Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in mdx mouse brain

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within its developing cerebral cortex. In this study, with the aim of elucidating the mechanisms of vascular involvement during brain impairment in Duchenne muscular distrophy (DMD), we have correlated the vascular density with VEGF and VEGF receptor-2 (VEGFR-2) expression in the brain cortex of normal and mdx mouse, an animal model with a genetic defect in a region homologous with the human DMD gene. Results showed that in mdx mouse, tissue area occupied by microvessels positive to factor VIII related antigen and VEGFR-2 increased in parallel to the tissue area occupied by neurons positive to VEGF. Our data suggest that increased vascularity in the brain of mdx mouse may be due, at least in part, to proliferation of endothelial cells in response to VEGF secreted by neuronal cells.     

Behavioral characterization of mdx3cv mice deficient in C-terminal dystrophins.

Cognitive deficits are frequently associated with Duchenne muscular dystrophy (DMD). They might be due to a deficiency in the brain isoforms of the 427 kDa full-length dystrophin, and/or to altered expression of other C-terminal dystrophin-gene products (Dp71, Dp140) also found in brain. Mdx mice, which only lack full-length dystrophin in both muscle and brain, were previously shown to have moderate learning and memory deficits. In the present study, we investigated behavioral responses in mdx3cv mutants, which have altered expression of all the dystrophin-gene products. Contrary to the original mdx mice, mdx3cv mice showed enhanced anxiety-related behaviors and reduced locomotion as compared to control mice. Although those perturbations might be related to the lack in C-terminal dystrophins, they do not seem sufficient to induce strong learning deficits in this mutant. Indeed, we showed that mdx3cv mice may display similar or weaker deficits during the learning of a bar-pressing task, as compared to mdx mice. The relevance of the mdx3cv mutant as a model to study the cognitive deficits associated with DMD is discussed.     

Development of new therapy on muscular dystrophy]

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder caused by a defect in the DMD gene. We have previously reported that micro-dystrophins, which have large deletions in rod repeat domain, successfully localize at the sarcolemma and stabilize dystroglycan-sarcoglycan complex in dystrophin-deficient mdx muscle. However, expression of a 3.7-kb micro-dystrophin cDNA, having only one rod repeat showed no effect on dystrophic phenotype. Further transgenic experiments are carrying to seek a functional but small-sized micro-dystrophin cDNA, which can be accommodated into Adeno-associated virus (AAV) vector. In normal muscle, AAV-LacZ vector expresses stably beta-gal for a long period, however, we noticed that immune response is evoked by AAV-LacZ vector in mdx muscle. Therefore, for successful gene therapy, it is required to reduce immune response against AAV-dystrophin vector and therapeutic proteins in mdx mice. We have already reported that utrophin was up-regulated at the sarcolemma of mdx mice, when a beta-galactosidase-expressing adenovirus vector, AxCALacZ was injected into the skeletal muscle. Moreover, up-regulated utrophin mitigated dystrophic phenotypes. Up-regulation of utrophin was induced by inflammatory response against adenovirus vector-mediated gene transfer and this up-regulation is one of promising tools for treatment of DMD.     

Facilitated CA1 hippocampal synaptic plasticity in dystrophin‐deficient mice: Role for GABAA receptors?

Duchenne muscular dystrophy (DMD) is associated with cognitive deficits that may result from a deficiency in the brain isoform of the cytoskeletal membrane-associated protein, dystrophin. CA1 hippocampal short-term potentiation (STP) of synaptic transmission is increased in dystrophin-deficient mdx mice, which has been attributed to a facilitated activation of NMDA receptors. In this study, extracellular recordings in the hippocampal slice preparation were used first to determine the consequences of this alteration on short-term depression (STD). STD induction was facilitated in mdx as compared with wild-type mice in a control medium. Because brain dystrophin deficiency results in a decreased number of gamma-aminobutyric acid A (GABAA)-receptor clusters, we tested the hypothesis that neuronal disinhibition contributes to the enhanced synaptic plasticity in mdx mice. We found that the GABAA receptor antagonist, bicuculline, increased basal neurotransmission in wild-type, but not in mdx mice and prevented the enhanced STP and STD in the CA1 area of slices from mdx mice. The possibility that altered GABA mechanisms underlie the facilitation of NMDA receptor-dependent synaptic plasticity in mdx mice is discussed.     

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.     

