Histological and mast cells evaluation after application of botulinum toxin type a in masseter muscles of rats.

IntroductionMusculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy.AimTo evaluate the induction of apoptosis and autophagy in BoNTA‐injected masseter muscle of adult mice.MethodologyUnilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC‐Universidad de Chile, #21446‐ODO‐UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2‐7d post‐injection and immunofluorescence at 7d post‐injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t‐test, one‐way ANOVA test, Mann‐Whitney test, or Kruskal‐Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4‐8; p <0.05).ResultsUnilateral injection of BoNTA did not change the relative levels of apoptosis‐related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86‐fold), non‐lipidated LC3 (1.66‐fold), and P62 (1.24‐fold) at 7 d post‐injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction.ConclusionsThe atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy‐ or apoptosis‐induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.

It has been reported that botulinum toxin type A (BoNT-A) produces structural changes in masticatory muscles. However, not all histomorphometric parameters affected by BoNT-A parameters have been assessed. This study investigated the histomorphometric changes in the masseter muscle of rats after a single injection of BoNT-A. Forty-four adult animals were randomly divided into control group (n = 22) and BoNT-A group (n = 22). Controls received a single dose of 0.14mL/kg of saline in masseter muscles, and the BoNT-A group received a 7U/Kg of BoNT-A. The groups received the same volume of injected substances. Animals were sacrificed on 7th (n = 5), 14th (n = 5), 21st (n = 5), 28th (n = 4) and 90th (n = 3) days post-treatment. Histological masseter tissue slides were obtained from hematoxylin-eosin treatment and analyzed in optical microscopy regarding muscle cross-sectional area, amount of connective tissue and quantity and diameter of myocytes. For statistical analysis, generalized linear models were used to compare the data (ANOVA). In all test, the significance level of 5% was set. BoNT-A values of cross-sectional area of the masseter muscle were significantly lower than controls (p < 0.01) throughout the study. Regarding myocytes quantity, BoNT-A subgroups presented higher values than controls (p < 0.0001) since the 14th day until the end of the study; however, the diameter of myocytes was smaller in all BoNT-A subgroups (p < 0.0001) in all assessment points. The amount of connective tissue was higher in BoNT-A subgroups (p < 0.0001) throughout the study. A single injection of BoNT-A altered the structure of masseter muscle of rats, regarding its histomorphometric parameters. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Botulinum toxin type A (BTX-A) reduces the muscular contractions by temporarily inhibiting the release of acetylcholine at the neuromuscular junction. The purpose of this study was to investigate the effects of the BTX-A injected into the masseter muscle of a developing rat mandible. Four-week-old male (no. 80) Sprague-Dawley rats were divided into four groups: control group, saline group, BTX-A group and baseline control group. Rats of baseline group were sacrificed at 0 day to provide baseline values of the mandibular measurements. The masseter muscle of rats in the saline and the BTX-A group were administered with saline and BTX-A solutions respectively. Experimental animals were sacrificed after 4 weeks. The BTX-A group demonstrated smaller mandibular dimension compared with the other groups (P < 0.05). Their condylar cartilages showed increased apoptosis at the proliferation stage of the reserve zone and masseter muscle fibers demonstrated atrophic changes. The result demonstrated BTX-A influence on inhibitory action of the developing mandible because of apoptosis at the proliferation stage of the reserve zone of the condylar cartilage in developing rat mandible.

To investigate the effects of botulinum toxin type A (BoNT-A) injection on jaw motor episodes during sleep in patients with or without orofacial pain who did not respond to oral splint treatment. Twenty subjects with a clinical diagnosis of SB completed this study. Ten subjects received bilateral BoNT-A injections (25 U per muscle) into the masseter muscles only (group A), and the other 10 received the injections into both the masseter and temporalis muscles (group B). Video-polysomnographic (vPSG) recordings were made before and at 4 weeks after injection. Rhythmic masticatory muscle activity (RMMA) and orofacial activity (OFA) were scored and analyzed for several parameters (e.g., frequency of episodes, bursts per episode, episode duration). The peak amplitude of electromyographic (EMG) activity in the two muscles was also measured. BoNT-A injection did not reduce the frequency, number of bursts, or duration for RMMA episodes in the two groups. The injection decreased the peak amplitude of EMG burst of RMMA episodes in the injected muscles (p < 0.001, repeated measure ANOVA) in both groups. At 4 weeks after injection, 9 subjects self-reported reduction of tooth grinding and 18 subjects self-reported reduction of morning jaw stiffness. A single BoNT-A injection is an effective strategy for controlling SB for at least a month. It reduces the intensity rather than the generation of the contraction in jaw-closing muscles. Future investigations on the efficacy and safety in larger samples over a longer follow-up period are needed before establishing management strategies for SB with BoNT-A. Shim YJ; Lee MK; Kato T; Park HU; Heo K; Kim ST. Effects of botulinum toxin on jaw motor events during sleep in sleep bruxism patients: a polysomnographic evaluation.

