Neuroprotection with hypothermia and allopurinol in an animal model of hypoxic-ischemic injury: Is it a gender question?

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig 1. Study diagram.

P10: 10 days of life, LCA: Left common carotid, ST: Sham-treated; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol, HE: Histological evaluation, MWMT: Water maze test.

Fig 2. Histological evaluation.

A: Graphical representation of the percentage of brain area lost in the affected hemisphere with respect to the contralateral hemisphere by experimental group (global), and by group and gender (Male, Female) B: Graphical representation of the hippocampal volume in the affected hemisphere by experimental group (global), and by group and gender (male, female) ST: Sham-treated; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol. Volume areas are expressed in arbitrary units. * Significant differences p<0.05.

Fig 3. Macroscopic-microscopic histologic evaluation.

A: Neuropathological brain scores. B: Representative photograph of perinatal coronal brain sections of the different experimental groups. ST: Sham-treated; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol.

Fig 4. Cleaved caspase-3 expression.

Western blot and densitometry analysis of cleaved caspase-3 protein in the hippocampal tissue of males and females from the different experimental groups. GAPDH was used as protein loading control. Abbreviatons: Control; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol. * Significant differences p<0.05.

Fig 5. Water maze test.

Plot representing the average escape latency in four trials performed each day. Results were expressed as mean of escape latency. Abbreviatons: ST: Sham-treated; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol.