Lymphocytic choriomeningitis virus injures the developing brain: effects and mechanisms.

Lymphocytic choriomeningitis virus (LCMV) is a prevalent pathogen, whose natural host and reservoir is the wild mouse. Humans can be infected when they contact the secretions of mice. Most infections of postnatal humans result in mild illness. However, the consequences can be severe when the infection occurs during pregnancy, as the virus crosses the placenta to infect the fetus. LCMV infection of the human fetus can lead to severe neuropathologic effects, including microencephaly, hydrocephalus, focal destructive lesions, and cerebellar hypoplasia. Outcomes among children with congenital LCMV are variable, but most are permanently and severely disabled. The neonatal rat inoculated with LCMV models human prenatal infection. The rat model has demonstrated that effects of LCMV depend on host age at the time of infection. Some effects, including encephalomalacia and neuronal migration disturbances, are immune-mediated and depend on the actions of T-lymphocytes. Other effects, including cerebellar hypoplasia, are virus-mediated and do not depend on T-lymphocytes. Cerebellar neuronal migration disturbances are caused by immune-mediated corruption of Bergmann glia structure. The rat pup inoculated with LCMV is a superb animal model for human congenital infection. All neuropathologic effects observed in human congenital LCMV infection can be recapitulated in the rat model. IMPACT: Lymphocytic choriomeningitis virus (LCMV) is a prevalent human pathogen that can cause serious neurologic birth defects when the infection occurs during pregnancy. The effects of the virus on the developing brain depend strongly on the age of the host at the time of infection. Some of the pathologic effects of LCMV are immune-mediated and are driven by T-lymphocytes, while other pathologic effects are due to the virus itself.

Measles Virus and the Central Nervous System: An Update.

MEASLES VIRUS AND ASSOCIATED CENTRAL NERVOUS SYSTEM: Sequelae Renee Buchanan, Daniel J. Bonthius Seminars in Pediatric Neurology Volume 19, Issue 3, September 2012, Pages 107-114 Worldwide, measles remains one of the most deadly vaccine-preventable diseases. In the United States, enrollment in the public schools requires that each child receives 2 doses of measles-containing vaccine before entry, essentially eliminating this once endemic disease. Recent outbreaks of measles in the United States have been associated with importation of measles virus from other countries and subsequent transmission to intentionally undervaccinated children. The central nervous system complications of measles can occur within days or years of acute infection and are often severe. These include primary measles encephalitis, acute postinfectious measles encephalomyelitis, measles inclusion body encephalitis, and subacute sclerosing panencephalitis. These measles associated central nervous system diseases differ in their pathogenesis and pathologic effects. However, all involve complex brain-virus-immune system interactions, and all can lead to severe and permanent brain injury. Despite better understanding of the clinical presentations and pathogenesis of these illnesses, effective treatments remain elusive.

Purkinje cell-specific deletion of CREB worsens alcohol-induced cerebellar neuronal losses and motor deficits.

