Therapeutic Hypothermia Combined with Hydrogen Sulfide Treatment Attenuated Early Blood-Brain Barrier Disruption and Brain Edema Induced by Cardiac Arrest and Resuscitation in Rat Model

Abstract

Brain injury remains a major problem in patients suffering cardiac arrest (CA). Disruption of the blood-brain barrier (BBB) is an important factor leading to brain injury. Therapeutic hypothermia is widely accepted to limit neurological impairment. However, the efficacy is incomplete. Hydrogen sulfide (H₂S), a signaling gas molecule, has protective effects after cerebral ischemia reperfusion injury. This study showed that combination of hypothermia and H₂S after resuscitation was more beneficial for attenuated BBB disruption and brain edema than that of hypothermia or H₂S treatment alone. CA was induced by ventricular fibrillation for 4 min. Hypothermia was performed by applying alcohol and ice bags to the body surface under anesthesia. We used sodium hydrosulphide (NaHS) as the H₂S donor. We found that global brain ischemia induced by CA and cardiopulmonary resuscitation (CPR) resulted in brain edema and BBB disruption; Hypothermia or H₂S treatment diminished brain edema, decreased the permeability and preserved the structure of BBB during the early period of CA and resuscitation, and more importantly, improved the neurologic function, increased the 7-day survival rate after resuscitation; the combination of hypothermia and H₂S treatment was more beneficial than that of hypothermia or H₂S treatment alone. The beneficial effects were associated with the inhibition of matrix metalloproteinase-9 expression, attenuated the degradation of the tight junction protein occludin, and subsequently protected the structure of BBB. These findings suggest that combined use of therapeutic hypothermia and hydrogen sulfide treatment during resuscitation of CA patients could be a potential strategy to improve clinical outcomes and survival rate.

Keywords: Blood–brain barrier; Cardiac arrest; Cardiopulmonary resuscitation; Hydrogen sulfide; Therapeutic hypothermia.

Fig. 1

Experimental time line. Rats were subjected to CA induced by electrical stimulation or sham operation. After four minutes of CA, rats were resuscitated by chest compression and mechanical ventilation. After successful ROSC, rats were randomized to one of four groups: Group S, Group CAR, Group H₂S, Group TH and Group H₂S + TH

Fig. 2

Effects of hypothermia and hydrogen sulfide treatment on survival rates and neurological function of rats with CA and resuscitation. A Kaplan–Meier plot of cumulative survival 7 days after CA and resuscitation in Group S (n = 5), Group CAR (n = 15), Group H₂S (n = 15), Group TH (n = 15), and Group H₂S + TH (n = 15). Data was presented as a percentage. B–D Time needed in the tape removal test at day 1, day 3, and day 7 after resuscitation. Date was presented as median (quartiles). ^*P < 0.05 versus Group CAR, ^#P < 0.05 versus Group H₂S, ^&P < 0.05 versus Group TH

Fig. 3

Effects of hypothermia and hydrogen sulfide treatment on brain edema and BBB integrity of rats with CA and resuscitation. A, B Brain water content in the cortex and hippocampus. Brain water content, an indicator of brain edema, was measured with wet-dry method at 24 h after resuscitation or sham operation (n = 5 rats per group). C–E EB and FITC–dextran permeability in the brain. BBB permeability was evaluated using EB in the whole brain, and FITC–dextran permeability in the cortex and hippocampus at 24 h after resuscitation or sham operation (n = 5 rats per group). Data are presented as mean ± SD. ^$P < 0.05 versus Group S, ^*P < 0.05 versus Group CAR, ^#P < 0.05 versus Group H₂S, ^&P < 0.05 versus Group TH

Fig. 4

The expression of occludin protein in the cortex and hippocampus at 24 h after resuscitation among groups (n = 5 rats per group). A, B Representative western blot images of occludin. C–D The bars of semi-quantitative. Results are expressed as the ratio of occludin and β-actin among groups. Data are presented as mean ± SD. ^$P < 0.05 versus Group S, ^*P < 0.05 versus Group CAR, ^#P < 0.05 versus Group H₂S, ^&P < 0.05 versus Group TH

Fig. 5

MMP-9 immunohistochemical staining and positive cell counts in the cortex and hippocampus in rats at 24 h after resuscitation (n = 5 rats per group). A, B MMP-9 immunohistochemical staining in the cortex and hippocampus, respectively. C, D MMP-9 positive cell counts in the prefrontal cortex and the cornu ammonis (CA-1) area of the hippocampus. Data are presented as mean ± SD. ^$P < 0.05 versus Group S, ^*P < 0.05 versus Group CAR, ^#P < 0.05 versus Group H₂S, ^&P < 0.05 versus Group TH

Fig. 6

Ultrastructure alteration of BBB in the hippocampus of rats at 24 h after resuscitation or sham operation. The representative transmission electron micrographs of BBB are displayed, the micrographs in a-e are the high magnification of the area inside the boxes in (A–E), respectively. Arrows indicate the location of the TJs. Scale bar = 2 μm (A–E) or 0.5 μm (a–e). AGroup S, B Group CAR, C Group H₂S, D Group TH, E Group H₂S + TH. F Quantification of the width of the TJs of BBB. (μm, mean ± SD). ^$P < 0.05 versus Group S, ^*P < 0.05 versus Group CAR, ^#P < 0.05 versus Group H₂S, ^&P < 0.05 versus Group TH