Administration of selective brain hypothermia using a simple cooling device in neonatal rats.

BACKGROUND: The interruption of oxygen and blood supply to the newborn brain around the time of birth is a risk factor for hypoxic-ischemic encephalopathy and may lead to infant mortality or lifelong neurological impairments. Currently, therapeutic hypothermia, the cooling of the infant's head or entire body, is the only treatment to curb the extent of brain damage. NEW METHOD: In this study, we designed a focal brain cooling device that circulates cooled water at a steady state temperature of 19 ± 1 °C through a coil of tubing fitted onto the neonatal rat's head. We tested its ability to selectively decrease brain temperature and offer neuroprotection in a neonatal rat model of hypoxic-ischemic brain injury. RESULTS: Our method cooled the brain to 30-33 °C in conscious pups, while keeping the core body temperature approximately 3.2 °C warmer. Furthermore, the application of the cooling device to the neonatal rat model demonstrated a reduction in brain volume loss compared to pups maintained at normothermia and achieved a level of brain tissue protection the same as that of whole-body cooling. COMPARISON WITH EXISTING METHODS: Prevailing methods of selective brain hypothermia are designed for adult animal models rather than for immature animals such as the rat as a conventional model of developmental brain pathology. Contrary to existing methods, our method of cooling does not require surgical manipulation or anaesthesia. CONCLUSION: Our simple, economical, and effective method of selective brain cooling is a useful tool for rodent studies in neonatal brain injury and adaptive therapeutic interventions.

Broccoli Sprouts Promote Sex-Dependent Cardiometabolic Health and Longevity in Long-Evans Rats.

Antioxidants and anti-inflammatory compounds are potential candidates to prevent age-related chronic diseases. Broccoli sprouts (BrSp) are a rich source of sulforaphane-a bioactive metabolite known for its antioxidant and anti-inflammatory properties. We tested the effect of chronic BrSp feeding on age-related decline in cardiometabolic health and lifespan in rats. Male and female Long-Evans rats were fed a control diet with or without dried BrSp (300 mg/kg body weight, 3 times per week) from 4 months of age until death. Body weight, body composition, blood pressure, heart function, and glucose and insulin tolerance were measured at 10, 16, 20, and 22 months of age. Behavioral traits were also examined at 18 months of age. BrSp feeding prolonged life span in females, whereas in males the positive effects on longevity were more pronounced in a subgroup of males (last 25% of survivors). Despite having modest effects on behavior, BrSp profoundly affected cardiometabolic parameters in a sex-dependent manner. BrSp-fed females had a lower body weight and visceral adiposity while BrSp-fed males exhibited improved glucose tolerance and reduced blood pressure when compared to their control counterparts. These findings highlight the sex-dependent benefits of BrSp on improving longevity and delaying cardiometabolic decline associated with aging in rats.

Bacteriophage carriers localize in the brain of a rat model of neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy arises from a reduction of oxygen and blood supply to the infant brain and can lead to severe brain damage and life-long disability. The damage is greatest at the irreversibly injured necrotic core, whereas the penumbra is the surrounding, potentially salvageable tissue populated with a mix of alive and dying cells. To date, there exists no method for targeting drugs to the brain damage. METHODS AND MAJOR RESULTS: Bacteriophages are viruses that propagate in bacteria but are biocompatible in humans and also amenable to genetic and chemical modification in a manner distinctive from conventional therapeutic nanoparticles. Here, a library of M13 bacteriophage was administered into a rat model of hypoxic-ischemic encephalopathy, and unique bacteriophage clones were confirmed to localize in healthy brain tissue versus the core and penumbra zones of injury. CONCLUSIONS: For the first time, there is a potential to directly deliver therapeutics to different regions of the neonatal brain injury.

Sulforaphane (SFA) protects neuronal cells from oxygen & glucose deprivation (OGD).