Distribution of dystrophin, nebulin and Ricinus communis I (RCA-I)-binding glycoprotein in tissues of normal and mdx mice

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene locus, appeared as an immunoreactive triplet of polypeptides in striated muscle tissues from normal mice on Western blot analysis. In smooth muscle tissues, an immunoreactive doublet of corresponding molecular weight was detected. No dystrophin was found in normal mouse brain, not even after enrichment for the Triton X-100 insoluble fraction. Dystrophin was absent from all corresponding tissues from the mdx mutant mouse strain which is known to lack dystrophin. The possibility that these immunoreactive bands represent isoforms is discussed. We have also investigated two other high molecular weight proteins which show secondary abnormalities in DMD muscle, namely nebulin and the 370 kDa Ricinus communis I lectin (RCA I)-binding glycoprotein. Nebulin levels were reduced in skeletal muscle from 6-week-old mdx mice but not in oesophagus from the same animals. By contrast, the RCA I-binding 370 kDa glycoprotein which is greatly reduced in DMD skeletal muscle was present in normal amounts in mdx skeletal muscle. These findings show, for the first time, that mdx myopathy differs from DMD myopathy not only morphologically, but also in its secondary biochemical abnormalities.     

Genetically dystrophic mdx/mdx mice exhibit decreased response to nicotine in passive avoidance

mdx mice are considered as a genetic homologous of human Duchenne muscular dystrophy. Recent evidence demonstrates that in mouse sympathetic ganglion dystrophin is involved in the stabilization of nicotinic acetylcholine receptor clusters. The purpose of this study was to verify possible effects of dystrophin alterations at the central level. This was assessed by evaluating the response to nicotine administration in mdx and wild-type mice. Thus the effects of post-training nicotine administrations (0.1, 0.25 and 0.5 mg/kg) were tested in mice subjected to a passive avoidance memory task, that measures the ability of mice to remember on test day a shock received 24 h before. Nicotine enhanced memory in wild-type as well as in mdx mice. However, the doses needed to increase memory in mdx were higher than in wild-type. These results are discussed in terms of possible functional changes in central nicotinic acetylcholine receptor in mdx mice.     

Heat shock pretreatment attenuates sepsis-associated encephalopathy in LPS-induced septic rats

Sepsis is the most common cause of mortality in intensive care units. Although sepsis-associated encephalopathy (SAE) is reported to be a leading manifestation of sepsis, its pathogenesis remains unclear. In our previous studies, we showed that heat shock pretreatment can reduce mortality in polymicrobial septic rats and protect the cerebral cortical function during hypoxia or drug-induced convulsion. In the present study, we investigated to what extent heat shock pretreatment might affect the development of SAE in septic rats and the possible mechanism behind its effect was discussed. To do this, we used lipopolysaccharide (LPS) to induce septic response in a SAE animal model. Heat shock pretreatment was performed and rectal temperature maintained between 41 and 42 °C for 15 min using an electric heating pad. Electroencephalography (EEG) activity, a sensitive electrophysiological recording of electrical activity in the brain, was used as an indicator of cerebral cortical dysfunction in SAE. In LPS rats not pretreated with heat shock, the EEG background activity decreased 10 min after intraperitoneal administration of LPS. However, in rats pretreated with heat shock, this decrease was significantly attenuated. Untreated septic rats were also found to have earlier, more frequent epileptic spikes. In summary, we found that heat shock could attenuate the electro-cortical dysfunction in rats with LPS-induced septic response, suggesting that heat shock response might potentially be used to prevent SAE in sepsis.     

GABAergic miniature spontaneous activity is increased in the CA1 hippocampal region of dystrophic mdx mice

Duchenne muscular dystrophy (DMD), a genetic disease due to dystrophin gene mutation and characterised by skeletal muscle failure, is associated with non-progressive cognitive deficits. In human and mouse brain, full-length dystrophin is localised postsynaptically in neocortical, hippocampal and cerebellar neurons. Evidence obtained in the CNS of dystrophic mice (mdx) suggested alterations of the GABAergic system. However, a direct functional evaluation of GABAergic synaptic transmission in mdx mice has not been conducted in the hippocampus, which is involved in cognitive processes and is rich in full-length dystrophin. Here, we investigated evoked and miniature inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons of mdx mice with patch clamp recording techniques. Results showed an increased frequency of miniature spontaneous IPSCs in mdx mice compared with controls, whereas evoked IPSCs did not show significant variations. Paired-pulse facilitation (PPF) analysis showed lack of facilitation at short intervals in mdx mice compared with that in wild-type mice. Analysis of density of synapses that innervate CA1 pyramidal cell bodies did not indicate significant differences between mdx mice and controls. Therefore, we suggest that increased miniature spontaneous IPSC frequency is due to altered pre-synaptic release probability. The present findings are discussed in the light of the accrued evidence for alterations of inhibitory synaptic transmission in the brain of dystrophic mice.     

