Engels Jonas, Elting Natalie, Braun Lisa, Bendix Ivo, Herz Josephine, Felderhoff-Müser Ursula, Dzietko Mark
Division of Neonatology, Department of Pediatrics I, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Dev Neurosci. 2017;39(1-4):287-297. doi: 10.1159/000457832. Epub 2017 Mar 25.
Hypoxic-ischemic (HI) injury to the developing brain occurs in 1 out of 1,000 live births and remains a major cause of significant morbidity and mortality. A large number of survivors suffer from long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after HI insult are not clearly understood, and preventive measures or clinical treatments are nonexistent or not sufficiently effective in the clinical setting. Sildenafil as a specific phosphodiesterase 5 inhibitor leads to increased levels of the second messenger cyclic guanosine monophosphate (cGMP) and promotes functional recovery and neurogenesis after ischemic injury to the adult brain.
Here, we investigated the effect of sildenafil treatment on activation of intracellular signaling pathways, histological and neurogenic response including functional recovery after an ischemic insult to the developing brain.
DESIGN/METHODS: Nine-day-old C57BL/6 mice were subjected either to sham operation or underwent ligation of the right common carotid artery followed by hypoxia (8%) for 60 min. Animals were either administered sildenafil (10 mg/kg, i.p.) or vehicle 2 h after hypoxia. A subgroup of animals received multiple injections of 10 mg/kg daily on 5 consecutive days. Pups were either perfusion fixed at postnatal days 14 or 47 for immunohistochemical analysis, or brains were dissected 2, 6, 12, and 24 h after the end of hypoxia and analyzed for cGMP, pAkt, pGSK-3β, and β-catenin by means of ELISA or immunoblotting. In addition, behavioral studies using the wire hang test and elevated plus maze were conducted 21 and 38 days after HI injury.
Based on cresyl violet staining, single or multiple sildenafil injections did not reveal any differences in injury scoring compared to sham animals. However, cerebral levels of cGMP were altered after sildenafil therapy. Treatment significantly increased numbers of immature neurons, as indicated by doublecortin immunoreactivity in the ipsilateral subventricular zone and striatum. In addition, animals treated with sildenafil after HI insult demonstrated improved functional recovery. pAkt, pGSK-3β, and β-catenin levels vary after HI injury but additional sildenafil treatment had no impact on protein expression compared to the level of sham controls.
Here, we report that treatment with sildenafil after HI insult did not improve histological brain injury scores. Nevertheless, our results suggest involvement of the cGMP and PI3K/Akt/GSK-3β signaling pathway with promotion of a neurogenic response and reduction of neurological deficits. In summary, sildenafil may have a role in promoting recovery from HI injury in the developing brain.
发育中的大脑发生缺氧缺血性（HI）损伤的情况在每1000例活产中出现1例，仍是导致严重发病和死亡的主要原因。大量幸存者患有包括癫痫发作和神经功能缺损在内的长期后遗症。然而，HI损伤后恢复的病理生理机制尚不清楚，在临床环境中不存在预防措施或临床治疗方法，或者这些方法效果不佳。西地那非作为一种特异性磷酸二酯酶5抑制剂，可导致第二信使环磷酸鸟苷（cGMP）水平升高，并促进成人大脑缺血损伤后的功能恢复和神经发生。
在此，我们研究了西地那非治疗对发育中大脑缺血损伤后细胞内信号通路激活、组织学和神经源性反应（包括功能恢复）的影响。
设计/方法：对9日龄的C57BL/6小鼠进行假手术或结扎右侧颈总动脉，随后进行60分钟的缺氧（8%）处理。在缺氧2小时后，给动物注射西地那非（10毫克/千克，腹腔注射）或赋形剂。一组动物连续5天每天接受10毫克/千克的多次注射。幼崽在出生后第14天或第47天进行灌注固定以进行免疫组织化学分析，或者在缺氧结束后2、6、12和24小时解剖大脑，通过酶联免疫吸附测定（ELISA）或免疫印迹法分析cGMP、磷酸化蛋白激酶B（pAkt）、磷酸化糖原合成酶激酶-3β（pGSK-3β）和β-连环蛋白。此外，在HI损伤后21天和38天使用悬线试验和高架十字迷宫进行行为学研究。
基于甲酚紫染色，与假手术动物相比，单次或多次注射西地那非在损伤评分上未显示出任何差异。然而，西地那非治疗后大脑中的cGMP水平发生了改变。治疗显著增加了未成熟神经元的数量，同侧脑室下区和纹状体中双皮质素免疫反应性表明了这一点。此外，HI损伤后接受西地那非治疗的动物功能恢复得到改善。HI损伤后pAkt、pGSK-3β和β-连环蛋白水平有所变化，但与假手术对照组相比，额外的西地那非治疗对蛋白质表达没有影响。
在此，我们报告HI损伤后用西地那非治疗并未改善大脑组织学损伤评分。然而，我们的结果表明cGMP和磷脂酰肌醇-3激酶/蛋白激酶B/糖原合成酶激酶-3β（PI3K/Akt/GSK-3β）信号通路参与促进神经源性反应和减少神经功能缺损。总之，西地那非可能在促进发育中大脑从HI损伤中恢复方面发挥作用。