BACKGROUND: Perinatal brain injury results in neurodevelopmental disabilities (neuroDDs) that include cerebral palsy, autism, attention deficit disorder, epilepsy, learning disabilities and others. Commonly, injury occurs when placental circulation, that is responsible for transporting nutrients and oxygen to the fetus, is compromised. Placental insufficiency (PI) is a reduced supply of blood and oxygen to the fetus and results in a hypoxic-ischemic (HI) environment. A significant HI state in-utero leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Given that over 80% of perinatal brain injuries that result in neuroDDs occur during gestation, prior to birth, preventive approaches are needed to reduce or eliminate the potential for injury and subsequent neuroDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that acts via cytoplasmic Nrf2 to enhance the production of anti-oxidants in the brain through the glutathione pathway. We have previously shown a profound in vivo neuro-protective effect of BrSps/SFA as a dietary supplement in pregnant rat models of both PI and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA has the ability to enhance cell survival, and with conditions of cancer, enhance cell death. Given our findings of the benefit of SFA/Broccoli Sprouts as a dietary supplement during pregnancy, with improvement to the fetus, it is important to determine the beneficial and toxic dosing range of SFA. We therefore explored, in vitro, the dosing range of SFA for neuronal and glial protection and toxicity in normal and oxygen/glucose deprived (OGD) cell cultures. METHODS: OGD simulates, in vitro, the condition experienced by the fetal brain due to PI. We developed a cell culture model of primary cortical neuronal, astrocyte and combined brain cell co-cultures from newborn rodent brains. The cultures were exposed to an OGD environment for various durations of time to determine the LD50 (duration of OGD required for 50% cell death). Using the LD50 as the time point, we evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects. Control cultures were exposed to normal media without OGD, and cytotoxicity of varying doses of SFA was also evaluated. Immunofluorescence (IF) and Western blot analysis of cell specific markers were used for culture characterization, and quantification of LD50. Efficacy and toxicity effect of SFA was assessed by IF/high content microscopy and by AlamarBlue viability assay, respectively. RESULTS: We determined the LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures. The protective effect of SFA was noticeable at 2.5 µM and 5 µM for neurons, although it was not significant. There was a significant protective effect of SFA at 2.5 µM (p<0.05) for astrocytes and co-cultures. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 µM (p<0.05) for astrocytes, ≥ 50 µM (p<0.01) for co-cultures, but not toxic in neurons; and toxic in control cultures as ≥ 100 µM (p<0.01) for neurons, and ≥ 50 µM (p<0.01) for astrocytes and co-cultures. One Way ANOVA and Dunnett's Multiple Comparison Test were used for statistical analysis. CONCLUSIONS: Our results indicate that cell death shows a trend to reduction in neuronal and astrocyte cultures, and is significantly reduced in co-cultures treated with low doses of SFA exposed to OGD. Doses of SFA that were 10 times higher were toxic, not only under conditions of OGD, but in normal control cultures as well. The findings suggest that: 1. SFA shows promise as a preventative agent for fetal ischemic brain injury, and 2. Because the fetus is a rapidly growing organism with profound cell multiplication, dosing parameters must be established to insure safety within efficacious ranges. This study will influence the development of innovative therapies for the prevention of childhood neuroDD.

Late-pregnancy uterine artery ligation increases susceptibility to postnatal Western diet-induced fat accumulation in adult female offspring.

Stressors during the fetal and postnatal period affect the growth and developmental trajectories of offspring, causing lasting effects on physiologic regulatory systems. Here, we tested whether reduced uterine artery blood flow in late pregnancy would alter body composition in the offspring, and whether feeding offspring a western diet (WD) would aggravate these programming effects. Pregnant rats underwent bilateral uterine artery ligation (BUAL) or sham surgery on gestational day (GD)18 (term = GD22). At weaning, offspring from each group received either a normal diet (ND) or a WD. BUAL surgery increased fetal loss and caused offspring growth restriction, albeit body weights were no longer different at weaning, suggesting postnatal catch-up growth. BUAL did not affect body weight gain, fat accumulation, or plasma lipid profile in adult male offspring. In contrast, while ND-fed females from BUAL group were smaller and leaner than their sham-littermates, WD consumption resulted in excess weight gain, fat accumulation, and visceral adiposity. Moreover, WD increased plasma triglycerides and cholesterol in the BUAL-treated female offspring without any effect on sham littermates. These results demonstrate that reduced uterine artery blood flow during late pregnancy in rodents can impact body composition in the offspring in a sex-dependent manner, and these effects may be exacerbated by postnatal chronic WD consumption.