INTRODUCTION: Exposure to alcohol during pregnancy can kill developing fetal neurons and lead to fetal alcohol spectrum disorder (FASD) in the offspring. However, not all fetuses are equally vulnerable to alcohol toxicity. These differences in vulnerability among individuals are likely due, at least in part, to genetic differences. Some genes encode neuroprotective molecules that act through signaling pathways to protect neurons against alcohol's toxic effects. One signaling pathway that can protect cultured neurons against alcohol-induced cell death in vitro is the cAMP pathway. A goal of this study was to determine whether the cAMP pathway can exert a similar neuroprotective effect against alcohol in vivo. A key molecule within the cAMP pathway is cAMP response element binding protein (CREB). In this study, CREB was specifically disrupted in cerebellar Purkinje cells to study its role in protection of cerebellar neurons against alcohol toxicity. METHODS: Mice with Purkinje cell-specific knockout of CREB were generated with the Cre-lox system. A 2 × 2 design was used in which Cre-negative and Cre-positive mice received either 0.0 or 2.2 mg/g ethanol by intraperitoneal (i.p.) injection daily over postnatal day (PD) 4-9. Stereological cell counts of cerebellar Purkinje cells and granule cells were performed on PD 10. Motor function was assessed on PD 40 using the rotarod. RESULTS: Purkinje cell-specific disruption of CREB alone (in the absence of alcohol) induced only a small reduction in Purkinje cell number. However, the loss of CREB function from Purkinje cells greatly increased the vulnerability of Purkinje cells to alcohol-induced cell death. While alcohol killed 20% of Purkinje cells in the Cre-negative (CREB-expressing) mice, alcohol killed 57% of Purkinje cells in the Cre-positive (CREB-nonexpressing) mice. This large loss of Purkinje cells did not lead to similar alcohol-induced losses of granule cells. In the absence of alcohol, lack of CREB function in Purkinje cells had no effect on rotarod performance. However, in the presence of alcohol, disruption of CREB in Purkinje cells substantially worsened rotarod performance. DISCUSSION: Disruption of a single gene (CREB) in a single neuronal population (Purkinje cells) greatly increases the vulnerability of that cell population to alcohol-induced cell death and worsens alcohol-induced brain dysfunction. The results suggest that the cAMP pathway can protect cells in vivo against alcohol toxicity and underline the importance of genetics in determining the neuropathology and behavioral deficits of FASD.

Structure-function relationships of TMJ lateral capsule-ligament complex.

The human temporomandibular joint (TMJ) lateral capsule ligament (LCL) complex is debated as a fibrous capsule with distinct ligaments or ligamentous thickening, necessitating further evaluation of the complex and its role in TMJ anatomy and mechanics. This study explores the ultrastructural arrangement, biomechanical tensile properties, and biochemical composition of the human LCL complex including region-specific differences to explore the presence of a distinct temporomandibular ligament and sex-specific differences to inform evaluations of potential etiological mechanisms. LCL complex ultrastructural arrangement, biomechanical properties, and biochemical composition were determined using cadaveric samples. Statistical modeling assessed sex- and region-specific effects on LCL complex tissue properties. Collagen fiber coherency, collagen fiber bundle size, and elastin fiber count did not differ between sexes, but females trended higher in elastin fiber count. LCL complex water and sGAG content did not differ between sexes or regions, but collagen content was higher in the anterior region (311.0 ± 185.6 µg/mg) compared to the posterior region (221.0 ± 124.9 µg/mg) (p = 0.045) across sexes and in males (339.6 ± 170.6 µg/mg) compared to females (204.5 ± 130.7 µg/mg) (p = 0.006) across regions. Anterior failure stress (1.1 ± 0.7 MPa) was larger than posterior failure stress (0.6 ± 0.4 MPa) (p = 0.024). Regional differences confirm the presence of a mechanically and compositionally distinct temporomandibular ligament. Baseline sex-specific differences are critical for etiological investigations of sex disparities in TMJ disorders. These results have important biomechanical and clinical ramifications, providing critical baseline tissue material properties, informing the development of TMJ musculoskeletal models, and identifying new areas for etiologic investigations for temporomandibular disorders.

Fatal Acute Hemorrhagic Leukoencephalitis Following Immunization Against Human Papillomavirus in a 14-Year-Old Boy.

Human papilloma virus (HPV) is a prevalent pathogen whose persistent infection can lead to a variety of cancers. To protect against this threat, an HPV vaccine has been developed and is routinely administered to adolescents. The HPV vaccine has a reassuring safety profile, but reports have emerged of acute disseminated encephalomyelitis following its administration. Acute hemorrhagic leukoencephalitis (AHLE) is a severe inflammatory disease of the central nervous system and the most fulminant form of ADEM. We report a previously healthy 14-year-old boy who developed headache, fatigue, focal weakness, and confusion 3 weeks after receiving the HPV vaccine. Neuroimaging demonstrated multifocal demyelination. Despite treatment with high-dose steroids, his encephalopathy worsened. He developed severe cerebral edema and died of cerebral herniation. Postmortem histology revealed perivenular sleeves of tissue damage, myelin loss surrounding small parenchymal vessels, and diffuse hemorrhagic necrosis, consistent with AHLE. This is the first report of AHLE following HPV vaccination.

