The Vannucci Model of Hypoxic-Ischemic Injury in the Neonatal Rodent: 40 years Later.

Perinatal hypoxic-ischemic (HI) brain damage has long been a major cause of acute mortality and chronic neurological morbidity in infants and children. Experimental animal models are essential to gain insights into the pathogenesis and management of perinatal HI brain damage. Prior to 1980, only large animal models were available. The first small animal model was developed in the postnatal 7 (P7) rat in 1981, now known as the Vannucci model. This model combines unilateral carotid artery ligation with subsequent hypoxia to produce transient hemispheric hypoxia-ischemia in the hemisphere ipsilateral to the ligation while the contralateral hemisphere is exposed to hypoxia only. This model has been characterized with studies of cerebral hemodynamics, cerebral metabolic changes, and acute and chronic neuropathology. Over the past 40 year, this animal model has been utilized in numerous laboratories around the world, has been adapted to the immature mouse, as well as to immature rodents at various stages of development. This brief review describes the validation and characterization studies of the original model and some of the adaptations. A discussion of all of the studies focused on specific cell types is beyond the scope of this review. Rather, we present the application of the model to the study of a specific cell type, the pre-oligodendrocyte, and the role this cell plays in the development of white matter injury in the preterm brain.

Cerebral asymmetry during development using linear measures from MRI.

Asymmetry of the human brain is a well-known phenomenon, but the nature and extent of these differences throughout postnatal development have not been examined. Accordingly, linear measurements of the brains of 121 infants, children, and adolescents were determined to ascertain cerebral hemispheric asymmetries. Using multiple statistical methods, the results showed that: 1) the frontal lobe is wider on the right, while the occipital lobe is wider on the left; 2) there are no side to side differences in cerebral hemispheric length or height; and 3) there are no major sex differences. Especially notable is the lack of any correlation between side to side differences in length, width, or height and increasing age, which was also the case for cerebral hemispheric area or volume with increasing age. Regarding petalias: 1) the right frontal petalia occurs in 61%, the left occipital in 60%, and both petalias in 36% of the cohort; 2) the right frontal and left occipital petalias are of similar lengths; 3) the distances of both petalias increase with advancing age but not when scaled to either cerebral hemispheric area or volume, indicating that petalias are equally prominent early in postnatal life compared to later development; and 4) there are no major sex differences in the frequency or magnitude of either petalia. These findings provide comprehensive new information regarding age and sex related cerebral hemispheric asymmetries during development.

Brain growth in modern humans using multiple developmental databases.

OBJECTIVE: Currently, there are several published articles detailing brain growth in modern humans. The contained databases were derived using disparate methodologies. The objective of the present investigation was to determine the level of agreement among several collections of immature modern human brains. MATERIALS AND METHODS: Twenty-one developmental collections of endocranial volume, brain weight, or brain volume were selected for analysis, including one skeletal, six autopsy, and 14 computed tomography/magnetic resonance imaging samples. Step-wise comparisons were determined, using conversion factors for brain specific gravity and size of the subarachnoid space. RESULTS: Derived brain weights are comparable and increase especially during the first year of postnatal life, with a further slight increase (+8-10%) between one and five years, and little change thereafter. The expansion in brain size occurs earlier than body size. Significant sex differences are apparent at all stages of development. Combining all datasets produced a composite database consisting of 3,491 brain weight values, with ages near birth through 18 years. Individual brain weights ranged from 190 to 1,792 g, and mean brain weights ranged from 457 to 1,365 g, with an overall mean and standard deviation of 897 ± 387 g. CONCLUSIONS: The investigation compares modern human collections regarding brain size trajectories from birth through 18 years of age. The 21 datasets are then incorporated into a single composite database. All major age groups and both sexes are well represented. The composite database should prove useful to other investigators interested in developmental aspects of the modern human brain.

The effects of adding prophylactic phenobarbital to therapeutic hypothermia in the term-equivalent hypoxic-ischemic rat.

BackgroundHypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal morbidity and mortality. Therapeutic hypothermia (TH) is the only available intervention, but neuroprotection is incomplete and variable. Seizures are common in infants with HIE undergoing TH and may worsen outcome. Phenobarbital (PB) is sometimes added, although use of prophylactic PB is controversial in the neonate. We hypothesize that prophylactic PB will not reduce, and may enhance, the neuroprotective effects of TH on brain injury and motor outcomes in the postnatal day 10 (P10) hypoxic-ischemic (HI) rat.MethodsP10 rat pups were subjected to unilateral HI and 4 h recovery with: normothermia (N); hypothermia (TH); and hypothermia with phenobarbital (TH+PB). Brain damage was assessed longitudinally at 24 h and 2 weeks using brain magnetic resonance imaging and 12 weeks using histochemical analysis. Motor function was assessed with the beam walk and cylinder tests.ResultsTH and TH+PB induced neuroprotection, as measured by global brain damage score and improved motor function. Exploratory analyses suggest that TH+PB may confer enhanced protection, especially to the extent of damage.ConclusionProphylactic PB with TH is not deleterious and may provide additional long-term neuroprotection, including improvement of motor outcomes following HI in the term-equivalent, neonatal rat.

Assessing Cerebral Metabolism in the Immature Rodent: From Extracts to Real-Time Assessments.

Brain development is an energy-expensive process. Although glucose is irreplaceable, the developing brain utilizes a variety of substrates such as lactate and the ketone bodies, ß-hydroxybutyrate and acetoacetate, to produce energy and synthesize the structural components necessary for cerebral maturation. When oxygen and nutrient supplies to the brain are restricted, as in neonatal hypoxia-ischemia (HI), cerebral energy metabolism undergoes alterations in substrate use to preserve the production of adenosine triphosphate. These changes have been studied by in situ biochemical methods that yielded valuable quantitative information about high-energy and glycolytic metabolites and established a temporal profile of the cerebral metabolic response to hypoxia and HI. However, these analyses relied on terminal experiments and averaging values from several animals at each time point as well as challenging requirements for accurate tissue processing.More recent methodologies have focused on in vivo longitudinal analyses in individual animals. The emerging field of metabolomics provides a new investigative tool for studying cerebral metabolism. Magnetic resonance spectroscopy (MRS) has enabled the acquisition of a snapshot of the metabolic status of the brain as quantifiable spectra of various intracellular metabolites. Proton (1H) MRS has been used extensively as an experimental and diagnostic tool of HI in the pursuit of markers of long-term neurodevelopmental outcomes. Still, the interpretation of the metabolite spectra acquired with 1H MRS has proven challenging, due to discrepancies among studies, regarding calculations and timing of measurements. As a result, the predictive utility of such studies is not clear. 13C MRS is methodologically more challenging, but it provides a unique window on living tissue metabolism via measurements of the incorporation of 13C label from substrates into brain metabolites and the localized determination of various metabolic fluxes. The newly developed hyperpolarized 13C MRS is an exciting method for assessing cerebral metabolism in vivo, that bears the advantages of conventional 13C MRS but with a huge gain in signal intensity and much shorter acquisition times. The first part of this review article provides a brief description of the findings of biochemical and imaging methods over the years as well as a discussion of their associated strengths and pitfalls. The second part summarizes the current knowledge on cerebral metabolism during development and HI brain injury.

Development of the Corpus Callosum: An MRI Study.

The size and shape of the corpus callosum and its major components (genu, body, and splenium) were measured by magnetic resonance imaging (MRI) in 118 normocephalic individuals aged from 1 postnatal week to 18.7 years. Genu, body, splenial, and total corpus callosal areas increased by 40-100% during the first year of life (p < 0.05). The genu expanded to a greater extent than the splenium during the first 6 years, while the splenium expanded to a greater extent between 7 and 18 years. The age-related difference in the maximal expansion of these structures indicated an anterior to posterior wave of corpus callosal enlargement during maturation, probably the consequence of differential axonal myelination. No sex differences existed during these two developmental phases for the genu, splenial, or total corpus callosal areas with or without scaling to the cerebral hemispheric volume. During infancy (0-24 months), however, the mean female splenial ratio (length/height) of 0.79 was greater than the male ratio of 0.65 (p = 0.024). The cerebral hemispheric length/height ratio was also greater in females, indicating that during infancy the female brain (and its component the corpus callosal splenium) is relatively longer than the male brain. This sex difference was confined to the splenium and disappeared with increasing age.

