Adenylyl cyclases types 1 and 8 promote pro-survival pathways after ethanol exposure in the neonatal brain.

Although a wide range of developmental disabilities following fetal alcohol exposure are observed clinically, the molecular factors that determine the severity of these sequelae remain undefined. In mice exposed to ethanol, deletion of adenylyl cyclases (ACs) 1 and 8 exacerbates the neuroapoptosis that occurs in a prolonged post-treatment period; however, it remains unclear whether AC1 and AC8 are critical to the primary or secondary mechanisms underlying ethanol-induced neurodegeneration. Here we demonstrate that mice lacking AC1 and AC8 (DKO) display significantly increased apoptosis in the striatum, a region sensitive to neuroapoptosis in the acute post-treatment period, compared to WT controls. The enhanced neuroapoptotic response observed in the striatum of DKO mice is accompanied by significant reductions in phosphorylation of known pro-survival proteins, insulin receptor substrate-1 (IRS-1), Akt and extracellular signal-regulated kinases (ERKs). These data suggest that AC1/AC8 are crucial activators of cell survival signaling pathways acutely following ethanol exposure and represent molecular factors that may directly modulate the severity of symptoms associated with Fetal Alcohol Syndrome.

Lithium protects against anesthesia-induced developmental neuroapoptosis.

BACKGROUND: Ethanol and anesthetic drugs trigger neuroapoptosis in the developing mouse brain. Recently, it was found that ethanol-induced neuroapoptosis is preceded by suppressed phosphorylation of extracellular signal-regulated protein kinase (ERK), and lithium counteracts both the phosphorylated ERK suppressant action and ethanol-induced neuroapoptosis. The current study was undertaken to address the following questions. (1) Do ketamine and propofol mimic ethanol in suppressing ERK phosphorylation? (2) If they do, does lithium prevent this suppressant action and also prevent these anesthetic drugs from triggering neuroapoptosis? METHOD: Postnatal day 5 mice were treated with propofol, ketamine, lithium, a combination of propofol or ketamine with lithium or saline, and their brains were prepared for Western blot analysis or histology. For Western blot, cytosolic lysates of caudate putamen were analyzed for expression of phosphorylated ERK and phosphorylated serine/threonine-specific protein kinase. For histology, brains were stained immunohistochemically with antibodies to activated caspase-3, and the density of activated caspase-3 positive cells was determined. RESULTS: Ketamine and propofol suppressed phosphorylated ERK, and lithium counteracted both the phosphorylated ERK suppressant action and neuroapoptotic action of these anesthetic drugs. CONCLUSION: If further testing finds lithium to be safe for use in pediatric/obstetric medicine, administration of a single dose of lithium before anesthesia induction may be a suitable means of mitigating the risk of anesthesia-induced developmental neuroapoptosis.

Cathepsin D deficiency induces persistent neurodegeneration in the absence of Bax-dependent apoptosis.

Neuronal ceroid lipofuscinosces/Batten disease (NCL) is a devastating group of neurodegenerative diseases caused by genetic disruptions in lysosomal function. Cathepsin D (CD) is a major lysosomal protease, and mutations in CD that render it enzymatically defective have been reported recently in subsets of NCL patients. The targeted deletion of CD in mice results in extensive neuropathology, including biochemical and morphological evidence of apoptosis and autophagic stress (aberrant autophagosome accumulation), effects that are similar to those observed in NCL. To determine the contribution of Bax-dependent apoptosis in this mouse model of NCL, combined Bax- and CD-deficient mice were generated. Morphological analysis of CD-deficient mouse brains indicated large numbers of pyknotic neurons and neurons with marked cytoplasmic swellings containing undigested lipofuscin. Cell death and apoptosis were evidenced by increases in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity and activation of caspase-3, respectively. DeOlmos silver-positive neurons were abundant in CD-deficient brain and correlated with neuron loss, as indicated by significant decreases in NeuN (neuronal nuclear antigen)-positive neurons. Lysosome dysfunction and autophagic stress were apparent in CD-deficient brain as indicated by the accumulation of autofluorescent storage material and by increased levels of LC3-II (light chain 3-II, a selective autophagosome marker), respectively. Bax deletion significantly inhibited caspase-3 activation and hippocampal TUNEL reactivity but did not prevent the majority of CD deficiency-induced neuropathology, including the persistence of pyknotic neurons, elevated cortical TUNEL reactivity, lysosome dysfunction and autophagic stress, neurodegeneration, and neuron loss. Together, these results suggest that CD deficiency-induced neuropathology does not require Bax-dependent apoptosis and highlights the importance of caspase-independent neuron death and neurodegeneration resulting from the genetic disruption of lysosome function.

