Differential brain region-specific expression of MeCP2 and BDNF in Rett Syndrome patients: a distinct grey-white matter variation.

INTRODUCTION AND OBJECTIVES: Rett Syndrome (RTT) is a neurodevelopmental disorder caused by Methyl CpG Binding Protein 2 (MECP2) gene mutations. Previous studies of MeCP2 in the human brain showed variable and inconsistent mosaic-pattern immunolabelling, which has been interpreted as a reflection of activation-state variability. We aimed to study post mortem MeCP2 and BDNF (MeCP2 target) degradation and brain region-specific detection in relation to RTT pathophysiology. METHODS: We investigated MeCP2 and BDNF stabilities in non-RTT human brains by immunohistochemical labelling and compared them in three brain regions of RTT and controls. RESULTS: In surgically excised samples of human hippocampus and cerebellum, MeCP2 was universally detected. There was no significantly obvious difference between males and females. However, post mortem delay in autopsy samples had substantial influence on MeCP2 detection. Immunohistochemistry studies in RTT patients showed lower MeCP2 detection in glial cells of the white matter. Glial fibrillary acidic protein (GFAP) expression was also reduced in RTT brain samples without obvious change in myelin basic protein (MBP). Neurons did not show any noticeable decrease in MeCP2 detection. BDNF immunohistochemical detection showed an astroglial/endothelial pattern without noticeable difference between RTT and controls. CONCLUSIONS: Our findings indicate that MeCP2 protein is widely expressed in mature human brain cells at all ages. However, our data points towards a possible white matter abnormality in RTT and highlights the importance of studying human RTT brain tissues in parallel with research on animal and cell models of RTT.

A de novo dominant mutation in ACTA1 causing congenital nemaline myopathy associated with a milder phenotype: expanding the spectrum of dominant ACTA1 mutations.

We describe the presentation and six-year follow up of a child with nemaline myopathy due to a de novo mutation in the skeletal muscle α-actin gene (ACTA1) characterized by dramatic improvement during the early childhood years. The presentation in this female patient was infantile-onset weakness in the facial, bulbar, respiratory and neck flexor muscles. A six-year follow-up revealed continued progressive improvement in her muscle strength. Based upon the histopathologic and ultrastructural features of nemaline rod disease, ACTA1 was sequenced. This revealed a mutation in exon 4 of ACTA1 (c.557A>G). Our report further expands the phenotypic spectrum associated with ACTA1 mutations. Although it is difficult to infer any genotype-phenotype correlation, this report stimulates the discussion regarding the pathophysiologic mechanism of the clinical improvement seen in some patients with ACTA1 mutations.

Multifocal haemorrhagic brain damage following hypoxia and blood pressure lability: case report and rat model.

AIMS: Haemorrhagic brain damage is frequently encountered as a complication of premature birth. Much less frequently, multifocal petechial haemorrhage is identified in asphyxiated term newborns. Our goal was to develop an experimental rat model to reproduce this pattern of brain damage. METHODS: Neonatal rat pups were exposed to a 24-h period of 10% or 8% hypoxia followed by a single dose of phenylephrine. Acute and subacute changes, as well as long-term outcomes, were investigated by histology, brain magnetic resonance imaging and behavioural assessment. Immunostaining for vascular endothelial growth factor and caveolin-1 was performed in the rat brains as well as in a 17-day human case. RESULTS: Small foci of haemorrhage were identified in almost all regions of the rat brain subjected to hypoxia plus phenylephrine, but not hypoxia alone. Exposure to 8% hypoxia was associated with more haemorrhagic foci than 10% hypoxia. With rare exceptions, the blood deposits were too small to be detected by magnetic resonance imaging. Altered immunohistochemical detection of vascular endothelial growth factor and caveolin-1 in the child and the rat model suggests a role for blood-brain barrier compromise. There were no clear behavioural changes and no residual morphological abnormalities in the 78-day follow-up of the rats. CONCLUSIONS: We conclude that transient hypoxia, in a dose-dependent manner, can weaken the vasculature and predispose to brain haemorrhage in the situation of labile blood pressure. Persistent hypoxia is likely to be important in the genesis of permanent severe brain damage.

Biochemical label-free tissue imaging with subcellular-resolution synchrotron FTIR with focal plane array detector.

The critical questions into the cause of neural degeneration, in Alzheimer disease and other neurodegenerative disorders, are closely related to the question of why certain neurons survive. Answers require detailed understanding of biochemical changes in single cells. Fourier transform infrared microspectroscopy is an excellent tool for biomolecular imaging in situ, but resolution is limited. The mid-infrared beamline IRENI (InfraRed ENvironmental Imaging) at the Synchrotron Radiation Center, University of Wisconsin-Madison, enables label-free subcellular imaging and biochemical analysis of neurons with an increase of two orders of magnitude in pixel spacing over current systems. With IRENI's capabilities, it is now possible to study changes in individual neurons in situ, and to characterize their surroundings, using only the biochemical signatures of naturally-occurring components in unstained, unfixed tissue. We present examples of analyses of brain from two transgenic mouse models of Alzheimer disease (TgCRND8 and 3xTg) that exhibit different features of pathogenesis. Data processing on spectral features for nuclei reveals individual hippocampal neurons, and neurons located in the proximity of amyloid plaque in TgCRND8 mouse. Elevated lipids are detected surrounding and, for the first time, within the dense core of amyloid plaques, offering support for inflammatory and aggregation roles. Analysis of saturated and unsaturated fatty acid ester content in retina allows characterization of neuronal layers. IRENI images also reveal spatially-resolved data with unprecedented clarity and distinct spectral variation, from sub-regions including photoreceptors, neuronal cell bodies and synapses in sections of mouse retina. Biochemical composition of retinal layers can be used to study changes related to disease processes and dietary modification.

Late infantile onset krabbe disease in siblings with cortical degeneration and absence of cerebral globoid cells.

Krabbe disease, a disorder caused by the deficiency of lysosomal galactosylceramidase, is typically associated with cerebral white matter degeneration, cortical sparing, accumulation of macrophages ("globoid cells"), and ultrastructural needle-shaped inclusions. Two sisters presented with progressive neurological deterioration beginning before the age of 2.5 years. The first, who died at the age of 9 years, exhibited profound destruction of cerebral white matter with sparing of subcortical fibers but no globoid cells. The brain of the second, who died at the age of 15 years and who had a proven galactosylceramidase deficiency, exhibited white matter destruction, previously undescribed circumscribed spongiform cortical degeneration (postcentral, inferior temporal, cingulate), and cerebellar atrophy, but no globoid cells. The peripheral nerve biopsies from both girls exhibited typical needle-shaped inclusions in Schwann cells. These observations confirm the rare reports that Krabbe disease is not always associated with globoid cells in the brain. Psychosine, which accumulates in the brain, might be toxic to cortical neurons following prolonged survival. The reason for the regional susceptibility in the cerebral cortex is unknown.

Preliminary attempts to establish a rat model of striatal injury in glutaric acidaemia type I.

Glutaric acidaemia type I (GA I) is caused by the deficiency of glutaryl-CoA dehydrogenase, resulting in accumulation of glutaric acid (GA) and 3- hydroxyglutaric acid (3-OH-GA) in blood and cerebrospinal fluid (CSF). Neuropathological changes with onset in childhood consist of severe neuronal loss in the caudate and putamen. An animal model is necessary to test possible intervention strategies, and prior reports suggested that GA or 3-OH-GA could be used to create specific neuron loss in adult rats. Adult, 3-week-old and 2-week-old rats received intrastriatal injections of GA and 3-OH-GA at a range of doses. High concentrations caused necrotic lesions in striatum. Low concentrations caused white-matter axonal damage and small areas of neuron loss. Injection of lipopolysaccharide prior to administration of 3-OH-GA was not associated with enhanced neuronal loss. Our findings contradict prior claims and we conclude that the simple model of a single GA or 3-OH-GA injection into rat brain does not replicate the neuropathological findings in humans.

Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers.

A neurological syndrome involving progressive action tremor with ataxia, cognitive decline and generalized brain atrophy has been described recently in some adult males with pre-mutation alleles of the fragile X syndrome (FXS) fragile X mental retardation gene (FMR1). Neurohistological studies have now been performed on the brains of four elderly premutation carriers, not reported previously, who displayed the neurological phenotype. Eosinophilic, intranuclear inclusions were present in both neuronal and astrocytic nuclei of the cortex in all four individuals. Systematic analysis of the brains of two of these carriers demonstrated the presence of the intranuclear inclusions throughout the cerebrum and brainstem, being most numerous in the hippocampal formation. The cerebellum displayed marked dropout of Purkinje cells, Purkinje axonal torpedoes and Bergmann gliosis. Intranuclear inclusions were absent from Purkinje cells, although they were present in a small number of neurones in the dentate nucleus and diffusely in cerebellar astrocytes. The presence of inclusions in the brains of all four FXS carriers with the neurological findings provides further support for a unique clinical entity associated with pre-mutation FMR1 alleles. The origin of the inclusions is unknown, although elevated FMR1 mRNA levels in these pre-mutation carriers may lead to the neuropathological changes.

Intracerebral infusion of a second-generation ciliary neurotrophic factor reduces neuronal loss in rat striatum following experimental intracerebral hemorrhage.

Neuronal and glial cell death in the striatum of a rat model of collagenase-induced intracerebral hemorrhage begins at 1 day and continues for at least 3 weeks. We hypothesized that administration of a neurotrophic agent would reduce neuronal loss in this experimental model. Because it has been shown to protect striatal neurons against excitotoxic injury, a second-generation ciliary neurotrophic factor (CNTF) (AXOKINE) was administered by continuous intracerebral infusion (2 microg/day) beginning 28 h after hemorrhage and continuing for 2 weeks. Magnetic resonance imaging showed that the hematoma size was comparable in control and treated rats prior to treatment. Counts of medium-sized striatal neurons within 320 microm of the hematoma 8 weeks after the hemorrhage revealed a slight but statistically significant benefit with a 42.5% loss in treated rats compared to 51.7% loss in controls. The results suggest that AXOKINE might be protective of striatal neurons in the vicinity of a hemorrhagic lesion.

Acute tissue damage after injections of thrombin and plasmin into rat striatum.

BACKGROUND AND PURPOSE: Extravasation of blood is associated with intracerebral hemorrhage and head trauma. The mechanism of brain cell injury associated with hemorrhage differs from that due to pure ischemia. The purpose of this study was to investigate the acute changes after intracerebral injections of proteins that are involved in blood clotting and clot lysis. METHODS: Sixty-eight adult rats were subjected to stereotaxic intrastriatal injections of normal saline (5 microL), low- (2.5 U/5 microL) and high-dose (25 U/5 microL) thrombin, low- (0.1 microgram/5 microL) and high-dose (1 microgram/5 microL) tissue plasminogen activator, low- (0.05 U/5 microL) and high-dose (0.5 U/5 microL) plasminogen, and low- (0.335 U/5 microL) and high-dose (3.35 U/5 microL) plasmin. Forty-eight hours later rats were perfusion fixed. Brain damage area, eosinophilic neurons, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells, infiltrating neutrophils, CD8a immunoreactive leukocytes, and reactive microglia were quantified. RESULTS: Damage area in striatum, dying cells, inflammatory cells, and microglial reaction were significantly greater after the high-dose plasminogen, plasmin, and thrombin injections. Tissue plasminogen activator injections were associated with mild inflammation. CONCLUSIONS: These results suggested that thrombin and plasmin are harmful to brain cells in vivo. Although the doses required to cause damage are relatively great in consideration of the plasma content of these proteins, their pathological effect might be enhanced through synergism with other mechanisms.

Pathophysiologic consequences of hydrocephalus.

Hydrocephalus-induced damage is dependent on the rate and magnitude of ventricular dilatation, the proximity to the ventricle, and the developmental stage at which the disturbance occurs. It is mediated through a combination of mechanical, ischemic, and metabolic-toxic disturbances. Developmental processes, including myelin production, can be impaired. Periventricular axons are the primary target, however. The potential for reversal of damage by shunting diminishes as the duration and severity of hydrocephalus increases. Ancillary pharmacologic means for preventing hydrocephalus-induced brain damage are worth pursuing.

T2-weighted MRI correlates with long-term histopathology, neurology scores, and skilled motor behavior in a rat stroke model.

