Children's Hospital Boston




		Research Description Research Summary Age-dependent mechanisms of perinatal brain injury and epileptogenesis The overall focus of this laboratory is to devise age-specific therapeutic strategies to prevent brain injury in perinatal period. We focus on understanding the molecular and cellular pathophysiology of 2 highly prevalent forms of injury: hypoxic encephalopathy and seizures in the full term infant and hypoxic/ischemic injury to the white matter (periventricular leukomalacia, PVL) in the premature infant. Our overall interest is on the interaction between brain development and injury. We have a specific interest in the role of glutamate receptor maturation in excitotoxicity and epileptogenesis in the immature brain. Clinically, the seizure incidence in infancy is higher than at any other time in life. Neonatal seizures occur primarily in the term infant, and the most common cause of seizures is lack of oxygen (hypoxia) and/or blood flow (ischemia), due to birth asphyxia, insufficient lung function, and infection, among other causes. Some infants go on to develop neurocognitive deficits, mental retardation, and/or epilepsy. When severe, seizures associated with hypoxic encephalopathy of the newborn can be refractory to conventional medications that are effective in older children and adults. The age-dependency and the lack of response to conventional medications suggest that this injury might have mechanisms different from those in a similarly affected mature brain. Our work thus far has identified a critical role of a specific neurotransmitter receptor, the AMPA subtype of glutamate receptors, in the vulnerability to and subsequent development of epilepsy. We use a combination of in vivo and in vitro models of this injury in the developing rodent brain. We are examining the sequence of changes that take place in the perinatal brain following an early life seizure, and how these can result in abnormalities of brain development and epilepsy. This information has been used to devise therapeutic strategies for both pre and post treatment in perinatal hypoxic encephalopathy. We currently are examining the potential clinical efficacy of two drugs that are in fact FDA approved for use for other indications and other age groups. If final preclinical studies are successful, we hope to implement them based on a clinical trial at Children's Hospital. The second research emphasis is on injury to the premature brain. Here, it appears that the white matter is selectively vulnerable to hypoxia/ischemia, and this results in PVL. PVL is the major antecedent to cerebral palsy, and to date no specific treatment exists. PVL occurs in premature infants that have respiratory distress, hypotension, sepsis and other complication of prematurity. Given the improved medical techniques, the number of infants born between 25-27 weeks and less than 1500 grams in birthweight is rising, yet these infants are those with the greatest risk of such complications. We have used in vivo and in vitro rodent models of injury to the primary cell type in the white matter, the oligodendrocyte. We and collaborators have found that the immature forms of oligodendrocytes that are present in the premature brain appear to be more sensitive to hypoxia/ischemia than mature forms that are present in brains of individuals of older ages. We examined the mechanism of toxicity, and found that these cells transiently express glutamate receptors during the window of vulnerability to hypoxia/ischemia. Subsequent studies showed that pharmacologic blockade of glutamate receptors on oligodendrocytes prevented the PVL-like injury in rodent models. We have extended our studies and found that a clinically available agent with glutamate blocking effects can also prevent the PVL-like damage to white matter, even when it is administered following the hypoxic/ischemic brain injury. Further studies on human autopsy tissue have shown that these glutamate receptors are likely present in the human white matter brain during the window of vulnerability to PVL, increasing the clinical relevance of our findings in our animal models. We also plan to examine the potential clinical efficacy of this therapeutic strategy, with the hope of its translation to a clinical trial. We hope that ongoing studies evaluating other mechanisms will produce additional therapeutic strategies for this disease process. A more recent area of interest has been the study of abnormal glutamate receptors in tuberous sclerosis comples(TSC). Tuberous sclerosis complex (TSC) is an autosomal dominant disorder, caused by mutations in either TSC1 or TSC2 genes and characterized by the development of hamartomas in multiple organs, including the brain. TSC-associated brain lesions include cortical tubers, cortical hypoplasia, radial lines, subependymal nodules and subependymal giant cell astrocytoma. We have recently found that specific glutamate receptors are overexpressed on dysplastic cells within these lesions, and may be a source of epileptogenesis. In addition, we have found similar abnormalities in transgenic mice bearing a TSC mutation. Current studies are examining electrophysiological correlates of these abnormalities in brain slices prepared from human biopsy material and transgenic mice. The overall aim of this research program is to identify unique receptors critical for epileptogenesis in the abnormal cells, and to use specific