Ethanol causes and lithium prevents neuroapoptosis and suppression of pERK in the infant mouse brain.

Transient exposure of immature animals during the brain growth spurt period to ethanol triggers neuroapoptosis in the developing brain. Here we report that lithium, when administered in a single, well-tolerated dose to infant mice, suppresses spontaneous neuroapoptosis that occurs naturally in the developing brain, and prevents ethanol from triggering neuroapoptosis. To explore lithium's mechanism of action, we focused on kinase signaling systems (ERK, Akt, JNK) that are believed to play a regulatory role in cell survival, and found that very rapidly after ethanol administration there is a suppression of ERK phosphorylation, and that lithium stimulates ERK phosphorylation and prevents ethanol from suppressing this phosphorylation process. Ethanol also suppressed pAKT, but lithium did not counteract this effect. We also found that ethanol activates the JNK system, but this cannot explain the neurotoxic action of ethanol, because JNK activation did not occur in the same neuronal populations that are killed by ethanol.

Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain.

Drugs that block N-methyl-d-aspartate glutamate receptors or that promote gamma-aminobutyric acid type A inhibition trigger neuroapoptosis in the developing rodent brain. Propofol reportedly interacts with both gamma-aminobutyric acid type A and N-methyl-d-aspartate glutamate receptors, but has not been adequately evaluated for its ability to induce developmental neuroapoptosis. Here we determined that the intraperitoneal (i.p.) dose of propofol required to induce a surgical plane of anesthesia in the infant mouse is 200 mg/kg. We then administered graduated doses of propofol (25-300 mg/kg i.p.) and found that doses >or=50 mg/kg induce a significant neuroapoptosis response. We conclude that propofol induces neuroapoptosis at 1/4 the dose required for surgical anesthesia.

Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice.

Drugs that suppress neuronal activity, including general anesthetics used in pediatric and obstetric medicine, trigger neuroapoptosis in the developing rodent brain. Exposure of infant rats for 6 hours to a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) reportedly causes widespread apoptotic neurodegeneration, followed by lifelong cognitive deficits. Isoflurane, the dominant ingredient in this triple cocktail, has not been evaluated individually for apoptogenic potential. It was recently reported that (1) the minimum alveolar concentration (MAC) for anesthetizing infant mice with isoflurane is 2.26%, and; (2) that infant mice, without assisted respiration, maintain normal arterial oxygen values but become hypoglycemic when exposed to isoflurane 3% for 30 minutes, then 1.8% for 1 hour (1.46 MAC-hours). In the present experiments, infant mice were exposed to isoflurane at various sub-MAC concentrations and durations, and the brains were evaluated quantitatively 5 hours after initiation of anesthesia exposure to determine the number of neuronal profiles undergoing apoptosis. Blood glucose values were also determined under each of these conditions. All conditions tested (isoflurane at 0.75% for 4 h, 1.5% for 2 h, 2.0% for 1 h) triggered a statistically significant increase in neuroapoptosis compared with the rate of spontaneous apoptosis in littermate controls. Blood glucose determinations ruled out hypoglycemia as a potential cause of the brain damage. It is concluded that exposure to sub-MAC concentrations of isoflurane for one or more hours triggers neuroapoptosis in the infant mouse brain. These findings are consistent with other recent evidence demonstrating that brief exposure to ethanol, ketamine, or midazolam triggers neuroapoptosis in the developing mouse brain.

Spatiotemporal evolution of apoptotic neurodegeneration following traumatic injury to the developing rat brain.

Closed head injury to the developing rat brain causes an acute excitotoxic lesion and axonal disruption at the impact site followed by a delayed pattern of apoptotic damage at various distant sites. Using an electromagnetic impact device to deliver a precisely controlled degree of mechanical deformation to the P7 infant rat skull, we studied the distribution of distant apoptotic lesions and the sequence and time course with which these lesions evolve following relatively mild closed head injury. The first major wave of apoptotic neurodegeneration occurred at 8 h postimpact in the retrosplenial cortex and pre- and parasubiculum. The next major wave occurred in the 16- to 24-h interval and was localized to the anterior thalamic nuclei. A third wave was detected at 36 to 48 h in the mammillary nuclei. We propose that the first and second waves were triggered by injury to a specific fiber tract, the corpus callosum/cingulum bundle that conveys reciprocal connections between the anterior thalamic nuclei and retrosplenial/pre- and parasubicular neurons. This fiber tract passes through a zone of maximum mechanical strain, as measured by tagged MRI. The third wave affecting mammillary neurons occurred because the principal synaptic targets of these neurons are the anterior thalamic neurons that were destroyed in the second wave of degeneration. Prevention of these apoptotic waves of brain damage is a realistic goal in view of the long delay between the impact event and onset of apoptotic degeneration.

ACTH therapy for Taiwanese children with West syndrome -- efficacy and impact on long-term prognosis.

To study the efficacy of adrenocorticotrophic hormone (ACTH) in treating Taiwanese children with West syndrome (WS) and the impact on long-term prognosis, 66 patients with WS (54 symptomatic and 12 cryptogenic) were collected from 1987 to 1998 in a medical center in Taiwan. A total of 53 patients were enrolled in this study and treated with ACTH at the dosage of 2.5IU/kg daily for 2 weeks with gradual tapering in subsequent 6 weeks. Immediate responses, side effects of ACTH and long-term outcomes of the patients including seizure and developmental status were evaluated during the average follow-up period of 35.6 months. The spasm-free percentage after one or two courses of ACTH treatment was 77.4%. Nine (17%) patients encountered severe side effects such as major infections, which prompted us to stop ACTH. At the end of follow-up, 22 (41.5%) patients had intractable seizures but 25 (47.2%) patients remained seizure free with or without anticonvulsants. The ACTH-responders had a better chance of remaining seizure free (P<0.05). Regarding the long-term developmental outcome, 12 (22.6%) patients had normal or borderline development; two thirds of them belonged to the crytpogenic group. Six (11.3%) patients expired and 24 (45.3%) were severely retarded; all but one of them belonged to the symptomatic group. The prognosis of WS heavily relies on whether a patient is cryptogenic or symptomatic (P<0.001). Good response to therapy or short treatment lag did not favorably affect the developmental outcomes of the symptomatic cases. We conclude that the long-term outcomes of WS in Taiwan were generally poor despite of treatment. Only cryptogenic patients had favorable prognosis. For symptomatic patients, ACTH therapy may be used to control the spasms and decrease the incidence of subsequent epilepsy, but it will not improve developmental outcome. Considering a high percentage of severe side effects in our study, a lower dosage of ACTH with adequate therapeutic efficacy but less side effects should be considered for treating Taiwanese children with WS.

Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain.

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one-time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase-3. It was found that either ketamine or midazolam caused a dose-dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Excitotoxic versus apoptotic mechanisms of neuronal cell death in perinatal hypoxia/ischemia.

Hypoxic/ischemic (H/I) neuronal degeneration in the developing central nervous system (CNS) is mediated by an excitotoxic mechanism, and it has also been reported that an apoptosis mechanism is involved. However, there is much disagreement regarding how excitotoxic and apoptotic cell death processes relate to one another. Some authors believe that an excitotoxic stimulus directly triggers apoptotic cell death, but this interpretation is largely speculative at the present time. Our findings support the interpretation that excitotoxic and apoptotic neurodegeneration are two separate and distinct cell death processes that can be distinguished from one another by ultrastructural evaluation. Here we review evidence supporting this interpretation, including evidence that H/I in the developing CNS triggers two separate waves of neurodegeneration, the first being excitotoxic and the second being apoptotic. The first (excitotoxic) wave destroys neurons that would normally provide synaptic inputs or synaptic targets for the neurons that die in the second (apoptotic) wave. Since neurons, during the developmental period of synaptogenesis, are programmed to commit suicide if they fail to achieve normal connectivity, this explains why neuroapoptosis occurs following H/I in the developing CNS. However, it does not support the interpretation that H/I directly triggers apoptotic neurodegeneration. Rather, it documents that H/I directly triggers excitotoxic neurodegeneration, and apoptotic neurodegeneration ensues subsequently as the natural response of developing neurons to a specific kind of deprivation - loss of the ability to form normal synaptic connections.