Sustained Release of Dexamethasone from Sulfobutyl Ether ß-cyclodextrin Modified Self-Assembling Peptide Nanoscaffolds in a Perinatal Rat Model of Hypoxia-Ischemia.

Inflammation plays a critical role in the development of hypoxia-ischemia (HI) induced newborn brain damage. A localized, sustained delivery of dexamethasone (Dex) through an intracerebral injection could reduce the inflammatory response in the injured perinatal brain while avoiding unnecessary side effects. Herein, investigated using anionic sulfobutyl ether ß-cyclodextrin (SBE-ß-CD) to load Dex in the (RADA)4 nanofiber networks as a means of reducing the inflammatory response to HI injury is investigated. The ionic interaction between SBE-ß-CD and (RADA)4 dramatically affects nanofiber formation and the stability of the nanoscaffold is highly dependent on the SBE-ß-CD/(RADA)4 ratio. It is observed that the Dex release rate is affected by the concentration of SBE-ß-CD and (RADA)4 peptide. A higher concentration of SBE-ß-CD or (RADA)4 results in a higher drug encapsulation efficiency and slower release rate of Dex. This phenomenon may be related to the structure of fiber bundles. Animal studies show that nanoscaffold loaded with Dex inhibits both microglia activation and glial scar formation compared to controls (Dex alone or nanoscaffold alone) within 2 days of injury. It is thought that this is a step toward building a multifaceted nanoscaffold that can be used to treat HI events in perinates.

Neurodevelopmental Reflex Testing in Neonatal Rat Pups.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Consumption of broccoli sprouts during late gestation and lactation confers protection against developmental delay induced by maternal inflammation.

BACKGROUND: The presence of a fetal inflammatory response is linked to cerebral palsy. Unfortunately no preventive therapies are available. In this study, we determined whether dietary supplementation with broccoli sprouts (BrSp), a phase-II enzyme inducer, would be effective in preventing the behavioural and pathologic manifestations in a rodent model of inflammation during late pregnancy. METHODS: Pregnant Long-Evans rats were administered i.p. Injections of saline (100µl) or lipopolysaccharide (LPS, 200µg/kg), every 12h on embryonic day (E) 19 and 20. In the treatment groups, dams were supplemented with 200mg/day of dried BrSp from E14 until postnatal day 21. Pups underwent a series of neurodevelopmental reflex tests from postnatal day 3-21 followed by neuropathological analyses. RESULTS: Pups born from the LPS group were significantly growth restricted (p<0.001) and delayed in hindlimb placing (p<0.05), cliff avoidance (p<0.05), and gait (p<0.001) compared to controls. In the open field behaviour analyses, LPS pups had an increase in grooming behaviour (p<0.05) and a decreased amount of time spent in the center of the box compared to controls. Dietary supplementation with BrSp to offspring exposed to LPS had increased birth weights (p<0.001), were no longer delayed in acquiring hindlimb placing, cliff avoidance, gait, and posture, and groomed less compared to LPS alone pups (p<0.01). Histological analyses revealed that LPS pups had reduced myelin basic protein compared to controls. CONCLUSIONS: Our data suggest that BrSp dietary supplementation during pregnancy may be effective in preventing growth restriction and neurodevelopmental delays.