The intraluminal suture model of transient middle cerebral artery occlusion (MCAO) in the Sprague Dawley strain of rats characteristically results in an inconsistently sized brain lesion. The purpose of the investigation reported here was to determine whether there were strong point-to-point correlations between the degree of cortical lesion size, as assessed in vivo using T2-weighted magnetic resonance imaging (MRI) and corresponding cortical lesion size using routine histopathological techniques. Moreover, we aimed to investigate if cortical lesion size as determined by these two modalities correlates with neurological and/or skilled motor deficits observed in individual animals. Baseline behavioral scores were obtained on the animals prior to receiving 60 min of transient MCAO. Following MCAO, animals were tested for 1-21 days for neurological deficits. T2-weighted MRIs of the cortex were taken at two and seven days post-MCAO. At 30 and 60 days the rats were retested for forelimb dexterity in the staircase test. Subsequently, the cortex was examined for histopathological damage. Indeed, there were highly significant correlations between lesion size determined by MRI and histopathology. The degree of cortical damage observed in the T2-weighted MRI, as well as the size of the histopathological lesions were, in turn, highly correlated with the degrees of deficiencies observed in the composite neurological assessments and with the deficits involving skilled use of the contralateral forepaw (damaged side).

Adenosine A2A receptor activation reduces proinflammatory events and decreases cell death following intracerebral hemorrhage.

The ubiquitous neuromodulator adenosine inhibits the production of several proinflammatory cytokines through activation of specific cell-surface adenosine receptors. We demonstrated recently that antisense oligonucleotides to tumor necrosis factor-alpha (TNF-alpha) are neuroprotective in a rat model of intracerebral hemorrhage. Therefore, we hypothesized that activation of adenosine receptors would provide protection against intracerebral hemorrhage-induced TNF-alpha production and inflammatory events. In vitro experiments showed that adenosine A1, A2A, and A3 receptor subtypes were present on U937 cells, and activation of these subtypes inhibited TNF-alpha production with a rank order of A2A > > A1 > A3. Prolonged treatment of U937 cells with the A2A receptor agonist 2-p-(carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) desensitized adenosine A2A, A1, and A3 receptors. CGS 21680 administration directly into the striatum immediately prior to the induction of intracerebral hemorrhage inhibited TNF-alpha mRNA and, 24 hours following induction, reduced parenchymal neutrophil infiltration (p < 0.001) and TUNEL-positive cells (p < 0.002) within and bordering the hematoma. These results suggest that pharmacological strategies targeting A2A receptors may provide effective inhibition of acute neurotoxic proinflammatory events that occur following intracerebral hemorrhage.

Protective effect of nimodipine on behavior and white matter of rats with hydrocephalus.

OBJECT: Hydrocephalus, a pathological dilation of the ventricles of the brain, causes damage to periventricular white matter, at least in part, through chronic ischemia. The authors tested the hypothesis that treatment with nimodipine, an L-type calcium channel-blocking agent with demonstrated efficacy in a range of cerebral ischemic disorders, would ameliorate the adverse effects of experimental hydrocephalus. METHODS: Hydrocephalus was induced in 3-week-old rats by injection of kaolin into the cisterna magna. The rats were treated by continuous administration of nimodipine or control vehicle for 2 weeks, beginning 2 weeks after induction of hydrocephalus. During the treatment period, the animals underwent repeated tests of motor and cognitive behavior. At the end of the treatment period, the rat brains were analyzed by histopathological and biochemical means. Nimodipine treatment prevented the declines in motor and cognitive behavior that were observed in untreated control rats. During the treatment period, ventricular enlargement, determined by magnetic resonance imaging, was equal in the two groups, although the corpus callosum was thicker in the treated rats. Myelin content in white matter and synaptophysin content in gray matter, an indicator of synapses, did not differ. CONCLUSIONS: The protective effect of nimodipine is most likely based on improved blood flow, although prevention of calcium influx-mediated proteolytic processes in axons cannot be excluded. Adjunctive pharmacological therapy may be beneficial to patients with hydrocephalus.

Future directions for therapy of childhood hydrocephalus: a view from the laboratory.

