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Histological changes in masticatory muscles of mdx mice
archives of oral biology 55 (2010) 318–324
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Histological changes in masticatory muscles of mdx mice Alexander Spassov a,*, Tomasz Gredes a, Tomasz Gedrange a, Silke Lucke a, Dragan Pavlovic b, Christiane Kunert-Keil a,c a
Department of Orthodontics, Faculty of Medicine, University of Greifswald, Rotgerber Str. 8, 17475 Greifswald, Germany Department of Anaesthesiology and Intensive Care Medicine, Faculty of Medicine, University of Greifswald, Friedrich-Loeffler Str. 23c, 17475 Greifswald, Germany c Institute of Pathophysiology, Faculty of Medicine, University of Greifswald, Greifswalder Str. 11C, 17495 Karlsburg, Germany b
article info
abstract
Article history:
Objective: Duchenne muscular dystrophy (DMD) patients have distorted dentofacial mor-
Accepted 6 February 2010
phology 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
Keywords:
patients and those of murine model of DMD – mdx mice – are both characterized by
Mdx mice
Ca2+ induced muscle damage, muscle weakness and characteristic histological changes.
Muscular dystrophy
Therefore, to determine the pathological changes in this animal model of DMD, we exam-
DMD
ined the masticatory muscles of the mdx mice for histological abnormalities including
Histopathology
nuclei localization, fibre diameters, and collagen expression.
Masticatory muscles
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 agematched control muscles (control vs. mdx; MAS: 33.44 0.49 mm vs. 37.76 0.68 mm, p < 0.005; TEM: 32.93 0.4 mm vs. 42.93 0.68 mm, p < 0.005; SOL: 33.15 0.29 mm vs. 40.62 0.55 mm, p < 0.005; TON: 13.44 0.68 mm vs. 15.63 0.18 mm, 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. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle degeneration caused by mutations in the gene encoding for the cytoskeletal protein dystrophin.1 The increased size of the calves and certain other limb muscle
groups, also known as pseudohypertrophy, is a well known feature of DMD.2 Histological changes found in this condition are degeneration, variation in fibre size, with the enlargement or atrophy of fibres and the internal migration of nuclei3 and subsequently the loss of fibres. Studies performed in the animal model of DMD, the mdx mice, reveal acute phases of
* Corresponding author. Tel.: +49 3834 867119; fax: +49 3834 867113. E-mail address: [email protected] (A. Spassov). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.02.005
archives of oral biology 55 (2010) 318–324
muscle necrosis at the age of 3–4 weeks, followed by regeneration and apparent stability with ageing.4 Degeneration and regeneration processes in mdx muscles are associated with histopathological features, such as centronucleated fibres, increased collagen expression and fibrosis, variation in muscle fibres size and the presence of phagocyte cells.5–7 Mdx skeletal muscles often present varying patterns of histopathology. For example adult mdx skeletal muscles revealed either absence (oesophagus), very mild (trunk and limb muscles), or severe (diaphragm) histopathological changes.8 Although many studies have compared various mdx muscles, including masticatory muscles, only few have been concentrated on the progression of the disease among muscles involved in mastication and swallowing. Existing animal and clinical studies imply a different timing for the onset of the dystrophic process in various masticatory muscles9,10 which may influence their coordinated activity during the masticatory process.11 Characteristic malocclusions and orofacial dysfunctions in DMD patients are most probably related to the altered masticatory and orofacial functions due to muscle dystrophy.12 In DMD patients the maximum biting force and mouth opening distance are significantly reduced as compared to controls.13 Furthermore, the lip incompetence, mouth breathing, macroglossia and tongue thrusting are common findings in these patients.14 Therefore, it is of importance to investigate histomorphometric changes in masticatory muscles in animal studies to be able to comprehend the muscle response to dystrophic changes. In humans, orofacial deformations occur at advanced stages of the disease. The importance of such investigations has grown, since the life expectancy of DMD patients has been extended. Most previous studies on mdx mice have almost exclusively investigated the masseter. However, mastication involves a group of muscles acting in synchrony, which must be taken into consideration, when studying the influence of dystrophy on the orofacial musculature. In 9-week-old mdx mice almost all skeletal muscles of the head, neck and trunk show degeneration and necrosis of muscle fibres. However, there is little information about histological parameters beyond 12 weeks in mdx mouse masticatory muscles, when the majority of fibres have already regenerated. Earlier studies investigated predominantly younger mdx mice. The aim of this present study was to obtain more information than is available from earlier studies. Therefore we analysed and compared histological parameters, such as fibre diameter, centralized nuclei, collagen expression and presence of inflammatory foci in three functionally important masticatory muscles: tongue (TON), masseter (MAS), temporal muscle (TEM), and compared them to the skeletal muscle soleus (SOL) in 100-day-old mdx mice. The data obtained will serve as background for functional measurements in future studies.
