Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor. Methods: Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC), the Hammersmith Functional Motor Scale (HFMS), and the Barthel Index. Results: After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion: Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Effects of high-dose creatine supplementation on kidney and liver responses in sedentary and exercised rats

This study evaluated the effects of high-dose of short-term creatine supplementation (5g.kg(-1). day(-1) to 1 week) and long-term creatine supplementation (1g.kg(-1). day(-1) to 4-8 weeks) on kidney and liver structure and function of sedentary and exercised Wistar rats ( Exercise sessions consisted of swimming at 80% of maximal work load supported during 5 days per week with daily sessions of 60 minutes throughout the duration of the supplementation). Seventy-two animals ( 245 +/- 5g) were divided into four groups (n = 18): control diet Sedentary ( SED), Creatine diet Sedentary (CRE), control diet Exercised (EXE), and Creatine diet Exercised (EXECRE). Histological and blood biochemical studies were performed after one, four, and eight weeks of creatine supplementation and exercise ( n = 6). No differences were found when comparing SED, EXE and EXECRE groups for kidney and liver structure and function at one, four and eight weeks. However, the CRE group showed higher levels of creatinine (1.1 +/- 0.2 vs. 0.4 +/- 0.1 mg.dl(-1); p < 0.05), and urea ( 37 +/- 3 vs. 19 +/- 1 mg. dl(-1); p < 0.05) when compared with all others groups at four and eight weeks. At eight weeks, the CRE group presented increased levels of ALT (41 +/- 7 vs. 23 +/- 7 U.L(-1); p < 0.05), AST (89 +/- 6 vs. 62 +/- 5 U. L(-1); p < 0.05), GGT (8.0 +/- 0.9 vs. 3.9 +/- 1.0 U. L(-1); p < 0.05), and AP (125 +/- 10 vs. 69 +/- 9 U. L(-1); p < 0.05) also when compared with all others groups. Moreover, the CRE group demonstrated some structural alterations indicating renal and hepatic damage at four and eight weeks, respectively. These results suggest that long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

Characterization of Fiber Types in Different Muscles of the Hindlimb in Female Weanling and Adult Wistar Rats

We analyzed lesser diameter and distribution of fiber types in different skeletal muscles from female Wistar rats using a histoenzymology Myofibrillar Adenosine Tri-phosphatase (mATPase) method. Fragments from muscles were frozen and processed by mATPase in different pH. Adult and weanling rat soleus muscles presented a predominance of type I fibers and larger fiber diameters. In the plantar muscle in adult rats, the type IIB fibers demonstrated greater lesser diameter while in the weanling animals, types I and IIB fibers were larger. The plantar muscle of animals of both ages was composed predominantly of the type IID fibers. The type IID fibers were observed in similar amounts in the lateral gastrocnemius and the medial gastrocnemius muscles. Type IIB fibers showed predominance and presented higher size in comparison with other types in the EDL muscle. The present study shows that data on fiber type distribution and fiber lesser diameter obtained in adult animals cannot always be applied to weanling animals of the same species. Using the mATPase, despite the difficult handling, is an important tool to determine the different characteristics of the specific fibers in the skeletal muscle tissue.

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O(2)/5% CO(2), pH = 7.4, 37 degrees C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F(2 alpha) (2 x 10(-6) M). Intraluminal perfusion of adenosine diphosphate (10(-5) M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10(-5) M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10(-5) M) can be inhibited by site-specific administration of atropine (10(-5) M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Effect of Titanium Tetrafluoride and Amine Fluoride Treatment Combined with Carbon Dioxide Laser Irradiation on Enamel and Dentin Erosion

