Neuroprotección con hipotermia y alopurinol en el daño hipóxico-isquémico. Papel del efecto antioxidante

La encefalopatía hipóxico-isquémica en el recién nacido constituye una importante causa de morbimortalidad. A pesar del tratamiento con hipotermia, la incidencia de muerte y discapacidad sigue siendo muy elevada, haciéndose necesario nuevas terapias que potencien su efecto. Uno de los mecanismos principales causantes del daño cerebral hipóxico-isquémico es el estrés oxidativo. Por ello, utilizando un modelo de EHI neonatal en crías de rata, se ha evaluado el efecto de la terapia conjunta hipotermia con el antioxidante alopurinol. Se ha observado un efecto protector de esta terapia combinada sobre el estrés oxidativo, evaluado a partir del estudio de los sistemas antioxidantes, así como de la oxidación de proteínas y lípidos

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

A New Technique for Collection of Cerebrospinal Fluid in Rat Pups

Background Neuroprotective strategies to prevent or decrease brain injury in hypoxic ischemic newborns are one of the main research lines in neonatology. Animal models have been used to assess the efficiency of new therapeutic strategies. Brain damage biomarkers in cerebrospinal fluid (CSF) are frequently used to evaluate the outcome at the bedside. Despite the importance of this approach in clinical practice, there are many difficulties in using it in small animals. The aim of this paper was to describe a new technique for collecting CSF in rat pups. Furthermore the reference values of S100β protein levels, commonly used in common clinical practice, were analyzed in animals between 7 to 12 days. Methods 42 Wistar rat pups aged 7 to 12 days were used. CSF was obtained by direct puncture of the cisterna magna with a 24-gauge needle. S100β protein levels were determined with enzyme-linked immunosorbent assay (ELISA). Results CSF was successfully obtained in 96% of the cases, with an average amount of 21.28 μl (5–40 μl). Normal values for S100β were described. HI animals presented higher S100β values than controls. Conclusions A simple, reproducible technique for CSF collection in rat pups has been described. This new method will allow study of brain injury biomarkers in newborn hypoxic ischemic animal models

Effects of Hypothermia and Allopurinol on Oxidative Status in a Rat Model of Hypoxic Ischemic Encephalopathy

Hypoxic ischemic encephalopathy (HIE) is one of the main causes of morbidity and mortality during the neonatal period, despite treatment with hypothermia. There is evidence that oxidative damage plays an important role in the pathophysiology of hypoxic-ischemic (HI) brain injury. Our aim was to investigate whether postnatal allopurinol administration in combination with hypothermia would reduce oxidative stress (OS) biomarkers in an animal model of HIE. Postnatal 10-day rat pups underwent unilateral HI of moderate severity. Pups were randomized into: Sham operated, hypoxic-ischemic (HI), HI + allopurinol (HIA), HI + hypothermia (HIH), and HI + hypothermia + allopurinol (HIHA). Biomarkers of OS and antioxidants were evaluated: GSH/GSSG ratio and carbonyl groups were tested in plasma. Total antioxidant capacity (TAC) was analyzed in plasma and cerebrospinal fluid, and 8-iso-prostaglandin F2α was measured in brain tissue. Plasma 2,20–azinobis- (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) levels were preserved in those groups that received allopurinol and dual therapy. In cerebrospinal fluid, only the HIA group presented normal ferric reducing ability of plasma (FRAP) levels. Protein oxidation and lipid peroxidation were significantly reduced in all groups treated with hypothermia and allopurinol, thus enhancing neuroprotection in HIE

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

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

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

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