Michael V. Johnston

Physiological and pathophysiological roles of excitatory amino acids during central nervous system development

Recent studies suggest that excitatory amino acids (EAAs) have a wide variety of physiological and pathophysiological roles during central nervous system (CNS) development. In addition to participating in neuronal signal transduction, EAAs also exert trophic influences affecting neuronal survival, growth and differentiation during restricted developmental periods. EAAs also participate in the development and maintenance of neuronal circuitry and regulate several forms of activity-dependent synaptic plasticity such as LTP and segregation of converging retinal inputs to tectum and visual cortex. Pre- and post-synaptic markers of EAA pathways in brain undergo marked ontogenic changes. These markers are commonly overexpressed during development; periods of overproduction often coincide with times when synaptic plasticity is great and when appropriate neuronal connections are consolidated. The electrophysiological and biochemical properties of EAA receptors also undergo marked ontogenic changes. In addition to these physiological roles of EAAs, overactivation of EAA receptors may initiate a cascade of cellular events which produce neuronal injury and death. There is a unique developmental profile of susceptibility of the brain to excitotoxic injury mediated by activation of each of the EAA receptor subtypes. Overactivation of EAA receptors is implicated in the pathophysiology of brain injury in several clinical disorders to which the developing brain is susceptible, including hypoxia-ischemia, epilepsy, physical trauma and some rare genetic abnormalities of amino acid metabolism. Potential therapeutic approaches may be rationally devised based on recent information about the developmental regulation of EAA receptors and their involvement in the pathogenesis of these disorders.

Neurotoxicity of N-methyl-d-aspartate is markedly enhanced in developing rat central nervous system

The neurotoxic lesion produced by direct injection of 25 nmol ofN-methyl-d-aspartate (NMDA) into the corpus striatum of 7-day-old rats was compared to the effects of injecting 75 nmol into the striatum or hippocampus of adults. The area of histopathology in the immature striatum was 21 × larger than the striatal lesions in adults. Damage from NMDA injected into the immature striatum also extended into the dorsal hippocampus and produced an area of destruction which was 16 × larger than observed after direct injection into the adult hippocampus. Several studies have implicated excessiveN-methyl-d-aspartate receptor activation in the pathogenesis of hypopoxic-ischemic and hypoglycemic injury and our results suggest that this neurotoxic mechanism is extremely active in the immature brain.

Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat.

SummaryElectrothermic lesion of the peri-pallidal region of the rat caused a marked reduction in the activity of choline acetyltransferase in the ipsilateral fronto-parietal cortex without affecting the activity of glutamate decarboxylase. Only lesions that involved the ventral globus pallidus significantly reduced cortical choline acetyltransferase activity; and lesions limited to the thalamus, internal capsule, pyriform cortex or zona incerta were ineffective. Excito-toxin lesions of the ventral globus pallidus caused 45–55% reductions in all presynaptic markers for cholinergic neurons but did not significantly decrease presynaptic markers for noradrenergic, serotonergic or histaminergic neurons in the cortex. The maximal reductions in cortical choline acetyltransferase activity achieved with the pallidal lesion was 70%; and enzyme activity reached its nadir by four days after placement of the lesion.The pallidal lesion, which ablated the large isodendritic acetylcholinesterase positive neuronal perikarya, resulted in a profound loss in histochemically stained acetylcholinesterase-reactive fibers in the fronto-parietal cortex but not in the cingulate, pyriform and occipital cortex or hippocampal formation; analysis of the subregions of the cortex revealed parallel reductions in choline acetyltransferase activity. The kainate lesion of the parietal cortex to ablate intrinsic neurons did not reduce the activity of tyrosine hydroxylase, a marker for noradrenergic terminals, but depressed glutamate decarboxylase by 68%; in contrast choline acetyltransferase activity fell only 29%. The results indicate that approximately 70% of the cholinergic innervation in the frontoparietal cortex is derived from acetylcholinesterase positive neurons in the peripallidal nucleus basalis, whereas the remainder appears to be localized in cortical intrinsic neurons.

Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat.