Objective: This in vitro study aimed to analyze the influence of carbon dioxide (CO(2)) laser irradiation on the efficacy of titanium tetrafluoride (TiF(4)) and amine fluoride (AmF) in protecting enamel and dentin against erosion. Methods: Bovine enamel and dentin samples were pretreated with carbon dioxide (CO(2)) laser irradiation only (group I), TiF(4) only (1% F, group II), CO(2) laser irradiation before (group III) or through (group IV) TiF(4) application, AmF only (1% F, group V), or CO(2) laser irradiation before (group VI) or through (group VII) AmF application. Controls remained untreated. Ten samples of each group were then subjected to an erosive demineralization and remineralization cycling for 5 days. Enamel and dentin loss were measured profilometrically after pretreatment, 4 cycles (1 day), and 20 cycles (5 days) and statistically analyzed using analysis of variance and Scheffe's post hoc tests. Scanning electron microscopy (SEM) analysis was performed in pretreated but not cycled samples (two samples each group). Results: After 20 cycles, there was significantly less enamel loss in groups V and IV and significantly less dentin loss in group V only. All other groups were not significantly different from the controls. Lased surfaces (group I) appeared unchanged in the SEM images, although SEM images of enamel but not of dentin showed that CO(2) laser irradiation affected the formation of fluoride precipitates. Conclusion: AmF decreased enamel and dentin erosion, but CO(2) laser irradiation did not improve its efficacy. TiF(4) showed only a limited capacity to prevent erosion, but CO(2) laser irradiation significantly enhanced its ability to reduce enamel erosion.

Low-Level Laser Intensity Application in Masseter Muscle for Treatment Purposes

Objective: This study evaluated with histochemical analysis how the number of laser applications can affect the masseter muscle. Background: In dentistry today, the laser is used in patients with temporomandibular disorders (TMDs), mainly for radiating pain in the masticatory muscles, whose origins may be associated with malocclusion, although the laser effects are not well understood on the cellular level. Materials and Methods: Thirty mice (HRS/J lineage) were randomly distributed into groups according to the number of laser applications (three, six, and 10). For each group of laser applications (experimental, n = 5), it was considered the control group (n = 5), which was not irradiated. All animals inhaled halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane, minimum 99%, Sigma Aldrich, India) before each laser irradiation performed on the left masseter muscle region, on alternate days with 20 J/cm(2), 40mW, for 20 sec. The muscle samples were collected for histochemical analysis with succinate dehydrogenase (SDH) enzyme 72 h after the last application. Results: (a) A decrease in area of light fibers type (35.91% +/- 6.9%; 32.08% +/- 6.3%, and 27.88% +/- 6.3%), according to the increase of laser applications (p < 0.05); (b) significant increase (p < 0.05) in the area of intermediate fibers, with an increase of laser application (11.08% +/- 3.9%; 16.52% +/- 5.7%, and 15.96% +/- 3.9%), although the increase with 10 applications was small; (c) area increase of dark fibers in the group with three laser applications (0.16% +/- 0.3%) (p < 0.05), and in groups with six and 10 laser applications, respectively (9.68% +/- 6.0% and 9.60% +/- 4.0%). Conclusions: The SDH enzyme activity revealed that the number of laser applications increases the metabolic pattern of the muscle fibers. A minimal difference in metabolic activity between six and 10 applications of a laser suggests that further analyses should be done to confirm that six applications are enough to produce the same clinical effects, thereby contributing data to professionals from different fields in regard to the cost-benefit ratio of this therapy.

Ultra-Structural Changes at the Apical Stop Irradiated with CO(2) Laser

Objective: This study evaluated ultra-structural dentine changes at the apical stop after CO(2) laser irradiation used during biomechanical preparation. Background: Most studies evaluating the sealing efficiency of CO(2) lasers have been carried out after apical root canal resections and retro-filling procedures. Methods: Sixty human canines were prepared with #1 to #6 Largo burs. The apical stops were established at 1 mm (n = 30) and 2 mm (n = 30) from the apex. Final irrigation was performed with 1% NaOCl and 15% EDTA followed by 20 ml of distilled and deionized water. Specimens were subdivided into three subgroups (n = 10 for each stop distance): GI-no radiation (n = 20); GII-3W potency (n = 20), GIII-5W potency (n = 20). After preparation, specimens were evaluated by scanning electron microscopy, with ultra-structural changes classified according to a scoring system based on six qualitatively different outcomes. Results: Statistical analysis using the Mann-Whitney test confirmed more intense results for the specimens irradiated at 5 W potency than at 3 W (p<0.0001). The Kruskal-Wallis test indicated that when using the same potencies (3 or 5 W) at 1 and 2 mm from the apex, there were no statistically significant differences in ultra-structural changes. Conclusions: Our results showed that ultra-structural changes ranged from smear layer removal to dentine fusion. As laser potency was increased from 3 to 5 W, ultra-structural changes included extensive fused lava-like areas sealing the apical foramen.