SOX3 duplication in a boy with 46,XX ovotesticular disorder of sex development and his 46,XX sister with atypical genitalia: Probable germline mosaicism.

Ovotesticular disorders of sex development (OT-DSD) are characterized by ovarian follicles and seminiferous tubules in the same individual, with a wide range of atypical genitalia. We report on two sibs with atypical genitalia and SRY-negative 46,XX DSD, OT-DSD was confirmed only in the boy, while the girl had bilateral ovaries. Chromosome microarray analysis (CMA) showed a 737-kb duplication at Xq27.1 including the entire SOX3 gene in both sibs, which was confirmed by quantitative real time PCR. Also, X chromosome inactivation assay showed random inactivation in both sibs. Whole exome sequencing revealed no pathogenic or likely pathogenic variant. CMA of the parents showed normal results for both, suggesting that germline mosaicism could be the reason of recurrence of this duplication in the siblings. Our results support a pathogenic role of SOX3 overexpression in 46,XX subjects leading to variable DSD phenotypes.

Articular cartilage degeneration and bone adaptation due to lack of dystrophin in mice.

INTRODUCTION: Duchenne muscular dystrophy is caused by the absence of dystrophin. This study aimed to investigate femoral morphological characteristics of lack of dystrophin in MDX mice, considering that this model, different from DMD patient, is not influenced by corticosteroids administration and limited ambulation. MATERIALS AND METHODS: Proximal femur of male 16-week-old Control and MDX mice were submitted to histological, morphometric (volume density of articular cartilage, compact bone, trabecular bone and bone marrow; articular cartilage layers area; articular cartilage cell area), and immunohistochemistry analysis for RUNX-2, RANK-L, MMP-2, MMP-9, Caspase-3 and KI-67. RESULTS: MDX showed loss of linearity of articular cartilage with subchondral bone transition and elevation of this subchondral bone to the articular surface when compared with control. In addition, MDX presented morphological difference in the pantographic network of collagen fibers. Volume density of trabecular bone tissue was higher in the MDX than Control, but volume density of articular cartilage was lower in MDX (p < 0.05). The articular cartilage layers and chondrocytes area were significantly smaller in MDX than Control. These results associated to MMPs and osteogenic markers of proximal femur revealed an adaptation process as a consequence of lack of dystrophin. CONCLUSIONS: The morphological changes observed in the bone tissue of the MDX may be not only secondary to muscle weakness or chronic use of corticosteroids but also our results indicate connections between decrease of cartilage thickness, collagen network alteration and consequent subchondral changes that may lead to articular cartilage degeneration and bone adaptation mechanism in MDX mice.

The Colombian Strain of Trypanosoma cruzi Induces a Proinflammatory Profile, Neuronal Death, and Collagen Deposition in the Intestine of C57BL/6 Mice Both during the Acute and Early Chronic Phase.

The objective of this study was to evaluate the histopathological changes caused by infection with the Colombian strain of Trypanosoma cruzi (T. cruzi) in the acute and chronic experimental phases. C57Bl/6 mice were infected with 1000 trypomastigote forms of the Colombian strain of T. cruzi. After 30 days (acute phase) and 90 days (early chronic phase) of infection, the animals were euthanized, and the colon was collected and divided into two parts: proximal and distal. The distal portion was used for histopathological analysis, whereas the proximal portion was used for quantification of pro- and anti-inflammatory cytokines. In addition, the weight of the animals and parasitemia were assessed. The infection induced gradual weight loss in the animals. In addition, the infection induced an increase in interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α) in the intestine in the acute phase, in which this increase continued until the early chronic phase. The same was observed in relation to the presence of intestinal inflammatory infiltrates. In relation to interleukin (IL)-10, there was an increase only in the early chronic phase. The Colombian strain infection was also able to induce neuronal loss in the myenteric plexus and deposition of the collagen fibers during the acute phase. The Colombian strain of T. cruzi is capable of causing histopathological changes in the intestine of infected mice, especially in inducing neuronal destructions. Thus, this strain can also be used to study the intestinal form of Chagas disease in experimental models.

COVID-19 and Lung Mast Cells: The Kallikrein-Kinin Activation Pathway.

