J Cerebrovasc Endovasc Neurosurg.  2021 Dec;23(4):304-313. 10.7461/jcen.2021.E2021.04.003.

Systemic macrophage depletion attenuates infarct size in an experimental mouse model of stroke

• Lee SW
• Song DJ
• Ryu HS
• Kim YS
• Kim TS
• Joo SP

• KMID: 2523888
• DOI: http://doi.org/10.7461/jcen.2021.E2021.04.003

Abstract

Objective
Macrophages have been shown to play important roles in various pathophysiological processes of the central nervous system via neuroinflammation, leading to an increased interest in macrophage biology. Circulating blood monocytes are among the first cells to infiltrate the brain after ischemic stroke; however, the role of innate immune cells such as monocytes and macrophages remains to be elucidated. Here, we investigated the association between blood monocytes and infarct size following ischemic stroke.
Methods
We induced stroke using a focal ischemia mouse model through middle cerebral artery suture occlusion. To deplete circulating blood monocytes, clodronate was injected intraperitoneally 24 h before the surgery. Animals were sacrificed at specified time points, and the infarct size and mRNA expression were then measured.
Results
The clodronate-injected mice showed significantly smaller infarct size than the control mice. Immunohistochemical staining revealed that monocyte depletion significantly blocked the infiltration of macrophages and microglia. The mRNA expression levels of macrophage and microglia markers were higher in the left infarcted brain than in the right non-infarcted brain.
Conclusions
In summary, monocyte depletion reduced the infarct size and mitigated neurological deficits in mice following ischemic stroke, likely by blocking the infiltration of inflammatory cells such as macrophages and microglia.

Keyword

Cerebral ischemia; Clodronate; Macrophages; Microglia; Infarction

Figure

• 

  Fig. 1. Experimental protocols, 2,3,5-triphenyltetrazolium chloride (TTC) staining, and infarct volume. (A) Both liposome-encapsulated phosphate-buffered saline and liposome-encapsulated clodronate were administered via intraperitoneal injection 24 h before the induction of stroke. After 24 h, focal ischemia was induced in the mice via transient middle cerebral artery occlusion for 60 min, followed by immediate reperfusion for 3 days. The mice (n=10 mice/group) were sacrificed on day 3 (black box). (B) TTC staining of infarcts; representative images. (C) Bar graph of the average infarct volume in each group. Comparison of infarct volume between the clodronate-injected and control mice showing that monocyte depletion had significant protective effects against infarction (54.73±3.21% vs. 26.25±2.07%, p=0.006). Data are presented as the mean±SEM. *p＜0.05, **p＜0.01 compared to the control group (n=10 mice/group). SEM, standard error of the mean.

• 

  Fig. 2. Experimental protocols, ionized calcium-binding adapter molecule 1(Iba-1)-stained cell counts, and infarct volume. (A) The mice were injected with either liposome-encapsulated phosphate-buffered saline (PBS) or liposome-encapsulated clodronate 24 h before the induction of stroke. After 24 h, focal ischemia was induced in the mice via transient middle cerebral artery occlusion for 60 min, followed by immediate reperfusion for 3 days. The mice (n=10 mice/group) were sacrificed on days 3, 5, and 7 after the induction of stroke (black box). (B) Iba-1 staining of infarcts on days 3, 5, and 7; representative images. (C) Cell counts of Iba-1-positive cells in representative infarcts (n=10 mice/group). The clodronate-injected mice showed significantly reduced numbers of resident microglia on days 3 and 5 (4.62±0.2 vs. 0.81±0.22%, p=0.003; 3.33±0.31 vs. 1.74±0.28%, p=0.048; 2.09±0.21% vs. 2.42±0.39%, p=0.76). Data are presented as the mean±SEM. *p＜0.05, **p＜0.01, and ***p＜0.001 compared to the control group. SEM, standard error of the mean.

• 

  Fig. 3. Experimental protocols and mRNA expression in both hemispheres. (A) Mice were injected either with liposome-encapsulated phosphate-buffered saline or liposome-encapsulated clodronate 24 h before the induction of stroke. After 24 h, focal ischemia was induced in the mice via transient middle cerebral artery occlusion for 60 min, followed by immediate reperfusion for 3 days. The mice (n=10 mice/group) were sacrificed on days 1, 3, 5, and 7 after stroke (black box). (B) Integrin subunit alpha M (ITGAM) and chemokine ligand 2 (CCL2) mRNA expression levels in the infarcted left brains on 1, 3, 5, and 7 days are shown. There was no significant difference between the groups on days 1 and 3; however, the control group showed higher ITGAM and CCL2 expression levels on days 5 and 7. (C) ITGAM and CCL2 mRNA expression levels in non-infarcted right brain sections on 1, 3, 5, and 7 days are shown. ITGAM expression levels were consistently decreased after the induction of stroke in the clodronate-injected mice. For days 3-7 after reperfusion, mRNA expression levels in the control and study groups were similar. *p＜0.05, **p＜0.01, and ***p＜0.001. SEM, standard error of the mean.

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