Systemic administration of dendrimer N-acetyl cysteine improves outcomes and survival following cardiac arrest

Abstract

Cardiac arrest (CA), the sudden cessation of effective cardiac pumping function, is still a major clinical problem with a high rate of early and long-term mortality. Post-cardiac arrest syndrome (PCAS) may be related to an early systemic inflammatory response leading to exaggerated and sustained neuroinflammation. Therefore, early intervention with targeted drug delivery to attenuate neuroinflammation may greatly improve therapeutic outcomes. Using a clinically relevant asphyxia CA model, we demonstrate that a single (i.p.) dose of dendrimer-N-acetylcysteine conjugate (D-NAC), can target "activated" microglial cells following CA, leading to an improvement in post-CA survival rate compared to saline (86% vs. 45%). D-NAC treatment also significantly improved gross neurological score within 4 h of treatment (p < 0.05) and continued to show improvement at 48 h (p < 0.05). Specifically, there was a substantial impairment in motor responses after CA, which was subsequently improved with D-NAC treatment (p < 0.05). D-NAC also mitigated hippocampal cell density loss seen post-CA in the CA1 and CA3 subregions (p < 0.001). These results demonstrate that early therapeutic intervention even with a single D-NAC bolus results in a robust sustainable improvement in long-term survival, short-term motor deficits, and neurological recovery. Our current work lays the groundwork for a clinically relevant therapeutic approach to treating post-CA syndrome.

Keywords: N‐acetyl cysteine; cardiac arrest; dendrimer; inflammation; rat.

FIGURE 1

Schematic diagrams of study design. (a) Timeline for cardiac arrest procedure in rats optimized to consistently generate clinically relevant cardiac arrest phenotypes. (b) Sequential outcome measurements collected at different time intervals for comparative effectiveness of therapeutic interventions

FIGURE 2

Dendrimer uptake in the primary motor cortex, striatum, and hippocampus. Schematic representation of the anatomical position of images. Representative images (40×) of dendrimer (red) co‐localized with Iba1 stained microglia (green) and DAPI (blue) stained cells in the (a) primary motor cortex, (b) striatum and (c) CA1 region of hippocampus. White arrows denote colocalization. Plot profile of immunofluorescence images acquired from (d) primary motor cortex, (e) striatum and (f) hippocampus. The “r” values indicate the Pearson correlation coefficients calculated for respective profile plots. Scale bars: 20 μm

FIGURE 3

Systemic D‐NAC improves survival from cardiac arrest. Post‐CA treatment with D‐NAC resulted in significant improvements in the survival rate (86%) compared to (45%) in saline‐treated animals (CA + Saline, n = 19; CA + D‐NAC group, n = 14, *p < 0.05). Fisher's Exact test was conducted. *p < 0.05, compared to saline‐treated animals

FIGURE 4

Systemic D‐NAC treatment improves neurologic deficit scores in CA rats. (a) NDS score was increased significantly in CA + D‐NAC group compared to CA + Saline at 4 h (CA + Saline, n = 19; CA + D‐NAC, n = 14; p = 0.007) and 48 h (CA + Saline, n = 19; CA + D‐NAC, n = 14; p = 0.04). (b) NDS subscores at 24 h revealed significant improvement in motor related functions with D‐NAC therapy (CA + Saline, n = 16; CA + D‐NAC group, n = 12; p = 0.002 in motor assessment, p = 0.02 in motor behavior, and p = 0.04 in behavior, respectively). (c) Comparison of NDS subscores at 48 h with surviving saline‐treated animals indicates a trend in improvement in motor assessments, albeit statistically nonsignificant (CA + Saline, n = 7; CA + D‐NAC, n = 11; p = 0.2). *p < 0.05, compared to saline‐treated animals

FIGURE 5

Systemic D‐NAC treatment improves cell survival in hippocampus. (a) Experimental timeline depicting single treatment intervention followed by histological procedures after 2 weeks of CA. Hippocampal sections were stained using cresyl violet. (b) Representative photomicrographs of the hippocampus (upper panel) and the corresponding CA1 and CA3 regions (lower panels) following cresyl violet staining from healthy control, CA + saline and CA + D‐NAC. D‐NAC treatment significantly prevented cardiac arrest induced neuronal cell death in the CA1 and CA3 subfield of the hippocampus, compared to that in the CA + Saline group (c) Optical density measurements (mean ± SEM) from CA1 stratum pyramidale consisting of primarily cell body of projection neurons revealed significant loss after cardiac arrest (healthy control, n = 5; CA + Saline, n = 6; p < 0.001). Single dosage of D‐NAC therapy substantially protected neuronal density (CA + Saline, n = 6; CA + D‐NAC, n = 7; p < 0.001). (d) CA3 stratum pyramidale optical density (mean ± SEM) showed significant decrease in saline‐treated group compared to control (healthy control, n = 5; CA + Saline, n = 6; p < 0.001) that could be recovered to an extent with D‐NAC treatment (CA + Saline, n = 6; CA + D‐NAC, n = 7; p < 0.001). **p < 0.001, compared to saline‐treated animals