Therapeutic hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity in survivors. Therapeutic hypothermia (TH) is the only available intervention, but the protection is incomplete. Preclinical studies of HIE/TH in the rodent have relied on the postnatal day (P) 7 rat whose brain approximates a 32-36 wk gestation infant, less relevant for these studies. We propose that HIE and TH in the term-equivalent P10 rat will be more translational. METHODS: P10-11 rat pups were subjected to unilateral hypoxia-ischemia (HI) and 4 h recovery in normothermic (N) or hypothermic (TH) conditions. Brain damage was assessed longitudinally at 24 h, 2 wk, and 12 wk. Motor function was assessed with the beam walk; recognition memory was measured by novel object recognition. RESULTS: Neuroprotection with TH was apparent at 2 and 12 wk in both moderately and severely damaged animals. TH improved motor function in moderate, but not severe, damage. Impaired object recognition occurred with severe damage with no evidence of protection of TH. CONCLUSION: This adaptation of the immature rat model of HI provides a reproducible platform to further study HIE/TH in which individual animals are followed up longitudinally to provide a useful translational preclinical model.

In vivo Monitoring of Cerebral Hemodynamics in the Immature Rat: Effects of Hypoxia-Ischemia and Hypothermia.

BACKGROUND: Neonatal hypoxic-ischemic (HI) encephalopathy occurs in 1-4 per 1,000 live term births and can cause devastating neurodevelopmental disabilities. Currently, therapeutic hypothermia (TH) is the only treatment with proven efficacy. Since TH is associated with decreased cerebral metabolism and cerebral blood flow (CBF), it is important to assess CBF at the bedside. Diffuse correlation spectroscopy (DCS) has emerged as a promising optical modality to noninvasively assess an index of CBF (CBFi) in both humans and animals. In this initial descriptive study, we employ DCS to monitor the evolution of CBFi following HI with or without TH in immature rats. We investigate potential relationships between CBF and subsequent cerebral damage. METHODS: HI was induced on postnatal day 10 or 11 rat pups by right common carotid artery ligation followed by 60-70 min hypoxia (8% oxygen). After HI, the pups recovered for 4 h under hypothermia (HI-TH group, n = 23) or normothermia (HI-N group, n = 23). Bilateral measurements of hemispheric CBFi were made with DCS in unanesthetized animals at baseline, before HI, and 0, 1, 2, 3, 4, 5, and 24 h after HI. The animals were sacrificed at either 1 or 4 weeks, and brain injury was scored on an ordinal scale of 0-5 (0 = no injury). RESULTS: Carotid ligation caused moderate bilateral decreases in CBFi. Following HI, an initial hyperemia was observed that was more prominent in the contralateral hemisphere. After initiation of TH, CBFi dropped significantly below baseline levels and remained reduced for the duration of TH. In contrast, CBFi in the HI-N group was not significantly decreased from baseline levels. Reductions in CBFi after 4 h of TH were not associated with reduced damage at 1 or 4 weeks. However, elevated ipsilateral CBFi and ipsilateral-to-contralateral CBFi ratios at 24 h were associated with worse outcome at 1 week after HI. CONCLUSIONS: Both HI and TH alter CBFi, with significant differences in CBFi between hypothermic and normothermic groups after HI. CBFi may be a useful biomarker of subsequent cerebral damage.

The role of inflammation in perinatal brain injury.

Inflammation is increasingly recognized as being a critical contributor to both normal development and injury outcome in the immature brain. The focus of this Review is to highlight important differences in innate and adaptive immunity in immature versus adult brain, which support the notion that the consequences of inflammation will be entirely different depending on context and stage of CNS development. Perinatal brain injury can result from neonatal encephalopathy and perinatal arterial ischaemic stroke, usually at term, but also in preterm infants. Inflammation occurs before, during and after brain injury at term, and modulates vulnerability to and development of brain injury. Preterm birth, on the other hand, is often a result of exposure to inflammation at a very early developmental phase, which affects the brain not only during fetal life, but also over a protracted period of postnatal life in a neonatal intensive care setting, influencing critical phases of myelination and cortical plasticity. Neuroinflammation during the perinatal period can increase the risk of neurological and neuropsychiatric disease throughout childhood and adulthood, and is, therefore, of concern to the broader group of physicians who care for these individuals.

Neonatal encephalopathy: pre-clinical studies in neuroprotection.

