BackgroundMotor neuron diseases (MND) represent a group of disorders that evolve with inexorable muscle weakness and medical management is based on symptom control. However, deeper characterization of non-motor symptoms in these patients have been rarely reported. MethodsThis cross-sectional study aimed to describe non-motor symptoms in MND and their impact on quality of life and functional status, with a focus on pain and sensory changes. Eighty patients (31 females, 55.712.9years old) with MND underwent a neurological examination, pain, mood, catastrophizing and psychophysics assessments [quantitative sensory testing (QST) and conditioned pain modulation (CPM)], and were compared to sex- and age-matched healthy controls (HC). ResultsChronic pain was present in 46% of patients (VAS=5.182.0). Pain of musculoskeletal origin occurred in 40.5% and was mainly located in the head/neck (51%) and lower back (35%). Neuropathic pain was not present in this sample. Compared to HC, MND patients had a lower cold detection threshold (p<0.002), and significantly lower CPM scores (4.9 +/- 0.2% vs. 22.1 +/- 0.2%, p=0.012). QST/CPM results did not differ between MND patients with and without pain. Pain intensity was statistically correlated with anxiety, depression and catastrophism, and spasticity scores were inversely correlated with CPM (=-0.30, p=0.026). ConclusionsPain is frequently reported by patients with MNDs. Somatosensory and CPM changes exist in MNDs and may be related to the neurodegenerative nature of the disease. Further studies should investigate the most appropriate treatment strategies for these patients. SignificanceWe report a comprehensive evaluation of pain and sensory abnormalities in motor neuron disease (MND) patients. We assessed the different pain syndromes present in MND with validated tools, and described the QST and conditioned pain modulation profiles in a controlled design.

Background: The expanded GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic abnormality in familial amyotrophic lateral sclerosis (FALS). Objective: To determine the C9ORF72 hexanucleotide repeat expansion in FALS patients from ALS Unit of São Paulo, Brazil. Patients and methods: Patients with FALS from the ALS Unit of Clinics Hospital, University of São Paulo Medical School, Brazil have been evaluated for the presence of an expanded (GGGGCC) in C9ORF72. A repeat-primed-PCR reaction was applied to provide a qualitative assessment of the expansions. PCR products were analyzed on an ABI3730 and visualized using GeneMapper-software. A cutoff of >30 repeats combined with a typical sawtooth pattern was considered pathologic. Results: Preliminaries results from 15 FALS patients (mean age of onset 51.40 ± 3.02 years) are shown. The repeat expansion was present in 5 FALS cases (33.3%) and 10 FALS did not present the pathologic expansion. One patient with C9ORF72 expansion presented a bulbar-onset and developed later a frontotemporal degeneration. Patients without C9ORF72 expansion had a spinal-onset disease. FALS patients with C9ORF72 expansion developed a later onset symptoms (54.80 ± 4.16 years) when compared to FALS without expansion (49.7 ± 4.07 years). A shorter lifespan was screen in C9ORF72 expansion carriers (5.0 ± 1.22 years) than C9ORF72 negative (9.10 ± 2.42 years). Conclusion: A high frequency of C9ORF7 expansion was detected in this partial report of a small FASL sample of São Paulo ALS Unit. Supported by FAPESP and CNPq, Brazil.

Myotubular and centronuclear myopathies (MTM/CNM) are congenital muscle diseases with a wide clinical spectrum and typical histopathological findings of nuclear centralizations. Four genes have been implicated in different clinical forms: MTM1 in severe X-linked cases, DNM2 in autosomal dominant adult-onset cases, BIN1 in autosomal recessive childhood-onset cases, and RYR1 in a few cases. Moreover, any of these genes can cause sporadic cases, and about half of all CNM/MTM cases end up without a molecular diagnosis. In this work, we present clinical, histopathological and preliminary molecular data of an original series of 27 Brazilian patients with CNM/MTM. Of these, 20 cases have features compatible with CNM and seven with MTM. All MTM patients had a typical history of X-linked inheritance with profound neonatal hypotonia and respiratory insufficiency and typical myotube-like features on muscle biopsy. None had acquired ambulation, and some are still ventilator-dependent. CNM cases had a wide phenotypical variation. Most cases were sporadic and had typical craniofacial weakness and hypotonia. However, age of onset varied from neonatal to adolescence and severity ranged from mild ambulatory cases to severe non ambulatory cases with respiratory compromise. In addition to typical CNM histopathological findings, we have found cases with fiber architecture disorganization (minicores, necklace fibers). Molecular study of these cases is still ongoing. A number of cases have mutations that have been previously described, such as E368K on DNM2 gene. Among the new mutations, we have found a nonsense mutation in MTM1 (c.139C>T) on a severe case of a 2-year old boy affected by MTM, and a heterozygous mutation in DNM2 (c.1279T>G) on a sporadic childhood-onset 17-year old girl. This work will help advance our knowledge of these diseases by establishing correlations between phenotypes and molecular mechanisms.