receptor antagonists to attenuate seizure activity. Abnormally expressed receptors may represent disease-specific antiepileptogenic agents with clinical potential Highlights of Major Accomplishments Discovery that perinatal hypoxia/ischemia causes acute downregulation of the GluR2 subunit of nonNMDA receptors, resulting in increased calcium permeability, enhanced excitability and seizures, Demonstration that immature oligodendrocytes (OLS) are sensitive to excitotoxic cell death via activation of nonNMDA receptors, Discovery that graded hypoxia/ischemia in the immature rat (P7) results in selective injury to immature OLS and that this injury can be prevented by systemic administration of nonNMDA antagonists, NBQX and topiramate, after the insult. Demonstration that immature neurons and oligodendrocytes in the human perinatal brain express calcium permeable AMPA receptors, suggesting they are age-specific therapeutic targets for the treatment of neontal brain injury and seizures.. Major Results 1. Mechanisms and sequelae of neonatal seizures with perinatal hypoxia 2. Non-NMDA receptors in oligodendrocytes and PVL 1. Mechanisms and sequelae of neonatal seizures with perinatal hypoxia 1.1 The role of the AMPA receptor in seizures induced by perinatal hypoxia. We have previously demonstrated that hypoxia causes seizures in immature rats during a discrete developmental window between postnatal days 10-12, but not at younger or older ages. Prior work revealed that seizures were selectively suppressed by the AMPA receptor antagonist NBQX, but not by NMDA antagonists or GABA agonists. Given the efficacy of the AMPA antagonist, we evaluated the antiepileptogenic effect of topiramate, a clinically available anticonvulsant with a known action as an AMPA antagonist. Topiramate (30 mg/kg i.p.) was highly effective at blocking acute and long term seizures induced by hypoxia at P10. These results suggest a potential use of this agent in the neonatal population, where seizures are often refractory to other conventional anticonvulsants. 1.2 Effects of perinatal hypoxia on neuronal excitability and glutamate receptor expression. Rats with hypoxic seizures during P10-12 develop long term increases in seizure susceptibility. Despite the increased excitability, there is no evidence of cell death either acutely or within 21 days following hypoxic seizures. As a goal of the laboratory was to determine the mechanisms underlying the long term epileptogenic effects of hypoxia, we evaluated whether cortical and hippocampal regions underwent intrinsic changes after hypoxia. Electrophysiological recordings in hippocampal slices removed from previously hypoxic rats reveal enhanced long term potentiation and kindling rates within 1 hour following hypoxia. Slices removed from rats in adulthood (P70) following hypoxia at P10 showed increased seizure susceptibility to chemical convulsants. In association with these electrophysiologic alterations, we have shown short term changes in expression of AMPA receptor subunits. GluR2 subunit expression is downregulated at 72h - 7 d following hypoxia, and a relative lack of GluR2 expression has been shown to increase calcium permeability at AMPA channels. Hippocampal slices compared between hypoxia and control rats using cobalt staining as a marker of calcium influx revealed that there was a significant increase in AMPA-induced cobalt influx in slices from hypoxia rats. Hence seizure-induced downregulation of GluR2 appears to have a functional effect on the neurons by increasing divalent cation permeability, notably in the absence of inducing cell death. Present studies are aimed at determining whether the GluR2 downregulation is a factor in the long term increases in neuronal excitability within the hippocampus. *2. Non-NMDA receptors in oligodendrocytes and PVL* We have recently developed an immature rat model of periventricular leukomalacia (PVL) where premyelinating OLs are specifically vulnerable to hypoxic/ischemic injury. Selective OL injury is greatest at P7, with less injury at younger (P4) or older (P11, P18) rats. OLs are known to express nonNMDA glutamate receptors, and given the critical role of glutamate in hypoxic/ischemic injury, we evaluated whether intracerebral AMPA injection also had a similar developmental profile. Similar to hypoxia, the greatest injury to OLs from AMPA was seen at P7. Furthermore immunocytochemistry revealed that nonNMDA receptor subunits are expressed at relatively higher concentrations on OLs at P7 than at older or younger ages, suggesting that the nonNMDA glutamate receptor expression may be an important factor in the age-specific vulnerability of OLs in PVL. To support this, we have recently shown that systemic administration of the AMPA antagonists NBQX or topiramate effectively block both hypoxic/ischemic and AMPA injection-induced OL injury at P7. Additional support emanates from in vitro observations that premyelinating OLs in culture, when AMPA and kainate receptor subunit expression is highest, exhibit greatest vulnerability to AMPA-mediated excitotoxicity, compared to the vulnerability exhibited by stage specific cultures of mature MBP+ OLs which are AMPA-resistant. Taken together, immature OLs appear to be selectively vulnerable to glutamate mediated excitotoxicity in vivo and in vitro, and this susceptibility may be due at least in part to their higher expression of nonNMDA glutamate receptors compared to mature OLs.