Drug-Drug Interactions Between Cannabidiol and Lithium.

Epidiolex® (Cannabidiol- CBD) is approved for epilepsy associated with Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS) in patients over 2 years of age. Common side effects include somnolence and diarrhea. Recent studies have demonstrated interactions between cannabidiol and several other antiseizure medications. However, little is known regarding interactions between cannabidiol and other classes of medications. We discuss an autistic patient with LGS and significant psychiatric comorbidities who was being treated with multiple antiseizure and psychiatric medications, including lithium, when CBD was added to his medical regimen. Several weeks after initiating CBD therapy, he developed hypersomnolence, ataxia and decreased oral intake and was found to have lithium toxicity. Lithium was discontinued and his symptoms resolved. He remains on CBD and 2 other antiseizure medications, seizure-free with improved behavior. We review mechanisms of action and pharmacokinetics of CBD and discuss possible explanations for lithium toxicity in this patient.

Varicella Vaccine Meningitis as a Complication of Herpes Zoster in Twice-Immunized Immunocompetent Adolescents.

Varicella-zoster virus vaccination is recommended for virtually all young children in the United States, Canada, and several other countries. Varicella vaccine is a live attenuated virus that retains some of its neurotropic properties. Herpes zoster caused by vaccine virus still occurs in immunized children, although the rate is much lower than in children who had wild-type varicella. It was commonly thought that 2 varicella vaccinations would protect children against the most serious complication of meningitis following herpes zoster; however, 2 meningitis cases have already been published. We now report a third case of varicella vaccine meningitis and define risk factors shared by all 3 immunized adolescents. The diagnosis in cerebrospinal fluid in this third case was verified by amplifying and sequencing portions of the viral genome, to document fixed alleles found only in the vaccine strain. Viral antibody was also detected in the cerebrospinal fluid by confocal microscopy. When compared with the other 2 cases, remarkably all 3 were 14 years old when meningitis occurred. All 3 were treated with intravenous acyclovir, with complete recovery. The adolescent in our case report also had recurrent asthma, which was treated with both prednisone tablets and beclomethasone inhaler before onset of meningitis. When the 3 cases were considered together, they suggested that immunity to varicella-zoster virus may be waning sufficiently in some twice-immunized adolescents to make them vulnerable to varicella vaccine virus reactivation and subsequent meningitis. This complication rarely happens in children after wild-type varicella.

Viral Strain Determines Disease Symptoms, Pathology, and Immune Response in Neonatal Rats with Lymphocytic Choriomeningitis Virus Infection.

When infection with lymphocytic choriomeningitis (LCMV) occurs during pregnancy, the virus can infect the fetus and injure the fetal brain. However, type, location, and severity of neuropathology differ among cases. One possible explanation for this diversity is that fetuses are infected with different viral strains. Using a rat model of congenital LCMV infection, we investigated how differences in LCMV strain (E350, WE2.2, and Clone 13) affect outcome. Rat pups received intracranial inoculations on postnatal day 4. E350 initially targeted glial cells, while WE2.2 and Clone 13 targeted neurons. The E350 strain induced focal destructive lesions, while the other strains induced global microencephaly. E350 attracted large numbers of CD8+ lymphocytes early in the disease course, while Clone 13 attracted CD4+ lymphocytes, and the infiltration occurred late. The E350 and WE2.2 strains induced large increases in expression of pro-inflammatory cytokines, while Clone 13 did not. The animals infected with E350 and WE2.2 became ataxic and performed poorly on the negative geotaxis assay, while the Clone 13 animals had profound growth failure. Thus, in the developing brain, different LCMV strains have different patterns of infection, neuropathology, immune responses and disease symptoms. In humans, different outcomes from congenital LCMV may reflect infection with different strains.

Severe Herpes Zoster Following Varicella Vaccination in Immunocompetent Young Children.