Passive avoidance behaviour deficit in the mdx mouse

Thirty per cent of boys with Duchenne muscular dystrophy (DMD) suffer from various degrees of mental retardation. Since dystrophin, the protein absent in muscles of boys with DMD, is produced also in the brain, it was postulated that the deficiency of brain dystrophin might account for the mental retardation found in DMD boys. The mdx mouse, a mouse model of DMD, fails to produce dystrophin in muscle and brain. This prompted us to study the cognitive function of these animals. Learning and memory processes were studied in 10 mdx females and 9 genetically matched controls using the passive avoidance test. Statistically significant differences in the retention of the passive avoidance response was detected between mdx and control mice, indicating an impairment in passive avoidance learning in mdx mice. Our data reinforce the view that brain dystrophin deficiency is correlated with cognitive dysfunction and indicate that mdx mice might be a model for the mental retardation found in DMD boys.     

Pharmacological control of cellular calcium handling in dystrophic skeletal muscle

Duchenne muscular dystrophy arises due to the lack of the cytoskeletal protein dystrophin. In Duchenne muscular dystrophy muscle, the lack of dystrophin is accompanied by alterations in the dystrophin-glycoprotein complex. We and others have found that the absence of dystrophin in cells of the Duchenne muscular dystrophy animal model, the mdx mouse, leads to elevated Ca(2+) influx and cytosolic Ca(2+) concentrations when exposed to stress. We have also shown that alpha-methylprednisolone, the only drug used successfully in the therapy of Duchenne muscular dystrophy, and creatine lowered cytosolic Ca(2+) levels in mdx myotubes. It is likely that chronic elevation of [Ca(2+)] in the cytosol in response to stress is an initiating event for apoptosis and/or necrosis in Duchenne muscular dystrophy or mdx muscle and that alterations in mitochondrial function and metabolism are involved. Other cellular signalling pathways (e.g. nitric oxide) might also be affected.     

Sustained poststimulus elevation in cerebral oxygen utilization after vascular recovery.

The brain's response to functional activation is characterized by focal increases in cerebral blood flow. It is generally assumed that this hyperemia is a direct response to the energy demands of activation, the so-called flow-metabolism coupling. Here we report experimental evidence that increases in oxygen metabolism can occur after activation without increases in flow. When using multimodality functional MRI (fMRI) to study visual activation in human brain, we observed a postactivation period of about 30 seconds during which oxygen consumption remained elevated, while blood flow and volume had already returned to baseline levels. The finding of such a prolonged and complete dissociation of vascular response and energy metabolism during the poststimulus period indicates that increased metabolic demand needs not per se cause a concomitant increase in blood flow. The results also show that the postactivation undershoot after the positive blood-oxygen-level-dependent hemodynamic response in fMRI should be reinterpreted as a continued elevation of oxygen metabolism, rather than a delayed blood volume compliance.     

A comparison of factors associated with collagen metabolism in different skeletal muscles from dystrophic (mdx) mice: impact of pirfenidone

Skeletal muscles in mdx mice exhibit differential degrees of pathological changes and fibrosis. The purpose of this study was to examine differences in various indices of collagen metabolism in skeletal muscles with widely different functions and activity profiles in mdx mice, and to determine whether pirfenidone would attenuate the development of fibrosis. Mice in the pirfenidone group were orally fed pirfenidone (500 mg/kg) daily for 4 weeks. Marked differences were noted in hydroxyproline concentration between muscles, which could not be explained solely by the level of type I collagen and transforming growth factor-beta1 (TGF-beta1) mRNA. In normal mice, matrix metalloproteinase (MMP)-2 mRNA was significantly higher in the gastrocnemius than in the diaphragm or genioglossus muscles, suggesting that collagen degradation plays an important role in regulating collagen accretion in skeletal muscle. In mdx mice, the levels of both MMP-2 and MMP-9 mRNA were significantly elevated relative to control, although the response was muscle specific. Pirfenidone treatment resulted in a significant reduction in the level of hydroxyproline concentration across all muscles, although the effect was small. Results from this study reveal intrinsic dissimilarities in collagen metabolism between functionally different skeletal muscles. Moreover, the pharmacological use of pirfenidone may be beneficial in preventing fibrosis in muscular dystrophy.     

Cerebral circulation and oxygen metabolism associated with subclinical periventricular hyperintensity as shown by magnetic resonance imaging

A combined magnetic resonance imaging and positron emission tomography study was performed on 21 patients with cerebrovascular risk factors but without neurological abnormalities. Our purpose was to investigate the hypothesis that periventricular hyperintensity (PVH) reflects ischemia. Periventricular hyperintensity was evaluated with a method we devised, and cerebral circulation and oxygen metabolism were evaluated with the oxygen-15 steady-state technique. We concluded that the brain with severe periventricular hyperintensity had abnormal circulation, although oxygen metabolism was not measurably affected. The role of a compensation mechanism under conditions of decreased oxygen supply was considered.     

