masks used to measure infarct volume. E, The performance in the grip test shows a reduction in forelimb strength after ischemia vs basal in each group with no differences between groups. F, The corner test showed that the percentage of right turns was significantly reduced in the high‐dose anakinra group (**P=0.008). G, The various components of the neurological score tests were not significantly affected by the treatment. The graphs with boxes show the 25th to 75th percentiles, the median line, and whiskers from the minimum to the maximum value, and all points. Other graphs show the mean±SD. AK indicates anakinra; MRI, magnetic resonance imaging; s.c., subcutaneous; and tMCAo, transient middle cerebral artery occlusion.

Figure 5. Effects of high‐dose anakinra in the permanent ischemia model in C57BL/6 mice.

A, Graphical description of the experimental design in C57BL/6J mice. B, Representative images of CBF (as assessed by arterial spin labeling) 15 minutes and 24 hours after the arterial occlusion, and corresponding measures in the contralateral and ipsilateral hemispheres of vehicle (n=10 mice; CBF 15 minutes: P=0.052; CBF 24 hours: *P=0.012) and high‐dose anakinra (n=8 mice; CBF 15 minutes: ***P=0.0005; CBF 24 hours: ***P=0.0002) groups. C, Brain lesions after pMCAo illustrated by T2w MRI at 24 hours and corresponding masks used to measure infarct volume in a representative mouse of each group. D, Values of infarct volume (P=0.073) and edema (P=0.102) 24 hours after ischemia for each treatment group. Values are expressed as the mean±SD. AK indicates anakinra; ASL, arterial spin labeling; CBF, cerebral blood flow; MRI, magnetic resonance imaging; and pMCAo, permanent middle cerebral artery occlusion.