Varicella vaccination is now virtually universal in North America, as well as in some European and Asian countries. Since varicella vaccine is a live attenuated virus, the virus replicates in the skin after administration and can travel via sensory nerves or viremia to become latent in the dorsal root ganglia. In some immunized children, virus reactivates within a few months to a few years to cause the dermatomal exanthem known as herpes zoster (shingles). Herpes zoster caused by vaccine virus often reactivates within the same dermatome as the site of the original varicella vaccine injection. We present evidence that occasional cases of herpes zoster following varicella vaccination in immunocompetent children can be as severe as herpes zoster following wild-type varicella. Analysis of the virus in one case disclosed that the vaccine virus causing herpes zoster was a wild-type variant with a mutation in ORF0. With regard to dermatomal localization of the viral eruption, we predict that herpes zoster of the lumbar dermatomes in children is likely to be caused by vaccine virus, because herpes zoster in those dermatomes is rare in children after wild-type varicella. One of the children with herpes zoster subsequently developed asthma, a known risk factor for herpes zoster, but none of the children had an autoimmune disease. Although postherpetic neuralgia is exceedingly rare, children who develop herpes zoster following varicella vaccination are at risk (albeit low) of developing meningoencephalitis and should be carefully observed for a few weeks.

Creation of a Porcine Kyphotic Model.

STUDY DESIGN: Large animal study. OBJECTIVE: Create a thoracic hyperkyphotic deformity in an immature porcine spine, so that future researchers may use this model to validate spinal instrumentation and other therapies used in the treatment of hyperkyphosis. SUMMARY OF BACKGROUND DATA: Although several scoliotic animal models have been developed, there have been no reports of a thoracic hyperkyphotic animal model creation in an immature animal. The present study was designed to produce a porcine hyperkyphotic model by the time the pig weighed 25 kg, which corresponds to the approximate weight of a child undergoing surgery for early-onset scoliosis (EOS). METHODS: Successful surgical procedures were performed in 6 consecutive 10-kg (male, 5-week-old) immature Yorkshire pigs. Procedure protocol consisted of 1) a left thoracotomy at T10-T11, 2) screw placement at T9 and T11, 3) partial vertebrectomy at T10, 4) posterior interspinous ligament transection, and 5) placement of wire loop around screws and tightening. Weekly x-ray imaging was performed preoperatively and postoperatively, documenting progressively increasing kyphosis as the pig grew. Necropsy was performed 5-6 weeks after surgery, with CT, slab section, and histologic analysis. RESULTS: Average T9-T11 kyphosis (measured by sagittal Cobb angle) was 6.1° ± 1.4° (mean ± SD) preoperatively, 30.5° ± 1.0° immediately postoperation, and significantly increased to 50.3° ± 7.2° (p < .0001) over 5-6 weeks in 6 consecutive pigs at time of necropsy. CONCLUSIONS: An animal model of relatively more rigid-appearing thoracic hyperkyphotic deformities in immature pigs has been created. Subsequent studies addressing management of early-onset kyphosis with spinal instrumentation are now possible. LEVEL OF EVIDENCE: Level V.

Effect of Measurement Technique on TMJ Mandibular Condyle and Articular Disc Morphometry: CBCT, MRI, and Physical Measurements.

PURPOSE: Accurate description of the temporomandibular size and shape (morphometry) is critical for clinical diagnosis and surgical planning and the design and development of regenerative scaffolds and prosthetic devices and to model the temporomandibular loading environment. The study objective was to determine the 3-dimensional morphometry of the temporomandibular joint (TMJ) condyle and articular disc using cone-beam computed tomography (CBCT), magnetic resonance imaging (MRI), and physical measurements of the same joints using a repeated measures design and to determine the effect of the measurement technique on temporomandibular size and shape. MATERIALS AND METHODS: Human cadaveric heads underwent a multistep protocol to acquire physiologically meaningful measurements of the condyle and disc. The heads first underwent CBCT scanning, and solid models were automatically generated. The superficial soft tissues were dissected, and intact TMJs were excised and underwent MRI scanning, with solid models generated after manual segmentation. After MRI, the intact joints were dissected, and physical measurements of the condyle and articular disc were performed. The CBCT-based model measurements, MRI-based model measurements, and physical measurements were standardized, and a repeated measures study design was used to determine the effect of the measurement technique on the morphometric parameters. RESULTS: Multivariate general linear mixed effects models showed significant effects for measurement technique for condylar morphometric outcomes (P < .001) and articular disc morphometric outcomes (P < .001). The physical measurements after dissection were larger than either the CBCT-based or MRI-based measurements. Differences in imaging-based morphometric parameters followed a complex relationship between imaging modality resolution and contrast between tissue types. CONCLUSIONS: Physical measurements after dissection are still considered the reference standard. However, owing to their inaccessibility in vivo, understanding how the imaging technique affects the temporomandibular size and shape is critical toward the development of high-fidelity solid models to be used in the design and development of regenerative scaffolds, surgical planning, prosthetic devices, and anatomic investigations.