Harlan Winkelmann (Borchen, Germany) and Charles River (Sulzfeld, Germany). Both strains were bred in the Department of Pathophysiology of the Medical Faculty at the University of Greifswald. Age-matched pairs of mdx and control animals (each n = 6, 100 days old) of either sex and of the same body weight (about 30 g) were killed using ether inhalation in a manner approved by the institutional animal ethics committee.
2.2.
Materials and methods
2.1.
Animals
Mice of the inbred strains C57Bl/10ScSn (control) and C57/ Bl10ScSn-Dmdmdx/J (mdx) were originally obtained from
Morphology
Masseter, temporal, tongue and soleus muscle were collected and mounted on cork supports using cryomatrix (Germany). The samples were snap-frozen in melting petroleum ether (Merck, Darmstadt, Germany) and stored at 80 8C. Samples (5 mm) were cryo cross-sectioned from the middle of all test muscles and the corpus linguae, and placed on slides. The slides were air dried, fixed with acetone for 10 min at room temperature and either stained with hemalaun/eosin for the visualisation of nuclei, or stained in Sirius Red F3B (Niepo¨tter Labortechnik, Bierstadt, Germany) for the visualisation of collagen. A blind test was conducted at the same time using identical staff, equipment, and chemicals.
2.3.
Image acquisition and analysis
Five digital pictures from each section of both staining methods were acquired at random of different places of the tissue (20-fold magnification; 3CCD colour camera; Hitachi HV-C20M; Hitachi Denshi Ltd., Japan, and Axiolab, Carl Zeiss, Go¨ttingen, Germany). For standardisation of the measurement in each picture the optical density of white background colour was attuned to 250 as described previously.15 Image analysis of up to 100 muscle fibres in each section was performed in two steps: (1) determination of the muscle fibre size according to the minimal ‘‘Feret’s diameter’’ – the minimum distance of parallel tangents at opposing borders of the muscle fibre16 and (2) determination of the percentage of muscle fibres containing centralized nuclei. Apart from this, the content of collagen between fibres was measured by determination of the quantity of pixels in each picture that had a positive reaction for collagen (mod: mean optical density). For fibre size measurement the smallest and the largest fibre diameter was determined and from these values the mean diameter was calculated for each fibre.17,18 Semiautomated analysis was performed using the image analysis program KSRun (Imaging system KS400, release 3.0; Zeiss, Vision GmbH, Munich, Germany) or ImageJ 1.42 for Windows (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997– 2009).
2.4.
2.
319
Statistical analysis
Statistical analysis was performed using the SigmaPlot Software (Systat Software, Inc., 1735, Technology Drive, Sn Jose, CA 95110, USA). The obtained values for the groups were compared using Student’s unpaired t-test. Data are given as means S.E.M. p < 0.05 was considered statistically significant.
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archives of oral biology 55 (2010) 318–324
Table 1 – Distribution of nuclei in masticatory muscles, soleus and tongue from age-matched control and mdx mice. Tissue
Centralized nuclei (%) control/mdx
Masseter Temporal Tongue Soleus
0.3/69.1 0/75.3 0.5/11.2 0/71.5
3.
Results
3.1.
Masticatory muscle morphology
Peripheral nuclei (%) control/mdx 99.7/30.9 100/24.7 99.5/88.8 100/28.5
The dystrophin deficient skeletal muscles of the masticatory system show several morphological abnormalities. The percentage of muscle fibres with non-peripheral nuclei, in sections from MAS, TEM, TON and SOL muscle, revealed that control muscles never had more than 0.5% of fibres containing centralized nuclei, whereas muscles from mdx mice contained 11–75% fibres with centralized nuclei depending on muscle specimen (Table 1). Numerous inflammatory foci were observed in mdx MAS, TEM, and SOL muscles; these were absent in the controls. In contrast, inflammatory cells were hardly detectable in the TON of mdx mice (Fig. 1A).
3.2.
Fibre diameters
The masticatory muscles, MAS and TEM, showed a wide range of fibre size comparable with the SOL muscle. In contrast, the superior longitudinal muscle fibres of the TON always presented only small fibre size between 10 and 30 mm. In control mice the mean fibre diameters were as follows: SOL, MAS and TEM, 33 mm; TON, 13 mm (mean S.E.M., MAS: 33.44 0.49 mm; TEM: 32.93 0.4 mm; SOL: 33.15 0.29 mm; TON: 13.44 0.68 mm; Fig. 1B-2). In the masticatory muscles of mdx mice, as presented in the histograms, demonstrate that large-calibre fibres are more abundant in all tested dystrophic muscles (Fig. 2). These differences in muscle fibre size distribution are also visible after calculation of the mean fibre diameters. In all dystrophic muscles of this study a significantly increased mean fibre diameter was observed as compared to the age-matched control muscles (mean S.E.M., mdx MAS: 37.76 0.68 mm; TEM: 42.93 0.68 mm; SOL: 40.62 0.55 mm; TON: 15.63 0.18 mm; Fig. 1B).
3.3.
Collagen expression
Levels of collagen staining in normal and mdx muscles were measured using image analysis. The Sirius Red staining of various masticatory muscles confirmed that collagen accumulates within the dystrophin deficient tissue. In mdx mice, there were no changes in the collagen level in TON, while the level of collagen was approximately three times higher in MAS muscle as compared to the controls (mod control vs. mdx: 1.34 vs. 3.99, p < 0.005). Furthermore, the TEM (mod control vs. mdx: 3.11 vs. 4.73, p < 0.01) and SOL muscle (mod control vs.
Fig. 1 – (A) Histopathological aspects of the muscle tissue from masseter (MAS), temporal (TEM), soleus (SOL) and tongue (TON) in control and mdx mice. Haematoxylin and eosin stained cryo-sections. IF, focus of inflammation. (B) Muscle fibre diameters of age-matched control and mdx mice (means W S.E.M; ***p < 0.005, unpaired Student’s ttest).
mdx: 2.36 vs. 3.49, p < 0.01) of the mdx group had significantly increased expression of collagen (Fig. 3).
4.
Discussion
The aim of the present work was to investigate and compare histological parameters in the masticatory muscles in 100day-old mdx and control mice. In this study we demonstrated that the dystrophin deficient masticatory muscles contained an increased number of fibres with centralized nuclei as
archives of oral biology 55 (2010) 318–324
Fig. 2 – Fibre diameter distribution in the four skeletal muscles from control and mdx mice illustrated in Fig. 1. The muscle fibres were grouped in size classes of 10 mm and the percentage of fibres in each class was plotted (control and mdx; n = 480 fibres each).
compared to the controls. Also all muscles, except TON, displayed numerous inflammatory foci and an accumulation of collagen, while a significantly increased mean fibre diameter was observed in all tested mdx muscles. These findings suggest that mdx masticatory muscles are unequally implicated in the development of the disease. The mdx mouse is the most widely studied animal model for Duchenne muscular dystrophy. In contrast to humans, the muscle pathology in mdx mice is minimally progressive and muscle fibrosis is absent except in the diaphragm.19,20 Mdx muscles display early necrosis, beginning at day 5 in the muscles of the head, trunk and girdle. Necrotic and apoptotic processes emerge in the limbs at approximately 3 weeks of age.5 Regeneration processes are initiated around the age of 6 weeks and continue until 12 weeks.21 At 3 months, 80–90% of the mdx skeletal fibres have central nuclei indicating a preceding cycle of degeneration and regeneration.22 At later stages mdx mice show a decline in their regeneration capacity
321
Fig. 3 – (A) Sirius Red stained cryo-sections from masseter (MAS), temporal (TEM), soleus (SOL) and tongue (TON) muscles in control and mdx mice. (B) Quantification of the collagen expression (mean optical density – mod) of agematched controls and mdx mice (mean W S.E.M; **p < 0.01; ***p < 0.005, unpaired Student’s t-test) was performed using the KSRun software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
while the necrotic processes persist,6 effects that lead to progressive muscle wasting and fibrosis. Regarding the above mentioned histopathological characteristics of mdx muscles (see Section 1), an increased number of fibres with central nucleation seemed to be a measurement of the degree of increased severity of the disease, since such cells, in reality, originate from regenerated cells.23–25 The high level of centronucleation is a result of an increased regeneration in an attempt to balance the muscle cell necrosis.26 Thus, the observed hypertrophic and small regenerated fibres in mdx may be explained as replacement of
322
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necrotized muscle cells.26 Their presence among normal sized cells increases the cell size variance, which may therefore reflect the degree of severity of disease.26–29 The results from the four studied dystrophic muscles clearly demonstrated that the degree of centronucleation did not significantly differ between MAS, TEM and SOL. However, the percentage of such cells in MAS, TEM and SOL was significantly greater than in TON. Accordingly, the MAS, TEM and SOL seem to be most influenced by the disease in the studied animals. In our histomorphometric study, the MAS revealed a high degree of centronucleated fibres which concords with a recent study that compared the mdx masseter with diaphragm and gastrocnemius.10 Moreover, the mentioned study refers to a heterogeneous alteration of mdx muscles, showing a milder dystrophy in MAS as compared to diaphragm and gastrocnemius. In our histomorphometric study, the MAS revealed a high degree of centronucleated fibres which agrees with a recent study that compared the mdx masseter with diaphragm and gastrocnemius.30 One recent study examined the centronucleation in regenerated MAS in mdx mice.11 It was revealed that regeneration, i.e. centronucleation, in mdx MAS occurred 15 weeks after necrosis. We found in a 13-week-old mdx MAS a high degree of centronucleation which is slightly earlier than what was found in the above mentioned study. Regarding the variation in cell size, it was obvious that the variation of the size of dystrophic muscle cells was considerably more than that of normal muscles.26 So, previous studies reported a wide range of fibre sizes consisting of abnormal proportions of small and large fibres in mdx mice muscles.5,6 We partially confirmed these findings for the masticatory muscles and SOL. A wider range of the fibre sizes was indeed found in MAS, TEM and SOL, which showed only minor differences between one another in their distribution of muscle fibre sizes. But, TON was markedly different, displaying only small fibres in both, normal and mdx mice. Interestingly, small-caliber fibres are reported to be more resistant to necrosis and it was discovered that no centronucleation occurred in fibres with diameters inferior to 20 mm.23 Our investigations are consistent with these findings showing that the mdx TON muscle displayed smaller fibres as compared to MAS or TEM, suggesting that small-caliber fibres in some muscles of mastication (TON) may be less susceptible to muscle damage. Furthermore, fibre size mean diameter was larger in all tested mdx muscles as compared to controls and this could indicate a hypertrophy that accompanied dystrophin deficiency in these animals. It is known that hypertrophy in mdx mice consists of a mixture of hypertrophic and small fibres but not of fibrosis.31 Replacement of muscle tissue by collagen and fatty deposits is a feature of both human DMD and, to a lesser extent, murine mdx dystrophy.32,33 A marked accumulation of collagen was shown in the mdx SOL muscle in mdx mice of from 3 weeks to 1 year old.32 Moreover, previous investigations of hind limb and trunk muscles have confirmed a high degree of fibrosis and increased collagen content in older, as opposed to younger mdx mice .34,35 Our results concord with these findings. In our study we found that the level of collagen
expression was higher in mdx MAS, TEM and SOL as compared to controls, while in TON the collagen expression remained unchanged. Our results confirmed higher collagen content in mdx masticatory muscles as opposed to that in normal mice. These findings were accompanied by signs of inflammation suggesting a link between the repairing process (high collagen expression) and an inflammatory reaction in the mdx muscles as previously suggested.36 The MAS and TEM muscles displayed the highest degrees of collagen proliferation, suggesting greater limitation of their functioning as compared to TON and confirming intrinsic dissimilarities in collagen metabolism of functionally different skeletal muscles in mdx mice.37 In regards to the investigated histomorphometric parameters, it can be concluded that the mdx MAS and TEM express similar histopathological features of higher collagen content and a higher degree of CNF, as well as a variance of cell sizes, in comparison to limb mdx muscles (SOL). In contrast, TON of mdx mice seem to be partially spared from dystrophic alterations, as compared to MAS, TEM and limb muscles. Previous studies found that laryngeal muscles of the mdx mice could be spared from the development of the disease through protective mechanisms.38 We suggested similar mechanisms for the mdx tongue, although the ‘‘specific protective mechanism’’ may not be a ‘‘mechanism,’’ in the strict sense of the term. It was recently found that, besides the diaphragm, the TON of 26-month-old mdx mice was the most severely implicated muscle.39 In contrast, in our study we found that TON was the least implicated muscle of all the muscles studied. But, the difference might be explained by the younger age of our study animals (3 months old). This would imply that with age, the mdx TON becomes progressively more implicated in the disease process. Similarly, studies on DMD have shown that some orofacial muscles become implicated later in the course of the dystrophic process, which may explain the fact that in humans orofacial deformations occur at advanced stages of the disease. The force developed by a muscle is based substantially on its anatomic structure and depends to a great extent on the geometry of supporting anatomical structures, while it can also influence the shape of these structures. Mandibular shape changes have already been reported for dystrophic dy/dy mice.40 The histopathological features found in the mdx mice in the presented study may be accompanied by the severe dysfunction and force reduction which in turn may alter the shape of supporting bone structures, as reported previously.41 But, the model has a disadvantage, in as much as the masticatory muscles of mdx mice are not directly comparable to those of human DMDs. Contrary to humans, muscle fibres of mdx mice do regenerate after muscle fibre necrosis. Furthermore, the mastication mechanics are fundamentally different. Human masseter and temporal muscles have also a heterogeneous fibre type composition, the proportion of the fibre type 2 being generally lower.42 However, our results imply heterogenous changes in the anatomic structure of masticatory muscles due to dystrophy, which may influence their functional properties. This work provides supplementary information on how muscles adapt to dystrophic diseases. The possible influence of the adapted orofacial muscular system on the functioning and bone development is a great
archives of oral biology 55 (2010) 318–324
challenge for modern dental medical care and orthodontics. New insights into the mechanisms of interaction of muscles and the supporting bony structures may contribute to efforts for improving the quality of life of DMD patients.
Acknowledgement We thank I. Pieper for excellent technical assistance. Funding: None. Competing interests: None declared. Ethical approval: Landesamt fu¨r Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Germany. LALLF M-V/TSD/7221.3-2.3-001/09.
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available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Histological changes in masticatory muscles of mdx mice Alexander Spassov a,*, Tomasz Gredes a, Tomasz Gedrange a, Silke Lucke a, Dragan Pavlovic b, Christiane Kunert-Keil a,c a
Department of Orthodontics, Faculty of Medicine, University of Greifswald, Rotgerber Str. 8, 17475 Greifswald, Germany Department of Anaesthesiology and Intensive Care Medicine, Faculty of Medicine, University of Greifswald, Friedrich-Loeffler Str. 23c, 17475 Greifswald, Germany c Institute of Pathophysiology, Faculty of Medicine, University of Greifswald, Greifswalder Str. 11C, 17495 Karlsburg, Germany b
article info
abstract
Article history:
Objective: Duchenne muscular dystrophy (DMD) patients have distorted dentofacial mor-
Accepted 6 February 2010
phology 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
Keywords:
patients and those of murine model of DMD – mdx mice – are both characterized by
Mdx mice
Ca2+ induced muscle damage, muscle weakness and characteristic histological changes.
Muscular dystrophy
Therefore, to determine the pathological changes in this animal model of DMD, we exam-
DMD
ined the masticatory muscles of the mdx mice for histological abnormalities including
Histopathology
nuclei localization, fibre diameters, and collagen expression.
Masticatory muscles
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 agematched control muscles (control vs. mdx; MAS: 33.44 0.49 mm vs. 37.76 0.68 mm, p < 0.005; TEM: 32.93 0.4 mm vs. 42.93 0.68 mm, p < 0.005; SOL: 33.15 0.29 mm vs. 40.62 0.55 mm, p < 0.005; TON: 13.44 0.68 mm vs. 15.63 0.18 mm, 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. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle degeneration caused by mutations in the gene encoding for the cytoskeletal protein dystrophin.1 The increased size of the calves and certain other limb muscle
groups, also known as pseudohypertrophy, is a well known feature of DMD.2 Histological changes found in this condition are degeneration, variation in fibre size, with the enlargement or atrophy of fibres and the internal migration of nuclei3 and subsequently the loss of fibres. Studies performed in the animal model of DMD, the mdx mice, reveal acute phases of
* Corresponding author. Tel.: +49 3834 867119; fax: +49 3834 867113. E-mail address: [email protected] (A. Spassov). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.02.005
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muscle necrosis at the age of 3–4 weeks, followed by regeneration and apparent stability with ageing.4 Degeneration and regeneration processes in mdx muscles are associated with histopathological features, such as centronucleated fibres, increased collagen expression and fibrosis, variation in muscle fibres size and the presence of phagocyte cells.5–7 Mdx skeletal muscles often present varying patterns of histopathology. For example adult mdx skeletal muscles revealed either absence (oesophagus), very mild (trunk and limb muscles), or severe (diaphragm) histopathological changes.8 Although many studies have compared various mdx muscles, including masticatory muscles, only few have been concentrated on the progression of the disease among muscles involved in mastication and swallowing. Existing animal and clinical studies imply a different timing for the onset of the dystrophic process in various masticatory muscles9,10 which may influence their coordinated activity during the masticatory process.11 Characteristic malocclusions and orofacial dysfunctions in DMD patients are most probably related to the altered masticatory and orofacial functions due to muscle dystrophy.12 In DMD patients the maximum biting force and mouth opening distance are significantly reduced as compared to controls.13 Furthermore, the lip incompetence, mouth breathing, macroglossia and tongue thrusting are common findings in these patients.14 Therefore, it is of importance to investigate histomorphometric changes in masticatory muscles in animal studies to be able to comprehend the muscle response to dystrophic changes. In humans, orofacial deformations occur at advanced stages of the disease. The importance of such investigations has grown, since the life expectancy of DMD patients has been extended. Most previous studies on mdx mice have almost exclusively investigated the masseter. However, mastication involves a group of muscles acting in synchrony, which must be taken into consideration, when studying the influence of dystrophy on the orofacial musculature. In 9-week-old mdx mice almost all skeletal muscles of the head, neck and trunk show degeneration and necrosis of muscle fibres. However, there is little information about histological parameters beyond 12 weeks in mdx mouse masticatory muscles, when the majority of fibres have already regenerated. Earlier studies investigated predominantly younger mdx mice. The aim of this present study was to obtain more information than is available from earlier studies. Therefore we analysed and compared histological parameters, such as fibre diameter, centralized nuclei, collagen expression and presence of inflammatory foci in three functionally important masticatory muscles: tongue (TON), masseter (MAS), temporal muscle (TEM), and compared them to the skeletal muscle soleus (SOL) in 100-day-old mdx mice. The data obtained will serve as background for functional measurements in future studies.
Harlan Winkelmann (Borchen, Germany) and Charles River (Sulzfeld, Germany). Both strains were bred in the Department of Pathophysiology of the Medical Faculty at the University of Greifswald. Age-matched pairs of mdx and control animals (each n = 6, 100 days old) of either sex and of the same body weight (about 30 g) were killed using ether inhalation in a manner approved by the institutional animal ethics committee.
2.2.
Materials and methods
2.1.
Animals
Mice of the inbred strains C57Bl/10ScSn (control) and C57/ Bl10ScSn-Dmdmdx/J (mdx) were originally obtained from
Morphology
Masseter, temporal, tongue and soleus muscle were collected and mounted on cork supports using cryomatrix (Germany). The samples were snap-frozen in melting petroleum ether (Merck, Darmstadt, Germany) and stored at 80 8C. Samples (5 mm) were cryo cross-sectioned from the middle of all test muscles and the corpus linguae, and placed on slides. The slides were air dried, fixed with acetone for 10 min at room temperature and either stained with hemalaun/eosin for the visualisation of nuclei, or stained in Sirius Red F3B (Niepo¨tter Labortechnik, Bierstadt, Germany) for the visualisation of collagen. A blind test was conducted at the same time using identical staff, equipment, and chemicals.
2.3.
Image acquisition and analysis
Five digital pictures from each section of both staining methods were acquired at random of different places of the tissue (20-fold magnification; 3CCD colour camera; Hitachi HV-C20M; Hitachi Denshi Ltd., Japan, and Axiolab, Carl Zeiss, Go¨ttingen, Germany). For standardisation of the measurement in each picture the optical density of white background colour was attuned to 250 as described previously.15 Image analysis of up to 100 muscle fibres in each section was performed in two steps: (1) determination of the muscle fibre size according to the minimal ‘‘Feret’s diameter’’ – the minimum distance of parallel tangents at opposing borders of the muscle fibre16 and (2) determination of the percentage of muscle fibres containing centralized nuclei. Apart from this, the content of collagen between fibres was measured by determination of the quantity of pixels in each picture that had a positive reaction for collagen (mod: mean optical density). For fibre size measurement the smallest and the largest fibre diameter was determined and from these values the mean diameter was calculated for each fibre.17,18 Semiautomated analysis was performed using the image analysis program KSRun (Imaging system KS400, release 3.0; Zeiss, Vision GmbH, Munich, Germany) or ImageJ 1.42 for Windows (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997– 2009).
2.4.
2.
319
Statistical analysis
Statistical analysis was performed using the SigmaPlot Software (Systat Software, Inc., 1735, Technology Drive, Sn Jose, CA 95110, USA). The obtained values for the groups were compared using Student’s unpaired t-test. Data are given as means S.E.M. p < 0.05 was considered statistically significant.
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Table 1 – Distribution of nuclei in masticatory muscles, soleus and tongue from age-matched control and mdx mice. Tissue
Centralized nuclei (%) control/mdx
Masseter Temporal Tongue Soleus
0.3/69.1 0/75.3 0.5/11.2 0/71.5
3.
Results
3.1.
Masticatory muscle morphology
Peripheral nuclei (%) control/mdx 99.7/30.9 100/24.7 99.5/88.8 100/28.5
The dystrophin deficient skeletal muscles of the masticatory system show several morphological abnormalities. The percentage of muscle fibres with non-peripheral nuclei, in sections from MAS, TEM, TON and SOL muscle, revealed that control muscles never had more than 0.5% of fibres containing centralized nuclei, whereas muscles from mdx mice contained 11–75% fibres with centralized nuclei depending on muscle specimen (Table 1). Numerous inflammatory foci were observed in mdx MAS, TEM, and SOL muscles; these were absent in the controls. In contrast, inflammatory cells were hardly detectable in the TON of mdx mice (Fig. 1A).
3.2.
Fibre diameters
The masticatory muscles, MAS and TEM, showed a wide range of fibre size comparable with the SOL muscle. In contrast, the superior longitudinal muscle fibres of the TON always presented only small fibre size between 10 and 30 mm. In control mice the mean fibre diameters were as follows: SOL, MAS and TEM, 33 mm; TON, 13 mm (mean S.E.M., MAS: 33.44 0.49 mm; TEM: 32.93 0.4 mm; SOL: 33.15 0.29 mm; TON: 13.44 0.68 mm; Fig. 1B-2). In the masticatory muscles of mdx mice, as presented in the histograms, demonstrate that large-calibre fibres are more abundant in all tested dystrophic muscles (Fig. 2). These differences in muscle fibre size distribution are also visible after calculation of the mean fibre diameters. In all dystrophic muscles of this study a significantly increased mean fibre diameter was observed as compared to the age-matched control muscles (mean S.E.M., mdx MAS: 37.76 0.68 mm; TEM: 42.93 0.68 mm; SOL: 40.62 0.55 mm; TON: 15.63 0.18 mm; Fig. 1B).
3.3.
Collagen expression
Levels of collagen staining in normal and mdx muscles were measured using image analysis. The Sirius Red staining of various masticatory muscles confirmed that collagen accumulates within the dystrophin deficient tissue. In mdx mice, there were no changes in the collagen level in TON, while the level of collagen was approximately three times higher in MAS muscle as compared to the controls (mod control vs. mdx: 1.34 vs. 3.99, p < 0.005). Furthermore, the TEM (mod control vs. mdx: 3.11 vs. 4.73, p < 0.01) and SOL muscle (mod control vs.
Fig. 1 – (A) Histopathological aspects of the muscle tissue from masseter (MAS), temporal (TEM), soleus (SOL) and tongue (TON) in control and mdx mice. Haematoxylin and eosin stained cryo-sections. IF, focus of inflammation. (B) Muscle fibre diameters of age-matched control and mdx mice (means W S.E.M; ***p < 0.005, unpaired Student’s ttest).
mdx: 2.36 vs. 3.49, p < 0.01) of the mdx group had significantly increased expression of collagen (Fig. 3).
4.
Discussion
The aim of the present work was to investigate and compare histological parameters in the masticatory muscles in 100day-old mdx and control mice. In this study we demonstrated that the dystrophin deficient masticatory muscles contained an increased number of fibres with centralized nuclei as
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Fig. 2 – Fibre diameter distribution in the four skeletal muscles from control and mdx mice illustrated in Fig. 1. The muscle fibres were grouped in size classes of 10 mm and the percentage of fibres in each class was plotted (control and mdx; n = 480 fibres each).
compared to the controls. Also all muscles, except TON, displayed numerous inflammatory foci and an accumulation of collagen, while a significantly increased mean fibre diameter was observed in all tested mdx muscles. These findings suggest that mdx masticatory muscles are unequally implicated in the development of the disease. The mdx mouse is the most widely studied animal model for Duchenne muscular dystrophy. In contrast to humans, the muscle pathology in mdx mice is minimally progressive and muscle fibrosis is absent except in the diaphragm.19,20 Mdx muscles display early necrosis, beginning at day 5 in the muscles of the head, trunk and girdle. Necrotic and apoptotic processes emerge in the limbs at approximately 3 weeks of age.5 Regeneration processes are initiated around the age of 6 weeks and continue until 12 weeks.21 At 3 months, 80–90% of the mdx skeletal fibres have central nuclei indicating a preceding cycle of degeneration and regeneration.22 At later stages mdx mice show a decline in their regeneration capacity
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Fig. 3 – (A) Sirius Red stained cryo-sections from masseter (MAS), temporal (TEM), soleus (SOL) and tongue (TON) muscles in control and mdx mice. (B) Quantification of the collagen expression (mean optical density – mod) of agematched controls and mdx mice (mean W S.E.M; **p < 0.01; ***p < 0.005, unpaired Student’s t-test) was performed using the KSRun software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
while the necrotic processes persist,6 effects that lead to progressive muscle wasting and fibrosis. Regarding the above mentioned histopathological characteristics of mdx muscles (see Section 1), an increased number of fibres with central nucleation seemed to be a measurement of the degree of increased severity of the disease, since such cells, in reality, originate from regenerated cells.23–25 The high level of centronucleation is a result of an increased regeneration in an attempt to balance the muscle cell necrosis.26 Thus, the observed hypertrophic and small regenerated fibres in mdx may be explained as replacement of
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necrotized muscle cells.26 Their presence among normal sized cells increases the cell size variance, which may therefore reflect the degree of severity of disease.26–29 The results from the four studied dystrophic muscles clearly demonstrated that the degree of centronucleation did not significantly differ between MAS, TEM and SOL. However, the percentage of such cells in MAS, TEM and SOL was significantly greater than in TON. Accordingly, the MAS, TEM and SOL seem to be most influenced by the disease in the studied animals. In our histomorphometric study, the MAS revealed a high degree of centronucleated fibres which concords with a recent study that compared the mdx masseter with diaphragm and gastrocnemius.10 Moreover, the mentioned study refers to a heterogeneous alteration of mdx muscles, showing a milder dystrophy in MAS as compared to diaphragm and gastrocnemius. In our histomorphometric study, the MAS revealed a high degree of centronucleated fibres which agrees with a recent study that compared the mdx masseter with diaphragm and gastrocnemius.30 One recent study examined the centronucleation in regenerated MAS in mdx mice.11 It was revealed that regeneration, i.e. centronucleation, in mdx MAS occurred 15 weeks after necrosis. We found in a 13-week-old mdx MAS a high degree of centronucleation which is slightly earlier than what was found in the above mentioned study. Regarding the variation in cell size, it was obvious that the variation of the size of dystrophic muscle cells was considerably more than that of normal muscles.26 So, previous studies reported a wide range of fibre sizes consisting of abnormal proportions of small and large fibres in mdx mice muscles.5,6 We partially confirmed these findings for the masticatory muscles and SOL. A wider range of the fibre sizes was indeed found in MAS, TEM and SOL, which showed only minor differences between one another in their distribution of muscle fibre sizes. But, TON was markedly different, displaying only small fibres in both, normal and mdx mice. Interestingly, small-caliber fibres are reported to be more resistant to necrosis and it was discovered that no centronucleation occurred in fibres with diameters inferior to 20 mm.23 Our investigations are consistent with these findings showing that the mdx TON muscle displayed smaller fibres as compared to MAS or TEM, suggesting that small-caliber fibres in some muscles of mastication (TON) may be less susceptible to muscle damage. Furthermore, fibre size mean diameter was larger in all tested mdx muscles as compared to controls and this could indicate a hypertrophy that accompanied dystrophin deficiency in these animals. It is known that hypertrophy in mdx mice consists of a mixture of hypertrophic and small fibres but not of fibrosis.31 Replacement of muscle tissue by collagen and fatty deposits is a feature of both human DMD and, to a lesser extent, murine mdx dystrophy.32,33 A marked accumulation of collagen was shown in the mdx SOL muscle in mdx mice of from 3 weeks to 1 year old.32 Moreover, previous investigations of hind limb and trunk muscles have confirmed a high degree of fibrosis and increased collagen content in older, as opposed to younger mdx mice .34,35 Our results concord with these findings. In our study we found that the level of collagen
expression was higher in mdx MAS, TEM and SOL as compared to controls, while in TON the collagen expression remained unchanged. Our results confirmed higher collagen content in mdx masticatory muscles as opposed to that in normal mice. These findings were accompanied by signs of inflammation suggesting a link between the repairing process (high collagen expression) and an inflammatory reaction in the mdx muscles as previously suggested.36 The MAS and TEM muscles displayed the highest degrees of collagen proliferation, suggesting greater limitation of their functioning as compared to TON and confirming intrinsic dissimilarities in collagen metabolism of functionally different skeletal muscles in mdx mice.37 In regards to the investigated histomorphometric parameters, it can be concluded that the mdx MAS and TEM express similar histopathological features of higher collagen content and a higher degree of CNF, as well as a variance of cell sizes, in comparison to limb mdx muscles (SOL). In contrast, TON of mdx mice seem to be partially spared from dystrophic alterations, as compared to MAS, TEM and limb muscles. Previous studies found that laryngeal muscles of the mdx mice could be spared from the development of the disease through protective mechanisms.38 We suggested similar mechanisms for the mdx tongue, although the ‘‘specific protective mechanism’’ may not be a ‘‘mechanism,’’ in the strict sense of the term. It was recently found that, besides the diaphragm, the TON of 26-month-old mdx mice was the most severely implicated muscle.39 In contrast, in our study we found that TON was the least implicated muscle of all the muscles studied. But, the difference might be explained by the younger age of our study animals (3 months old). This would imply that with age, the mdx TON becomes progressively more implicated in the disease process. Similarly, studies on DMD have shown that some orofacial muscles become implicated later in the course of the dystrophic process, which may explain the fact that in humans orofacial deformations occur at advanced stages of the disease. The force developed by a muscle is based substantially on its anatomic structure and depends to a great extent on the geometry of supporting anatomical structures, while it can also influence the shape of these structures. Mandibular shape changes have already been reported for dystrophic dy/dy mice.40 The histopathological features found in the mdx mice in the presented study may be accompanied by the severe dysfunction and force reduction which in turn may alter the shape of supporting bone structures, as reported previously.41 But, the model has a disadvantage, in as much as the masticatory muscles of mdx mice are not directly comparable to those of human DMDs. Contrary to humans, muscle fibres of mdx mice do regenerate after muscle fibre necrosis. Furthermore, the mastication mechanics are fundamentally different. Human masseter and temporal muscles have also a heterogeneous fibre type composition, the proportion of the fibre type 2 being generally lower.42 However, our results imply heterogenous changes in the anatomic structure of masticatory muscles due to dystrophy, which may influence their functional properties. This work provides supplementary information on how muscles adapt to dystrophic diseases. The possible influence of the adapted orofacial muscular system on the functioning and bone development is a great
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challenge for modern dental medical care and orthodontics. New insights into the mechanisms of interaction of muscles and the supporting bony structures may contribute to efforts for improving the quality of life of DMD patients.
Acknowledgement We thank I. Pieper for excellent technical assistance. Funding: None. Competing interests: None declared. Ethical approval: Landesamt fu¨r Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Germany. LALLF M-V/TSD/7221.3-2.3-001/09.
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