Abstract Body: Introduction
Secondary brain injury following intracerebral hemorrhage (ICH) is largely driven by neuroinflammation, which is exacerbated by neutrophil infiltration and their release of neutrophil extracellular traps (NETs). This process disrupts the blood-brain barrier and increases neuronal death. The present study investigates the efficacy of Ir-CPI, an inhibitor of neutrophil-mediated thromboinflammation, in reducing neutrophil-driven damage and promoting functional recovery post-ICH.

Methods
ICH was induced in mice via bacterial collagenase injection into the right striatum. Mice were then treated with either PBS, Ir-CPI or enoxaparin. Treatments were administered intravenously immediately after stroke. Cerebral lesion and hemorrhage volumes were assessed up to 14 days by MRI. Neutrophil infiltration, NET release, and neuronal degeneration were evaluated histologically. Functional recovery was assessed using the Corner test and the Bederson’s scale.

Results
After ICH induction, Ir-CPI did not increase cerebral lesion or hemorrhage volumes compared to PBS. Ir-CPI resulted in lower hemorrhage volumes than the enoxaparin group on Day 1. By Day 14, the volume of hemorrhage in the Ir-CPI group was significantly reduced in comparison to Day 1. Additionally, Ir-CPI decreased neutrophil infiltration and NETs release in the hemorrhagic area, along with a reduction in neuronal degeneration. In contrast, enoxaparin had no significant impact on reducing neutrophils or neuronal damage. Functional recovery was superior in the Ir-CPI group, with the Corner test showing a significant improvement from Day 1 to Day 5 compared to the PBS and enoxaparin groups. By Day 14, the Ir-CPI group's performance did not significantly differ from baseline, indicating enhanced long-term motor function and spatial awareness. The Bederson’s score also demonstrated a significant improvement in the Ir-CPI group, with lower scores between Day 1 and Day 5 compared to the other groups, thereby highlighting Ir-CPI's effectiveness in reducing neurological deficits.

Conclusions
Ir-CPI is a promising therapeutic agent for patients suffering from ICH, exhibiting both safety and efficacy in reducing neutrophil-driven brain damage and enhancing functional recovery. These findings indicate that Ir-CPI may be beneficial in mitigating secondary brain injury and improving patient outcomes following ICH. The potential of Ir-CPI is currently being evaluated in the Phase IIa BIRCH trial (NCT05970224).