Neonatal encephalopathy resulting from HI (hypoxia-ischaemia) continues to be a significant cause of mortality and morbidity in infants and children, affecting 1-2/1000 live term births and up to 60% of pre-term births. In order to understand the pathophysiology of this insult, as well as design therapeutic interventions, it is important to establish a relevant animal model for pre-clinical studies. One of the most frequently used models of HI-induced brain damage in immature animals is the unilateral carotid ligation/hypoxia model, initially developed in our laboratory more than 30 years ago. The original model employed the postnatal day 7 rat, whose brain is representative of that of a late gestation, pre-term [32-36 weeks GA (gestational age)] human infant. We, and others, have employed this model to characterize the pathophysiological, biochemical/energetic and neuropathological events following HI, as well as the determination of the unique characteristics of the immature brain that define its vulnerability to, and outcome from, HI. In defining the cascade of events following HI, it has become possible to identify potential targets for intervention and neuroprotection. Currently, the only available therapeutic intervention for neonatal encephalopathy in the term asphyxiated infant is therapeutic hypothermia, although this must be initiated within 6 h of birth and is at best partially effective in moderately injured infants. Ongoing pre-clinical studies are necessary to determine the basis for the partial protection afforded by hypothermia as well as the design of adjunct therapies to improve the outcome. The present review highlights the importance of using a well-characterized and relevant animal model to continue to pursue translational research in neuroprotection for the infant brain.

Mast cell isolation from the immature rat brain.

Mast cells are immune cells of hematopoietic origin that circulate as precursor cells prior to migration into vascularized tissues where they mature and undergo terminal differentiation in response to different cytokines within the local environment. Mast cells are well known as important regulators of inflammatory processes in peripheral tissues and recent studies support the involvement of mast cells in mediating the inflammatory response to cerebral hypoxia-ischemia in both the neonatal and adult brain. To better study mast cell function in vivo, it is important to be able to identify their environment-specific phenotype, as well as to study their interaction with other neural cells in vitro. Previous such studies of mast cells have relied on mast cells isolated from gut or bone marrow, or on a number of mast cell lines, all of which may behave differently from brain mast cells. The purpose of this study was to develop a technique for the isolation of mast cells from neonatal rat brain and to characterize these cells following hypoxia and hypoxia-ischemia. We adapted a previously described technique of coupling an antibody to the mast cell-specific FcεR1 receptor to a MACS microbead for the selective removal of intact mast cells from a neonatal brain preparation. We have isolated toluidine blue-positive brain mast cells that provide substrate for both protein analysis and in vitro studies. These cells express proteins previously used to specifically identify microglia in the brain, Iba-1 and coronin-1a. A subpopulation of mast cells in vivo also expresses Iba-1. Thus, we report a novel method for isolation of brain mast cells suitable for the study of mast cell phenotype under a variety of conditions. Further, we suggest that the use of proteins such as Iba-1 for the identification of microglia in the brain includes the caveat that mast cells may also be detected.

N-3 fatty acid rich triglyceride emulsions are neuroprotective after cerebral hypoxic-ischemic injury in neonatal mice.

We questioned if acute administration of n-3 fatty acids (FA) carried in n-3 rich triglyceride (TG) emulsions provides neuroprotection in neonatal mice subjected to hypoxic-ischemic (H/I) brain injury. We examined specificity of FA, optimal doses, and therapeutic windows for neuroprotection after H/I. H/I insult was induced in C57BL/6J 10-day-old mice by right carotid artery ligation followed by exposure to 8% O(2) for 15 minutes at 37°C. Intraperitoneal injection with n-3-rich TG emulsions, n-6 rich TG emulsions or saline for control was administered at different time points before and/or after H/I. In separate experiments, dose responses were determined with TG containing only docosahexaenoic acid (Tri-DHA) or eicosapentaenoic acid (Tri-EPA) with a range of 0.1-0.375 g n-3 TG/kg, administered immediately after H/I insult. Infarct volume and cerebral blood flow (CBF) were measured. Treatment with n-3 TG emulsions both before- and after- H/I significantly reduced total infarct volume by a mean of 43% when administered 90 min prior to H/I and by 47% when administered immediately after H/I. In post-H/I experiments Tri-DHA, but not Tri-EPA exhibited neuroprotective effects with both low and high doses (p<0.05). Moreover, delayed post-H/I treatment with Tri-DHA significantly decreased total infarct volume by a mean of 51% when administered at 0 hr, by 46% at 1 hr, and by 51% at 2 hr after H/I insult. No protective effect occurred with Tri-DHA injection at 4 hr after H/I. There were no n-3 TG related differences in CBF. A significant reduction in brain tissue death was maintained after Tri-DHA injection at 8 wk after the initial brain injury. Thus, n-3 TG, specifically containing DHA, is protective against H/I induced brain infarction when administered up to 2 hr after H/I injury. Acute administration of TG-rich DHA may prove effective for treatment of stroke in humans.

Craniometric measures of microcephaly using MRI.

