Inflammatory profile in a canine model of hypothermic circulatory arrest.

BACKGROUND: Hypothermic circulatory arrest (HCA) is a technique used for complex repair of the aorta, but it can be associated with neurologic morbidity. To better understand the molecular changes that underlie ischemic brain injury, we assessed gene expression and cytokine/chemokine polypeptide concentration in brain tissue and cerebrospinal fluid (CSF) of canines that underwent two hours of HCA. MATERIALS AND METHODS: Adult male canines were cannulated peripherally for cardiopulmonary bypass, cooled to 18°C, and arrested for two hours. Animals were euthanized two, eight, or 24 hours post-HCA (n = 8 per group), and their brains were compared to brains from eight normal canines, using gene expression microarray analysis, cytokine assay, and histopathology. RESULTS: Two to eight hours after HCA, pro-inflammatory cytokine mRNAs increased markedly, and gene expression was enriched within signaling pathways related to neuroinflammation or ischemic injury. Concentrations of pro-inflammatory cytokine polypeptides IL-6, IL-8, IL-1ß, and CCL2 were very low in normal canine brain, whereas anti-inflammatory IL-10 and TGF-ß1 were expressed at moderate levels. Pro-inflammatory cytokine concentrations rose robustly in cerebral tissue and CSF after HCA. IL-6 and IL-8 peaked at eight hours and declined at 24 hours, while IL-1ß and CCL2 remained elevated. Concentrations of anti-inflammatory IL-10 and TGF-ß1 were maintained after HCA, with a significant increase in TGF-ß1 at 24 hours. CONCLUSIONS: These cytokines represent potential diagnostic markers for ischemic neurologic injury that could be used to assess neurologic injury in patients undergoing HCA. The cellular mechanisms underlying this pro-inflammatory, ischemic-induced injury represent potential targets for neuroprotection in the future.

NMDA Receptor Antagonism for Neuroprotection in a Canine Model of Hypothermic Circulatory Arrest.

BACKGROUND: Hypothermic circulatory arrest (HCA) is associated with neurologic morbidity, in part mediated by activation of the N-methyl-D-aspartate glutamate receptor causing excitotoxicity and neuronal apoptosis. Using a canine model, we hypothesized that the N-methyl-D-aspartate receptor antagonist MK801 would provide neuroprotection and that MK801 conjugation to dendrimer nanoparticles would improve efficacy. MATERIALS AND METHODS: Male hound dogs were placed on cardiopulmonary bypass, cooled to 18°C, and underwent 90 min of HCA. Dendrimer conjugates (d-MK801) were prepared by covalently linking dendrimer surface OH groups to MK801. Six experimental groups received either saline (control), medium- (0.15 mg/kg) or high-dose (1.56 mg/kg) MK801, or low- (0.05 mg/kg), medium-, or high-dose d-MK801. At 24, 48, and 72 h after HCA, animals were scored by a standardized neurobehavioral paradigm (higher scores indicate increasing deficits). Cerebrospinal fluid was obtained at baseline, eight, 24, 48, and 72 h after HCA. At 72 h, brains were examined for histopathologic injury in a blinded manner (higher scores indicate more injury). RESULTS: Neurobehavioral deficit scores were reduced by low-dose d-MK801 on postoperative day two (P < 0.05) and by medium-dose d-MK801 on postoperative day 3 (P = 0.05) compared with saline controls, but free drug had no effect. In contrast, high-dose free MK801 significantly improved histopathology scores compared with saline (P < 0.05) and altered biomarkers of injury in cerebrospinal fluid, with a significant reduction in phosphorylated neurofilament-H for high-dose MK801 versus saline (P < 0.05). CONCLUSIONS: Treatment with MK-801 demonstrated significant improvement in neurobehavioral and histopathology scores after HCA, although not consistently across doses and conjugates.

Uptake of dendrimer-drug by different cell types in the hippocampus after hypoxic-ischemic insult in neonatal mice: Effects of injury, microglial activation and hypothermia.

