Changes in caveolin-1, caveolin-3 and vascular endothelial growth factor expression and protein content after botulinum toxin A injection in the right masseter muscle of dystrophin deficient (mdx-) mice.

Progressive muscle wasting, frequently associated with inflammation, muscle fibre degeneration and fibrosis, is a characteristic of DMD (Duchenne muscular dystrophy). Its most common used animal model, the mdx mouse, however can overcome muscle degeneration by regeneration processes and is for this reason not suitable to answer all scientific questions. The aim of this study was to evaluate the ability of botulinum toxin A (BTX-A) in breaking down muscle regeneration in mdx mice. For this purpose, the right masseter muscle of 100 days old mdx and healthy mice was paralyzed by a single specific intramuscular injection of BTX-A. After 21 days, right and left masseter and temporal muscles as well as tongue muscle were carefully dissected, and gene and protein expression of caveolin-1, caveolin-3 and vascular endothelial growth factor (VEGF) were determined using quantitative RT-PCR and Western blot technique. Statistics were performed using Student's t-test and Mann Whitney U-test (significance level: P ≤ 0.05). After BTX-A injection, in both mice strains and for all three studied genes, no significant differences in mRNA amount could be detected between treated and untreated masseter muscles. A significant increase in caveolin-1, caveolin-3 and VEGF mRNA expression could only be found in the right temporal muscle of control mice compared to the left side. All three investigated proteins were more frequent to be found in dystrophic masseter muscle samples compared to the corresponding control samples, whereas significant decreased caveolin-3 protein levels could only be detected in the treated masseter versus untreated masseter muscle of controls. In contrast to previous conclusions, with this study it was not possible to prove a BTX-A-induced dystrophic phenotype in control animals, in which only the known decreases of caveolin-3 protein expression could be verified due to denervation. At the same time, however, gene and protein expression in dystrophic mice was not changed after BTX-A injection.

Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice.

Duchenne Muscular Dystrophy (DMD) and its murine model, mdx, are characterized by Ca(2+) induced muscle damage and muscle weakness followed by distorted dentofacial morphology. In both, DMD patients and in mdx mice, could be proven so far that only the extraocular muscles (EOM) are not affected by muscular dystrophy. The EOMs are protected against calcium overload by enhanced expression of genes involved in the Ca(2+) homeostasis. We could recently demonstrate that masticatory muscles of mdx mice are differentially affected by muscle dystrophy. The dystrophic masseter and temporalis shows muscle histology comparable to all other skeletal muscles in this animal model, whereas dystrophic tongue muscles seem to develop a milder phenotype. Due to this fact it is to hypothesize that an altered Ca(2+) homeostasis seems to underlie the mdx masticatory muscle pathology. Aim of this study was to examine the mRNA and protein levels of the sarcoplasmic reticulum Ca(2+) ATPases SERCA1 and SERCA2, the plasma membrane Ca(2+) ATPases Atp2b1 and Atp2b4, the sodium/calcium exchanger NCX1, the ryanodine receptor 1, parvalbumin, sarcolipin, phospholamban and the L-type Ca(2+) channel alpha-1 subunit (Cacna1s) in Musculus masseter, temporalis, and tongue of 100 day old control and mdx mice. In mdx masseter muscle significant increased mRNA levels of NCX1 and Cacna1s were found compared to control mice. In contrast, the mRNA amount of RYR1 was significant reduced in mdx temporalis muscle, whereas ATP2b4 was significant increased. In mdx tongue a down-regulation of the ATP2b1, sarcolipin and parvalbumin mRNA expression was found, whereas the phospholamban mRNA level was significantly increased compared to controls. These data were verified by western blot analyses. Our findings revealed that mdx masticatory muscles showed an unequally altered expression of genes involved in the Ca(2+) homeostasis that can support the differences in masticatory muscles response to dystrophin deficiency.

Histological changes and changes in the myosin mRNA content of the porcine masticatory muscles after masseter treatment with botulinum toxin A.

OBJECTIVES: Botulinum toxin A (Botox) is increasingly used for treatment of muscle hyperfunction. For a better understanding of the possible morphologic and chewing changes in patients induced by a therapy with Botox, muscle fiber and myosin heavy chain (MyHC) mRNA alterations were examined in this animal study. MATERIALS AND METHODS: The investigation was carried out on 14-week-old pigs (seven treated animals, eight controls; calculated animal size with a power of 0.5). To initialise the total immobilisation of the right masseter, the Botox injection was distributed into ten areas. After a 56-day period, muscle tissue was taken from the left and right side of the masseter (three regions), temporal (two regions), medial pterygoid and geniohyoid muscles using a standardized method. The muscle fiber cross sections were examined immunohistochemically. Fiber staining was accomplished with antibodies to specific MyHC isoforms. The MyHC mRNA changes were analysed using real-time RT-PCR. RESULTS: Muscles adapt to such stress by changing fiber types and MyHC mRNA content. Paralysed masseters display atrophic changes while other masticatory muscles show hypertrophic changes. The results indicated that the typical distributions of type IIa und IIb fiber types in masticatory muscles were increased in the masseter muscles due to Botox application. On the other hand, the masseters without Botox in the treated group showed a significant increase of type I MyHC. CONCLUSIONS: Application of Botox may lead to uncontrolled structural changes in affected and unaffected muscles. CLINICAL RELEVANCE: Treatment of muscle hypertrophy with Botox may cause muscle imbalance.

Comparison of reference points in different methods of temporomandibular joint imaging.

PURPOSE: Conventional radiography is a well-established method for imaging of the temporomandibular joint (TMJ) structures. However, the dental computer tomography becomes more important for the visualization of teeth in the jaw-bone. The applicability of dental computer tomography for the visualization of the TMJ it not yet been proven. The aim of the study was to identify TMJ structures using reference points with the magnetic resonance imaging (MRI) and the computed tomography (CT). METHODS: In order to compare the visualization and measurement of the TMJ a total of eight human cadaver heads was examined with CT and MRI and analysed using reference points. RESULTS: In both imaging techniques the selected reference points and distances are well definable and allow objective evaluation of anatomical structures. The CT images display a clearly better contrast to noise ratio than the MR images. The distance measurement of different width and length showed significant correlation of both images techniques. CONCLUSIONS: In TMJ diagnostics, maximum information could be obtained using both imaging techniques together due to synergistic effects.

Low NaCl intake elevates renal medullary endothelin-1 and endothelin A (ETA) receptor mRNA but not the sensitivity of renal Na+ excretion to ETA receptor blockade in rats.

AIMS: This study was performed to investigate the effects of NaCl intake on renal mRNA expression of pre-pro-endothelin-1 (ET-1), endothelin A (ET(A)) and endothelin B (ET(B)) receptors as well as on renal ET-1 content in rats. We further tested for NaCl intake-dependent differences in the contribution of the ET system to renal sodium handling. METHODS: Male Sprague-Dawley rats with telemetric devices were randomized to 0.15%, 0.60% and 1.80% NaCl diets with or without losartan. Renal sodium balance and arterial pressure were monitored. Renal blood flow and fractional sodium excretion (FENa) were measured in response to acute infusion of ET(A) and ET(B) blockers into the inner stripe of the outer renal medulla. RESULTS: Medullary pre-pro-ET-1, ET(A) and ET(B) receptor mRNA was 50%, 81% and 33% higher in rats on 0.15% vs. 1.80% NaCl. Losartan reduced medullary gene expression in rats on 0.15% NaCl. Medullary ET-1 content was 983 +/- 88 and 479 +/- 42 ng mg(-1) protein in rats on 0.15% and 1.80% NaCl (P < 0.001). Chronic ET(A) receptor blocker treatment reduced arterial pressure by 8-10 mmHg in rats on 0.15% vs. 1.80% NaCl without affecting renal sodium balances. Acute medullary ET(A) or ET(B) receptor blockade did not alter medullary blood flow and FENa in animals on either diet. CONCLUSION: In rats renal medullary ET-1 content and mRNA expression of three ET system components are inversely related to NaCl intake. Higher expression levels on low NaCl intake are AT(1) receptor dependent but are not associated with increased sensitivity of renal sodium handling to ET(A) receptor blockade.

Expression of glycodelin A in decidual tissue of preeclamptic, HELLP and intrauterine growth-restricted pregnancies.

During pregnancy, the placenta produces a variety of proteins that are responsible for the establishment of the foeto-maternal tolerance and circulation. The aim of this study was to investigate the expression of glycodelin A (formerly named PP14) in decidual tissue of placentas with intrauterine growth restriction (IUGR), preeclamptic patients, hemolysis, elevated liver, low-platelet (HELLP) patients and normal decidual tissue. Slides of paraffin-embedded decidual tissue of patients with IUGR, preeclamptic patients, HELLP patients and normal-term placentas were incubated with either polyclonal or monoclonal antibodies against glycodelin A. Staining reaction was performed with the ABC reagent. Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score. In addition, expression of glycodelin mRNA was analysed by in situ hybridisation. Expression of glycodelin A was significantly reduced in decidual cells of placentas with IUGR and HELLP, as investigated with both monoclonal and polyclonal antibodies and in situ hybridisation. However, preeclamptic decidual tissue showed no significantly different expression of intensity of glycodelin mRNA compared with normal placental tissue controls. A reduced expression of glycodelin A by decidual cells seems to be related to IUGR and HELLP. Therefore, glycodelin A might play an important role in the pathogeneses of these diseases.

Expression of multidrug transporters in dysembryoplastic neuroepithelial tumors causing intractable epilepsy.

OBJECTIVE: Dysembryoplastic neuroepithelial tumors (DNT) are relatively benign brain lesions that often cause medically intractable epilepsy. There is mounting evidence that multidrug transporters such as P-glycoprotein (P-gp) or multidrug resistance-associated proteins (MRP) play an important role in the development of resistance to antiepileptic drugs (AED). MATERIAL AND METHODS: In the present study, we examined the expression of several multidrug transporters in 14 cases of DNT. The peritumoral brain tissue as well as 9 cases of arteriovenous malformations (AVM) served as controls. P-gp, MRP2, MRP5 and breast cancer resistance protein (BCRP) expression was evaluated qualitatively and quantitatively using immunohistochemistry. RESULTS: All transporters were overexpressed quantitatively in DNT, but each revealed a different labeling pattern. P-gp and BCRP were predominantly located in the endothelium of brain vessels. MRP5 was detected primarily in endothelial cells, but notably also in neurons. The expression of P-gp, MRP2 and MRP5 was low in AVM, whereas BCRP demonstrated strong staining. Examination of MDR1 gene polymorphisms revealed no correlation with P-gp expression whereas the MRP2 exon 10 G1249A polymorphism was associated with different MRP2 labelling. CONCLUSIONS: Our results show that multidrug transporters are overexpressed in DNT. This finding supports the view that several of these transport proteins may play an important role in the mechanisms of drug resistance in epileptic brain tissue.

Verapamil regulates activity and mRNA-expression of human beta-glucuronidase in HepG2 cells.

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil. To further explore this phenomenon, we investigated the effect of verapamil on beta-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2. Treatment of HepG2 cells with verapamil revealed down-regulation of beta-gluc activity, protein, and mRNA level down to 50% of the control with EC(50) values of 25 microM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human beta-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by beta-glucuronidase.