BACKGROUND: Microcephalic brains exhibit varying sizes, shapes, and dimensions when compared to normocephalic counterparts, but the extent of these differences is unresolved. AIMS: To ascertain developmental changes in brain morphology using craniometric (linear brain) measures derived from MRI in microcephalic individuals and in normocephalic controls. STUDY DESIGN: A retrospective, cross-sectional cohort study. SUBJECTS: Twenty-one primary and secondary microcephalic individuals ages 2 postnatal weeks to 8.5 years with occipito-frontal circumference<2nd percentile for age; 83 age-equivalent normocephalic controls. OUTCOME MEASURES: Age, sex, weight, height, body mass index, occipito-frontal circumference, and diagnosis prompting the MRI scan. Sixteen craniometric measures to determine specific ratios and age-related changes in brain shape and size. RESULTS: Microcephalic infants and children not only have abnormally small brains but also proportionately lower weights and heights. The brain volumes of both primary and secondary microcephalics were quite variable, ranging from 266 to 723 cm(3) and 440 to 730 cm(3), respectively (p=0.34). Despite their smaller sizes, the shapes of 15/21 (71%) microcephalic brains were similar to those of age-equivalent controls. Cerebral hemispheric configurations were not consistent among the 6 misshapen brains, which included 2 primary, 3 secondary, and 1 unknown microcephalics. Older microcephalic brains could be distinguished from their normocephalic counterparts by two specific craniometric ratios (frontal cerebellar pole/sagittal cerebral length; axial temporal width/axial cerebellar width), each incorporating cerebral and cerebellar dimensions in either length or width. CONCLUSIONS: The findings should provide useful information for distinguishing the characteristics of both modern and ancient microcephalic from normocephalic brains.

Pre-conditioning induces the precocious differentiation of neonatal astrocytes to enhance their neuroprotective properties.

Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

Temporal changes in mRNA expression of the brain nutrient transporters in the lithium-pilocarpine model of epilepsy in the immature and adult rat.

The lithium-pilocarpine model mimics most features of human temporal lobe epilepsy. Following our prior studies of cerebral metabolic changes, here we explored the expression of transporters for glucose (GLUT1 and GLUT3) and monocarboxylates (MCT1 and MCT2) during and after status epilepticus (SE) induced by lithium-pilocarpine in PN10, PN21, and adult rats. In situ hybridization was used to study the expression of transporter mRNAs during the acute phase (1, 4, 12 and 24h of SE), the latent phase, and the early and late chronic phases. During SE, GLUT1 expression was increased throughout the brain between 1 and 12h of SE, more strongly in adult rats; GLUT3 increased only transiently, at 1 and 4h of SE and mainly in PN10 rats; MCT1 was increased at all ages but 5-10-fold more in adult than in immature rats; MCT2 expression increased mainly in adult rats. At all ages, MCT1 and MCT2 up-regulation was limited to the circuit of seizures while GLUT1 and GLUT3 changes were more widespread. During the latent and chronic phases, the expression of nutrient transporters was normal in PN10 rats. In PN21 rats, GLUT1 was up-regulated in all brain regions. In contrast, in adult rats GLUT1 expression was down-regulated in the piriform cortex, hilus and CA1 as a result of extensive neuronal death. The changes in nutrient transporter expression reported here further support previous findings in other experimental models demonstrating rapid transcriptional responses to marked changes in cerebral energetic/glucose demand.

Craniometric measures during development using MRI.

Developmental changes in brain volume and shape in infants, children, and adolescents were ascertained with MRI, using craniometric (linear brain) measures in 118 individuals, ages 1 postnatal week to 18.7years. Collected clinical data included age, sex, weight, height, body mass index, occipito-frontal circumference (OFC), and diagnosis prompting the MRI scan. Twenty craniometric measures were obtained to allow for the determination of specific ratios as well as sex and age-related changes in brain shape and size. Analysis of the cohort showed that OFC is larger today than 40years ago, likely related to a concomitant increase in body stature. The data indicated a wide variation in the maturational pattern of several specific craniometric ratios, which reflects changes in the volume and configuration of the brain with advancing age. The increases in brain volume and changes in brain shape were most dramatic during infancy, with continued minor escalations in volume and reshaping during childhood and adolescence. Sex differences existed both in brain volume and shape, as well as evidence of sexual dimorphism. Changes in cerebellar volume and shape lagged behind the corresponding changes in the cerebral hemispheres. These collective data in living developing individuals allow for comparisons of clinical or craniometric measures in distant and more recent humans.

Response to 'comment on recent modeling studies of astrocyte-neuron metabolic interactions': much ado about nothing.

