Extracellular chromatin is an important mediator of ischemic stroke in mice

Abstract

Objective: Recently, a growing number of studies have revealed a prothrombotic and cytotoxic role for extracellular chromatin. Cerebral ischemia/reperfusion injury is characterized by a significant amount of cell death and neutrophil activation, both of which may result in the release of chromatin. The goal of this study was to assess the effect of extracellular chromatin in ischemic stroke using a mouse model of transient middle cerebral artery occlusion.

Methods and results: Similar to reports in stroke patients, we observed increased levels of circulating nucleosomes and DNA after ischemic stroke in mice. In addition, we observed that general hypoxia also augmented extracellular chromatin. We hypothesized that targeting extracellular chromatin components would be protective in ischemic stroke. Indeed, treatment with recombinant human DNase 1 significantly improved stroke outcome. Neutralization of histones using an antihistone antibody was also protective as evidenced by smaller infarct volumes, whereas increasing levels of extracellular histones via histone infusion exacerbated stroke outcome by increasing infarct size and worsening functional outcome.

Conclusions: Our results indicate that extracellular chromatin is generated and is detrimental during cerebral ischemia/reperfusion in mice. Targeting DNA and histones may be a new therapeutic strategy to limit injury resulting from ischemic stroke.

Figure 1. Nucleosome levels are increased after hypoxia

Female (A) and male (B) wild-type animals were kept under normoxic (room air) or hypoxic (hypoxia chamber, 6% 0₂) conditions for 24 h, after which plasma samples were collected. Nucleosome levels were determined in the plasma samples and expressed as nucleosome specific absorbance levels (Abs 405). Hypoxia significantly increased the levels of circulating nucleosomes. (** p<0.005; **** p<0.0001)

Figure 2. DNA and nucleosome levels are increased after cerebral ischemia/reperfusion

Mice were subjected to 2 h of tMCAO, followed by 22 h of reperfusion. Before the surgical procedure (baseline) and after 22 h of reperfusion (post-treatment), blood samples were collected and plasma was prepared. Sham-operated animals underwent the same surgical procedures but without occlusion of the right MCA. DNA levels (A, n=9 for each group) and nucleosome levels (B, n=6 for each group) were measured and normalized over baseline levels of sham-treated mice. Both DNA and nucleosome levels slightly increased in some animals after sham treatment but this was not significant (NS). DNA and nucleosome levels were significantly higher in all animals that experienced stroke. (* p<0.05)

Figure 3. rhDNase 1 improves outcome after ischemic stroke

Wild-type mice treated with rhDNase 1 or vehicle were subjected to 1 h of tMCAO and 23 h of reperfusion after which mice behavior was tested and brains analyzed. (A) Early treatment with rhDNase 1 (started before reperfusion). Representative 2,3,5-triphenyl tetrazolium chloride stains of 3 consecutive coronal brain sections (left), on which ischemic infarctions appear white. Brain infarct volumes as measured by planimetry (right). Mice receiving early treatment with rhDNase 1 developed significantly smaller infarctions than control (vehicle) mice. (B) Neurological Bederson score, grip test score and corner test results of the experiment shown in A. On all three parameters, mice treated early with rhDNase 1 showed a significantly better neurologic outcome when compared to control mice (* p<0.05). (C) Delayed treatment with rhDNase 1 (started 1 h after reperfusion). Representative 2,3,5-triphenyl tetrazolium chloride stains of 3 consecutive coronal brain sections (left). Brain infarct volumes as measured by planimetry (right). Mice receiving the delayed treatment with rhDNase 1 also developed significantly smaller infarctions than control (vehicle) mice.

Figure 4. Histone neutralization is protective in ischemic stroke

Wild-type mice treated with a histone-neutralizing antibody (BWA3) or isotype control antibody (IgG1) were subjected to 1 h of tMCAO and 23 h of reperfusion after which mice behavior was tested and brains analyzed. (A) Representative 2,3,5-triphenyl tetrazolium chloride stains of 3 consecutive coronal brain sections (left), on which ischemic infarctions appear white. Brain infarct volumes as measured by planimetry (right). Mice treated with BWA3 developed significantly smaller infarctions in comparison to mice treated with the isotype control (* p<0.05). (B) Functional Bederson and grip test scores. Neutralization of histones (BWA3) showed a tendency for a better neurologic outcome when compared to IgG1 control treatment. Treatment with vehicle gave the same results as treatment with IgG1 antibody (not shown, p=0.12).

Figure 5. Infusion of histones exacerbates ischemic stroke

Wild-type mice treated with histones or saline (vehicle) were subjected to 1 h of tMCAO and 23 h of reperfusion after which mice behavior was tested and brains analyzed. (A) Representative 2,3,5-triphenyl tetrazolium chloride stains of 3 consecutive coronal brain sections (left), on which ischemic infarctions appear white. Brain infarct volumes as measured by planimetry (right). Mice treated with histones developed significantly larger infarctions in comparison to mice treated with vehicle (* p<0.05). (B) Functional Bederson and grip test scores. Infusion of histones led to worsening of neurologic outcome, as seen by increased Bederson score, when compared to control treatment.