Ethanol-induced apoptosis in the developing visual system during synaptogenesis.

PURPOSE: Ethanol is known to have deleterious effects on the human fetal nervous system (fetal alcohol syndrome), including components of the visual system, but only modest progress has been made in understanding these effects. The authors have recently demonstrated that, during the period of synaptogenesis, a single episode of ethanol intoxication lasting for several hours triggers a massive wave of apoptotic neurodegeneration in several regions of the developing rat or mouse forebrain. The present study was undertaken to determine to what extent the developing visual system is vulnerable to the apoptogenic effects of ethanol. METHODS: Infant rats and mice at ages from birth to 21 days were treated subcutaneously with a single dose of ethanol or with two doses, 2 hours apart, on a single day. Blood alcohol levels were determined, and the retinas and visual centers in the brain were examined by light and electronmicroscopy at various times from 4 to 24 hours after treatment. RESULTS: Retinal ganglion cells and neurons in the lateral geniculate nucleus, superior colliculus, and visual cortex were all highly susceptible to ethanol's apoptogenic action, the period of peak sensitivity being postnatal days 1 to 4 for ganglion cells and 4 to 7 for the other visual neurons. A transient elevation of blood alcohol to approximately 120 mg/dL was sufficient to activate the cell death program in visual neurons. CONCLUSIONS: During synaptogenesis, a single ethanol intoxication episode triggers apoptotic cell death of neurons at all levels of the visual system from retina to the visual cortex.

Bilateral central retinal vein occlusion with multiple intracerebral hemorrhage in a neonate.

Central retinal vein occlusion and intracerebral hemorrhage are rare diseases during infancy and are both related to venous thrombosis. We present the case of a full-term male hydrops infant without specific neurologic symptoms initially but later demonstrating bilateral central retinal vein occlusion and intracerebral hemorrhage. We conclude that routine funduscopic examination in high-risk newborns should be seriously considered.

Agyria-pachygyria: clinical, neuroimaging, and neurophysiologic correlations.

Agyria-pachygyria complex is a disorder of neuronal migration and organization. Patients suffer either motor or intellectual retardation. We report our experiences of 10 patients with agyria-pachygyria complex and evaluate their clinical features, electroencephalography, and evoked potentials. Of nine electroencephalography examinations, five patients demonstrated characteristically high-amplitude fast activity. One of nine patients had an abnormal brainstem auditory-evoked potential. Three of seven patients had abnormal goggled visual-evoked potential. Six patients received somatosensory-evoked potential examinations, and five of these were abnormal, including four with prolonged central conduction times. Of the 10 patients, eight survived with variable intellectual and motor retardation; two died of sepsis. Patients with grades 1-4 agyria-pachygyria had high incidences of somatosensory-evoked potential abnormalities and also suffered worse neurologic outcomes. Normal brainstem auditory-evoked potential but abnormal cortical somatosensory-evoked potential components and prolonged central conduction time in these patients indicate that agyria-pachygyria is a supratentorial disease. We conclude that somatosensory-evoked potential examination is supplemental to neuroimaging in predicting the neurologic prognosis of patients with agyria-pachygyria.

Hypopituitarism associated with neurofibromatosis type 1: report of one case.

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder with a wide range of clinical manifestations. Hydrocephalus unrelated to brain tumors is rare in neurofibromatosis type 1. A 16-year-and-6-month-old girl with primary amenorrhea was found to have hydrocephalus associated with neurofibromatosis type 1. After endocrine and brain imaging study, the cause of primary amenorrhea was proven to be hypopituitarism due to hydrocephalus. She entered puberty soon after relieving the hydrocephalus with a ventriculoperitoneal shunt. However, arrest of puberty was noted three months later. Therefore estrogen replacement therapy was given. Early detection of hydrocephalus was difficult in this patient. From the experience of our case, MRI study of brain is indicated in patient with neurofibromatosis type 1 and endocrine dysfunction.

Schwartz-Jampel syndrome: report of one case.

We report a case of Schwartz-Jampel syndrome in a 2-year-9-month-old Taiwanese girl and her clinical response to treatment. She had a history of generalized muscle stiffness and hypertrophy since birth. Clinical and electromyographic myotonia were noted. Other clinical features included short stature, unusual facial appearance, ophthalmoplegia, elbow joint contractures, and developmental hip dysplasia. Muscle stiffness and myotonia improved after infusion of lidocaine. She then received oral mexiletine and the symptoms significantly improved.