For many years, a tenet of cerebral metabolism held that glucose was the obligate energy substrate of the mammalian brain and that neuronal oxidative metabolism represented the majority of this glucose utilization. In 1994, Pellerin and Magistretti formulated the astrocyte-neuron lactate shuttle (ANLS) hypothesis, in which astrocytes, not neurons, metabolized glucose, with subsequent transport of the glycolytically derived lactate to fuel the energy needs of the neuron during neurotransmission. By considering the concentrations and kinetic characteristics of the nutrient transporter proteins, Simpson et al later supported the opposite view, in which lactate flows from neurons to astrocytes, thus leading to the neuron-astrocyte lactate shuttle (NALS). Most recently, a commentary was published in this journal attempting to discredit the NALS. This challenge has stimulated the present response in which we detail the inaccuracies of the commentary and further model several different possibilities. Although our simulations continue to support the predominance of neuronal glucose utilization during activation and neuronal to astrocytic lactate flow, the most important result is that, regardless of the direction of the flow, the overall contribution of lactate to cerebral glucose metabolism is found to be so small as to make this ongoing debate 'much ado about nothing'.

Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia-ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia-ischemia. Only C1q(-/-) mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q(-/-) brain mitochondria and preserved activity of the respiratory chain. Compared with C1q(+/+) neurons, cortical C1q(-/-) neurons exhibited resistance to oxygen-glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q(-/-) neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation.

The PPAR-gamma agonist, darglitazone, restores acute inflammatory responses to cerebral hypoxia-ischemia in the diabetic ob/ob mouse.

Diabetes is an increased risk factor for stroke and results in increased brain damage in experimental animals and humans. The precise mechanisms are unclear, but our earlier studies in the db/db mice suggested that the cerebral inflammatory response initiating recovery was both delayed and diminished in the diabetic mice compared with the nondiabetic db/+ mice. In this study, we investigated the actions of the peroxisome proliferator-activated receptor (PPAR)-gamma agonist darglitazone in treating diabetes and promoting recovery after a hypoxic-ischemic (H/I) insult in the diabetic ob/ob mouse. Male ob/+ and ob/ob mice received darglitazone (1 mg/kg) for 7 days before induction of H/I. Darglitazone restored euglycemia and normalized elevated corticosterone, triglycerides, and very-low-density lipoprotein levels. Darglitazone dramatically reduced the infarct size in the ob/ob mice at 24 h of recovery compared with the untreated group (30+/-13% to 3.3+/-1.6%, n=6 to 8) but did not show any significant effect in the ob/+ mice. Microglial and astrocytic activation monitored by cytokine expression (interleukin-1beta and tumor necrosis factor-alpha) and in situ hybridization studies (bfl1 and glial fibrillary acidic protein) suggest a biphasic inflammatory response, with darglitazone restoring the compromised proinflammatory response(s) in the diabetic mouse at 4 h but suppressing subsequent inflammatory responses at 8 and 24 h in both control and diabetic mice.

Moderate recurrent hypoglycemia during early development leads to persistent changes in affective behavior in the rat.

Recurrent hypoglycemia is a common problem among infants and children that is associated with several metabolic disorders and insulin-dependent diabetes mellitus. Although studies have reported a relationship between a history of juvenile hypoglycemia and psychological health problems, the direct effects of recurrent moderate hypoglycemia have not been fully determined. Thus, in this study, we used an animal model to examine the effects of recurrent hypoglycemia during the juvenile period on affective, social, and motor function (assessed under euglycemic conditions) across development. To model recurrent hypoglycemia, rats were administered 5 U/kg of insulin or saline twice per day from postnatal day (P)10 to P19. Body weight gain was retarded in insulin-treated rats during the treatment period, but recovered by the end of treatment. However, insulin-treated rats displayed increases in affective reactivity that emerged early during treatment and persisted after treatment into early adulthood. Specifically, insulin-treated pups showed increased maternal separation-induced vocalizations as infants, and an exaggerated acoustic startle reflex as juveniles and young adults. Moreover, young adult rats with a history of recurrent juvenile hypoglycemia exhibited increased fear-potentiated startle and increases in behavioral and hormonal responses to restraint stress. Some of these effects were sex-dependent. The changes in affective behavior in insulin-exposed pups were accompanied by decreases in adolescent social play behavior. These results provide evidence that recurrent, transient hypoglycemia during juvenile development can lead to increases in fear-related behavior and stress reactivity. Importantly, these phenotypes are not reversed with normalization of blood glucose and may persist into adulthood.