Pioglitazone attenuates hepatic inflammation and fibrosis in phosphatidylethanolamine N-methyltransferase-deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-ß (Tgf-ß). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.

Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain.

PURPOSE: Apoptosis is implicated in mild-moderate ischemic injury. Cell death pathways in the severely ischemic brain are not characterized. We sought to determine the role of apoptosis in the severely ischemic immature brain. METHODS: Seven-day old rats were randomly assigned to mild-moderate or severe cerebral hypoxia-ischemia (HI) group. After ligating the right common carotid artery, animals were subjected to hypoxia for 90min in the mild-moderate HI or 180min in the severe HI. The core and peri-infarct area were measured in H&E stained brain sections using NIS Elements software. Brain sections were processed for caspase-3, AIF and RIP3 immuno-staining. Number of positive cells were counted and compared between the two groups. RESULTS: The core constituted a significantly higher proportion of the ischemic lesion in the severely compared to the moderately injured brain (P<0.04) up to 7 days post-injury. Apoptotic cell death was significantly higher (P<0.05) in the core than the peri-infarct of the severe HI brain. In the peri-infarct area of severe HI, AIF-induced cell death increased over time and caspase-3 and AIF equally mediated neuronal death. Necroptosis was significantly higher (P=0.02) in the peri-infarct of the severe HI lesion compared to the moderate HI lesion. In males, but not in females, apoptosis was higher in moderate compared to severe HI. CONCLUSIONS: Caspase-independent cell death plays an important role in severe ischemic injury. Injury severity, timing of intervention post-injury and sex of the animal are important determinants in designing neuroprotective intervention for the severely ischemic immature brain.

Evidence for therapeutic intervention in the prevention of cerebral palsy: hope from animal model research.

Knowledge translation, as defined by the Canadian Institute of Health Research, is defined as the exchange, synthesis, and ethically sound application of knowledge--within a complex system of interactions among researchers and users--to accelerate the capture of the benefits of research through improved health, more effective services and products, and a strengthened healthcare system. The requirement for this to occur lies in the ability to continue to determine mechanistic actions at the molecular level, to understand how they fit at the in vitro and in vivo levels, and for disease states, to determine their safety, efficacy, and long-term potential at the preclinical animal model level. In this regard, particularly as it relates to long-term disabilities such as cerebral palsy that begin in utero, but only express their full effect in adulthood, animal models must be used to understand and rapidly evaluate mechanisms of injury and therapeutic interventions. In this review, we hope to provide the reader with a background of animal data upon which therapeutic interventions for the prevention and treatment of cerebral palsy, benefit this community, and increasingly do so in the future.

Housing environment and sex affect behavioral recovery from ischemic brain damage.

In previous work from our laboratory, we have developed a model of stroke that allows the comparison of stroke injury across age groups (10-day old, 63-day old, 180-day old rats). In this current study, we incorporated environmental enrichment to determine whether this form of rehabilitation alters behavioral recovery, and whether age and/or sex interacts with enrichment. Our results indicated that ischemic male rats that were housed in standard cages (shoebox housed) performed the poorest on two tasks of motor ability, and that this performance was related to the size of the lesion. With the exception of lesion size, in which 10-day old enriched rats had significantly smaller lesions than either the enriched 63-day old rats or the 180-day old rats, we observed no significant effects of age on behavioral recovery. Thus, our results are consistent with the observation that male rats may be more vulnerable to stroke and that they may differentially benefit from enrichment.

Treatment of the term newborn with brain injury: simplicity as the mother of invention.

Neonatal brain injury remains a common cause of developmental disability, despite tremendously enhanced obstetrical and neonatal care. The timing of brain injury occurs throughout gestation, labor, and delivery, providing an evolving form of brain injury and a moving target for therapeutic intervention. Nonetheless, markedly improved methods are available to identify those infants injured at birth, via clinical presentation with neonatal encephalopathy and neuroimaging techniques. Postischemic hypothermia has been shown to be of tremendous clinical promise in several completed and ongoing trials. As part of this approach to the treatment of the newborn, other parameters of physiologic homeostasis can and should be attended to, with strong animal and clinical evidence that their correction will have dramatic influence on the outcome of the newborn infant. This review addresses aspects of newborn care to which we can direct our attention currently, and which should result in a safe and efficacious improvement in the prognosis of the newborn with neonatal encephalopathy.