We estimated the number of mast cells in monkey lungs by both quantitative histologic examination and measurement of total lung histamine, and showed that monkey lungs contain between 10(7) and 10(8) mast cells, with approximately 83% of these being located in conducting airways, and 17% in the parenchyma. The number of mast cells found in each airway generation increased from approximately 60,000 in the trachea to 8 million in the terminal bronchioles. In airways from different generations the number of mast cells superficial to the basement membrane in the epithelium and lumen (EMC) was compared to the number of mast cells found in the submucosa between basement membrane and cartilage and to the number of those found outside the cartilage. The number of EMC varied between animals and ranged from 0-0.4% of the total number of mast cells in the trachea, to 0-27% of the total in the terminal bronchioles. On the average, EMC accounted for 12% of the total number of mast cells in conducting airways, where we calculate that there is approximately one EMC for every 100,000 epithelial cells. Eosinophils were distributed in close relation to mast cells in the mucosa and submucosa, but were rare outside the cartilage. We conclude that the number of mast cells increases from central to peripheral airways and that this may account for the marked peripheral airway response observed after antigen challenge.

Chronic allograft nephropathy is the major cause of long-term graft failure in human allografted kidney transplantation. In addition to macrophages and T lymphocytes, mast cells have been shown to increase in chronic allograft nephropathy. The present study examined tryptase-positive mast cells and microvessels in the allografted kidney. We selected 131 biopsy specimens obtained from 100 allografted, 14 non-grafted renal biopsy specimens and nine nephrectomy specimens due to renal cell carcinomas. Formalin-fixed, paraffin- embedded specimens were immunostained using primary antibodies for mast cell tryptase, mast cell chymase and CD34. The number of the mast cells and microvessels was counted in at least 20 high-power fields (10 x 40). Tryptase-positive mast cells outnumbered chymase, toluidine blue or naphthol-AS-D choloacetate-positive mast cells. The mean number of the tryptase-positive mast cells was significantly higher in the 36 specimens with chronic allograft nephropathy (5.1 +/- 3.5) among the grafted kidneys with other disease categories (P < 0.001). In the chronic allograft nephropathy, the mean numbers of mast cells was significantly higher in Ci 2 + Ci 3 (n = 20; 6.4 +/- 3.89) than in Ci 1 (n = 16; 3.6 +/- 2.65) (P < 0.01). In the non-grafted kidney, the number of mast cells was highest in the four specimens with diabetic nephropathy. Mast cells and microvessels were analysed in the two representative cases, which subsequently developed chronic allograft nephropathy. Both of the cases showed the highest number of mast cells in chronic allograft nephropathy. In contrast, the mean number of microvessels tended to decrease along with interstitial fibrosis. This study demonstrated clearly a close association between renal interstitial fibrosis and mast cells, which might play an important role in the development of chronic allograft nephropathy.

This study assessed the long-term effects of botulinum toxin type A (BoNT-A) in subjective pain, pain sensibility, and muscle thickness in persistent myofascial temporomandibular-disorder pain (MFP-TMD) patients. Fourteen female subjects with persistent MFP received BoNT-A treatment with different doses (10U-25U for temporalis muscle and 30U-75U for masseter muscle). The treatment was injected bilaterally in the masseter and anterior temporalis muscles in a single session. Clinical measurements included: self-perceived pain (VAS), pain sensibility (PPT), and muscles thickness (ultrasonography). Follow-up occurred 1, 3, 6, and 72 months after treatment for VAS and PPT and 1, 3, and 72 months for ultrasonography. For statistical analysis, the Friedman test with the Bonferroni test for multiple comparisons as a post hoc test was used for non-parametric repeated measures comparisons among the evaluation times. A 5% probability level was considered significant in all tests. VAS values presented a significant decrease throughout the study (p < 0.05). Regarding PPT values, a significant increase was found when comparing baseline data with post-treatment follow-ups (p < 0.05), and even though a significant decrease was found in muscle thickness when baseline values were compared with the 1- and 3-months assessments, no differences were found when compared with the 72 months follow-up (p > 0.05). A single injection of BoNT-A presents long-term effects in reducing pain in persistent MFP-TMD patients, and a reversibility of adverse effects on masticatory-muscle thickness.

BackgroundThe aim of this study was to investigate the effect of a botulinum toxin type A (BTX-A) injection in the masseter muscle using electromyography (EMG) in an animal model.MethodsTen male adult (>3 months of age) New Zealand white rabbits were used. Muscle activity was continuously recorded from 8 hours before to 8 hours after BTX-A injection. The rabbits received unilateral BTX-A injections of either 5 units (group 1, n = 5) or 20 units (group 2, n = 5).ResultsThe masseter muscle activity of the rabbits was significantly reduced immediately after BTX-A injection (P < 0.05 for both groups). When the results from group 1 were compared with those from group 2, only the peak voltage was significantly decreased in group 2 (P = 0.013).ConclusionMasseter muscle activity measured by EMG was immediately decreased after a BTX-A injection.

This study was performed in order to assess whether acute stress can increase mast cell and enterochromaffin (EC) cell numbers, and proteinase-activated receptor-2 (PAR2) expression in the rat colon. In addition, we aimed to investigate the involvement of corticotrophin-releasing factor in these stress-related alterations. Eighteen adult rats were divided into 3 experimental groups: 1) a saline-pretreated non-stressed group, 2) a saline-pretreated stressed group, and 3) an astressin-pretreated stressed group. The numbers of mast cells, EC cells, and PAR2-positive cells were counted in 6 high power fields. In proximal colonic segments, mast cell numbers of stressed rats tended to be higher than those of non-stressed rats, and their PAR2-positive cell numbers were significantly higher than those of non-stressed rats. In distal colonic segments, mast cell numbers and PAR2-positive cell numbers of stressed rats were significantly higher than those of non-stressed rats. Mast cell and PAR2-positive cell numbers of astressin-pretreated stressed rats were significantly lower than those of saline-pretreated stressed rats. EC cell numbers did not differ among the three experimental groups. Acute stress in rats increases mast cell numbers and mucosal PAR2 expression in the colon. These stress-related alterations seem to be mediated by release of corticotrophin-releasing factor.

Because mast cell proliferation has been reported in disorders of abnormal bone remodeling, we quantitated the number of mast cells in undecalcified section of iliac crest bone from 16 untreated women with postmenopausal osteoporosis and contrasted the findings to values from 10 normal women and 12 normal men. The mean number of marrow mast cells was greater in normal women than men (1.17 vs 0.40 cells per mm2, P less than 0.05). Compared to the normal women, osteoporotic women had a greater number of mast cells in the marrow (3.38 vs 1.17 cells per mm2, P less than 0.01). However, there was no statistically significant correlation between the number of marrow mast cells and either the patient's age or the severity of the bone lesions, as assessed by histomorphometry. Our findings confirm the association between increased numbers of mast cells and postmenopausal osteoporosis.

An association was found in rats of different strains between the susceptibility to EAU and the number of mast cells in the choroid of the eye. High responder rats (Lewis, CAR) had strikingly more choroidal mast cells than the low responder BN animals, whereas intermediate numbers of mast cells were found in the F1 hybrids of Lewis and BN (LBNF), which exhibited an average level of susceptibility to EAU. LeR rats, derived from the Lewis strain, developed EAU only when treated with B. pertussis, and their number of choroidal mast cells was only about 1/5 of that found in the Lewis rats. Unlike the differences in the number of choroidal mast cells, small variations were found in the skin mast cell numbers of the tested rats. It is proposed that the number of local mast cells may be one mechanism by which the susceptibility to an organ-specific autoimmune disease is genetically regulated.

Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymase- and tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72-73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activity in the upper dermis may lead to an imbalance in the biochemical regulatory systems.

Botulinum Toxin as a medical therapy was introduced by Dr Alan Scott more than 20 years ago. The first clinical applications of Botulinum Toxin type A (BT-A) were for the treatment of strabismus and for periocular spasms. Botulinum Toxin type A is often effective in small to moderate angle convergent strabismus (esotropia) of any cause, and may be an alternative to surgery in these cases. Botulinum Toxin type A may have a role in acute or chronic fourth and sixth nerve palsy, childhood strabismus and thyroid eye disease. The use of BT-A for strabismus varies enormously in different cities and countries for no apparent reason. Botulinum Toxin type A may be particularly useful in situations where strabismus surgery is undesirable. This may be in elderly patients unfit for general anaesthesia, when the clinical condition is evolving or unstable, or if surgery has not been successful. Botulinum Toxin type A can give temporary symptomatic relief in many instances of bothersome diplopia irrespective of the cause. Ptosis and acquired vertical deviations are the commonest complications encountered. Vision-threatening complications are rare. Repeated use of BT-A is safe.

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