A personal perspective on the study of experimental models of hydrocephalus is offered. Many animal models are available; each has its own advantages and disadvantages. Detailed study of more than one model is needed to clarify the pathogenesis of hydrocephalus-induced brain damage in the immature nervous system. Further information is needed about the mechanism of axonal injury in periventricular tissue, changes in the extracellular compartment, water dynamics within brain tissue, the role of neurotrophic factors in hydrocephalus, and the sites of injury in chronic 'arrested' hydrocephalus. Insight into the multifactorial nature of the brain damage may allow us to develop supplemental pharmacologic therapies, which could protect the brain and promote recovery in the pre- and postshunt period.

Correlation of cerebral hypoxic-ischemic T2 changes with tissue alterations in water content and protein extravasation.

BACKGROUND AND PURPOSE: Age-dependent changes in T2-weighted MR images have been reported in cerebral hypoxia-ischemia. However, the biophysical mechanisms responsible for the image changes remain poorly defined. We investigated whether cerebral hypoxia-ischemia-induced T2 changes correlate with alterations in either water content or protein extravasation. METHODS: One- and 4-week-old rats were subjected to unilateral carotid artery occlusion plus hypoxia in 8% oxygen. T2 images were acquired before, during, and 1 or 24 hours after hypoxia-ischemia. Blood-brain barrier disruption and brain edema were evaluated by immunohistological detection of IgG extravasation and measurement of water content by dry-wet weight and specific gravity methods. RESULTS: In 1-week-old rats, T2 values, areas of hyperintensity on T2-weighted images, and water content in the ipsilateral hemisphere increased during hypoxia-ischemia, recovered at 1 hour after hypoxia-ischemia, and increased again at 24 hours after hypoxia-ischemia. Extravasation of IgG occurred during hypoxia-ischemia and remained detectable 24 hours after hypoxia-ischemia. In 4-week-old rats, an increase in T2 or extravasation of IgG did not occur until 24 hours after hypoxia-ischemia despite a comparable elevation in water content during and soon after hypoxia-ischemia. CONCLUSIONS: T2 imaging appears reliable for detecting edema associated with disruption of the blood-brain barrier but not necessarily an increase in cerebral water or plasma proteins alone. The different hypoxic-ischemic changes in T2 in immature and older brain are associated with differences in alterations in water content plus extravasation of protein, consistent with age-dependent differences in hypoxic-ischemic alterations in vascular permeability.

Efficacy of disodium 4-[(tert-butylimino)methyl]benzene-1,3-disulfonate N-oxide (NXY-059), a free radical trapping agent, in a rat model of hemorrhagic stroke.

Because free radical mechanisms may contribute to brain injury in hemorrhagic stroke, the effect of the free radical trapping agent disodium 4-[(tert-butylimino)methyl]benzene-1,3-disulfonate N-oxide (NXY-059) was investigated on outcome following intracerebral hemorrhage (ICH) in rat. ICH was induced in 20 adult rats by infusion of collagenase into the caudate-putamen. Thirty minutes later rats were treated with NXY-059 (50 mg/kg subcutaneous plus 8.8 mg/kg/h for 3 days subcutaneous delivered via implanted osmotic pumps) or saline (equivalent volumes). Magnetic resonance imaging 24 h after ICH confirmed that the hemorrhage was uniform in the two groups, and subsequent imaging at 7 and 42 days post-ICH showed that the hematoma resolved similarly in the two groups. Behavioral testing on days 1, 3, 7, 14, and 21 after ICH showed that rats treated with NXY-059 had significantly decreased neurological impairment at all times. Deficits in skilled forelimb use 4-5 weeks post-ICH, and in striatal function 6 weeks post-ICH, were not reduced by treatment with NXY-059. Treatment with NXY-059 significantly reduced the neutrophil infiltrate observed 48 h post-hemorrhage in the vicinity of the hematoma, and the number of TUNEL-positive cells 48 h post-hemorrhage at the hematoma margin. However, by 6 weeks there were no differences in neuronal densities in treated and control rats.

Aboriginals with multiple sclerosis: HLA types and predominance of neuromyelitis optica.