Neuraminidase-1 (Neu1) regulates the catabolism of sialoglycoconjugates in lysosomes. Congenital Neu1 deficiency in children is the basis of sialidosis, a neurosomatic disorder whose symptoms include hypotonia, muscle weakness and osteoskeletal deformities. Mice with Neu1 deficiency develop an atypical form of muscle degeneration characterized by abnormal fibroblast proliferation and expanded extra cellular matrix (ECM), with invasion of muscle fibers by ECM components, cytosolic fragmentation, vacuolar formation and muscle atrophy. The aim of this study is to investigate the role of Neu1 on muscle regeneration process. Experimentally, inflammatory response, muscle fiber maturation and fibrosis development during the muscle regeneration process induced by intramuscular administration of cardiotoxin were assessed in Neu1 deficient mice. Muscle fiber maturation was not affected in Neu1 mice during regeneration. However, there were an accumulation of inflammatory cells and increase of ECM components that might affect the normal muscle tissue morphology during the muscle regeneration process. The determination of Neu1 role on muscle physiology is important to understand the neuromuscular clinical manifestations reported in patients with Neu1 deficiency and the importance of lysosomes and the sialic acid metabolism on the physiopathogenesis of muscle diseases (sponsored by FAPESP no 2009/02937-4).

Sarcoglycanopathies are a group of autossomal recessive limb girdle muscular dystrophies (LGMD) caused by deficiency in one of the four alfa-, beta-, gama- and delta-sarcoglycans (SG) at muscle fiber sarcolemma. Mutations in individual sarcoglycans are responsible for LGMD-2C (gama-SG), LGMD-2D (alfa-SG), LGMD-2E (beta-SG) and LGMD-2F (delta-SG). The disease is characterized by progressive weakness leading to loss of ambulation, difficulties in breathing and often premature death, associated to high serum creatine kinase (CK) level. Diagnosis of sarcoglycanopathies is based on the histopathological and immunohistochemical analysis of muscle biopsy. Our aim was to report the clinical and histopathological findings in 17 Brazilian children (two pairs of siblings), four males and 13 females, with SG deficiency detected by immunohistochemical analysis. The age at onset of the disease ranged from 2 to 11 years, and the most common clinical presentation were difficulty in climbing stairs, frequent falls and walking on tiptoe. Three patients lost walking ability at 10, 14 and 12 years of age. The weakness predominated in pelvic girdles, and winging of the scapula, calf hypertrophy and scoliosis were also common. CK levels were markedly increased in all patients. Four patients had restrictive respiratory pattern, and three of them presented associated cardiac abnormalities without clinical symptoms. A dystrophic pattern on muscle biopsy was noted in all patients. In 15 of the 17 patients, muscle immunohistochemical analysis was available: all showed reduction or absence of sarcolemmal expression of one, two or more of the four sarcoglycans. Sarcoglycanopathies are severe forms of LGMD affecting predominantly children, and the diagnosis might be confirmed by the detection of the SG deficiency on muscle tissue. Additional molecular testing is required to determine the specific SG deficiency (Sponsored by FAPESP no. 2010/08902-5 and CNPq).

Secondary neurodegeneration takes place in the surrounding tissue of spinal cord trauma and modifies substantially the prognosis, considering the small diameter of its transversal axis. We analyzed neuronal and glial responses in rat spinal cord after different degree of contusion promoted by the NYU Impactor. Rats were submitted to vertebrae laminectomy and received moderate or severe contusions. Control animals were sham operated. After 7 and 30 days post surgery, stereological analysis of Nissl staining cellular profiles showed a time progression of the lesion volume after moderate injury, but not after severe injury. The number of neurons was not altered cranial to injury. However, same degree of diminution was seen in the caudal cord 30 days after both severe and moderate injuries. Microdensitometric image analysis demonstrated a microglial reaction in the white matter 30 days after a moderate contusion and showed a widespread astroglial reaction in the white and gray matters 7 days after both severities. Astroglial activation lasted close to lesion and in areas related to Wallerian degeneration. Data showed a more protracted secondary degeneration in rat spinal cord after mild contusion, which offered an opportunity for neuroprotective approaches. Temporal and regional glial responses corroborated to diverse glial cell function in lesioned spinal cord.

