Article, Neurology

Moderate brain hypothermia started before resuscitation improves survival and neurobehavioral outcomes after CA/CPR in mice

a b s t r a c t

Aim of the study: No definitive experimental or clinical evidence exists whether brain hypothermia before, rather than during or after, resuscitation can reduce hypoxic-ischemic brain injury following car- diac arrest/cardiopulmonary resuscitation (CA/CPR) and improve outcomes. We examined the effects of moderate brain hypothermia before resuscitation on survival and histopathological and neurobehavioral outcomes in a mouse model.

Methods: Adult C57BL/6 male mice (age: 8-12 weeks) were subjected to 8-min CA followed by CPR. The animals were randomly divided into sham, normothermia (NT; brain temperature 37.5 ?C), and extracra- nial hypothermia (HT; brain temperature 28-32 ?C) groups. The hippocampal CA1 was assessed 7 day after resuscitation by histochemical staining. Neurobehavioral outcomes were evaluated by the Barnes maze (BMT), openfield (OFT), rotarod, and light/dark (LDT) tests. Cleaved caspase-3 and heat shock pro- tein 60 (HSP70) levels were investigated by Western blotting.

Results: The HT group exhibited higher survival and lower CA1 neuronal injury than did the NT group. HT mice showed improved spatial memory in the BMT compared with NT mice. NT mice travelled a shorter distance in the OFT and tended to spend more time in the light compartment in the LDT than did sham and HT mice. The levels of cleaved caspase-3 and HSP70 were non-significantly higher in the NT than in the sham and HT groups.

Conclusions: Moderate brain hypothermia before resuscitation improved survival and reduced histolog- ical neuronal injury, spatial memory impairment, and anxiety-like behaviours after CA/CPR in mice.

(C) 2019

Introduction

Despite many years of research on Early defibrillation, car- diopulmonary resuscitation (CPR), and CPR medication, out-of- hospital cardiac arrest (OHCA) remains a significant cause of mor- bidity and mortality worldwide, with an estimated global annual incidence of sudden cardiac death of 4-5 million [1]. Therapeutic hypothermia is the only neuroprotective therapy shown to increase survival and decrease morbidity in adult OHCA patients [2]. Although the protective mechanisms of TH remain unclear, reports have suggested that ischemic apoptosis and reactive oxy-

* Corresponding author at: Department of Emergency Medicine, Chungbuk National University, 1, Chungdae-ro, Seowon-gu, Cheongju, Republic of Korea.

E-mail address: [email protected] (H. Kim).

1 Mun-Sun Jang and Suk Woo Lee contributed equally to this work.

gen species production during post-ischemic reperfusion are reduced by hypothermia [3].

Extensive research has focused on the implementation and effectiveness of TH as a post-resuscitation therapy. Recent data suggest that early TH application during CPR is superior to cooling initiated after resuscitation, resulting in both increased survival and improved neurologic outcomes [4]. Thus, experimental extracranial hypothermia during resuscitation after cardiac arrest reduced hippocampal injury [5], and TH during CPR signifi- cantly reduced myocardial infarction [6]. Ruttmann et al. recently reported survival with favourable neurological outcomes in about a third of all accidental hypothermic non-avalanche OHCA patients [7], and several case series yielded similar survival rates [8]. Brown et al. estimated the survival rate of OHCA patients to be approxi- mately 50% for primary hypothermic CA [9].

These findings suggest that inducing hypothermia in the brain

parenchyma before, rather than during or after, resuscitation

https://doi.org/10.1016/j.ajem.2019.01.027

0735-6757/(C) 2019

mitigates Ischemic injury and improves outcomes after CA. There- fore, the authors hypothesized that brain cooling before resuscita- tion from CA/CPR achieves protective levels of brain hypothermia and results in improved neurological outcomes mimicking conven- tional TH after resuscitation. However, no definitive experimental or clinical evidence exists for this hypothesis. We used a well- established mouse model in which the brain and body tempera- tures can be independently controlled to analyse neurobehavioral and histopathological outcomes of CA/CPR started at different temperatures.

Materials and methods

Experimental animals

This study conformed to the National Institutes of Health guide- lines for the care and use of animals in research. All experimental protocols were approved by the Chungbuk National University Institutional Animal Care and Use Committee (CBNUA-845-02) and reported in accordance with the ARRIVE guidelines. Male C57BL/6 adult mice (age: 8-12 weeks) were used. Mice were housed under a standard 12/12 h light/dark cycle and had free access to food and water.

Animal preparation

Anaesthesia was induced with 3% isoflurane and maintained with 1.5-2% isoflurane in oxygen-enriched air (fraction of inspired oxygen [FiO2], 30%) via a face mask. Animals were endotracheally intubated using an intravenous 22G catheter connected to a mouse ventilator (Minivent, Hugo Sachs Elektronik, March-Hugstetten, Germany) set to a respiratory rate of 150 breaths/min. A PE-10 catheter was inserted into the Right internal jugular vein for drug and fluid administration. Needle electrodes were placed subcuta- neously on the chest for electrocardiogram (ECG) monitoring throughout the experimental procedures. Temperature probes were placed into the left ear canal and rectum. The auricular canal temperature has been shown to be similar to the brain parenchy- mal temperature during CA/CPR [5]. Rectal temperature was main- tained near 37 ?C during surgery with a heating lamp and pad.