BACKGROUND: MS is common in people of northern European ethnicity who live in northern geographic areas; however, MS is rarely identified among aboriginal peoples living in the same areas. OBJECTIVES: To determine the prevalence, clinical features, HLA type, and viral infections associated with MS among aboriginals in Manitoba, Canada. METHODS: A retrospective study was performed in which the clinical features of all aboriginal patients with MS together with HLA type and human herpesvirus-6, HIV-1, human T-cell lymphotropic virus-1, and endogenous retrovirus associated with MS (MSRV) infections were analyzed and compared with results from nonaboriginal patients with MS. RESULTS: Seven aboriginals with MS were identified with a period prevalence among aboriginals of 40:100,000. Clinical features included relapsing-remitting (n = 6) or primary progressive (n = 1) phenotypes with aggressive disease courses and frequent involvement of optic nerves and spinal cord (n = 5) compared with nonaboriginal patients. Autopsy of one patient showed necrosis and eosinophil infiltrates in a cervical spinal cord lesion and a demyelinated optic nerve. Analysis of HLA alleles at the DRB1 and DQB1 loci indicated that the HLA types detected were common in aboriginals, but there were no HLA alleles previously associated with the development of MS. Analysis of the copy number of MSRV did not show differences among aboriginals and nonaboriginals with or without MS. CONCLUSIONS: Aboriginals of Algonkian background are at increased risk for an aggressive type of MS, resembling neuromyelitis optica, which is resistant to conventional MS treatments and occurs independently of HLA alleles previously associated with MS.

Long-term deficits following cerebral hypoxia-ischemia in four-week-old rats: correspondence between behavioral, histological, and magnetic resonance imaging assessments.

We examined whether following a hypoxic-ischemic insult in young animals there are long-lasting functional deficits that correlate either to histological tissue damage or to potential compensatory plasticity changes. Four-week-old rats were subjected to an episode of cerebral hypoxia-ischemia (right carotid artery occlusion + 30 min of hypoxia) or a sham operation. In hypoxic-ischemic animals there were gross neurological deficits 1, 24, and 48 h postinsult with recovery by 1 week. Behavioral deficits were observed in both the acquisition and the performance of a response duration differentiation test and a fine motor control test (staircase test) 3 months after the hypoxia-ischemia. Functional magnetic resonance imaging studies demonstrated less activation in the sensory-motor cortex of hypoxic-ischemic animals in response to left vs right forepaw stimulation 4 months postinsult. Histological assessment delineated striatal, cortical, and hippocampal damage in the hypoxic-ischemic hemisphere and a reduction in cortical thickness, bilaterally. GFAP immunoreactivity was increased in injured striatum and cortex. Neurofilament heavy chain (NF200) immunoreactivity was normally most intense in white matter and decreased in areas of ipsilateral cortical damage. Synaptophysin immunoreactivity was reduced around areas of infarction and somewhat increased in adjacent undamaged striatum and in layer IV of parietal cortex. The histological damage or chronic degenerative changes could account for much of the variance in functional outcome detected with sensitive behavioral tests so that overall the compensatory or plasticity changes evident within the juvenile brain are rather modest.

Effect of FK-506 on inflammation and behavioral outcome following intracerebral hemorrhage in rat.

Beginning 15 min after induction of intracerebral hemorrhage (ICH) by intrastriatal administration of collagenase, rats were treated intramuscularly with FK-506 (3 mg/kg) or with vehicle. Treatment was repeated daily for 7 days. MR imaging 1, 7, and 28 days post-ICH showed that treatment did not affect hematoma size or its subsequent resolution. Two days post-ICH, neutrophil infiltration around the hematoma was decreased in the FK-506-treated rats, as was the number of TUNEL-positive cells at the edge of the hematoma and in the peripheral region. The decreased inflammatory response was accompanied by functional improvement in the treated rats. The neurological deficit induced by the ICH (beam walking ability, postural reflex, spontaneous circling) was significantly decreased from 3 to 21 days post-ICH by treatment with FK-506. Skilled use of the forelimb ipsilateral to the ICH was improved and sensory neglect of the same limb was decreased 8-9 weeks post-ICH in rats treated with FK-506. However, neuronal loss assessed 9 weeks post-ICH was not different in the treated and untreated rats.