Regional Patterns of Alcohol-Induced Neuronal Loss Depend on Genetics: Implications for Fetal Alcohol Spectrum Disorder.

BACKGROUND: Alcohol exposure during pregnancy can kill developing neurons and lead to fetal alcohol spectrum disorder (FASD). However, affected individuals differ in their regional patterns of alcohol-induced neuropathology. Because neuroprotective genes are expressed in spatially selective ways, their mutation could increase the vulnerability of some brain regions, but not others, to alcohol teratogenicity. The objective of this study was to determine whether a null mutation of neuronal nitric oxide synthase (nNOS) can increase the vulnerability of some brain regions, but not others, to alcohol-induced neuronal losses. METHODS: Immunohistochemistry identified brain regions in which nNOS is present or absent throughout postnatal development. Mice genetically deficient for nNOS (nNOS-/- ) and wild-type controls received alcohol (0.0, 2.2, or 4.4 mg/g/d) over postnatal days (PD) 4 to 9. Mice were sacrificed in adulthood (~PD 115), and surviving neurons in the olfactory bulb granular layer and brain stem facial nucleus were quantified stereologically. RESULTS: nNOS was expressed throughout postnatal development in olfactory bulb granule cells but was never expressed in the facial nucleus. In wild-type mice, alcohol reduced neuronal survival to similar degrees in both cell populations. However, null mutation of nNOS more than doubled alcohol-induced cell death in the olfactory bulb granule cells, while the mutation had no effect on the facial nucleus neurons. As a result, in nNOS-/- mice, alcohol caused substantially more cell loss in the olfactory bulb than in the facial nucleus. CONCLUSIONS: Mutation of the nNOS gene substantially increases vulnerability to alcohol-induced cell loss in a brain region where the gene is expressed (olfactory bulb), but not in a separate brain region, where the gene is not expressed (facial nucleus). Thus, differences in genotype may explain why some individuals are vulnerable to FASD, while others are not, and may determine the specific patterns of neuropathology in children with FASD.

T-Cells Underlie Some but Not All of the Cerebellar Pathology in a Neonatal Rat Model of Congenital Lymphocytic Choriomeningitis Virus Infection.

Lymphocytic choriomeningitis virus (LCMV) infection during pregnancy injures the human fetal brain. Neonatal rats inoculated with LCMV are an excellent model of congenital LCMV infection because they develop cerebellar injuries similar to those in humans. To evaluate the role of T-lymphocytes in LCMV-induced cerebellar pathology, congenitally athymic rats, deficient in T-lymphocytes were compared with euthymic rats. Peak viral titers and cellular targets of infection were similar, but viral clearance from astrocytes was impaired in the athymic rats. Cytokines and chemokines rose to higher levels and for a greater duration in the euthymic rats than in their athymic counterparts. The euthymic rats developed an intense lymphocytic infiltration, accompanied by destructive lesions of the cerebellum and a neuronal migration defect because of T-cell-mediated alteration of Bergmann glia. These pathologic changes were absent in the athymic rats but were restored by adoptive transfer of lymphocytes. Athymic rats were not free of pathologic effects, however, as the virus induced cerebellar hypoplasia. Thus, T-lymphocytes play key roles in LCMV clearance, cytokine/chemokine responses, and pathogenesis of destructive lesions and neuronal migration disturbances but not all pathology is T-lymphocyte-dependent. Cerebellar hypoplasia from LCMV occurs even in the absence of T-lymphocytes and is likely due to the viral infection itself.