Potential of xenon to induce or to protect against neuroapoptosis in the developing mouse brain.

PURPOSE: Drugs that suppress neuronal activity, including all general anesthetics that have been tested thus far (ketamine, midazolam, isoflurane, propofol, and a cocktail of midazolam, nitrous oxide and isoflurane), trigger neuroapoptosis in the developing rodent brain. Combinations of nitrous oxide and isoflurane, or ketamine and propofol, cause more severe neuroapoptosis than any single agent by itself, which suggests a positive correlation between increased levels of anesthesia and increased severity of neuroapoptosis. In contrast, there is evidence that the rare gas, xenon, which has anesthetic properties, protects against isoflurane-induced neuroapoptosis in the infant rat brain, while not inducing neuroapoptosis by itself. The present study was undertaken to evaluate the potential of xenon to induce neuroapoptosis or to protect against neuroapoptosis induced by isoflurane in the infant mouse brain. METHODS: Seven-day-old C57BL/6 mice were exposed to one of four conditions: air (control); 0.75% isoflurane; 70% xenon; or 0.75% isoflurane + 70% xenon for four hours. For histopathological evaluation of the brains, all pups were euthanized two hours later using activated caspase-3 immunohistochemical staining to detect apoptotic neurons. Under each condition, quantitative assessment of the number of apoptotic neurons in the cerebral cortex (CC) and in the caudate/putamen (C/P) was performed by unbiased stereology. RESULTS: The combination of xenon + isoflurane produced a deeper level of anesthesia than either agent alone. Both xenon alone (p < 0.003 in CC; p < 0.02 in C/P) and isoflurane alone (p < 0.001 in both CC and C/P) induced a significant increase in neuroapoptosis compared to controls. The neuroapoptotic response to isoflurane was substantially more robust than the response to xenon. When xenon was administered together with isoflurane, the apoptotic response was reduced to a level lower than that for isoflurane alone (p < 0.01 in CP; marginally non-significant in CC). CONCLUSIONS: We conclude that xenon, in the infant mouse brain, has paradoxical properties. It triggers neuroapoptosis, and when combined with isoflurane, it increases the depth of anesthesia, and retains its own apoptogenic activity. However, it suppresses, rather than augments, isoflurane's apoptogenic activity.

Neuroapoptosis in the infant mouse brain triggered by a transient small increase in blood alcohol concentration.

Exposure of infant rats or mice to ethanol on a single occasion during the period of rapid synaptogenesis can cause extensive apoptotic neurodegeneration throughout the developing CNS. Prior studies were designed to assess the effects of large doses of ethanol (comparable to heavy binge drinking), whereas in the present study, we sought to determine what magnitude and duration of blood ethanol elevation are required to trigger a minimal neuroapoptotic response. We found that a rise in blood ethanol to a level in the range of 50 mg/dl for a duration of 30 to 45 min was sufficient to trigger a significant neuroapoptosis response deleting approximately 20,000 neurons per infant mouse brain. Since blood ethanol elevations in this range are commonly achieved by humans in a social drinking context, a mother with only a moderate drinking habit might expose her fetus to such elevations on multiple occasions during pregnancy.

Role of caspase-3 in ethanol-induced developmental neurodegeneration.

Acute, transient exposure to ethanol causes a widespread pattern of caspase-3 activation and neuroapoptosis in the developing rodent brain. To determine whether caspase-3 activation is an essential step in ethanol-induced developmental neuroapoptosis, we treated homozygous caspase-3 knockout mice or wild-type mice on postnatal day 7 with an apoptosis-inducing dose of ethanol and examined the brains at appropriate survival times for evidence of apoptotic neurodegeneration. In caspase-3 knockout mice, the cell death process evolved more slowly than in wild-type mice, and morphological changes observed were not those typically associated with apoptosis. However, neuronal cell counts performed 2 weeks post-treatment revealed that the extent of neuron loss was similar in wild-type and caspase-3-deficient mice. We conclude that absence of functional caspase-3 alters the time course and morphological characteristics of the neurodegenerative process but does not prevent ethanol-induced neuron death.