Abnormal brain response of chronic schizophrenia patients despite normal performance during a visual vigilance task

Deficits of attention are common among individuals with schizophrenia (SZ) and are related both to genetic liability to the disorder and to functional outcome among patients. To explore the brain systems underlying these attentional abnormalities, we compared the response of nine patients with chronic SZ or schizoaffective disorder to that of 10 matched healthy individuals performing a simple visual vigilance task during functional magnetic resonance imaging. The two groups performed equivalently on the task. When the blood oxygen level dependent (BOLD) signal during identification of a target letter among similar-looking letters was compared to the response during fixation trials, both groups showed multiple clusters of significant brain response in widespread cortical regions. Compared with healthy participants, SZ patients showed a diminished response in the inferior frontal cortex and an abnormally enhanced response in right postcentral gyrus, right medial temporal lobe and left cerebellum. The results suggest that abnormalities of functional brain response to attentional tasks can be observed among patients with SZ even when behavioral performance is unimpaired, and provide further evidence that brain systems related to attention are likely to be involved in the pathophysiology of the disorder.     

Abnormal brain response of chronic schizophrenia patients despite normal performance during a visual vigilance task

Deficits of attention are common among individuals with schizophrenia (SZ) and are related both to genetic liability to the disorder and to functional outcome among patients. To explore the brain systems underlying these attentional abnormalities, we compared the response of nine patients with chronic SZ or schizoaffective disorder to that of 10 matched healthy individuals performing a simple visual vigilance task during functional magnetic resonance imaging. The two groups performed equivalently on the task. When the blood oxygen level dependent (BOLD) signal during identification of a target letter among similar-looking letters was compared to the response during fixation trials, both groups showed multiple clusters of significant brain response in widespread cortical regions. Compared with healthy participants, SZ patients showed a diminished response in the inferior frontal cortex and an abnormally enhanced response in right postcentral gyrus, right medial temporal lobe and left cerebellum. The results suggest that abnormalities of functional brain response to attentional tasks can be observed among patients with SZ even when behavioral performance is unimpaired, and provide further evidence that brain systems related to attention are likely to be involved in the pathophysiology of the disorder.     

Decreased blood flow and oxygen metabolism in the cerebellum, brain stem and thalamus in a case with Menkes kinky hair disease

Cerebral blood flow and oxygen metabolism were measured in a five-year-old boy with atypical Menkes kinky hair disease (MKHD) by using positron emission tomography (PET). The patient was diagnosed as having atypical MKHD because of low serum and urinary copper levels, and clinical symptoms. The CT revealed mild to moderate degrees of brain atrophy predominantly in the cerebellum. The PET demonstrated marked decreases of cerebral blood flow and oxygen metabolism in the cerebellum, brain stem and thalamus. These findings seem to reflect the neuropathological abnormalities observed in MKHD. PET seems to be more sensitive than CT in detecting abnormalities in the affected structures. However, because this case is atypical the question of whether typical cases show similar features on the PET remains.     

Regional cerebral blood flow reductions, heart failure and Alzheimer's disease

OBJECTIVES: To discuss whether there are similarities between the functional brain abnormalities detectable in association with the diagnoses of heart failure (HF) and Alzheimer's disease (AD), focusing particularly on neuroimaging findings in vivo. METHODS: Using an electronic database (Medline), we reviewed imaging studies that have evaluated resting cerebral blood flow (CBF), resting glucose metabolism or amyloid deposition in groups of subjects suffering AD or HF compared with healthy controls. RESULTS: Single photon emission computed tomography (SPECT) investigations have reported global CBF reductions in HF groups compared with controls. In one recent SPECT study using modern voxel-based methods for image analysis, regional CBF deficits in the pre-cuneus and posterior cingulate gyrus were detected in a sample of HF sufferers relative to controls. The regional distribution of functional deficits in the latter study was similar to that found in many positron emission tomography (PET) investigations of glucose metabolism at early AD stages, as well as in recent PET investigations of amyloid deposition in AD. DISCUSSION: Imaging studies have rarely investigated whether there are localized functional brain deficits in association with HF. Recent regional CBF SPECT data provide preliminary anatomic support to a view that AD-like brain changes may develop in HF patients, possibly as a consequence of chronic CBF reductions. Additional studies of larger HF samples are needed to confirm this possibility, preferably using PET measures that have afforded greater sensitivity and specificity to identify brain functional abnormalities associated with the diagnosis of AD, such as indices of glucose metabolism and amyloid deposition.