Alexander Disease: A Novel Mutation in GFAP Leading to Epilepsia Partialis Continua.

Alexander disease is a genetically induced leukodystrophy, due to dominant mutations in the glial fibrillary acidic protein (GFAP ) gene, causing dysfunction of astrocytes. We have identified a novel GFAP mutation, associated with a novel phenotype for Alexander disease. A boy with global developmental delay and hypertonia was found to have a leukodystrophy. Genetic analysis revealed a heterozygous point mutation in exon 6 of the GFAP gene. The guanine-to-adenine change causes substitution of the normal glutamic acid codon (GAG) with a mutant lysine codon (AAG) at position 312 (E312 K mutation). At the age of 4 years, the child developed epilepsia partialis continua, consisting of unabating motor seizures involving the unilateral perioral muscles. Epilepsia partialis continua has not previously been reported in association with Alexander disease. Whether and how the E312 K mutation produces pathologic changes and clinical signs that are unique from other Alexander disease-inducing mutations in GFAP remain to be determined.

Defensive Perimeter in the Central Nervous System: Predominance of Astrocytes and Astrogliosis during Recovery from Varicella-Zoster Virus Encephalitis.

UNLABELLED: Varicella-zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types (neurons, microglia, oligodendrocytes, and astrocytes). The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytium formation within the aggregates included the continuity of cytoplasm positive for the VZV glycoprotein H (gH) fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis. IMPORTANCE: Investigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples with which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons.

Transient activation of microglia following acute alcohol exposure in developing mouse neocortex is primarily driven by BAX-dependent neurodegeneration.

Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in the developing brain in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice, we found that alcohol-induced neuroapoptosis in the neocortex is closely correlated in space and time with the appearance of activated microglia near dead cells. The timing and molecular pattern of microglial activation varied with the level of cell death. Although microglia rapidly mobilized to contact and engulf late-stage apoptotic neurons, apoptotic bodies temporarily accumulated in neocortex, suggesting that in severe cases of alcohol toxicity the neurodegeneration rate exceeds the clearance capacity of endogenous microglia. Nevertheless, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, there was a transient increase in expression of pro-inflammatory factors, TNFα and IL-1ß, after severe (5 g/kg) but not moderate (3 g/kg) EtOH levels. Alcohol-induced microglial activation and pro-inflammatory factor expression were largely abolished in BAX null mice lacking neuroapoptosis, indicating that microglial activation is primarily triggered by apoptosis rather than the alcohol. Therefore, acute alcohol exposure in the developing neocortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain.

Genetic absence of nNOS worsens fetal alcohol effects in mice. II: microencephaly and neuronal losses.

BACKGROUND: Prenatal alcohol exposure can kill developing neurons, leading to microencephaly and mental retardation. However, not all fetuses are equally vulnerable to alcohol's neurotoxic effects. While some fetuses are severely affected and are ultimately diagnosed with fetal alcohol syndrome (FAS), others have no evidence of neuropathology and are behaviorally normal. These widely different outcomes among alcohol-exposed fetuses are likely due, in part, to genetic differences. Some fetuses possess genotypes that make them much more vulnerable than others to alcohol's teratogenic effects. However, to date, only 1 gene has been identified whose mutation can worsen alcohol-induced behavioral deficits in an animal model of FAS. That gene is neuronal nitric oxide synthase (nNOS). The purpose of this study was to determine whether mutation of nNOS can likewise worsen alcohol-induced microencephaly and lead to permanent neuronal deficits. METHODS: Wild-type and nNOS(-/-) mice received alcohol (0.0, 2.2, or 4.4 mg/g) daily over postnatal days (PDs) 4 to 9. Beginning on PD 85, the mice underwent a series of behavioral tests; the results of which are reported in the companion paper. The brains were then weighed, and stereological cell counts were performed on the cerebral cortex and hippocampal formation, which are the brain regions that mediate the aforementioned behavioral tasks. RESULTS: Alcohol caused dose-dependent microencephaly, but only in the nNOS(-/-) mice and not in wild-type mice. Alcohol-induced neuronal losses were more severe in the nNOS(-/-) mice than in the wild-type mice in all of the brain regions examined, including the cerebral cortex, hippocampal CA3 subregion, hippocampal CA1 subregion, and dentate gyrus. CONCLUSIONS: Targeted mutation of the nNOS gene increases the vulnerability of the developing brain to alcohol-induced growth restriction and neuronal losses. This increased neuropathology is associated with worsened behavioral dysfunction. The results demonstrate the critical importance of genotype in determining the outcome of developmental alcohol exposure.