Birth asphyxia can cause moderate to severe brain injury. It is unclear to what degree apoptotic or necrotic mechanisms of cell death account for damage after neonatal hypoxia–ischemia (HI). In a 7-d-old rat HI model, we determined the contributions of apoptosis and necrosis to neuronal injury in adjacent Nissl-stained, hematoxylin and eosin-stained, and terminal deoxynucleotidyl transferase-mediated UTP nick end-labeled sections. We found an apoptotic–necrotic continuum in the morphology of injured neurons in all regions examined. Eosinophilic necrotic neurons, typical in adult models, were rarely observed in neonatal HI. Electron microscopic analysis showed “classic” apoptotic and necrotic neurons and “hybrid” cells with intermediate characteristics. The time course of apoptotic injury varied regionally. In CA3, dentate gyrus, medial habenula, and laterodorsal thalamus, the density of apoptotic cells was highest at 24–72 hr after HI and then declined. In contrast, densities remained elevated from 12 hr to 7 d after HI in most cortical areas and in the basal ganglia. Temporal and regional patterns of neuronal death were compared with expression of caspase-3, a cysteine protease involved in the execution phase of apoptosis. Immunocytochemical and Western blot analyses showed increased caspase-3 expression in damaged hemispheres 24 hr to 7 d after HI. A p17 peptide fragment, which results from the proteolytic activation of the caspase-3 precursor, was detected in hippocampus, thalamus, and striatum but not in cerebral cortex. The continued expression of activated caspase-3 and the persistence of cells with an apoptotic morphology for days after HI suggests a prolonged role for apoptosis in neonatal hypoxic ischemic brain injury.

Neurobiology of hypoxic-ischemic injury in the developing brain.

Hypoxic ischemia is a common cause of damage to the fetal and neonatal brain. Although systemic and cerebrovascular physiologic factors play an important role in the initial phases of hypoxic-ischemic injuries, the intrinsic vulnerability of specific cell types and systems in the developing brain may be more important in determining the final pattern of damage and functional disability. Excitotoxicity, a term applied to the death of neurons and certain other cells caused by overstimulation of excitatory, mainly glutamate, neurotransmitter receptors, plays a critical role in these processes. Selected neuronal circuits as well as certain populations of glia such as immature periventricular oligodendroglia may die from excitotoxicity triggered by hypoxic ischemia. These patterns of neuropathologic vulnerability are associated with clinical syndromes of neurologic disability such as the extrapyramidal and spastic diplegia forms of cerebral palsy. The cascade of biochemical and histopathologic events triggered by hypoxic ischemia can extend for days to weeks after the insult is triggered, creating the potential for therapeutic interventions.

Plasticity in the developing brain: implications for rehabilitation

Neuronal plasticity allows the central nervous system to learn skills and remember information, to reorganize neuronal networks in response to environmental stimulation, and to recover from brain and spinal cord injuries. Neuronal plasticity is enhanced in the developing brain and it is usually adaptive and beneficial but can also be maladaptive and responsible for neurological disorders in some situations. Basic mechanisms that are involved in plasticity include neurogenesis, programmed cell death, and activity-dependent synaptic plasticity. Repetitive stimulation of synapses can cause long-term potentiation or long-term depression of neurotransmission. These changes are associated with physical changes in dendritic spines and neuronal circuits. Overproduction of synapses during postnatal development in children contributes to enhanced plasticity by providing an excess of synapses that are pruned during early adolescence. Clinical examples of adaptive neuronal plasticity include reorganization of cortical maps of the fingers in response to practice playing a stringed instrument and constraint-induced movement therapy to improve hemiparesis caused by stroke or cerebral palsy. These forms of plasticity are associated with structural and functional changes in the brain that can be detected with magnetic resonance imaging, positron emission tomography, or transcranial magnetic stimulation (TMS). TMS and other forms of brain stimulation are also being used experimentally to enhance brain plasticity and recovery of function. Plasticity is also influenced by genetic factors such as mutations in brain-derived neuronal growth factor. Understanding brain plasticity provides a basis for developing better therapies to improve outcome from acquired brain injuries.

Randomized Phase III Trial of Vinorelbine Plus Cisplatin Compared With Observation in Completely Resected Stage IB and II Non–Small-Cell Lung Cancer: Updated Survival Analysis of JBR-10