Effect of caffeine and morphine on the developing pre-mature brain.

Apnea of pre-maturity is common, occurring in 85% of infants born less than 34 week gestation. Oral caffeine is the most frequent form of therapy, often in conjunction with the use of intubation and intermittent ventilation. Morphine is used to reduce the pain believed to be associated with the latter. Little information is available on the effects of caffeine, morphine or their combination, on the developing brain. We determined the effect of caffeine and morphine alone and in combination of cell death on the developing brain of the rat. Cell death, measured by Fluoro-jade B and activated caspase-3, was significantly increased at 12 and 24 hour post-caffeine injection (P < 0.05) in the cortex, caudate, nucleus accumbens, hypothalamus, hippocampus and superior colliculus. No alterations were seen following morphine injection alone. However, in the thalamus, the combination of caffeine and morphine did increase cell death to a significantly greater extent than caffeine alone. Further research is required to determine the long-term pathologic and functional effects of caffeine and the combination of caffeine and morphine on the developing immature brain.

Enriched environment and the effect of age on ischemic brain damage.

Stroke affects all age groups from the newborn to the elderly. Previous work from our laboratory has shown that despite a greater susceptibility to brain damage, the immature brain recovers more rapidly and to a greater extent than does the more mature nervous system. In the current study, we examined the influence of environmental enrichment on the effects of age on the brain damaging effects of stroke. Rats aged 10, 63, and 180 days received ischemic insults following stereotactic intra-cerebral injection of endothelin-1, and resulting in injury to the right middle cerebral artery territory. Rats were then housed in either environmentally enriched cages, or standard cages for 60 days, after which they were sacrificed, and brain volumes determined for the extent of neurologic injury. Rats receiving the insult at 10 days of age showed a reduction of pathologic injury when housed in the enriched cages compared to standard. Conversely, rats receiving the insult at 180 days and housed environmentally enriched cages actually showed an increased volume of brain damage compared to controls. Our findings clearly indicate the dramatic influence of age on the extent of stroke and the influence of rehabilitative therapies. Behavioral correlation to morphologic alterations is required. Attempts at therapeutic interventions clearly need to be age-specific.

The influence of aging on recovery following ischemic brain damage.

Stroke is a health hazard that affects all age groups, however the impact of age on brain injury following ischemia remains largely unexamined. We examined the extent to which age, from the newborn to mature adult, affects behavioral recovery following similar degrees of ischemic brain damage. We utilized a model that produces comparable volumes of brain damage between the different ages. Endothelin-1, a potent vasoconstrictor, was injected into the brain of 10, 63 and 180-day-old rats, at the level of the right middle cerebral artery. On days 3, 7, 14, 28 and 56 post-insult, behavioral tests including rota-rod, foot-fault, open-field, inclined screen, tape-removal test and postural reflex were performed. Control animals underwent sham surgery, but ischemia was not induced. Neuropathology was assessed on day 63 post-insult. Volume of damage was determined for each brain as a percentage of the contralateral hemisphere (which remains undamaged). Our results indicated that the volume of damage for each age group was 22.97, 19.97, and 18.85% for 10, 63 and 180-day-old rats, respectively, and were not significantly different from each other. Overall, ischemic animals did significantly more poorly on behavioral testing than did controls. When broken down by age, the difference between ischemics and controls was only evident in the 63 and 180-day-old animals. The tape-removal test revealed main effects of age, group, and day (p<0.001). In addition, significant interactions were noted for day of testing by age (p<0.001), day of testing by group with ischemics performing more slowly than controls, and an age by group interaction which indicated that the 63 and 180-day-old ischemic rats did not recover completely during the testing period and remained significantly slower than their controls (p<0.001). In the foot-fault task, the 63 and 180-day-old ischemic animals performed significantly more poorly on days 3, 7, and 14 of recovery, returning to control values by day 28. The 180-day olds performed more poorly on day 3 of recovery, but then returned to control values. For open-field testing, the results indicate an overall difference between ischemics and controls, with the 63 and 180-day-old animals improving with time though they did not achieve control values. In conclusion our data suggest functional performance is poorly and inconsistently correlated with the extent of morphologic injury across all age groups. The immature rat clearly recovers more completely and more rapidly than do older, more mature rats. The findings may imply a greater degree of brain plasticity in the infant rat compared to the adult, and have important implications related to the underlying mechanisms of recovery and the association between brain damage and functional improvement.