S100, a calcium-binding protein, and basic fibroblast growth factor (bFGF, FGF-2) are found predominantly in astrocytes in the central nervous system. Those molecules show trophic properties to neurons and are upregulated after brain lesions. The present study investigated the changes in the S100b and bFGF immunoreactivities after a partial lesion of the rat midbrain ascending dopamine pathways induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Stereological method revealed increases in the estimated total number and density of bFGF immunoreactive astroglial profiles in the ipsilateral pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA). Increases in the counts of astroglial S100b immunoreactive profiles were found in the striatum, SNc, and VTA mainly ipsilateral but also in the contralateral nuclei. These results open up the possibility that interactions between astroglial S100b and bFGF may be relevant to paracrine events related to repair and maintenance of remaining dopamine neurons following striatal 6-OHDA induced partial lesion of ascending midbrain dopamine pathway.

Recent findings have pointed out the role of neurotrophic factors in the survival and maintenance of neurons of the auditory system. Basic fibroblast growth factor (bFGF, FGF-2) is a potent neurotrophic molecule whose actions can be seen in the central and peripheral nervous systems. In the present study, FGF-2 immunoreactivity was analyzed in the auditory pathways of the adult rat, employing a well-characterized polyclonal antibody against FGF-2. In the cochlea, FGF-2 immunoreactivity was observed in the inner and outer hair cells of the organ of Corti, spiral ganglion neurons, spiral limbus, and stria vascularis. Stereological methods employing optical fractionator revealed the presence of 84.5, 15, and 0.5% of spiral ganglion neurons possessing FGF-2 immunoreactivity of strong, moderate, and weak intensity, respectively. In the central auditory pathways, FGF-2 immunoreactivity was found in the cytoplasm of the neurons of the cochlear nuclei, trapezoid body nuclei, medial geniculate nucleus, and inferior colliculus. The two-color immunoperoxidase method showed FGF-2 immunoreactivity in the nuclei of astrocytes throughout the central auditory pathway. Computer-assisted microdensitometric image analysis revealed higher levels of specific mean gray values of FGF-2 immunoreactivity in the trapezoid body and ventral cochlear nucleus and also in the spiral ganglion and inner hair cells. Sections incubated with FGF-2 antibody preabsorbed with human recombinant FGF-2 showed no immunoreaction in the majority of the studied regions, exhibiting only a slight immunoreactive product in the hair cells of the organ of Corti. Furthermore, no changes in immunoreactivity were observed in sections incubated with FGF-2 antiserum preincubated with human recombinant acidic FGF (FGF-1). The findings suggest that FGF-2 may exert paracrine and autocrine actions on neurons of the central and peripheral auditory systems and may be of importance in the mechanism of hearing diseases.

The authors have previously described astroglial activation in the ipsilateral nigrostriatal system and ventral tegmental area following small doses of 6-hydroxydopamine (6-OHDA) injected unilaterally in the striatum. This article further evaluated astroglial reactivity in several brain regions after striatal 6-OHDA-induced punctate lesion in the nigrostriatal pathway. Adult male Wistar rats received a unilatera1 stereotaxical injection of the 6-OHDA (8 μg/4 μl) in the neostriatum and sacrificed 1 or 3 weeks later. Control animals received only solvent. Immunohistochemistry was employed for visualization of the tyrosine hydroxylase (TH), marker for dopamine cells, and glial fibrillary acidic protein (GFAP), marker for astrocytes. TH immunoreactive terminals disappeared in the striatum close to the injection site and a disappearance of a small number of a defined population of dopamine cell bodies was observed in the ipsilateral pars compacta of the substantia nigra (SNc). No dopamine lesion was detected in the contralateral nigrostriatal pathway. Astroglial reaction was seen close to the lesion in the neostriatum and in the ipsilateral SNc of the 1 week 6-OHDA lesioned rats. Specific stereological tools employing point intercepts and rotator, revealed an increased presence of reactive astrocytes in many forebrain regions like frontal, parietal and piriform cortex, septum, neostriatum and SNc, bilaterally, and also corpus callosum after 1 week of 6-OHDA injection. The astroglial activation was characterized by increases in the size of the cell body and/or processes. Astrocytic reaction was found only in the ipsilateral nigrostriatal pathway by 3 weeks of 6-OHDA, a slight activation also remaining in the ipsilateral septum and piriform cortex. Astrocytic reaction was seen in the solvent-injected rats only in the neostriatum close to the needle track. The transient widespread astroglial reaction observed in many brain regions following a striatal injection of 6-OHDA may represent a global paracrine trophic response in the brain.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.