Perinatal hypoxic-ischemic encephalopathy (HIE) can result in neurodevelopmental disability, including cerebral palsy. The only treatment, hypothermia, provides incomplete neuroprotection. Hydroxyl polyamidoamine (PAMAM) dendrimers are being explored for targeted delivery of therapy for HIE. Understanding the biodistribution of dendrimer-conjugated drugs into microglia, neurons and astrocytes after brain injury is essential for optimizing drug delivery. We conjugated N-acetyl-L-cysteine to Cy5-labeled PAMAM dendrimer (Cy5-D-NAC) and used a mouse model of perinatal HIE to study effects of timing of administration, hypothermia, brain injury, and microglial activation on uptake. Dendrimer conjugation delivered therapy most effectively to activated microglia but also targeted some astrocytes and injured neurons. Cy5-D-NAC uptake was correlated with brain injury in all cell types and with activated morphology in microglia. Uptake was not inhibited by hypothermia, except in CD68+ microglia. Thus, dendrimer-conjugated drug delivery can target microglia, astrocytes and neurons and can be used in combination with hypothermia for treatment of HIE.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

OBJECTIVE: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. METHODS: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation. RESULTS: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals. INTERPRETATION: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.

Ischemia-induced neuroinflammation is associated with disrupted development of oligodendrocyte progenitors in a model of periventricular leukomalacia.

Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time window can produce long-lasting defects in oligodendroglial maturation, myelination deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long-lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy. Here, we report that permanent unilateral carotid ligation on postnatal day 5 in CD-1 mice induces an inflammatory response, as defined by microglial activation and recruitment, as well as significant changes in cytokine expression (increased IL-1ß, IL-6, TGF-ß1, and TNF-α) following ischemia. Transient reduction in counts of oligodendrocyte progenitor cells (OPCs) at 24 and 48 h after ischemia, a shift in OPC cell size and morphology towards the more immature form, as well as likely migration of OPCs were found. These OPC changes were topographically associated with areas showing microglial activation, and OPC counts negatively correlated with increased microglial staining. The presented data show a striking neuroinflammatory response in an ischemia-induced model of PVL, associated with oligodendroglial injury. Future studies modulating the neuroinflammatory response in this model may contribute to a better understanding of the interaction between microglia and OPCs in PVL and open opportunities for future therapies.

Ketamine Mitigates Neurobehavioral Deficits in a Canine Model of Hypothermic Circulatory Arrest.

Hypothermic circulatory arrest is a protective technique used when complete cessation of circulation is required during cardiac surgery. Prior efforts to decrease neurologic injury with the NMDA receptor antagonist MK801 were limited by unacceptable side effects. We hypothesized that ketamine would provide neuroprotection without dose-limiting side effects. Canines were peripherally cannulated for cardiopulmonary bypass, cooled to 18°C, and underwent 90 minutes of circulatory arrest. Ketamine-treated canines (n = 5; total dose 2.85 mg/kg) were compared to untreated controls (n = 10). A validated neurobehavioral deficit score was obtained at 24, 48, and 72 hours (0 = no deficits/normal exam; higher score represents increasing deficits). Biomarkers of neuronal injury in the cerebrospinal fluid were examined at baseline and at 8, 24, 48, and 72 hours. Brain histopathologic injury was scored at 72 hours (higher score indicates more necrosis and apoptosis). Ketamine-treated canines had significantly improved, lower neurobehavioral deficit scores compared to controls (overall P = 0.003; 24 hours: median 72 vs 112, P = 0.030; 48 hours: 47 vs 90, P = 0.021; 72 hours: 30 vs 89, P = 0.069). Although the histopathologic injury scores of ketamine-treated canines (median 12) were lower than controls (16), there was no statistical difference (P = 0.10). Levels of phosphorylated neurofilament-H and neuron specific enolase, markers of neuronal injury, were significantly lower in ketamine-treated animals (P = 0.010 and = 0.039, respectively). Ketamine significantly reduced neurologic deficits and biomarkers of injury in canines after hypothermic circulatory arrest. Ketamine represents a safe and approved medication that may be useful as a pharmacologic neuroprotectant during cardiac surgery with circulatory arrest.

Mutant IDH1 promotes phagocytic function of microglia/macrophages in gliomas by downregulating ICAM1.