A new model for determining the influence of age and sex on functional recovery following hypoxic-ischemic brain damage.

Stroke is a disorder affecting the lives of all age groups, and particularly those at the opposite ends of the age spectrum. It is generally believed that the immature brain is more resistant to damage resulting from a hypoxic/ischemic injury, and that it is also more 'plastic' in terms of its ability to recover. Evidence from our laboratory, and a host of others, has indicated, however, that the developing brain may in fact be more sensitive to injury resulting from hypoxia-ischemia. The question remains, however, whether the immature brain has a greater capacity for recovery. In order to determine the relative capability for functional recovery between age groups, a stroke model of comparable injury is required. This paper describes a new rodent model of ischemic injury allowing for comparisons of behavioral recovery spanning the spectrum of ages between newborn and the elderly. Endothelin-1, a potent vasoconstrictor, was stereotactically injected into the brains of 10-, 63-, and 180-day-old Wistar rats, immediately adjacent to the middle cerebral artery. Regionally, the cortex, caudate, and thalamus were most significantly affected, with sparing of the hippocampus. Pathologic assessment indicated a similar degree of injury across age groups affecting the territorial distribution of the middle cerebral artery, with a predominance of damage in the anterior sections of the cortex and caudate (p < 0.05), compared to the posterior sections including the cortex and thalamus. There were no regional differences in the extent of damage between age groups. Interestingly, however, there were significant differences between males and females regarding the overall extent of brain damage (p < 0.05), with males showing greater damage than females. In addition, there were significant regional differences in the extent of damage between males and females, particularly regarding cortical damage (p < 0.05), both anteriorly and posteriorly, and the caudate anteriorly (p < 0.05). Our findings provide an important new model for comparison of brain damage among the entire spectrum of ages affected by stroke. Importantly, this will allow for further investigations regarding both functional recovery and gender difference comparisons. This may have important ramifications for the development of therapeutic interventions that are age and gender specific.

Preventing hyperthermia decreases brain damage following neonatal hypoxic-ischemic seizures.

Neonatal seizures are the most common manifestation of underlying cerebral dysfunction. Hypoxic-ischemic encephalopathy is the cause of seizures in 40-60% of newborns. Previous work from our laboratory demonstrates that seizures associated with a hypoxic-ischemic insult results in aggravation of neuronal cell death, specifically within the hippocampus. The latter occurs in the setting of spontaneously occurring hyperthermia of 1.5 degrees C. The purpose of this study was to determine whether preventing the onset of seizure induced hyperthermia would be neuroprotective. Three groups of 10-day old rat pups received unilateral hypoxic-ischemic insults for 30 min followed by KA-induced seizures. Hyperthermia was prevented by lowering the environmental temperature ("relative hypothermia") to 29 degrees C such that the seizuring rat pups were normothermic. In one group, the prevention of hyperthermia occurred immediately following hypoxia-ischemia, whereas in the other group it occurred at the onset of seizures. The third group of rat pups (controls) remained at their nesting temperature and therefore became hyperthermic during seizures. Early (3 days) and late (20 days) neuropathology was assessed. Rat pups in whom hyperthermia was prevented during seizures displayed a significant reduction in brain damage compared to controls (p<0.05). Assessment of hippocampal brain damage also showed a significant improvement in neuronal necrosis at 20 days of recovery compared to 3 days of recovery (p<0.05). The results indicate that preventing spontaneous hyperthermia in this model of hypoxic-ischemic seizures in the newborn is neuroprotective.

