Gupta Bhavna, Hornick Mary G, Briyal Seema, Donovan Ramona, Prazad Preetha, Gulati Anil
Division of Neonatology, Department of Pediatrics, Advocate Children's Hospital, Park Ridge, IL, United States.
Chicago College of Pharmacy, Midwestern University, Downers Grove, IL, United States.
Front Pediatr. 2020 Feb 27;8:65. doi: 10.3389/fped.2020.00065. eCollection 2020.
Neonatal HIE is associated with high morbidity and mortality. Current research, is focused on developing alternative treatments to therapeutic hypothermia for treatment of HIE. The endocannabinoid system is known to be influential in neuronal protection. Activation of brain CB2 receptors, has been shown to reduce inflammatory markers and decrease infarct volume in adult cerebral ischemic models. Rat pups were divided into six groups: 1-Placebo; 2-JWH133; 3-HIE + Placebo; 4-HIE + JWH133; 5-HIE + Hypothermia + Placebo; and 6-HIE + Hypothermia + JWH133. HIE was induced in in groups 3-6 by right carotid ligation on postnatal day 7 followed by placement in a hypoxic chamber. Pups in groups 5 and 6 were treated with hypothermia. Western blot analysis was used to analyze brain tissue for acute inflammatory markers (IL-6, TNFα, MIP1α, and RANTES), immunoregulatory cytokines (TGFβ and IL-10), and CB2 receptor expression. DNA fragmentation in the brains of pups was determined via TUNEL staining post HIE. The combination of JWH133 and hypothermia significantly reduced tumor necrosis factor α (TNFα) (-57.7%, = 0.0072) and macrophage inflammatory protein 1α (MIP1α) (-50.0%, = 0.0211) as compared to placebo. DNA fragmentation was also significantly reduced, with 6.9 ± 1.4% TUNEL+ cells in HIE+JWH133 and 12.9 ± 2.2% in HIE+Hypothermia + JWH133 vs. 16.6 ± 1.9% in HIE alone. No significant difference was noted between groups for the expression of interleukins 6 and 10, RANTES, or TGFβ. After 8 h, CB2 receptor expression increased nearly 2-fold in the HIE and HIE + JWH133 groups (+214%, = 0.0102 and +198%, = 0.0209, respectively) over placebo with no significant change in the hypothermia groups. By 24 h post HIE, CB2 receptor expression was elevated over five times that of placebo in the HIE ( < 0.0001) and HIE + JWH133 ( = 0.0002) groups, whereas hypothermia treatment maintained expression similar to that of placebo animals. These results indicate that the combination of CB2 agonist and hypothermia may be neuroprotective in treating HIE, opening the door for further studies to examine alternative or adjuvant therapies to hypothermia.
新生儿缺氧缺血性脑病（HIE）与高发病率和死亡率相关。目前的研究集中在开发治疗HIE的亚低温替代疗法。已知内源性大麻素系统对神经元保护有影响。在成人大脑缺血模型中，脑CB2受体的激活已被证明可降低炎症标志物并减少梗死体积。将新生大鼠幼崽分为六组：1-安慰剂组；2-JWH133组；3-HIE+安慰剂组；4-HIE+JWH133组；5-HIE+亚低温+安慰剂组；6-HIE+亚低温+JWH133组。在出生后第7天通过右颈动脉结扎并随后置于缺氧舱中，在3-6组中诱导HIE。5组和6组的幼崽接受亚低温治疗。采用蛋白质免疫印迹分析来分析脑组织中的急性炎症标志物（IL-6、TNFα、MIP1α和RANTES）、免疫调节细胞因子（TGFβ和IL-10）以及CB2受体表达。通过HIE后TUNEL染色测定幼崽大脑中的DNA片段化。与安慰剂相比，JWH133和亚低温联合使用显著降低了肿瘤坏死因子α（TNFα）（-57.7%，P=0.0072）和巨噬细胞炎性蛋白1α（MIP1α）（-50.0%，P=0.0211）。DNA片段化也显著减少，HIE+JWH133组TUNEL+细胞为6.9±1.4%，HIE+亚低温+JWH133组为12.9±2.2%，而单独HIE组为16.6±1.9%。各亚组间白细胞介素6和10、RANTES或TGFβ的表达无显著差异。8小时后，与安慰剂相比，HIE组和HIE+JWH133组的CB2受体表达增加了近2倍（分别为+214%，P=0.0102和+198%，P=0.0209），亚低温组无显著变化。HIE后24小时，HIE组（P<0.0001）和HIE+JWH133组（P=0.0002）的CB2受体表达比安慰剂组升高了五倍多，而亚低温治疗使表达维持在与安慰剂动物相似的水平。这些结果表明，CB2激动剂与亚低温联合使用在治疗HIE时可能具有神经保护作用，为进一步研究亚低温的替代或辅助疗法打开了大门。