Tumor-associated microglia/macrophages (TAMs) are the main innate immune effector cells in malignant gliomas and have both pro- and anti-tumor functions. The plasticity of TAMs is partially dictated by oncogenic mutations in tumor cells. Heterozygous IDH1 mutation is a cancer driver gene prevalent in grade II/III gliomas, and IDH1 mutant gliomas have relatively favorable clinical outcomes. It is largely unknown how IDH mutation alters TAM phenotypes to influence glioma growth. Here we established clinically relevant isogenic glioma models carrying monoallelic IDH1 R132H mutation (IDH1R132H/WT) and found that IDH1R132H/WT significantly downregulated immune response-related pathways in glioma cells, indicating an immunomodulation role of mutant IDH1. Co-culturing IDH1R132H/WT glioma cells with human macrophages promoted anti-tumor phenotypes of macrophages and increased macrophage migration and phagocytic capacity. In orthotopic xenografts, IDH1R132H/WT decreased tumor growth and prolonged animal survival, accompanied by increased TAM recruitment and upregulated phagocytosis markers, suggesting the induction of anti-tumor TAM functions. Using human cytokine arrays that query 36 proteins, we identified significant downregulation of ICAM-1/CD54 in IDH1R132H/WT gliomas, which was further confirmed by ELISA and immunoblotting analyses. ICAM1 gain-of-function studies revealed that ICAM1 downregulation in IDH1R132H/WT cells played a mechanistic role to mediate the immunomodulation function of IDH1R132H/WT. ICAM-1 silencing in IDH1 wild-type glioma cells decreased tumor growth and increased the anti-tumor function of TAMs. Together, our studies support a new TAM-mediated phagocytic function within IDH1 mutant gliomas, and improved understanding of this process may uncover novel approaches to targeting IDH1 wild type gliomas.

Extracellular Matrix Protein Tenascin C Increases Phagocytosis Mediated by CD47 Loss of Function in Glioblastoma.

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47-SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47-/-) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47-/- tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway-mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47-/- xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47-/- tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells. SIGNIFICANCE: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system-based therapies for brain tumors.

Early Detection of Hypothermic Neuroprotection Using T2-Weighted Magnetic Resonance Imaging in a Mouse Model of Hypoxic Ischemic Encephalopathy.

Perinatal hypoxic-ischemic encephalopathy (HIE) can lead to neurodevelopmental disorders, including cerebral palsy. Standard care for neonatal HIE includes therapeutic hypothermia, which provides partial neuroprotection; magnetic resonance imaging (MRI) is often used to assess injury and predict outcome after HIE. Immature rodent models of HIE are used to evaluate mechanisms of injury and to examine the efficacy and mechanisms of neuroprotective interventions such as hypothermia. In this study, we first confirmed that, in the CD1 mouse model of perinatal HIE used for our research, MRI obtained 3 h after hypoxic ischemia (HI) could reliably assess initial brain injury and predict histopathological outcome. Mice were subjected to HI (unilateral carotid ligation followed by exposure to hypoxia) on postnatal day 7 and were imaged with T2-weighted MRI and diffusion-weighted MRI (DWI), 3 h after HI. Clearly defined regions of increased signal were comparable in T2 MRI and DWI, and we found a strong correlation between T2 MRI injury scores 3 h after HI and histopathological brain injury 7 days after HI, validating this method for evaluating initial injury in this model of HIE. The more efficient, higher resolution T2 MRI was used to score initial brain injury in subsequent studies. In mice treated with hypothermia, we found a significant reduction in T2 MRI injury scores 3 h after HI, compared to normothermic littermates. Early hypothermic neuroprotection was maintained 7 days after HI, in both T2 MRI injury scores and histopathology. In the normothermic group, T2 MRI injury scores 3 h after HI were comparable to those obtained 7 days after HI. However, in the hypothermic group, brain injury was significantly less 7 days after HI than at 3 h. Thus, early neuroprotective effects of hypothermia were enhanced by 7 days, which may reflect the additional 3 h of hypothermia after imaging or effects on later mechanisms of injury, such as delayed cell death and inflammation. Our results demonstrate that hypothermia has early neuroprotective effects in this model. These findings suggest that hypothermia has an impact on early mechanisms of excitotoxic injury and support initiation of hypothermic intervention as soon as possible after diagnosis of HIE.

Vascular response to hypoxic preconditioning in the immature brain.

