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

<div><p>Background</p><p>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.</p><p>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.</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>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.</p><p>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.</p><p>Conclusions</p><p>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.</p></div

Water maze test.

<p>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.</p

Histological evaluation.

<p>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.</p

Influence of catch up growth on spatial learning and memory in a mouse model of intrauterine growth restriction

<div><p>Background</p><p>Intrauterine growth restriction (IUGR) and rapid postnatal weight gain or catch up growth (CUG) increase the susceptibility to metabolic syndrome during adult life. Longitudinal studies have also revealed a high incidence of learning difficulties in children with IUGR. The aim of the present study was to investigate the effect of nutrition and CUG on learning memory in an IUGR animal model. We hypothesized that synaptic protein expression and transcription, an essential mechanism for memory consolidation, might be affected by intrauterine undernutrition.</p><p>Methods</p><p>IUGR was induced by 50% maternal caloric undernutrition throughout late gestation. During the suckling period, dams were either fed <i>ad libitum</i> or food restricted. The pups were divided into: Normal prenatal diet-Normal postnatal diet (NN), Restricted prenatal diet- Normal postnatal diet + catch up growth (RN+), Normal prenatal diet-Restricted postnatal diet (NR) and Restricted prenatal diet-Restricted postnatal diet (RR). At 4 weeks of age, memory was assessed via a water maze test. To evaluate synaptic function, 2 specific synaptic proteins (postsynaptic density-95 [PSD95], synaptophysin) as well as insulin receptors (IR) were tested by Western Blot and quantitative polymerase chain reaction (qPCR). Brain-derived neurotrophic factor and serum insulin levels were also studied.</p><p>Results and conclusions</p><p>The RN+ group presented a learning curve similar to the NN animals. The RR animals without CUG showed learning disabilities. PSD95 was lower in the RR group than in the NN and RN+ mice. In contrast, synaptophysin was similar in all groups. IR showed an inverse expression pattern to that of the PSD95. In conclusion, perinatal nutrition plays an important role in learning. CUG after a period of prenatal malnutrition seems to improve learning skills. The functional alterations observed might be related to lower PSD95 activity and a possible dysfunction in the hormone regulation of synaptic plasticity.</p></div

Macroscopic-microscopic histologic evaluation.

<p>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.</p

BDNF and synaptic protein expression.

<p>A. BDNF mRNA expression was increased in the groups with better learning results. B. PSD95 and synaptophysin transcription. C. PSD95 and synaptophysin expression. There was a reduction in RR PSD95 expression. Significant differences compared to NN and NR+ *p < .05. No differences were observed in synaptophysin expression or trasncription.</p

Graphical scheme of the experimental groups and procedures.

<p>A) <b>Animal model:</b> The diagram of the experiment is represented, with the different groups. B) <b>Functional evaluation:</b> The WMT is performed from 25 to 35 days. <b>C) Molecular tests:</b> Different test are performed for the molecular evaluation: Hormonal tests (ELISA), Protein quantification (Western blot) and RNA quantification (qPCR).</p

Insulin metabolism.

<p><b>A</b>. Serum insulin levels were increased in NN and RN+ animals. Significant differences between groups *p < .05. <b>B.</b> Brain insulin receptor transcription was increased in RR and RN animals. <b>C.</b> Brain insulin receptor expression was increased in RR and NR animals. Significant differences compared to NN and NR+ *p < .05.</p

Pup growth evaluation.

<p>Pup weight two days after birth, by group. Pup weight at 21 days of life, by group.</p