Rodríguez-Fanjul Javier, Durán Fernández-Feijóo Cristina, Lopez-Abad Míriam, Lopez Ramos Maria Goretti, Balada Caballé Rafael, Alcántara-Horillo Soledad, Camprubí Camprubí Marta
Department of Neonatology, BCNatal, Sant Joan de Déu-Hospital Clínic, Barcelona, Spain.
Department of Neonatology, Hospital Álvaro Cunqueiro, EOXI, Vigo, Spain.
PLoS One. 2017 Sep 20;12(9):e0184643. doi: 10.1371/journal.pone.0184643. eCollection 2017.
Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Therapeutic hypothermia (TH) is the standard treatment for term newborns after perinatal hypoxic ischemic injury (HI). Despite this, TH does not provide complete neuroprotection. Allopurinol seems to be a good neuroprotector in several animal studies, but it has never been tested in combination with hypothermia. Clinical findings show that male infants with (HI) fare more poorly than matched females in cognitive outcomes. However, there are few studies about neuroprotection taking gender into account in the results. The aim of the present study was to evaluate the potential additive neuroprotective effect of allopurinol when administrated in association with TH in a rodent model of moderate HI. Gender differences in neuroprotection were also evaluated.
P10 male and female rat pups were subjected to HI (Vannucci model) and randomized into five groups: sham intervention (Control), no treatment (HI), hypothermia (HIH), allopurinol (HIA), and dual therapy (hypothermia and allopurinol) (HIHA). To evaluate a treatment's neuroprotective efficiency, 24 hours after the HI event caspase3 activation was measured. Damaged area and hippocampal volume were also measured 72 hours after the HI event. Negative geotaxis test was performed to evaluate early neurobehavioral reflexes. Learning and spatial memory were assessed via Morris Water Maze (MWM) test at 25 days of life.
Damaged area and hippocampal volume were different among treatment groups (p = 0.001). The largest tissue lesion was observed in the HI group, followed by HIA. There were no differences between control, HIH, and HIHA. When learning process was analyzed, no differences were found. Females from the HIA group had similar results to the HIH and HIHA groups. Cleaved caspase 3 expression was increased in both HI and HIA. Despite this, in females cleaved caspase-3 was only differently increased in the HI group. All treated animals present an improvement in short-term (Negative geotaxis) and long-term (WMT) functional tests. Despite this, treated females present better long-term outcome. In short-term outcome no sex differences were observed.
Our results suggest that dual therapy confers great neuroprotection after an HI event. There were functional, histological, and molecular improvements in all treated groups. These differences were more important in females than in males. No statistically significant differences were found between HIHA and HIH; both of them present a great improvement. Our results support the idea of different regulation mechanisms and pathways of cell death, depending on gender.
缺氧缺血性脑病（HIE）是新生儿脑损伤的最重要原因之一。治疗性低温（TH）是足月新生儿围产期缺氧缺血性损伤（HI）后的标准治疗方法。尽管如此，TH并不能提供完全的神经保护作用。在多项动物研究中，别嘌醇似乎是一种良好的神经保护剂，但从未与低温联合进行过测试。临床研究结果表明，患有HI的男婴在认知结果方面比匹配的女婴更差。然而，很少有研究在结果中考虑性别因素对神经保护的影响。本研究的目的是评估在中度HI啮齿动物模型中，别嘌醇与TH联合应用时潜在的附加神经保护作用。同时也评估了神经保护中的性别差异。
将出生后10天的雄性和雌性大鼠幼崽进行HI造模（Vannucci模型），并随机分为五组：假干预组（对照组）、不治疗组（HI组）、低温治疗组（HIH组）、别嘌醇治疗组（HIA组）和联合治疗组（低温与别嘌醇）（HIHA组）。为了评估治疗的神经保护效率，在HI事件发生24小时后测量caspase3的激活情况。在HI事件发生72小时后还测量了损伤面积和海马体积。进行负趋地性试验以评估早期神经行为反射。在出生后25天通过莫里斯水迷宫（MWM）试验评估学习和空间记忆。
各治疗组之间的损伤面积和海马体积存在差异（p = 0.001）。HI组观察到最大的组织损伤，其次是HIA组。对照组、HIH组和HIHA组之间没有差异。在分析学习过程时，未发现差异。HIA组的雌性大鼠与HIH组和HIHA组的结果相似。HI组和HIA组中裂解的caspase 3表达均增加。尽管如此，在雌性大鼠中，仅HI组裂解的caspase-3有不同程度的增加。所有接受治疗的动物在短期（负趋地性）和长期（WMT）功能测试中均有改善。尽管如此，接受治疗的雌性大鼠长期预后更好。在短期预后方面未观察到性别差异。
我们的结果表明，联合治疗在HI事件后具有强大的神经保护作用。所有治疗组在功能、组织学和分子方面均有改善。这些差异在雌性中比在雄性中更明显。HIHA组和HIH组之间未发现统计学上的显著差异；两者均有很大改善。我们的结果支持根据性别存在不同细胞死亡调节机制和途径的观点。