We hypothesized that hypoxic preconditioning (PC) modifies the microvasculature in the immature brain and thereby affects the cerebral blood flow (CBF) during a subsequent hypoxic-ischemic (HI) insult. On postnatal day 6 rats were exposed to hypoxia (36 degrees C, 8.0% O2) or normoxia for 3 h. Unilateral HI (unilateral carotid ligation and 8% hypoxia) was induced 24 h later. Cortical CBF was measured with the 14C-iodoantipyrine technique (at the end of HI) or with laser Doppler flowmetry (Perimed PF5001) before and during HI. At 0, 2, 8, and 24 h cerebral cortex was sampled and analyzed with gene arrays (Affymetrix 230 2.0). L-nitroarginine or vehicle was administrated before hypoxic PC or 30 mins before HI followed by CBF measurement (laser Doppler) during subsequent HI. Twenty-four hours after PC animals were perfusion-fixed and brains immunolabeled for von Willebrand factor and vascular density was determined by stereological quantification. The decrease in CBF during HI was attenuated significantly in PC versus control animals (P<0.01), as detected by both techniques. Several vascular genes (Angpt2, Adm, Apln, Vegf, Flt1, Kdr, Pdgfra, Agtrap, Adora2a, Ednra, serpine1, caveolin, Id1, Prrx1, Ero1l, Acvrl1, Egfl7, Nudt6, Angptl4, Anxa2, and NOS3) were upregulated and a few (Csrp2, Adora2b) were downregulated after PC. A significant increase in vascular density (P<0.05) was seen after PC. Nitric oxide synthase inhibition did not affect CBF during HI after PC. In conclusion, hypoxic PC upregulates vascular genes, increases vascular density and attenuates the decrease of CBF during a subsequent HI, which could contribute to tolerance.

Generation-6 hydroxyl PAMAM dendrimers improve CNS penetration from intravenous administration in a large animal brain injury model.

Hypothermic circulatory arrest (HCA) provides neuroprotection during cardiac surgery but entails an ischemic period that can lead to excitotoxicity, neuroinflammation, and subsequent neurologic injury. Hydroxyl polyamidoamine (PAMAM) dendrimers target activated microglia and damaged neurons in the injured brain, and deliver therapeutics in small and large animal models. We investigated the effect of dendrimer size on brain uptake and explored the pharmacokinetics in a clinically-relevant canine model of HCA-induced brain injury. Generation 6 (G6, ~6.7nm) dendrimers showed extended blood circulation times and increased accumulation in the injured brain compared to generation 4 dendrimers (G4, ~4.3nm), which were undetectable in the brain by 48h after final administration. High levels of G6 dendrimers were found in cerebrospinal fluid (CSF) of injured animals with a CSF/serum ratio of ~20% at peak, a ratio higher than that of many neurologic pharmacotherapies already in clinical use. Brain penetration (measured by drug CSF/serum level) of G6 dendrimers correlated with the severity of neuroinflammation observed. G6 dendrimers also showed decreased renal clearance rate, slightly increased liver and spleen uptake compared to G4 dendrimers. These results, in a large animal model, may offer insights into the potential clinical translation of dendrimers.

Dextromethorphan protects male but not female mice with brain ischemia.

The non-competitive N-methyl-D-aspartate receptor antagonist dextromethorphan is protective against some types of brain injury. Unilateral carotid ligation in postnatal day 12 CD1 mice produces ischemic brain injury. To evaluate the neuroprotective potential of dextromethorphan against ischemic injury in the immature brain, seven litters of postnatal day 12 CD1 mice received either dextromethorphan or vehicle after a unilateral carotid ligation. Only the male pups were protected, and brain injury was unchanged in the female pups treated with dextromethorphan. These results suggest that dextromethorphan neuroprotection against ischemic injury in the immature brain is sex-dependent.

Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration.

Immature mouse unilateral carotid ligation model of stroke.

Cerebral palsy in humans results from a diverse group of disorders that produce nonprogressive motor impairments in the developing brain. Stroke is an important cause of hemiparetic cerebral palsy in neonates and young children. We recently developed a new immature mouse model of stroke that demonstrates seizures, the severity of which correlates with brain injury. This model has strengths compared with other immature rodent models of ischemic injury, such as relative technical ease and the presence of seizures, as is seen in humans. This model also has relative weaknesses, such as the inability to titrate the severity of the injury with different periods of hypoxia. In addition, more work is needed to delineate the long-term consequences of the insult in this new model.

Protective effect of erythropoietin in neonatal hypoxic ischemia in mice.

The effect of systemic erythropoietin pretreatment on hypoxic ischemic injury was examined in neonatal mice. Injury was significantly less in cortex, hippocampus, striatum and thalamus of erythropoietin-treated animals (5 U/g vs vehicle) 24 h after hypoxic ischemia and in all of these regions except hippocampus at 7 days. Activated caspase-3- and activated NFkappaB-immunoreactive neurons were observed in the injured areas; these areas were smaller in the erythropoietin group. To our knowledge, this is the first report demonstrating persistent neuroprotective effects of erythropoietin in neonatal mice.

A new model of stroke and ischemic seizures in the immature mouse.