The neuronal nitric oxide synthase (nNOS) gene and neuroprotection against alcohol toxicity.

When a mother abuses alcohol during pregnancy, the offspring can suffer a myriad of abnormalities, collectively known as fetal alcohol spectrum disorder (FASD). Foremost among these abnormalities is central nervous system dysfunction, which commonly manifests itself as mental retardation, clumsiness, hyperactivity, and poor attention span. These behavior problems are due, in large part, to alcohol-induced neuronal losses in the developing fetal brain. However, not all fetuses are equally affected by maternal alcohol consumption during pregnancy. While some fetuses are severely affected and develop hallmarks of FASD later in life, others exhibit no evident neuropathology or behavioral abnormalities. This variation is likely due, at least in part, to differences in fetal genetics. This review focuses on one particular gene, neuronal nitric oxide synthase, whose mutation worsens alcohol-induced neuronal death, both in vitro and in vivo. In addition, ectopic expression of the neuronal nitric oxide synthase gene protects neurons against alcohol toxicity. The gene encodes an enzyme that produces nitric oxide (NO), which facilitates the protective effects of neuronal growth factors and which underlies the ability of neurons to resist alcohol toxicity as they mature. Nitric oxide exerts its protective effects against alcohol via a specific signaling pathway, the NO-cGMP-PKG pathway. Pharmacologic manipulation of this pathway could be of therapeutic use in preventing or ameliorating FASD.

Genetic absence of nNOS worsens fetal alcohol effects in mice. I: behavioral deficits.

BACKGROUND: Alcohol abuse during pregnancy often induces neuropsychological problems in the offspring, including learning disorders, attention deficits, and behavior problems, all of which are prominent components of fetal alcohol spectrum disorders (FASD). However, not all children who were exposed to alcohol in utero are equally affected by it. While some children have major deficits, others are spared. This unequal vulnerability is likely due largely to differences in fetal genetics. Some fetuses appear to have certain genotypes that make them much more prone to FASD. However, to date, no gene has been identified that worsens alcohol-induced brain dysfunction. Nitric oxide (NO) is a gaseous molecule that can protect developing neurons against alcohol-induced death. In the brain, NO is produced by neuronal nitric oxide synthase (nNOS). In this study, we examined whether homozygous mutation of the nNOS gene in mice worsens the behavioral deficits of developmental alcohol exposure. METHODS: Wild-type and nNOS(-/-) mice received alcohol (0.0, 2.2, or 4.4 mg/g) daily over postnatal days (PDs) 4 to 9. Beginning on PD 85, the mice underwent a series of behavioral tests, including open field activity, the Morris water maze, and paired pulse inhibition. RESULTS: For the wild-type mice, alcohol impaired performance only in the water maze. In contrast, for the nNOS(-/-) mice, alcohol impaired performance on all 3 tasks. Furthermore, the nNOS(-/-) mice were substantially more impaired than wild-type mice in their performance on all 3 of the behavioral tests and at both the low (2.2) and high (4.4) doses of alcohol. CONCLUSIONS: Targeted disruption of the nNOS gene worsens the behavioral impact of developmental alcohol exposure and allows alcohol-induced learning problems to emerge that are not seen in wild type. This is the first demonstration that a specific genotype can interact with alcohol to worsen functional brain deficits in an animal model of FASD.