Alexander Spassov

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.

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

IntroductionCannabinoid 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.MethodsIn 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.ResultsHU308 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.ConclusionCB2R 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.

Histological changes in masticatory muscles of mdx mice

OBJECTIVE Duchenne muscular dystrophy (DMD) patients have distorted dentofacial morphology that could be a result of changed force balance of masticatory muscles due to unequal dystrophic changes in various masticatory muscles. Skeletal muscles of DMD patients and those of murine model of DMD - mdx mice - are both characterized by Ca(2+) induced muscle damage, muscle weakness and characteristic histological changes. Therefore, to determine the pathological changes in this animal model of DMD, we examined the masticatory muscles of the mdx mice for histological abnormalities including nuclei localization, fibre diameters, and collagen expression. DESIGN Muscle sections from masseter (MAS), temporal (TEM), tongue (TON) and soleus (SOL) of mdx and control normal mice were stained with hemalaun/eosin or with Sirius Red and morphometrically analysed. Levels of collagen staining in normal and mdx muscles were measured using image analysis and the mean optical density (mod) was determined. RESULTS Dystrophin deficient masticatory muscles contained 11-75% fibres with centralised nuclei. In mdx mice an increased mean fibre diameter was observed as compared to the age-matched control muscles (control vs. mdx; MAS: 33.44+/-0.49microm vs. 37.76+/-0.68microm, p<0.005; TEM: 32.93+/-0.4microm vs. 42.93+/-0.68microm, p<0.005; SOL: 33.15+/-0.29microm vs. 40.62+/-0.55microm, p<0.005; TON: 13.44+/-0.68microm vs. 15.63+/-0.18microm, p<0.005). Increased expression of collagen was found in MAS (mod control vs. mdx: 1.34 vs. 3.99, p<0.005), TEM (mod control vs. mdx: 3.11 vs. 4.73, p<0.01) and SOL (mod control vs. mdx: 2.36 vs. 3.49, p<0.01). CONCLUSION Our findings revealed that mdx masticatory muscles are unequally affected by the disease process. The masticatory muscles of the mdx mice could present a useful model for further investigating the influence of dystrophin deficiency on muscles function.

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.

Caveolin-1, caveolin-3 and VEGF expression in the masticatory muscles of mdx mice.

Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx), its murine model, are characterized by muscle damage and muscle weakness associated with inflammation and new vessel formation. Caveolins, dystrophin-associated proteins, are involved in the pathogenesis of DMD, because increased numbers of caveolae are found in DMD and mdx hindlimb muscles. Caveolae influence angiogenesis due to their content of vascular endothelial growth factor (VEGF) receptors. Orofacial muscles in mdx mice undergo muscle necrosis followed by muscle regeneration. To ascertain the role of caveolins and VEGF in the pathogenesis of dystrophic masticatory muscles, we examined the expression of caveolin-1 (cav-1), caveolin-3 (cav-3) and VEGF in control and mdx mice. In mdx masticatory muscles, no changes in transcript and protein levels of VEGF were found, whereas cav-1 and cav-3 expression was increased. Using immunohistochemistry, a strong sarcolemmal staining of caveolin-3 in regenerated muscle fibers was found. Furthermore, immunohistochemistry with the caveolin-1 antibody showed an increase in the amount of blood vessels in areas with regenerating muscle fibers. Dystrophic masticatory muscles showed changes comparable to those of hindlimb muscles in the expression of cav-1 and cav-3. The angiogenesis seems to be unaffected in the jaw muscles of mdx mice. We speculate that the increased caveolin expression could cause extensive and efficient muscle regeneration.

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.

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), Students 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.

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.

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.

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 (mechanogrowth 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.