Ischemic brain injury from stroke is an important cause of disability in infants and children, but current experimental models for the disorder are complex. These preparations require occlusion of small intracerebral vessels or common carotid artery ligation combined with exposure to reduced levels of oxygen. Unilateral carotid artery ligation alone was sufficient to cause brain injury in more than 70% of 12-day-old CD1 mice. Using a blinded behavioral rating scale of seizure activity in mice, a direct, highly significant correlation between the severity of seizures over the 4-hour period after ligation and the severity of histologic brain injury 7 days later (Spearman's rho = 0.835, P < 0.001) was documented. This study presents the first model of stroke in immature mice produced by unilateral carotid artery ligation alone, and the first to demonstrate a clear correlation between acute ischemia-induced seizures and brain injury. This new model should be useful for examining the pathogenesis of stroke in the immature brain and the potential contribution of seizures to final outcome.

Brain injury in canine models of cardiac surgery.

Neuropathology and neurologic impairment were characterized in a clinically relevant canine model of hypothermic (18°C) circulatory arrest (HCA) and cardiopulmonary bypass (CPB). Adult dogs underwent 2 hours of HCA (n = 39), 1 hour of HCA (n = 20), or standard CPB (n = 22) and survived 2, 8, 24, or 72 hours. Neurologic impairment and neuropathology were much more severe after 2-hour HCA than after 1-hour HCA or CPB; histopathology and neurologic deficit scores were significantly correlated. Apoptosis developed as early as 2 hours after injury and was most severe in the granule cells of the hippocampal dentate gyrus. Necrosis evolved more slowly and was most severe in amygdala and pyramidal neurons in the cornu ammonis hippocampus. Neuronal injury was minimal up to 24 hours after 1-hour HCA, but 1 dog that survived to 72 hours showed substantial necrosis in the hippocampus, suggesting that, with longer survival time, the injury was worse. Although neuronal injury was minimal after CPB, we observed rare apoptotic and necrotic neurons in hippocampi and caudate nuclei. These results have important implications for CPB in humans and may help explain the subtle cognitive changes experienced by patients after CPB.

Potential for treatment of severe autism in tuberous sclerosis complex.

The Food and Drug Administration (FDA) has approved two mechanism-based treatments for tuberous sclerosis complex (TSC)-everolimus and vigabatrin. However, these treatments have not been systematically studied in individuals with TSC and severe autism. The aim of this review is to identify the clinical features of severe autism in TSC, applicable preclinical models, and potential barriers that may warrant strategic planning in the design phase of clinical trial development. A comprehensive search strategy was formed and searched across PubMed, Embase and SCOPUS from their inception to 2/21/12, 3/16/12, and 3/12/12 respectively. After the final search date, relevant, updated articles were selected from PubMed abstracts generated electronically and emailed daily from PubMed. The references of selected articles were searched, and relevant articles were selected. A search of clinicaltrials.gov was completed using the search term "TSC" and "tuberous sclerosis complex". Autism has been reported in as many as 60% of individuals with TSC; however, review of the literature revealed few data to support clear classification of the severity of autism in TSC. Variability was identified in the diagnostic approach, assessment of cognition, and functional outcome among the reviewed studies and case reports. Objective outcome measures were not used in many early studies; however, diffusion tensor imaging of white matter, neurophysiologic variability in infantile spasms, and cortical tuber subcategories were examined in recent studies and may be useful for objective classification of TSC in future studies. Mechanism-based treatments for TSC are currently available. However, this literature review revealed two potential barriers to successful design and implementation of clinical trials in individuals with severe autism-an unclear definition of the population and lack of validated outcome measures. Recent studies of objective outcome measures in TSC and further study of applicable preclinical models present an opportunity to overcome these barriers.

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation.

Neuronal death pathways following hypoxia-ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.

Treatment advances in neonatal neuroprotection and neurointensive care.

Knowledge of the nature, prognosis, and ways to treat brain lesions in neonatal infants has increased remarkably. Neonatal hypoxic-ischaemic encephalopathy (HIE) in term infants, mirrors a progressive cascade of excito-oxidative events that unfold in the brain after an asphyxial insult. In the laboratory, this cascade can be blocked to protect brain tissue through the process of neuroprotection. However, proof of a clinical effect was lacking until the publication of three positive randomised controlled trials of moderate hypothermia for term infants with HIE. These results have greatly improved treatment prospects for babies with asphyxia and altered understanding of the theory of neuroprotection. The studies show that moderate hypothermia within 6 h of asphyxia improves survival without cerebral palsy or other disability by about 40% and reduces death or neurological disability by nearly 30%. The search is on to discover adjuvant treatments that can further enhance the effects of hypothermia.