Inhibition of CXCL12 Signaling Attenuates the Postischemic Immune Response and Improves Functional Recovery after Stroke

Abstract

After stroke, brain inflammation in the ischemic hemisphere hampers brain tissue reorganization and functional recovery. Housing rats in an enriched environment (EE) dramatically improves recovery of lost neurologic functions after experimental stroke. We show here that rats housed in EE after stroke induced by permanent occlusion of the middle cerebral artery (pMCAO), showed attenuated levels of proinflammatory cytokines in the ischemic core and the surrounding peri-infarct area, including a significant reduction in the stroke-induced chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor-1 (CXCL12). To mimic beneficial effects of EE, we studied the impact of inhibiting CXCL12 action on functional recovery after transient MCAO (tMCAO). Rats treated with the specific CXCL12 receptor antagonist 1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclo-tetradecan (AMD3100) showed improved recovery compared with saline-treated rats after tMCAO, without a concomitant reduction in infarct size. This was accompanied by a reduction of infiltrating immune cells in the ischemic hemisphere, particularly cluster of differentiation 3-positive (CD3 þ ) and CD3 þ /CD4 þ T cells. Spleen atrophy and delayed death of splenocytes, induced by tMCAO, was prevented by AMD3100 treatment. We conclude that immoderate excessive activation of the CXCL12 pathway after stroke contributes to depression of neurologic function after stroke and that CXCR4 antagonism is beneficial for the recovery after stroke.

Preconditioning-induced CXCL12 upregulation minimizes leukocyte infiltration after stroke in ischemia-tolerant mice

“…16 We also found this CD4 T cell-specific reduction in our AMD3100 cohort. In our RHP-treated mice, the diapedesis of several other leukocyte subsets, 11 including neutrophils and macrophages, was naturally attenuated at the time Ruscher et al 17 administered AMD3100. Our findings are therefore consonant with the notion that post-stroke AMD3100 recapitulates the protective effect of an RHP-induced downregulation of chemokines, selectins, and integrins, all of which minimize inflammatory mechanisms in the ischemia-tolerant brain, whereas pre-stroke AMD3100 administration to RHP-treated animals blocks the ability of RHP to establish this endogenously protective, anti-inflammatory phenotype.…”

“…CXCL12 and CXCR4 are upregulated in a delayed manner following initial post-stroke depletion, 26 coincident with a neuroprotective timeframe for acute AMD3100 administration that limits CD4 T cell diapedesis and improves functional recovery in other studies. 17 In contrast, viral delivery of CXCL12 to the ischemic brain one week after permanent focal stroke reduced long-term cortical atrophy while increasing functional recovery, neurogenesis, and angiogenesis -all of which were blocked with longterm AMD3100 administration. 27 Future studies should therefore investigate how CXCL12-CXCR4 signaling affects the evolution of long-term neurovascular recovery in the CNS, and whether these mechanisms are also modulated by preconditioning or post-conditioning.…”

“…Therefore, the use of neurotherapeutics to enhance and/or expand CXCL12 expression by BBB endothelial cells prior to stroke, either via an engineered CXCL12 analog 38 or viral-mediated delivery, 27,39 could eventually be used in lieu of RHP to create a neuroprotective phenotype in individuals at identified risk for stroke, as well as enhance the recruitment of EPCs to promote cortical angiogenesis prior to injury. Considering the potential long-term use of CXCR4 antagonists in clinical populations, 40 and the efficacy of acute post-stroke antagonism of CXCL12 in animal models, [16][17][18] it is imperative to continue to probe how CXCL12 contributes to the maintenance of immune privilege during resting, physiologic conditions, as well as during the progression of injury and recovery following acute and chronic CNS injury. …”

“…[16][17][18] We thus sought causal evidence for enhanced CXCL12-CXCR4 signaling in the RHPinduced, post-stroke anti-inflammatory phenotype we observed. 10,11 Two separate cohorts of RHP-treated mice were used: An experimental group that received AMD3100 after the completion of RHP, and a control group that received PBS vehicle after RHP.…”

“…This hypothesis was confirmed by blocking CXCL12-CXCR4 signaling with AMD3100 beginning two days after stroke, which reduced the extent of leukocyte infiltration and improved functional recovery. 16,17 Given that RHP also results in reduced leukocyte diapedesis at one and two days after transient focal stroke, 10,11 we sought to determine whether this phenotype was secondary to an RHP-mediated reduction in post-stroke CXCL12 expression. Stroke reduced CXCL12b mRNA one day later in both untreated (p < 0.05) and RHPtreated (p < 0.001) mice compared to pre-stroke values (Figure 1(b)).…”