Minocycline fails to improve neurologic and histologic outcome after ventricular fibrillation cardiac arrest in rats

Abstract

Background: Prolonged cardiac arrest (CA) produces extensive neuronal death and microglial proliferation and activation resulting in neuro-cognitive disabilities. Among other potential mechanisms, microglia have been implicated as triggers of neuronal death after hypoxic-ischemic insults. Minocycline is neuroprotective in some brain ischemia models, either by blunting the microglial response or by a direct effect on neurons.

Aim: To improve survival, attenuate neurologic deficits, neuroinflammation, and histological damage after ventricular fibrillation (VF) CA in rats.

Methods: Adult male isoflurane-anesthetized rats were subjected to 6 min VF CA followed by 2 min resuscitation including chest compression, epinephrine, bicarbonate, and defibrillation. After return of spontaneous circulation (ROSC), rats were randomized to two groups: (1) Minocycline 90 mg/kg intraperitoneally (i.p.) at 15 min ROSC, followed by 22.5 mg/kg i.p. every 12 h for 72 h; and (2) Controls, receiving the same volume of vehicle (phosphate-buffered saline). The rats were kept normothermic during the postoperative course. Neurologic injury was assessed daily using Overall Performance Category (OPC; 1 = normal, 5 = dead) and Neurologic Deficit Score (NDS; 0% = normal, 100% = dead). Rats were sacrificed at 72 h. Neuronal degeneration (Fluoro-Jade C staining) and microglia proliferation (anti-Iba-1 staining) were quantified in four selectively vulnerable brain regions (hippocampus, striatum, cerebellum, cortex) by three independent reviewers masked to the group assignment.

Results: In the minocycline group, 8 out of 14 rats survived to 72 h compared to 8 out of 19 rats in the control group (P = 0.46). The degree of neurologic deficit at 72 h [median, (interquartile range)] was not different between survivors in minocycline vs controls: OPC 1.5 (1-2.75) vs 2 (1.25-3), P = 0.442; NDS 12 (2-20) vs 17 (7-51), P = 0.328) or between all studied rats. The number of degenerating neurons (minocycline vs controls, mean ± SEM: Hippocampus 58 ± 8 vs 76 ± 8; striatum 121 ± 43 vs 153 ± 32; cerebellum 20 ± 7 vs 22 ± 8; cortex 0 ± 0 vs 0 ± 0) or proliferating microglia (hippocampus 157 ± 15 vs 193 cortex 0 ± 0 vs 0 ± 0; 16; striatum 150 ± 22 vs 161 ± 23; cerebellum 20 ± 7 vs 22 ± 8; cortex 26 ± 6 vs 31 ± 7) was not different between groups in any region (all P > 0.05). Numerically, there were approximately 20% less degenerating neurons and proliferating microglia in the hippocampus and striatum in the minocycline group, with a consistent pattern of histological damage across the individual regions of interest.

Conclusion: Minocycline did not improve survival and failed to confer substantial benefits on neurologic function, neuronal loss or microglial proliferation across multiple brain regions in our model of rat VF CA.

Keywords: Cardiopulmonary resuscitation; Heart arrest/pathology; Microglia/drug effects; Minocycline/pharmacology; Neurons/drug effects; Survival rate.

©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.

Figures

Figure 1

A flow-chart of the study. Please refer to the text for details on randomization protocol. ROSC: Return of spontaneous circulation.

Figure 2

Overall performance categories after cardiac arrest. Each dot represents one rat. No difference between groups. OPC: Overall performance category.

Figure 3

Neurologic deficit score at 72 h in all rats studied (left panel) and in survivors only (right panel). Boxes represent interquartile ranges. The line across each box indicates the median, and the whiskers are the highest and lowest values. No differences between groups. C: Control group; M: Minocycline group; NDS: Neurologic deficit score.

Figure 4

Kaplan-Maier survival plot. No differences between groups.

Figure 5

Regional neuronal degeneration (top) and microglial proliferation (bottom) after cardiac arrest. Regional neuronal loss and microglial proliferation after cardiac arrest in controls and rats treated with minocycline were not different between groups in any region at 72 h. HIP: Hippocampus; STRI: Striatum; CEREB: Cerebellum; CTX: Cortex. Mean ± SEM values are displayed.

Figure 6

Representative samples of neuronal degeneration after 6 min ventricular fibrillation cardiac arrest at 72 h. Blue staining is 4',6-diamidino-2-phenylindole, visualizing neurons, and green staining is Fluoro-Jade C, visualizing degenerating neurons. Hippocampal neuronal loss is visible in the cardiac arrest 1 sector and in hilar region of the dentate gyrus. The inset shows the mid-section of cardiac arrest 1 in closer detail. Marked neuronal degeneration of the medium spiny neurons is seen in the striatum. Selectively vulnerable neuronal loss of Purkinje neurons visualized in the cerebellum. No neurodegeneration is observed in the cortex. Magnification × 10 except the panoramic view of the hippocampus, magnification × 4. The scale bars in the far left and far right lower panels represent 10 µm.

Figure 7

Representative samples of microglial activation and proliferation after 6 min ventricular fibrillation cardiac arrest at 72 h. Sections are stained with hematoxylin. Brown staining is anti-Iba-1 staining, visualizing microglia, counterstained with diaminobenzamide. Magnification × 10. The scale bars in the far left and far right lower panels represent 10 µm.