CA and resuscitation

CA and CPR were performed as previously described with the addition of independent extracranial Temperature control [5,10,11]. Briefly, mice were subjected to 8-min CA, which induced global cerebral ischemia and caused selective, delayed cell death of hippocampal CA1 neurons and neurobehavioral abnormalities in adult C57BL/6 mice [10,12], using active cooling or warming via a separate temperature control system to keep the brain hypother- mic or normothermic before resuscitation. The animals were ran- domly divided into 3 groups. In the hypothermic brain group (HT), rapid cooling was started 15 min before CA using a polyethy- lene tubing coil connected to a temperature-controlled water bath (4.1 +- 2.3 ?C) and a pump. The pericranial temperature was main- tained at 31.8 +- 1.7 ?C before resuscitation. In the normothermic brain group (NT), both the brain and body temperatures were maintained within the normal range throughout CA/CPR and early recovery (Fig. 1). CA was induced by injection of 50 lL of 0.5 M KCl via the jugular catheter, and confirmed by the appearance of asys- tole on the ECG and spontaneous breathing cessation. The endotra- cheal tube was disconnected from the ventilator, and anaesthesia was stopped. CPR was begun 8 min after CA induction by slow injection of 0.5-1.0 mL of epinephrine (16 lL of epinephrine per mL of 0.9% saline), chest compressions (approximately 300 per min), and ventilation with 100% oxygen (200 breaths/min). As soon as return to spontaneous circulation (ROSC) was achieved, defined as ECG activity with visible cardiac contractions, chest compres-

sions were stopped. Five minute post-resuscitation, the FiO2 was decreased to 50%. When the spontaneous breathing rate reached 60 breaths/min, the endotracheal tube was removed. The animal was then placed into its home cage for Complete recovery. Mice in the Sham group (n = 8) underwent all procedures except for CA induction, cardiac compressions, and epinephrine injection.

Measurements and outcomes

Health assessment score

Mice were weighed and their health assessed daily for 3 days after CA/CPR. The graded scoring systems ranged from 0 to 2, 0 to 3, or 0 to 5 depending on the behaviour assessed, with 0 indicat- ing no deficit and the upper limit indicating the most impairment. The behaviours assessed included consciousness (0-3), interaction (0-2), ability to grab a wire top (0-2), motor function (0-5), and activity (0-2) [10]. The individual category scores were summated to generate an overall health assessment score.

Survival

Survival was monitored for 7 days after weaning of mechanical ventilation.

Neurobehavioral outcomes

Behavioural testing was carried out between the hours of 8 a.m. and 7 p.m. and performed by a blinded observer.

  • Barnes maze test. The Barnes maze test (BMT) was con- ducted as previously described with minor modifications [13]. The maze consisted of a white acrylic circular platform (91 cm in diameter) with 20 equally spaced holes and a black acrylic escape box (20 x 5 x 6 cm) along the perimeter. The maze was sur- rounded by 4 spatial cues at the height of the maze.

    Acquisition trials. Each mouse was subjected to 4 acquisi- tion trials per day for 3 days with an inter-trial interval of 10- 15 min. Immediately prior to the first trial, the mouse was placed in the middle of the maze in a black starting cylinder (10 cm in diameter), and a buzzer (80-90 dB) was turned on. After 10 s, the chamber was lifted, and the mouse was pre-trained to enter the escape box by being guided to, and remaining in it for 2 min. Each acquisition trial began as the pre-training trial but the mouse was free to explore the maze. The trial ended when the mouse entered the goal tunnel or after 3 min had elapsed. Immediately after the mouse entering the tunnel, the buzzer was turned off and the mouse was allowed to stay in the tunnel for 1 min. After each trial, the maze was cleaned with 70% alcohol and rotated to eliminate intra-maze cues. The trials were recorded using a video Tracking system (SMART; Panlab, Barcelona, Spain).

  • Probe trial. During a 90-s probe trial, conducted on POD 7, the escape tunnel leading to the target box was closed. The mice were allowed to explore the maze. Target and adjacent hole visits, path length, and latency to reach the target hole for the first time were recorded.
  • Open-field test. Locomotor activity was measured in a white open-top acrylic box (40 x 40 x 40 cm) with an illumination inten- sity of 20 lx at the box floor level for 30 min. The activity was auto- matically recorded using a video tracking system with the SMART

3.0 software. Distance travelled, time spent in the centre (25%) and area margins, and mean walking speed were measured.

  • Light/dark preference test. The apparatus consisted of black and white compartments (20 x 40 x 40 cm) separated by a con- necting gate (5 x 8 cm). Each animal was individually placed in the centre of the bright compartment (facing away from the door), and the following parameters were measured for 5 min: latency of the initial movement from the light to dark area (transition), total number of transitions, and total time spent in the light area.

Fig. 1. Experimental design & Kaplan-Meier survival analysis. C57BL6 adult mice were subjected to extracranial hypothermia (HT) or normothermia (NT) before resuscitation. (A) Experimental timelines illustrate the CA/CPR process and the timing of histological, behavioural, and biochemical analyses. (B) Auricular canal temperature was measured as a proxy for brain parenchymal temperature throughout the CA/CPR sequence and plotted by time (e.g., ROSC10, 10 min after ROSC). (C-F) Neurobehavioral tests included the Barnes maze (C), open field (D), light/dark (E), and rotarod (F) tests. (G) Statistically significant improvement in survival was seen in the extracranial hypothermia (HT) group compared with that in the extracranial normothermia (NT) group. The log-rank statistic was 19.05 (P < 0.0001). BL, baseline; CA, cardiac arrest; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation.