Congenital muscle dystrophy and diet consistency affect mouse skull shape differently.

The bones of the mammalian skull respond plastically to changes in masticatory function. However, the extent to which muscle function affects the growth and development of the skull, whose regions have different maturity patterns, remains unclear. Using muscle dissection and 3D landmark-based geometric morphometrics we investigated the effect of changes in muscle function established either before or after weaning, on skull shape and muscle mass in adult mice. We compared temporalis and masseter mass and skull shape in mice with a congenital muscle dystrophy (mdx) and wild type (wt) mice fed on either a hard or a soft diet. We found that dystrophy and diet have distinct effects on the morphology of the skull and the masticatory muscles. Mdx mice show a flattened neurocranium with a more dorsally displaced foramen magnum and an anteriorly placed mandibular condyle compared with wt mice. Compared with hard diet mice, soft diet mice had lower masseter mass and a face with more gracile features as well as labially inclined incisors, suggesting reduced bite strength. Thus, while the early-maturing neurocranium and the posterior portion of the mandible are affected by the congenital dystrophy, the late-maturing face including the anterior part of the mandible responds to dietary differences irrespective of the mdx mutation. Our study confirms a hierarchical, tripartite organisation of the skull (comprising neurocranium, face and mandible) with a modular division based on development and function. Moreover, we provide further experimental evidence that masticatory loading is one of the main environmental stimuli that generate craniofacial variation.

Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis.

INTRODUCTION: Cannabinoid receptor 2 (CB2R) expression is upregulated during sepsis. However, there are conflicting results regarding the effects of CB2R modulation in the hyperinflammatory phase of the disease. The aim of this study was therefore to investigate the effects of CB2R manipulation on leukocyte activation within the intestinal microcirculation in two acute experimental sepsis models. METHODS: In the endotoxemia model we studied four groups of Lewis rats: controls, lipopolysaccharide (LPS), LPS + CB2R agonist HU308 (2.5 mg/kg), and LPS + CB2R antagonist AM630 (2.5 mg/kg). In the colon ascendens stent peritonitis (CASP)-induced sepsis model we also studied four groups: sham group, CASP and CASP + CB2R agonist (HU308, 2.5 or 10 mg/kg). Intravital microscopy was performed 2 hours following LPS/placebo administration or 16 hours following CASP/sham surgery to quantify intestinal leukocyte recruitment. Additionally, hemodynamic monitoring, histological examinations and measurements of inflammatory mediators were performed. RESULTS: HU308 administration significantly reduced intestinal leukocyte adhesion in both acute sepsis models. The systemic levels of inflammatory mediators were significantly reduced by 10 mg/kg HU308 treatment in CASP animals. CONCLUSION: CB2R activation reduces leukocyte activation and systemic release of inflammatory mediators in acute experimental sepsis. Drugs targeting the CB2R pathway may have therapeutic potential in sepsis.

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (mdx) mice.

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechano-growth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.

Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P < 0.01; mean ± standard error of the mean (SEM), Student's unpaired t-test], as well as in the tongue muscle (65.7 versus 73.8 per cent; P < 0.05). Similarly, the content of MyHC-2x isoforms in mdx tongue muscle was lower than in the controls (25.9 versus 30.8 per cent; P < 0.05). The observed down-regulation of the MyHC-2x and MyHC-2b mRNA in the masticatory muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

Physostigmine reverses disturbances of the intestinal microcirculation during experimental endotoxemia.

Intestinal microcirculatory disturbances play an important role in the pathophysiology of sepsis. A neural anti-inflammatory pathway has been suggested as a potential target for therapy that may dampen systemic inflammation. The aim of this study is to investigate the effects of physostigmine, a cholinesterase inhibitor, on the intestinal microcirculation and vascular contractility in experimental endotoxemia. Endotoxemia was induced in Lewis rats by intravenous lipopolysaccharide (LPS) administration. Animals were treated with either physostigmine or saline (control) following LPS challenge. The intestinal microcirculation, including leukocyte-endothelial interaction, functional capillary density (FCD) and non-perfused capillary density (NCD), was examined by intravital microscopy (IVM) 2 hours after LPS administration. The impact of physostigmine on vascular contractility of rat aortic rings was examined by in vitro myography. Physostigmine significantly reduced the number of adhering leukocytes in intestinal submucosal venules (V1 venules: -61%, V3 venules: -36%) of LPS animals. FCD was significantly increased by physostigmine treatment (circular muscle layer: +180%, longitudinal muscle layer: +162%, mucosa: +149%). Low concentrations of physostigmine produced significant contraction of aortic ring preparations, whereas high concentrations produced relaxation. In conclusion, physostigmine treatment significantly improved the intestinal microcirculation in experimental endotoxemia by reducing leukocyte adhesion and increasing FCD.

3D assisted morphological analysis of lingual upper central and lateral incisor surfaces.

OBJECTIVE: To determine the lingual surface morphology of central and lateral upper incisors evaluating constant morphological regions for better adhesion of industrial prefabricated lingual brackets. MATERIAL AND METHODS: A total of 102 randomly selected patients at the end of the first phase of second dentition with intact central and lateral upper incisors participated in this study. After impression taking and cast model preparation, 3D laser scans of the lingual surface of the upper central and lateral incisors were taken (Laserscan 3D®, Willytec, Munich, Germany), digitalised, and transferred into CAD software to analyse the surface morphology by superimposition. For better comparison of morphological variations and determination of the most constant lingual regions, the surface was divided into five parts: incisal edge, mesial ridge, lateral ridge, cingulum, and medial sector. Statistical analysis was performed by the paired t-test. RESULTS: Statistically significant differences were found in all surfaces, with cingulum as the most inconstant region. The most constant region was the medial sector and the mesial ridge. CONCLUSION: As expected, the lingual surface underlies a high intra-individual variation complicating industrial prefabricated lingual brackets adhesion. However, the mesial ridge and the medial sector seem to be the most constant regions within intra-individual morphological variations.

Talin, vinculin and nestin expression in orofacial muscles of dystrophin deficient mdx mice.

The activity of cytoskeletal proteins like talin, vinculin and nestin increases in muscle that regenerates. Little is known about their role or at least their expression in the process of regeneration in masticatory muscles of mdx mice, a model of Duchenne muscular dystrophy. To determine a potential role of cytoskeletal proteins in the regeneration process of mdx masticatory muscles, we examined the expression of talin 1, talin 2, vinculin and nestin in 100-day-old control and mdx mice using quantitative RT-PCR, Western blot analyses and histochemistry. The protein expression of talin 1, talin 2, nestin and vinculin in mdx muscles remained unchanged as compared with normal mice. However, in mdx masseter it was found a relative increase of nestin compared to controls. The protein expression of talin 1 and vinculin tended to be increased in mdx tongue and talin 2 to diminish in mdx masseter and temporal muscle. In mdx mice, we found significantly lower percentage of transcripts coding for nestin, talin 1, talin 2 and vinculin in masseter (p < 0.05) and temporal muscle (p < 0.001). In contrast, the mRNA expression of nestin was found to be increased in mdx tongue. Activated satellite cells, myoblasts and immature regenerated muscle fibres in mdx masseter and temporal revealed positive staining for nestin. The findings of the presented work suggest dystrophin-lack-associated changes in the expression of cytoskeletal proteins in mdx masticatory muscles could be compensatory for dystrophin absence. The expression of nestin may serve as an indicator for the regeneration in the orofacial muscles.

Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs.

UNLABELLED: Adaptive remodelling of the mandibular condyle in response to mandibular advancement is the mechanism exploited by orthodontic forward displacement devices. OBJECTIVE: This work investigated the expression of collagens, matrix metalloproteinases and vascular endothelial growth factor during this process. DESIGN: Twenty juvenile pigs were randomly divided into two experimental groups, where the treatment group was fitted with mandibular advancement splints, and the control group was not. Changes in the mRNA content of condylar cartilage tissue was then were measured by real-time PCR using specific primers after 4weeks of treatment. RESULTS: The temporal pattern of the expression of Col1 and MMP13 during condylar adaptation coincided with that during natural condylar growth. The amount of the expression of Col10 during condylar adaptation was significantly lower (p<0.05), whereas the expression of Col2, MMP8 and VEGF was significantly higher compared to natural growth (p<0.05). CONCLUSIONS: It is suggested that condylar adaptation in growing pigs triggered by mandibular forward positioning results not only from passive adaptation of cartilage, but also involves growth affected processes. Our results showed that mechanical strain produced by mandibular advancement induced remodelling and revascularization in the posteriocranial mandibular condyle. These results are mostly consistent with former published histological and histomorphometrical analyses.

Bone substitution materials on the basis of BONITmatrix® up-regulate mRNA expression of IGF1 and Col1a1.

The aim of this study was to investigate the effects of BONITmatrix(®) and OSSA NOVA on the expression of growth factors and osteogenic differentiation. For this purpose, the mRNA expression of VEGF, IGF1, IGF2, collagen-1, collagen-2 and MMP8 was analysed in surgically created defects on the crania of adult male rats. Cranial samples were collected after implantation of BONITmatrix(®) or OSSA NOVA scaffolds for 4 weeks and determinations of gene expression were performed by quantitative RT-PCR. Real-time RT-PCR analyses showed a significantly higher expression of IGF1 in both groups treated with BONITmatrix(®) and OSSA NOVA compared to untreated controls, whereas type I collagen mRNA expression only increased in BONITmatrix(®) treated rats compared to controls. No changes in transcript expression of IGF2, VEGF, collagen-2 and MMP8 were detectable between the analysed groups. In conclusion, BONITmatrix(®) and OSSA NOVA stimulate the expression of growth factor IGF1, but only the granular dosage form is able to stimulate osteoblast differentiation.

Experimental and histological investigations of the bone using two different oscillating osteotomy techniques compared with conventional rotary osteotomy.

Over the past decade, coinciding with the appearance of a number of new ultrasonic surgical devices, there has been a marked increase in interest in the use of ultrasound in oral surgery and implantology as alternative osteotomy method. The aim of this study was the comparison of the effect of osteotomies performed using ultrasonic surgery (Piezosurgery(®)), sonic surgery SONICflex(®) and the conventional bur method on the heat generation within the bone underneath the osteotomy and light-microscopy observations of the bone at different cutting positions in porcine mandibular segments. It was found that the average heat generated by SONICflex(®) sonic device was close to that by conventional rotary bur (1.54-2.29°C), whereas Piezosurgery(®) showed a high generated heat up to 18.17°C. Histological investigations of the bone matrix adjacent to the defect radius showed intact osteocytes with all three instruments and similar wide damage diameter at the bottom region. SONICflex(®) showed smooth cutting surfaces with minimal damage in the upper defect zone. Finally, presented results showed that sonic surgery performed with SONICflex(®) is an alternative osteotomy method and can be used as an alternative to the conventional bur method.

Intravenous free and dipeptide-bound glutamine maintains intestinal microcirculation in experimental endotoxemia.

OBJECTIVE: The administration of glutamine (Gln), which is depleted in critical illness, is associated with an improvement of gut metabolism, structure, and function. The aim of the present study was to evaluate the effects of intravenous Gln and its galenic formulation, l-alanyl-l-glutamine dipeptide (AlaGln), on the intestinal microcirculation during experimental endotoxemia using intravital fluorescence microscopy. Gln or AlaGln administration was performed as pretreatment or post-treatment, respectively. To identify further the underlying mechanisms, amino acid levels were studied. METHODS: Sixty male Lewis rats were randomly divided into six groups (n = 10/group): control, LPS (lipopolysaccharide 5 mg/kg intravenously), Gln/LPS (LPS animals pretreated with Gln 0.75 g/kg Gln intravenously), AlaGln/LPS (LPS animals pretreated with AlaGln intravenously, 0.75 g/kg Gln content), LPS/Gln (LPS animals post-treated with Gln 0.75 g/kg intravenously), and LPS/AlaGln (LPS animals post-treated with AlaGln intravenously, 0.75 g/kg Gln content). Two hours after the endotoxin challenge, the microcirculation of the terminal ileum was studied using intravital fluorescence microscopy. Blood samples were drawn at the beginning, during, and the end of the experiment to determine the amino acid levels. RESULTS: The Gln and AlaGln pre- and post-treatment, respectively, prevented the LPS-induced decrease in the functional capillary density of the intestinal muscular and mucosal layers (P < 0.05). The number of adherent leukocytes in the submucosal venules was significantly attenuated after the Gln and AlaGln pre- and post-treatment (P < 0.05). CONCLUSION: The Gln and AlaGln administrations improved the intestinal microcirculation by increasing the functional capillary density of the intestinal wall and decreasing the submucosal leukocyte activation.

Increased oxidative stress in dystrophin deficient (mdx) mice masticatory muscles.

BACKGROUND: It has been suggested that increased oxidative stress and the glutathione antioxidant system play an important role in the pathogenesis of Duchenne muscular dystrophy. However, there is still a lack of data about the oxidative status in dystrophic masticatory muscles. METHODS: In the masticatory muscles of the mouse model of Duchenne muscular dystrophy (mdx and controls; 100 days old, n=8-10 each group) we examined the GSH and GSSG content (glutathione reduced/oxidized form) and the level of lipid peroxidation (LPO) as measured by the thiobarbituric acid-reaction. RESULTS: In the mdx mice masticatory muscles we found increased oxidative stress as compared to the controls. The GSH values in mdx muscles were decreased (mean±SEM; masseter 339.8±37.6 µg/g vs. 523.1±36.1 µg/g, temporal 304.1±49.6 µg/g vs.512.6±60.6 µg/g, tongue muscle 243.3±28. 8 µg/g vs. 474.9±40.1 µg/g; Fig. 1) as compared to normal mice. The GSH/GSSG ratio in mdx mice was consequently decreased. No significant differences in GSSG content and LPO levels were found between mdx and control mice. CONCLUSIONS: The results imply that oxidative stress is present in all three studied mdx mouse masticatory muscles.

Myogenic differentiation factor 1 and myogenin expression not elevated in regenerated masticatory muscles of dystrophic (mdx) mice.

BACKGROUND: Orofacial muscles in mdx mice, a model of Duchenne muscular dystrophy (DMD), undergo muscle necrosis followed by muscle regeneration. The activity of myogenic regulatory factors (MRF) in muscles that regenerate may reveal specific changes. Little is known about the role of MRF, particularly their expression after muscle necrosis in the orofacial muscles of mdx mice and in DMD. Patients suffering from DMD present characteristic malocclusions in association with orofacial dysfunctions. Investigating the role of MRFs in mdx masticatory muscles may help to develop preventive and therapeutic strategies for DMD. MATERIAL AND METHODS: Using Western Blot analysis, we examined the protein expression of MRFs (myogenin and MyoD1) in masticatory muscles such as masseter, temporal, and tongue muscle and one hindlimb muscle, the soleus of control and mdx mice (n = 6-7). The mean optical density (MOD) of proteins was measured for quantification. RESULTS: Myogenin and MyoD1 were detected in mdx and control mice. The amount of myogenin in masseter (MOD, mean ± standard error of the mean (SEM), control vs. mdx: 3.08 ± 0.67 vs. 1.83 ± 0.33), tongue (MOD control vs. mdx: 1.53 ± 0.22 vs. 1.41 ± 0.14), temporal (MOD control vs. mdx: 1.23 ± 0.16 vs. 1.43 ± 0.35), and soleus muscles (MOD, control vs. mdx: 1.95 ± 0.26 vs. 2.31 ± 0.42) did not differ between the mouse strains. MyoD1 amounts in mdx, similar to that of myogenin, remained unchanged when compared to control mice (MOD control vs. mdx: masseter 0.75 ± 0.09 vs. 0.86 ± 0.13; tongue 1.55 ± 0.25 vs. 1.41 ± 0.28; temporal 0.71 ± 0.10 vs. 0.73 ± 0.11; soleus 1.09 ± 0.26 vs. 1.03 ± 0.24). CONCLUSION: The results indicate that protein expression of MyoD1 and myogenin in mdx mice does not differ from controls, suggesting a secondary role of MyoD1 and myogenin in the regeneration stage of mdx orofacial muscles.