The Effect of Early Application of Synthetic Peptides 19-2.5 and 19-4LF to Improve Survival and Neurological Outcome in a Mouse Model of Cardiac Arrest and Resuscitation

**Background:** Cardiac arrest (CA) leads to global ischemia-reperfusion injury, triggering a systemic inflammatory response known as post-cardiac arrest syndrome, which contributes to high mortality and neurological deficits. Synthetic antimicrobial peptides (sAMPs) such as Pep19-2.5 and Pep19-4LF have shown anti-inflammatory and immunomodulatory effects in vitro and in vivo, including reducing pro-inflammatory cytokines and organ injury in sepsis and hemorrhagic shock models. This study hypothesized that early administration of these peptides after CA and cardiopulmonary resuscitation (CPR) could attenuate inflammation, improve survival, and enhance neurological outcomes in a mouse model.

**Methods:** A total of 164 female C57BL/6J mice (4-5 months old) were used, with 127 included in the final analysis. Mice were anesthetized, intubated, and mechanically ventilated. Cardiac arrest was induced by intravenous injection of 80 µg/g potassium chloride, and resuscitation was initiated after 8 minutes with chest compressions (450/min) and epinephrine (0.4 µg/g). After return of spontaneous circulation (ROSC), animals were randomized into three groups: control (saline), Pep19-2.5 (4 µg), or Pep19-4LF (7 µg), administered intravenously over 2 hours. Long-term survival and neurological outcomes were assessed over 28 days using NeuroScore, RotaRod, tape removal test, and water maze. Plasma cytokines (IL-6, IL-1β, TNFα, VEGF-A) and the neuronal damage biomarker UCH-L1 were measured at 4 hours and 28 days post-ROSC. Brain tissue mRNA expression of inflammatory and neuronal markers was analyzed at 4 hours.

**Key Results:** Survival at 28 days was 21.6% (8/37) in controls, 20% (7/35) in Pep19-2.5, and 13.5% (5/37) in Pep19-4LF, with no significant difference between groups (p=0.474). At 16 hours post-ROSC, survival was significantly lower in Pep19-2.5 (34.3%) vs. controls (62.2%, p=0.029), but this difference disappeared by 32 hours. Neurological assessments (NeuroScore, RotaRod, water maze) showed no significant differences between groups. In the tape removal test, Pep19-4LF-treated mice had significantly faster sensory perception on Day 28 compared to Pep19-2.5 (left paw: 2.3±0.8 s vs. 13.14±3.0 s, p=0.009; right paw: 1.25±0.2 s vs. 5.00±1.4 s, p=0.009). Plasma cytokines at 4 hours were elevated but not significantly different between groups. UCH-L1 levels at 4 hours were significantly higher in Pep19-4LF (14.00±4.09 pg/mL) vs. controls (5.72±2.5 pg/mL, p=0.025). Brain mRNA expression showed significantly lower IL-6 in Pep19-2.5 vs. controls (p=0.009) and Pep19-4LF (p=0.037), and lower ICAM-1 in Pep19-2.5 vs. controls (p=0.018).

**Clinical Implications:** This study demonstrates that early administration of Pep19-2.5 and Pep19-4LF does not improve survival or neurological outcomes in a mouse model of cardiac arrest and resuscitation, despite prior evidence of anti-inflammatory effects in other conditions. The lack of benefit may be due to the short duration of peptide administration (2 hours) compared to longer infusions in other studies. The increased UCH-L1 levels in the Pep19-4LF group suggest potential neuronal injury, warranting caution. These findings highlight the complexity of post-cardiac arrest syndrome and the need for further research on optimal dosing, timing, and duration of anti-inflammatory interventions. The study also confirms the utility of UCH-L1 as an early biomarker of brain injury in this model.