Prolonged neonatal seizures exacerbate hypoxic-ischemic brain damage: correlation with cerebral energy metabolism and excitatory amino acid release.

BACKGROUND: Perinatal hypoxia-ischemia (HI) is the most common precipitant of seizures in the first 24-48 h of a newborn's life. In a previous study, our laboratory developed a model of prolonged, continuous electrographic seizures in 10-day-old rat pups using kainic acid (KA) as a proconvulsant. Groups of animals included those receiving only KA, or HI for 15 or 30 min, followed by KA infusion. Our results showed that prolonged electrographic seizures following 30 min of HI resulted in a marked exacerbation of brain damage. We have undertaken studies to determine alterations in hippocampal high-energy phosphate reserves and the extracellular release of hippocampal amino acids in an attempt to ascertain the underlying mechanisms responsible for the damage promoted by the combination of HI and KA seizures. METHODS: All studies were performed on 10-day-old rats. Five groups were identified: (1) group I--KA alone, (2) group II--15 min of HI plus KA, (3) group III--15 min of HI alone, (4) group IV--30 min of HI plus KA, and (5) group VI--30 min of HI alone. HI was induced by right common carotid artery ligation and exposure to 8% oxygen/balance nitrogen. Glycolytic intermediates and high-energy phosphates were measured. Prior to treatment, at the end of HI (both 15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7, 24 and 48 h, blood samples were taken for glucose, lactate and beta-hydroxybutyrate. At the same time points, animals were sacrificed by decapitation and brains were rapidly frozen for subsequent dissection of the hippocampus and measurement of glucose, lactate, beta-hydroxybutyrate, adenosine triphosphate (ATP) and phosphocreatine (PCr). In separate groups of rats as defined above, microdialysis probes (CMA) were stereotactically implanted into the CA2-3 region of the ipsilateral hippocampus for measurement of extracellular amino acid release. Dialysate was collected prior to any treatment, at the end of HI (15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7 and 9 h. Determination of glutamate, serine, glutamine, glycine, taurine, alanine, and GABA was accomplished using high-performance liquid chromatography with EC detection. RESULTS: Blood and hippocampal glucose concentrations in all groups receiving KA were significantly lower than control during seizures (p < 0.05). beta-Hydroxybutyrate values displayed the inverse, in that values were significantly higher (p < 0.01) in all KA groups compared with pretreatment controls during seizure activity. Values returned to control by 2 h following the cessation of seizures. Lactate concentrations in brain and blood mimicked those of beta-hydroxybutyrate. ATP values declined to 0.36 mmol/l in both the 15 and 30 min hypoxia groups compared with 1.85 mmol/l for controls (p < 0.01). During seizures, ATP and PCr values declined significantly below their homologous controls. Following seizures, ATP values only for those animals receiving KA plus HI for 30 min remained below their homologous controls for at least 24 h. Determination of amino acid release revealed elevations of glutamate, glycine, taurine, alanine and GABA above pretreatment control during HI, with a return to normal prior to KA injections. During seizures and for the 4 h of recovery monitored, only glutamate in the combined HI and KA group rose significantly above both the 15 min of HI plus KA and the KA alone group (p < 0.05). CONCLUSION: Under circumstances in which there is a protracted depletion of high-energy phosphate reserves, as occurs with a combination of HI- and KA-induced seizures, excess amounts of glutamate become toxic to the brain. The latter may account for the exacerbation of damage to the newborn hippocampus, and serve as a target for future therapeutic intervention.