Mast cells (MCs) have relevant participation in inflammatory and vascular hyperpermeability events, responsible for the action of the kallikrein-kinin system (KKS), that affect patients inflicted by the severe form of COVID-19. Given a higher number of activated MCs present in COVID-19 patients and their association with vascular hyperpermeability events, we investigated the factors that lead to the activation and degranulation of these cells and their harmful effects on the alveolar septum environment provided by the action of its mediators. Therefore, the pyroptotic processes throughout caspase-1 (CASP-1) and alarmin interleukin-33 (IL-33) secretion were investigated, along with the immunoexpression of angiotensin-converting enzyme 2 (ACE2), bradykinin receptor B1 (B1R) and bradykinin receptor B2 (B2R) on post-mortem lung samples from 24 patients affected by COVID-19. The results were compared to 10 patients affected by H1N1pdm09 and 11 control patients. As a result of the inflammatory processes induced by SARS-CoV-2, the activation by immunoglobulin E (IgE) and degranulation of tryptase, as well as Toluidine Blue metachromatic (TB)-stained MCs of the interstitial and perivascular regions of the same groups were also counted. An increased immunoexpression of the tissue biomarkers CASP-1, IL-33, ACE2, B1R and B2R was observed in the alveolar septum of the COVID-19 patients, associated with a higher density of IgE+ MCs, tryptase+ MCs and TB-stained MCs, in addition to the presence of intra-alveolar edema. These findings suggest the direct correlation of MCs with vascular hyperpermeability, edema and diffuse alveolar damage (DAD) events that affect patients with a severe form of this disease. The role of KKS activation in events involving the exacerbated increase in vascular permeability and its direct link with the conditions that precede intra-alveolar edema, and the consequent DAD, is evidenced. Therapy with drugs that inhibit the activation/degranulation of MCs can prevent the worsening of the prognosis and provide a better outcome for the patient.

Consequences of ankle joint immobilisation: insights from a morphometric analysis about fibre typification, intramuscular connective tissue, and muscle spindle in rats.

Orthosis immobilisations are routinely used in orthopaedic procedures. This intervention is applicable in bone fractures, ligament injuries, and tendonitis, among other disorders of the musculoskeletal system. We aimed to evaluate the effects of ankle joint functional immobilisation on muscle fibre morphology, connective tissue, muscle spindle and fibre typification triggered by a novel metallic orthosis. We developed a rodent-proof experimental orthosis able to hold the tibiotalar joint in a functional position for short and long terms. The tibialis anterior muscles of free and immobilised legs were collected and stained by histology and histochemistry techniques to investigate general muscle morphology, connective tissue and muscle fibre typification. Morphometric analysis of muscle cross-section area, fibre type cross-section area, fibre type density, percentage of intramuscular connective tissue, and thickness of the muscle spindle capsule were obtained to gain insights into the experimental protocol. We found that short- and long-term immobilisation decreased the cross-section area of the muscles and induced centralisation of myonuclei. The connective tissue of immobilised muscle increased after 2 and 4 weeks mainly by deposition of type III and type I collagen fibres in the perimysium and endomysium, respectively, in addition to muscle spindle capsule thickening. Type IIB muscle fibre was severely affected in our study; the profile assumed odd shapes, and our data suggest interconversion of these fibre types within long-term immobilisation. In conclusion, our protocol has produced structural and histochemical changes in muscle biology. This method might be applied to various rodent models that enable genetic manipulation for the investigation of muscle degeneration/regeneration processes.

Insulin Modulates Myogenesis and Muscle Atrophy Resulting From Skin Scald Burn in Young Male Rats.

BACKGROUND: Burn injuries (BIs) due to scalding are one of the most common accidents among children. BIs greater than 40% of total body surface area are considered extensive and result in local and systemic response. We sought to assess morphological and myogenic mechanisms through both short- and long-term intensive insulin therapies that affect the skeletal muscle after extensive skin BI in young rats. MATERIALS AND METHODS: Wistar rats aged 21 d were distributed into four groups: control (C), control with insulin (C + I), scald burn injury (SI), and SI with insulin (SI + I). The SI groups were submitted to a 45% total body surface area burn, and the C + I and SI + I groups received insulin (5 UI/Kg/d) for 4 or 14 d. Glucose tolerance and the homeostatic model assessment of insulin resistance index were determined. Gastrocnemius muscles were analyzed for histopathological, morphometric, and immunohistochemical myogenic parameters (Pax7, MyoD, and MyoG); in addition, the expression of genes related to muscle atrophy (MuRF1 and MAFbx) and its regulation (IGF-1) were also assessed. RESULTS: Short-term treatment with insulin favored muscle regeneration by primary myogenesis and decreased muscle atrophy in animals with BIs, whereas the long-term treatment modulated myogenesis by increasing the MyoD protein. Both treatments improved histopathological parameters and secondary myogenesis by increasing the MyoG protein. CONCLUSIONS: Treatment with insulin benefits myogenic parameters during regeneration and modulates MuRF1, an important mediator of muscle atrophy.

Are visual inspection and digital palpation reliable methods to assess ability to perform a pelvic floor muscle contraction? An intra-rater study.

AIMS: To investigate the intrarater reliability of visual inspection and digital palpation to classify women's ability to perform a voluntary pelvic floor muscle (PFM) contraction and the association between the two methods. METHODS: This was a test-retest clinical study including 44 women. The ability to perform a PFM voluntary contraction was evaluated two times in all participants using visual inspection and digital palpation. All analyzed participants were assessed with a 7-day interval between the two assessments and by the same examiner. Kappa's agreement coefficient was used to estimate the intrarater reliability, and Fisher's exact test was used to analyze association between the two methods. RESULTS: This study found a substantial intrarater reliability of visual inspection (k = 0.73; p < .001) and digital palpation (k = 0.74; p < .001). A significant association between visual inspection and digital palpation was found at both time points (p < .001). CONCLUSION: Both visual inspection and digital palpation have substantial intrarater reliability and visual inspection can be recommended when vaginal palpation is not tolerated.

Sleep Deprivation Interferes with JAK/STAT Signaling Pathway and Myogenesis in the Masseter Muscle of Rats.

OBJECTIVES: The aim of the study was to study the Janus kinase/tyrosine kinase-activated transduction factor (JAK/STAT) signaling pathway and myogenesis on the masseter muscle after sleep deprivation and to investigate the role of stress in this scenario. SUBJECTS AND METHODS: A total of 18 male Wistar rats were divided into the following groups: control (n = 6): animals were not submitted to any procedures, and paradoxical sleep deprivation and vehicle (PSD + V; n = 6): animals were subjected to PSD for 96 h and (PSD + MET; n = 6): animals were subjected to PSD for 96 h with administration of metyrapone. Paradoxical sleep deprivation was performed by the modified multiple platforms method. Histopathological analysis, histomorphometry, and immunohistochemistry were performed. RESULTS: The results showed the presence of inflammatory infiltrate in the PSD + V and PSD + MET groups and atrophy. Histomorphometry showed that the cellular profile area decreased, while cellular density increased in both experimental groups. Expression of p-STAT 3, MyoD, and MyoG increased in the PSD + V group, while the PSD + MET group showed increased expression of IL-6 and p-STAT 3. CONCLUSION: Our results suggest that sleep deprivation induces an inflammatory response and atrophy in the masseter muscle of rats.

Phosphatidylserine controls calcium phosphate nucleation and growth on lipid monolayers: A physicochemical understanding of matrix vesicle-driven biomineralization.

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.

Extensive burn injury causes bone collagen network alteration and growth delay related to RANK-L immunoexpression change.

PURPOSE: Extensive burn injury mainly affects children, and hypermetabolic state can lead to growth delay. This study aimed to investigate bone histopathological and morphometric aspects, collagen fibers network and the immunoexpression of biological markers related to bone development in a young experimental model for extensive burn. MATERIALS AND METHODS: A total of 28 male Wistar rats were distributed into Control (C) and subjected to scald burn injury (SBI) groups. Sham or injured animals were euthanized 4 or 14 days post-lesion and proximal epiphyses of the femur were submitted to histological, morphometric (thickness epiphyseal plate), and RUNX-2 and receptor activator of nuclear factor kappa- ß ligand (RANK-L) immunoexpression methods. RESULTS: Histopathological femoral findings showed delayed appearance of the secondary ossification center in SBI, 14 days post-injury. Collagen fibers 4 days after injury were observed in articular cartilage as a pantographic network with a transversally oriented lozenge-shaped mesh, but this network was thinner in SBI. Fourteen days after the injury, the pantographic network of collagen presented square-shaped mesh in C, but this aspect was changed to a wider mesh in SBI. Morphometric analysis of epiphyseal plate revealed that the SBI group had less thickness than the respective controls (p<0.05). RUNX-2 showed no difference between groups, but RANK-L score was higher in all SBI groups. CONCLUSIONS: Extensive burn injury causes delayed bone growth and morphological changes. Alterations in collagen network and enhancement in immunoreactivity of RANK-L result in increased osteoclastogenesis.

Paradoxical sleep deprivation induces differential biological response in rat masticatory muscles: Inflammation, autophagy and myogenesis.

BACKGROUND: The aim of this study was to evaluate whether sleep deprivation (SD) induces inflammation, autophagy and myogenesis in the following masticatory muscles: masseter and temporal. METHODS: In this study, 18 animals were randomly distributed into three groups: control group (CTL, n = 6), SD for 96 hours (SD96, n = 6), and SD for 96 hours and more 96 hours of sleep recovery (SD96 + R, n = 6). RESULTS: In the histopathological analysis, SD 96 was able to induce inflammation in masseter and temporal. Nevertheless, the lack of inflammatory process was evidenced to the masseter in the group SD96 + R. Upregulation of TNF-alpha production was detected in the SD96 group, while SD96 + R decreased TNF immunoexpression for both skeletal muscles evaluated. MyoD and myogenin increased in rats submitted to SD96. By contrast, the levels of MyoD decreased in the group SD96 + R. Myogenin pointed out high immunoexpression in SD96 + R groups. In temporal, pAkt decreased in animals submitted to SD96, but it increased in the group SD96 + R. The levels of LC3 protein increased in both skeletal muscles studied, and masseter decreased LC3 protein expression in the SD96 + R. CONCLUSION: In summary, our results demonstrate that SD is able to induce inflammation, atrophy and myogenesis in rat masticatory muscles, being more intense in temporal when compared to masseter.

Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites.

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8 pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.

Therapeutic effects of vaccine derived from amastigote surface protein-2 (ASP-2) against Chagas disease in mouse liver.

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi (T. cruzi) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C, caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdßGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

Short time insulin treatment post burn improves elastic-collagen rearrangement and reepithelization.

Extensive burn may cause acute resistance to insulin, which accentuates hypermetabolism, impairs glucose metabolism, immune dysfunction and risks of sepsis. To minimize these effects, insulin is used as a treatment. The purpose was to analyze the collagen-elastic arrangement effects of insulin on the burned skin. Wistar rats were assigned in groups: control (C); control with insulin (C + I); scald burn injury (SBI); and SBI with insulin (SBI+ I). SBI were submitted to 45% total body surface area burn and the insulin-treated groups received insulin (5 UI/Kg/day) for 4 or 14 days (d). Insulin levels, glucose tolerance test and HOMA index were determined. The skin sections were analyzed for histophatological and morphoquantitative data. Histopathological findings showed increased reepithelization of SBI+ I and formation of a new muscle layer after 14 days. In the collagen-elastic arrangement, insulin for 4 days increased the volume fraction (Vv) of thin collagen and elastic fibers. After 14 days, independently of injury, insulin decreased the elastic fibers. Insulin was able to reverse damages in the collagen-elastic rearrangement and stimulate reepithelization after 4 days. Untreated scald-burned animals showed higher Vv of thick collagen after 4 days, while those treated had a higher Vv of thin collagen. The Vv of elastic fibers was increased in SBI+ I for 4 days. In conclusion, insulin treatment was able to stimulate reepithelization. It also reversed the damages to the collagen-elastic arrangement in the scald-burned group, improving the organization of thin collagen and increasing the Vv of elastic fibers in the injured group treated with insulin for a short time, that is, for 4 days.

Angiotensin-(1-7) and Alamandine Promote Anti-inflammatory Response in Macrophages In Vitro and In Vivo.

The renin-angiotensin system (RAS) peptides play an important role in inflammation. Resolution of inflammation contributes to restore tissue homeostasis, and it is characterized by neutrophil apoptosis and their subsequent removal by macrophages, which are remarkable plastic cells involved in the pathophysiology of diverse inflammatory diseases. However, the effects of RAS peptides on different macrophage phenotypes are still emerging. Here, we evaluated the effects of angiotensin-(1-7) (Ang-(1-7)) and the most novel RAS peptide, alamandine, on resting (M0), proinflammatory M(LPS+IFN-γ), and anti-inflammatory M(IL-4) macrophage phenotypes in vitro, as well as on specific immune cell populations and macrophage subsets into the pleural cavity of LPS-induced pleurisy in mice. Our results showed that Ang-(1-7) and alamandine, through Mas and MrgD receptors, respectively, do not affect M0 macrophages but reduce the proinflammatory TNF-α, CCL2, and IL-1ß transcript expression levels in LPS+IFN-γ-stimulated macrophages. Therapeutic administration of these peptides in LPS-induced inflammation in mice decreased the number of neutrophils and M1 (F4/80lowGr1+CD11bmed) macrophage frequency without affecting the other investigated macrophage subsets. Our data suggested that both Ang-(1-7) and alamandine, through their respective receptors Mas and MrgD, promote an anti-inflammatory reprogramming of M(LPS+IFN-γ)/M1 macrophages under inflammatory circumstances and potentiate the reprogramming induced by IL-4. In conclusion, our work sheds light on the emerging proresolving properties of Ang-(1-7) and alamandine, opening new avenues for the treatment of inflammatory diseases.

Sleep deprivation induces pathological changes in rat masticatory muscles: Role of Toll like signaling pathway and atrophy.

The aim of this study was to evaluate the Toll like signaling pathway and atrophy after sleep deprivation (SD) in rat masticatory muscles: masseter and temporal. A total of 24 animals was distributed into three groups: Control group (CTL, n = 8), subjected to SD for 96 h (SD96, n = 8) and subjected to SD for 96 h more 96 h of sleep recovery (SD96 + R, n = 8). Histopathological analysis revealed the presence of acute inflammatory cells, congested vessels, fibrosis, and high cellularity in the skeletal muscle fibers from masseter and temporal submitted to SD. These morphological alterations were not observed in the control group since neither inflammatory cells nor congested vessels were observed to this group. In the group SD96 + R, the absence of inflammation was noticed to the masseter only. In this group, COX-2 and TNF-alpha downregulation were detected when comparing to control group. MyD88 and pIKK decreased in SD96 and SD96 + R groups being pNFKBp50 downregulatated in SD96 + R. MyD88 expression increased in rats submitted to SD96 and SD96 + R in temporal when compared to control group. On the other hand, pIKK decreased the protein expression in groups SD96 and SD96 + R while pNFKBp50 showed a decreased protein expression in group SD96 only. The activation of atrophy by means of MAFbx upregulation was detected in temporal muscle in SD96 and SD96 + R when compared to control. In summary, our results show that SD is able to induce morphological alterations in rat masticatory muscles. Toll like signaling pathway and atrophy play important roles in ethiopathogenesis induced by SD, being dependent of skeletal muscle type.

The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

Spontaneous physical activity and mediators of energy homeostasis in the hypothalamus of mice from 4 to 10 months of age.

NEW FINDINGS: What is the central question of this study? Is the initial decline of spontaneous physical activity (SPA) in mice related to impaired insulin and leptin signalling or brain-derived neurotrophic factor expression in the hypothalamus? What is the main finding and its importance? We showed that SPA started to decline at an early stage, concomitantly with an impairment of hypothalamic leptin signalling. Consequently, energy expenditure decreased and glucose tolerance worsened. Our results demonstrate the need to counteract the initial decline in SPA to avoid metabolic impairments and indicate the possible involvement of central leptin in the reduction in SPA with age. The biological control of physical activity is poorly understood. Age decreases insulin, leptin and brain-derived neurotrophic factor (BDNF) signalling in the hypothalamus, and all have been shown to modulate spontaneous physical activity (SPA). We investigated the age at which SPA starts to decline and whether this is associated with the emergence of hypothalamic insulin and leptin resistance and reduced BDNF expression. Spontaneous physical activity (and other parameters of locomotion) and energy expenditure were determined monthly in mice from the 4th to the 10th month of age. Metabolic and hypothalamic analyses were performed in 4-, 6- and 10-month-old mice. Spontaneous physical activity, distance travelled and speed of locomotion started to decrease in 6-month-old mice. The reduction in SPA became more evident from 8 months of age. Energy expenditure decreased from the 8th month. Hypothalamic BDNF protein expression and insulin signalling did not change throughout the time span studied. Leptin signalling decreased at 6 and 10 months compared with 4 months. Also, compared with 4 months, 6- and 10-month-old mice were glucose intolerant. In conclusion, SPA begins to decline in parallel with reduced hypothalamic leptin signalling. Metabolic impairment also manifests as SPA decreases, highlighting the need to understand the regulation of SPA in order to combat its decline.

Effects of different fluences of low-level laser therapy in an experimental model of spinal cord injury in rats.

The aim of this study was to evaluate the in vivo response of different fluences of low-level laser therapy (LLLT) on the area of the injury, inflammatory markers, and functional recovery using an experimental model of traumatic spinal cord injury (SCI). Thirty two rats were randomly divided into four experimental groups: control group (CG), laser-treated group 500 J/cm2 (L-500), laser-treated group 750 J/cm2 (L-750), and laser-treated group 1000 J/cm2 (L-1000). SCI was performed by an impactor equipment (between the ninth and tenth thoracic vertebrae), with a pressure of 150 kdyn. Afterwards, the injured region was irradiated daily for seven consecutive sessions, using an 808-nm laser, at the respective fluence of each experimental groups. Motor function and tactile sensitivity were performed on days 1 and 7 post-surgery. Animals were euthanized on the eighth day after injury, and the samples were retrieved for histological and immunohistochemistry analyses. Functional evaluation and tactile sensitivity were improved after LLLT, at the higher fluence. Additionally, LLLT, at 750 and 1000 J/cm2, reduces the lesion volume and modulates the inflammatory process with decrease of CD-68 protein expression. These results suggest that LLLT at higher doses was effective in promoting functional recovery and modulating inflammatory process in the spinal cord of rats after SCI.

Validation of the Italian version of the UNESP-Botucatu unidimensional composite pain scale for the assessment of postoperative pain in cattle.

OBJECTIVE: The aim of this study was to validate the Italian version of the UNESP-Botucatu Unidimensional Composite Pain Scale (UCPS-IV) for assessing postoperative pain in cattle. STUDY DESIGN: Video analysis and psychometric testing. ANIMALS: A total of 40 Nellore cattle, age 2-3 years, weighing 365±51 kg. METHODS: An English version of the scale was translated to Italian and back translated to English to ensure semantic equivalence. A total of four Italian observers and the researcher who developed the original scale (considered as the gold standard) analysed videos of 40 cattle previously subjected to orchiectomy (recorded for each cattle at four predetermined timepoints during the perioperative period) unaware of the videos' timepoint. They assigned a pain score [ranging from no pain (0) to severe pain (10)] using the Italian version of the scale and verified the need for analgesic treatment for each animal. The obtained scores were subjected to psychometric validity, responsiveness and reliability tests. RESULTS: The factor analysis of the scale resulted in a one-factor solution. Significant changes in pain scores in response to surgery and analgesics confirmed not only the content and construct validity, but also responsiveness. The agreement between the researcher who developed the original scale and the blinded observers and the correlation between the pain scores determined by the scale and those determined by three classical unidimensional pain scales confirmed criterion and concurrent validity. Internal consistency of the scale was excellent. Inter- and intrarater reliability ranged from moderate to good and from moderate to very good for all scale items, respectively, supporting reproducibility and stability. The cut-off point for rescue analgesia calculated by the receiver operating characteristic curve was > 3. CONCLUSIONS AND CLINICAL RELEVANCE: The results confirm that the UNESP-Botucatu UCPS-IV appears to be a valid and reliable tool for the assessment of postorchiectomy pain in the bovine species. The determination of a cut-off point for rescue analgesia is also helpful for guiding analgesic therapy.