PURPOSE Adjuvant cisplatin-based chemotherapy (ACT) is now an accepted standard for completely resected stage II and III A non-small-cell lung cancer (NSCLC). Long-term follow-up is important to document persistent benefit and late toxicity. We report here updated overall survival (OS) and disease-specific survival (DSS) data. PATIENTS AND METHODS Patients with completely resected stage IB (T2N0, n = 219) or II (T1-2N1, n = 263) NSCLC were randomly assigned to receive 4 cycles of vinorelbine/cisplatin or observation. All efficacy analyses were performed on an intention-to-treat basis. Results Median follow-up was 9.3 years (range, 5.8 to 13.8; 33 lost to follow-up); there were 271 deaths in 482 randomly assigned patients. ACT continues to show a benefit (hazard ratio [HR], 0.78; 95% CI, 0.61 to 0.99; P = .04). There was a trend for interaction with disease stage (P = .09; HR for stage II, 0.68; 95% CI, 0.5 to 0.92; P = .01; stage IB, HR, 1.03; 95% CI, 0.7 to 1.52; P = .87). ACT resulted in significantly prolonged DSS (HR, 0.73; 95% CI, 0.55 to 0.97; P = .03). Observation was associated with significantly higher risk of death from lung cancer (P = .02), with no difference in rates of death from other causes or second primary malignancies between the arms. CONCLUSION Prolonged follow-up of patients from the JBR.10 trial continues to show a benefit in survival for adjuvant chemotherapy. This benefit appears to be confined to N1 patients. There was no increase in death from other causes in the chemotherapy arm.

Excitotoxicity in perinatal brain injury.

Excitotoxicity is an important mechanism involved in perinatal brain injuries. Glutamate is the major excitatory neurotransmitter, and most neurons as well as many oligodendrocytes and astrocytes possess receptors for glutamate. Perinatal insults such as hypoxia‐ischemia, stroke, hypoglycemia, kernicterus, and trauma can disrupt synaptic function leading to accumulation of extracellular glutamate and excessive stimulation of these receptors. The activities of certain glutamate receptor/channel complexes are enhanced in the immature brain to promote activity‐dependent plasticity. Excessive stimulation of glutamate receptor/ion channel complexes triggers calcium flooding and a cascade of intracellular events that results in apoptosis and/or necrosis. Recent research suggests that some of these intracellular pathways are sexually dimorphic. Age dependent expression of different glutamate receptor subtypes with varying abilities to flux calcium has been associated with special patterns of selective vulnerability at different gestational ages. For example, selective injury to the putamen, thalamus and cerebral cortex from near total asphyxia in term infants may be related to excessive activation of neuronal NMDA and AMPA type glutamate receptors, while brain‐stem injury may be related primarily to stimulation of neuronal AMPA/kainate receptors. In contrast, periventricular leukomalacia in premature infants has been linked to expression of AMPA/kainate receptors on immature oligodendrocytes. Insight into the molecular pathways that mediate perinatal brain injuries could lead to therapeutic interventions.

Sex and the pathogenesis of cerebral palsy

Cerebral palsy (CP) and related developmental disorders are more common in males than in females, but the reasons for this disparity are uncertain. Males born very preterm also appear to be more vulnerable to white matter injury and intraventricular hemorrhage than females. Experimental studies in adult animals and data from adult patients with stroke indicate that sex hormones such as estrogens provide protection against hypoxic‐ischemic injury, and the neonatal brain is also influenced by these hormones. However, hormonal influences on the fetus and neonates are substantially different from those on adults. Recent data from neonatal rodents subjected to hypoxia‐ischemia also demonstrate differences between males and females. Knockout of the gene for poly (ADP‐ribose) polymerase (PARP‐1), a major step in the cascade of injury, protected male but not female mouse pups from hypoxic‐ischemic injury. Other reports demonstrated major differences between male and female neurons grown separately in cell culture, suggesting that sex differences in the fetal or neonatal period result from intrinsic differences in cell death pathways. This new information indicates that there are important neurobiological differences between males and females with respect to their response to brain injuries. This information is relevant to understanding the pathogenesis of CP as well as to the design of future clinical trials of potential neuroprotective strategies.

PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury

Poly(ADP‐ribose) polymerase‐1 is over‐activated in the adult brain in response to ischemia and contributes to neuronal death, but its role in perinatal brain injury remains uncertain. To address this issue, 7‐day‐old wild‐type (wt) and PARP‐1 gene deficient (parp+/– and parp–/–) Sv129/CD‐1 hybrid mice were subjected to unilateral hypoxia‐ischemia and histologic damage was assessed 10 days later by two evaluators. Poly(ADP‐ribose) polymerase‐1 knockout produced moderate but significant (p < 0.05) protection in the total group of animals, but analysis by sex revealed that males were strongly protected (p < 0.05) in contrast to females in which there was no significant effect. Separate experiments demonstrated that PARP‐1 was activated over 1–24 h in both females and males after the insult in neonatal wt mice and rats using immnocytochemistry and western blotting for poly(ADP‐ribose). Brain levels of NAD+ were also significantly reduced, but the decrease of NAD+ during the early post‐hypoxia‐ischemia (HI) phase was only seen in males. The results indicate that hypoxia‐ischemia activates Poly(ADP‐ribose) polymerase‐1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury.