The ultrastructure of muscle fibers and satellite cells in experimental autoimmune encephalomyelitis after treatment with transcranial magnetic stimulation.

In this study, we investigated the effect of transcranial magnetic stimulation (TMS) on the ultrastructure of muscle fibers and satellite cells in rats with experimental autoimmune encephalomyelitis (EAE). EAE-induced animals were treated with TMS (60 Hz at 0.7 mT) for 2 hours in the morning, once a day, 5 days a week, for 3 weeks, starting on day 15 post-immunization. The rats were sacrificed on day 36 post-immunization, and the soleus muscles were evaluated by light microscopy and transmission electron microscopy. Findings were compared with a non-treated EAE group. Electron microscopy analysis showed the presence of degenerated mitochondria, autophagic vacuoles, and altered myofibrils in non-treated EAE group. This correlates with the presence of acid phosphatase activity in muscle fibers and core-targetoid lesions with desmin immunohistochemistry. Most myonuclei in the EAE group showed apoptotic features. In contrast, EAE induced-TMS treated animals had less ultrastructural changes in the mitochondria and the myofibrils, together with less frequent apoptotic nuclear features. Peripheral desmin+ protrusions, as a marker of active satellite cells, were significantly increased in TMS-treated group. This correlates ultrastructurally with the presence of active features in satellite cells in the TMS group. In conclusion, the attenuation of ultrastructural alterations in muscle fibers and activation response of satellite cells caused by EAE indicated that skeletal muscle had a regenerative response to TMS.

Transcranial Magnetic Stimulation Improves Muscle Involvement in Experimental Autoimmune Encephalomyelitis.

Skeletal muscle is affected in experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis that produces changes including muscle atrophy; histological features of neurogenic involvement, and increased oxidative stress. In this study, we aimed to evaluate the therapeutic effects of transcranial magnetic stimulation (TMS) on the involvement of rat skeletal muscle and to compare them with those produced by natalizumab (NTZ). EAE was induced by injecting myelin oligodendrocyte glycoprotein (MOG) into Dark Agouti rats. Both treatments, NTZ and TMS, were implemented from day 15 to day 35. Clinical severity was studied, and after sacrifice, the soleus and extensor digitorum longus muscles were extracted for subsequent histological and biochemical analysis. The treatment with TMS and NTZ had a beneficial effect on muscle involvement in the EAE model. There was a clinical improvement in functional motor deficits, atrophy was attenuated, neurogenic muscle lesions were reduced, and the level of oxidative stress biomarkers was lower in both treatment groups. Compared to NTZ, the best response was obtained with TMS for all the parameters analyzed. The myoprotective effect of TMS was higher than that of NTZ. Thus, the use of TMS may be an effective strategy to reduce muscle involvement in multiple sclerosis.

Ultrasonographic and Histological Correlation after Experimental Reconstruction of a Volumetric Muscle Loss Injury with Adipose Tissue.

Different types of scaffolds are used to reconstruct muscle volume loss injuries. In this experimental study, we correlated ultrasound observations with histological findings in a muscle volume loss injury reconstructed with autologous adipose tissue. The outcome is compared with decellularized and porous matrix implants. Autologous adipose tissue, decellularized matrix, and a porous collagen matrix were implanted in volumetric muscle loss (VML) injuries generated on the anterior tibial muscles of Wistar rats. Sixty days after implantation, ultrasound findings were compared with histological and histomorphometric analysis. The muscles with an autologous adipose tissue implant exhibited an ultrasound pattern that was quite similar to that of the regenerative control muscles. From a histological point of view, the defects had been occupied by newly formed muscle tissue with certain structural abnormalities that would explain the differences between the ultrasound patterns of the normal control muscles and the regenerated ones. While the decellularized muscle matrix implant resulted in fibrosis and an inflammatory response, the porous collagen matrix implant was replaced by regenerative muscle fibers with neurogenic atrophy and fibrosis. In both cases, the ultrasound images reflected echogenic, echotextural, and vascular changes compatible with the histological findings of failed muscle regeneration. The ultrasound analysis confirmed the histological findings observed in the VML injuries reconstructed by autologous adipose tissue implantation. Ultrasound can be a useful tool for evaluating the structure of muscles reconstructed through tissue engineering.

Structural abnormalities in the human diaphragm in drowning and hanging deaths: preliminary results.

PURPOSE: Previous experiments in rats have indicated that there are histological changes in skeletal muscle in drowning deaths; these changes include muscle fibers that contain ragged red fibers (RRF). The purpose of this study was to examine whether these changes also occur in humans. METHODS: Histologic and histochemical examinations of three muscles (diaphragm, pectoralis, and psoas) were performed on 24 cadavers with three different causes of death: 8 drowning, 8 hanging, and 8 sudden cardiac disease. Muscle samples were stained with hematoxylin-eosin, MGT, nicotinamide adenine dinucleotide-tetrazolium reductase, succinate dehydrogenase, ATPase, and acid phosphatase via standard staining procedures. RESULTS: There were statistically significant differences in the detection of RRFs in these cohorts. Additionally, several other cytoarchitectural changes (whorled and core-like fibers) were observed in the diaphragm in the drowning cohort and to a lesser extent in the hangings. These structural abnormalities were not observed in the sudden cardiac disease deaths, thus suggesting a common mechanism for the production of these muscular changes that is not shared in the cardiac death group. The mechanism is most likely intense hypoxia and mechanical trauma of the respiratory muscles in the setting of active blood circulation with intense muscle contraction. CONCLUSIONS: Our results confirmed that there are histological changes in the diaphragm in drownings and, to a lesser extent, in hangings.

Histological Changes in Skeletal Muscle During Death by Drowning: An Experimental Study.

A diagnosis of drowning is a challenge in legal medicine as there is generally a lack of pathognomonic findings indicative of drowning. This article investigates whether the skeletal muscle undergoes structural changes during death by drowning. Eighteen Wistar rats were divided into 3 equal groups according to the cause of death: drowning, exsanguination, and cervical dislocation. Immediately after death, samples of the masseter, sternohyoid, diaphragm, anterior tibial, soleus, and extensor digitorum longus muscles were obtained and examined by light and electron microscopy.In the drowning group, all muscles except the masseter displayed scattered evidence of fiber degeneration, and modified Gomori trichrome staining revealed structural changes in the form of abnormal clumps of red material and ragged red fibers. Under the electron microscope, there was myofibrillar disruption and large masses of abnormal mitochondria. In the exsanguination group, modified Gomori trichrome staining disclosed structural changes and mitochondrial abnormalities were apparent under light microscopy; however, there was no evidence of degeneration. No alterations were observed in the cervical dislocation group.As far as we know, this is the first time that these histological findings are described in death by drowning and are consistent with rhabdomyolysis and intense anoxia of skeletal muscle.

Influence of Physical Exercise on the Rehabilitation of Volumetric Muscle Loss Injury Reconstructed with Autologous Adipose Tissue.

BACKGROUND: In volumetric muscle loss (VML) injuries, spontaneous muscle regeneration capacity is limited. The implantation of autologous adipose tissue in the affected area is an option to treat these lesions; however, the effectiveness of this therapy alone is insufficient for a complete recovery of the damaged muscle. This study examined the influence of treadmill exercise on the rehabilitation of VML injuries reconstructed with autologous adipose tissue, as a strategy to counteract the limitations of spontaneous regeneration observed in these injuries. METHODS: Forty adult male Wistar rats were divided into eight groups of five individuals each: normal control (NC), regenerative control (RC), VML control (VML), VML injury reconstructed with fresh autologous adipose tissue (FAT), exercise-rehabilitated control (RNC), exercise-rehabilitated regenerative control (RRC), exercise-rehabilitated VML injury (RVML), and exercise-rehabilitated VML injury reconstructed with fresh autologous adipose tissue (RFAT). Histological and histochemical staining techniques were used for the analysis of structural features and histomorphometric parameters of the tibialis anterior muscle. Grip strength tests were conducted to assess muscle force. RESULTS: Exercise rehabilitation decreased the proportion of disoriented fibers in RFAT vs. FAT group. The percentage of fibrosis was significantly higher in FAT and RFAT groups versus NC and RNC groups but did not vary significantly between FAT and RFAT groups. Overall, muscle grip strength and fiber size increased significantly in the exercise-rehabilitated groups compared to control groups. CONCLUSIONS: To conclude, rehabilitation with physical exercise tended to normalize the process of muscle repair in a model of VML injury reconstructed with fresh autologous adipose tissue, but it did not reduce the intense fibrosis associated with these injuries.

Experimental muscle injury: correlation between ultrasound and histological findings.

INTRODUCTION: In this study we correlated ultrasound findings with histological changes taking place during experimentally induced degeneration-regeneration in rat skeletal muscle. METHODS: Gastrocnemius muscles were injected with mepivacaine, and the progress of the muscle injury was monitored by ultrasound from day 1 to day 20. Muscles were extracted on the same days for histological examination. RESULTS: The degenerative phase was characterized by increased echogenicity in the injured area; thereafter, echogenicity gradually diminished during the regenerative phase, attaining normal levels by 20 days postinjection. By this stage, histological examination revealed that regeneration was complete. The heteroechoic texture observed from day 4 to day 10 appeared to reflect the coexistence of degenerative and regenerative processes. CONCLUSIONS: The results suggest that the degenerative and regenerative phases of muscle injury may be distinguished sonographically through differences in echogenicity and echotexture and, using Doppler ultrasound, differences in the degree of vascularization.

Antioxidant-like effects and protective action of transcranial magnetic stimulation in depression caused by olfactory bulbectomy.

We studied the effects of transcranial magnetic stimulation (TMS, 60 Hz and 0.7 mT for 4 h/day for 14 days) on oxidative and cell damage caused by olfactory bulbectomy (OBX) in Wistar rats. The levels of lipid peroxidation products and caspase-3 were enhanced by OBX, whereas it prompted a reduction in reduced glutathione (GSH) content and antioxidative enzymes activities. The treatment with TMS reverted towards normality the biomarkers indicative of oxidative stress and apoptosis. In conclusion, our data show that TMS induced a protection against cell and oxidative damage induced by OBX, as well as they support the hypothesis that oxidative stress may play an important role in depression.

Histology of skeletal muscle reconstructed by means of the implantation of autologous adipose tissue: an experimental study.

The purpose of this study was to determine the histological characteristics of a skeletal muscle reconstructed by means of the implantation of autologous adipose tissue following an experimentally-induced volumetric muscle loss. A cylindrical piece in the belly of the rat anterior tibial muscle was removed. In the hole, inguinal subcutaneous adipose tissue of the same rat was grafted. Animals were sacrificed 7, 14, 21, 28 and 60 days posttransplantation. Histological, histochemical, immunohistochemical and morphometric techniques were used. At all times analyzed, the regenerative muscle fibers formed from the edges of the muscle tissue showed histological, histochemical and immunohistochemical differences in comparison with the control group. These differences are related to delays in the maturation process and are related to problems in reinnervation and disorientation of muscle fibers. The stains for MyoD and desmin showed that some myoblasts and myotubes seem to derive from the transplanted adipose tissue. After 60 days, the transplant area was 20% occupied by fibrosis and by 80% skeletal muscle. However, the neo-muscle was chaotically organized showing muscle fiber disorientation and centronucleated fibers with irregular shape and size. Our results support the hypothesis that, at least from a morphological point of view, autologous adipose tissue transplantation favors reconstruction following a volumetric loss of skeletal muscle by combining the inherent regenerative response of the organ itself and the myogenic differentiation of the stem cells present in the adipose tissue. However, in our study, the formed neo-muscle exhibited histological differences in comparison with the normal skeletal muscle.

Denervated muscle extract promotes recovery of muscle atrophy through activation of satellite cells. An experimental study.

PURPOSE: The objective of the present study was to determine whether a denervated muscle extract (DmEx) could stimulate satellite cell response in denervated muscle. METHODS: Wistar rats were divided into 4 groups: normal rats, normal rats treated with DmEx, denervated rats, and denervated rats treated with DmEx. The soleus muscles were examined using immunohistochemical techniques for proliferating cell nuclear antigen, desmin, and myogenic differentiation antigen (MyoD), and electron microscopy was used for analysis of the satellite cells. RESULTS: The results indicate that while denervation causes activation of satellite cells, DmEx also induces myogenic differentiation of cells localized in the interstitial space and the formation of new muscle fibers. Although DmEx had a similar effect in nature on innervated and denervated muscles, this response was of greater magnitude in denervated vs. intact muscles. CONCLUSION: Our study shows that treatment of denervated rats with DmEx potentiates the myogenic response in atrophic denervated muscles.

Effect of testosterone on oxidative stress and cell damage induced by 3-nitropropionic acid in striatum of ovariectomized rats.

This paper evaluates the effects of testosterone (0.5 mg/kg subcutaneously (s.c.) for 8 days) on oxidative stress and cell damage induced by 3-nitropropionic acid (20 mg/kg intraperitoneally (i.p.) for 4 days) in ovariectomized rats. Gonadectomy triggered oxidative damage and cell loss, evaluated by the detection of caspase-3, whereas 3-nitropropionic acid increased the levels of oxidative stress induced by ovariectomy and prompted cell damage characterized by enhanced levels of lactate dehydrogenase. These changes were blocked by testosterone administration. Our results support the following conclusions: i) ovariectomy triggers oxidative and cell damage via caspase-3 in the striatum; ii) 3-nitropropionic acid exacerbates oxidative stress induced by ovariectomy and leads to cell damage characterized by increased levels of lactate dehydrogenase; iii) testosterone administration decreases oxidative stress and cell damage. Additionally, these data support the hypothesis that testosterone might play an important role in the onset and development of neurodegenerative diseases.

17 beta-Estradiol may affect vulnerability of striatum in a 3-nitropropionic acid-induced experimental model of Huntington's disease in ovariectomized rats.

The aim of present study was to clarify the role of female sex hormones in the development and course of neurodegenerative disease in an experimental model of Huntington's disease induced by 3-nitropropionic acid (NPA) (30 mg/kg intraperitoneally (i.p.)/day for 4 days) in ovariectomized rat. Gonadectomy prompted oxidative stress and cell death evaluated by the detection of caspase-3, whereas 3-nitropropionic acid enhanced the oxidative stress induced by ovariectomy and it triggered cell damage characterized by increases of LDH levels. These changes were prevented by administration of 17 beta-estradiol. Our findings suggested that: (i) ovariectomy induced oxidative stress and apoptosis in the brain; (ii) 3-nitropropionic acid exacerbated oxidative stress induced by ovariectomy and shifting cell to cell death; and (iii) 17 beta-estradiol administration decreased oxidative stress and cell death induced by ovariectomy and 3-nitropropionic acid. These results revealed that sex ovarian hormones play a important role in onset and development of neurodegenerative diseases, as well as neuroprotective effects of 17 beta-estradiol against the changes induced ovariectomy and ovariectomy plus 3-nitropropionic acid.

Muscular hypertrophy and atrophy in normal rats provoked by the administration of normal and denervated muscle extracts.

This study was conducted to determine the effects of extracts obtained from both normal and denervated muscles on different muscle types. Wistar rats were used and were divided into a control group and four experimental groups. Each experimental group was treated intraperitoneally during 10 consecutive days with a different extract. These extracts were obtained from normal soleus muscle, denervated soleus, normal extensor digitorum longus, and denervated extensor digitorum longus. Following treatment, the soleus and extensor digitorum longus muscles were obtained for study under optic and transmission electron microscope; morphometric parameters and myogenic responses were also analyzed. The results demonstrated that the treatment with normal soleus muscle and denervated soleus muscle extracts provoked hypertrophy and increased myogenic activity. In contrast, treatment with extracts from the normal and denervated EDL had a different effect depending on the muscle analyzed. In the soleus muscle it provoked hypertrophy of type I fibers and increased myogenic activity, while in the extensor digitorum longus atrophy of the type II fibers was observed without changes in myogenic activity. This suggests that the muscular responses of atrophy and hypertrophy may depend on different factors related to the muscle type which could be related to innervation.

Diatoms in forensic analysis: A practical approach in rats.

A diagnosis of drowning is a challenge in legal medicine, as there is generally a lack of pathognomonic findings indicative of drowning. Diatom analysis has been considered very supportive for a diagnosis of drowning, although the test is still controversial for some investigators. We assessed diatoms association with drowning in the peripheral tissues of drowned rats and the effects of the drowning medium on the diatom yield. A modified acid digestion method was optimised for diatom recovery in water and rat tissues. Eighteen adult Wistar rats were employed for the study, subdivided into six groups of three rats. Groups 1, 3 and 5 were drowned in seawater, lake water, or river water respectively, while groups 2, 4 and 6 were controls (immersed after death in seawater, lake water or river water respectively). Water samples were taken from the sea, lake and river in Málaga and Córdoba (Spain) for the purposes of diatomological mapping and drowning of the rats. Diatoms were successfully recovered from all water samples and matched with tissues of the drowned rats. There were significant differences in diatom numbers between control and test samples for all the tissues studied, as well as within test samples. Histological investigations conducted on lung samples obtained from drowned rats provided complementary and valuable information. This study demonstrates the feasibility of the diatom test as a reliable method for the diagnosis of drowning, especially if adequate precautions are taken to avoid contamination, and if interpretation of the analysis is performed in light of other complementary investigations.

Skeletal muscle findings in experimental autoimmune encephalomyelitis.

INTRODUCTION: Skeletal muscle is a target organ in multiple sclerosis, a chronic debilitating disease of the central nervous system caused by demyelination and axonal deterioration. Since the experimental autoimmune encephalomyelitis model reproduces the relapsing-remitting course found in most multiple sclerosis patients, this model was used to compare the histological features of skeletal muscle at onset with those observed at the start of the second relapse. MATERIAL AND METHODS: Histological, histochemical and ultrastructural changes, as well as biochemical oxidative damage and antioxidant-system markers, were examined in the soleus and extensor digitorum longus muscles of Dark Agouti rats in which experimental autoimmune encephalomyelitis had been induced by active immunization using myelin oligodendrocyte glycoprotein. RESULTS: Histological examination at disease onset revealed ragged-red fibers and ultrastructural evidence of mitochondrial degeneration. At the second relapse, neurogenic changes included a wide range of cytoarchitectural lesions, skeletal muscle atrophy and the appearance of intermediate fibers; however, differences were observed between soleus and extensor digitorum longus lesions. Biochemical tests disclosed an increase in oxidative stress markers at onset, which was more pronounced at the second relapse. CONCLUSIONS: Microscopic findings suggest that two patterns can be distinguished at disease onset: an initial phase characterized by muscle mitochondrial alterations, and a second phase dominated by a histological muscle pattern of clearly neurogenic origin.

Melatonin improves 3-nitropropionic acid induced behavioral alterations and neurotrophic factors levels.

OBJECTIVE: This study sought to determine whether melatonin causes changes in neurotrophic factors and it protects against the mycotoxin 3-nitropropionic acid (3-NP) in brain tissue. METHODS: Rats were given 3-NP over four consecutive days (20 mg/kg BW), while melatonin was administered over 21 days (1 mg/kg/BW), starting after the last injection of 3-NP. RESULTS: Rats treated with 3-NP displayed significant changes in neurotrophic factor (BDNF and GDNF) levels, together with alterations in behavior; they also displayed extensive oxidative stress and a massive neuronal damage. CONCLUSIONS: Melatonin improved behavioral alterations, reduced oxidative damage, lowered neurotrophic factor levels and neuronal loss in 3-NP-treated rats. These results suggest that melatonin exerts a neuroprotective action.

Protective effect of nicotine on oxidative and cell damage in rats with depression induced by olfactory bulbectomy.

We evaluated the effects of nicotine on cell and oxidative damage caused by olfactory bulbectomy (OBX). The rats were divided into seven groups as follows: i) control; ii) vehicle (6% ethanol); iii) treated with nicotine; iv) sham operated; v) olfactory bulbectomy (OBX); vi) OBX+vehicle; and vii) OBX+Nic. The OBX was performed using the trepanation of frontal bone. The olfactory bulbs were cut and removed without damage to the frontal cortex. Two weeks after surgery nicotine was administered chronically once daily for 14 days, intraperitoneally (i.p.) in doses of 1.5 mg/kg, two weeks after surgery. OBX caused an increase in lipid peroxidation products and caspase-3 but prompted a reduction in reduced glutathione (GSH) content and antioxidative enzyme activity. All these changes were reverted by treatment of nicotine (14 days). In conclusions: i) OBX induces oxidative stress and cell death by apoptosis; and ii) nicotine presents antidepressant and antioxidant effect. All these findings suggest that nicotine would be a therapeutic tool for depression, although more studies are needed in this area to define the appropriate treatment regime.

Effect of 17beta-estradiol on olfactory bulbectomy-induced oxidative stress and behavioral changes in rats.

The present study evaluated 17beta-estradiol (17betaE(2)) (2.5 mg/kg sc) effects on bilateral OBX-induced behavioral changes and oxidative stress. OBX in male Wistar rats produced an increase in lipid peroxidation products and a decline in reduced glutathione (GSH) content and glutathione peroxidase (GSH-Px) activity, together with an increase in caspase-3 activity. Additionally, OBX triggered changes of behavior such as an enhancement of immobility time in the forced swim test and hyperactivity in the open field test. These changes were reversed by treatment with 17betaE(2) (14 days). Our results reveled that 17betaE(2) has a protective effect against oxidative stress, cell damage and behavioral changes induced by OBX, and present antidepressant and antianxiety properties.

Transcranial magnetic stimulation attenuates cell loss and oxidative damage in the striatum induced in the 3-nitropropionic model of Huntington's disease.

An investigation was conducted on the effect of transcranial magnetic field stimulation (TMS) on the free radical production and neuronal cell loss produced by 3-nitropropionic acid in rats. The effects of 3-nitropropionic acid were evaluated by examining the following changes in: the quantity of hydroperoxides and total radical-trapping antioxidant potential (TRAP), lipid peroxidation products, protein carbonyl groups, reduced glutathione (GSH) content, glutathione peroxidase (GSH-Px), catalase and succinate dehydrogenase (SDH) activities; total nitrite and cell death [morphological changes, quantification of neuronal loss and lactate dehydrogenase (LDH) levels]. Our results reveal that 3-nitropropionic acid induces oxidative and nitrosative stress in the striatum, prompts cell loss and also shows that TMS prevents the harmful effects induced by the acid. In conclusion, the results show the ability of TMS to modify neuronal response to 3-nitropropionic acid.