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Acta Histochemica
journal homepage:www.elsevier.com/locate/acthis
Short communication
Presence of metalloproteinases 2 and 9 and 8-OHdG in the
fibrotic process in
skeletal muscle of Mdx mice
Lidiane Begalli de Souza, Carla Maziero, Mariana Cruz Lazzarin, Hananiah Tardivo Quintana,
Tabata de Carvalho Tomé, Vivianne Izabelle de Araújo Baptista, Flavia de Oliveira
*
Departamento de Biociências, Universidade Federal de São Paulo, Campus Baixada Santista, SP, Brazil
A R T I C L E I N F O
Keywords:
Duchenne muscular dystrophy Skeletal muscle
MMP-2 MMP-9 8-OHdG
A B S T R A C T
Inflammation and oxidative stress occurs in muscle of Duchenne muscular dystrophy (DMD). The relationship between a panel of biomarkers and the DMD outcome is necessary to indicate of disease progression and re-sponse to rehabilitation programs. The aim was to analyze the connective tissue of muscle of Mdx mice and immunoexpression of MMP-2, MMP-9, and 8-OHdG, which signalizes oxidative stress related to DNA damage. Biceps brachii of male C57BL/10 and C57BL/10-Dmdmdxmice was submitted to Hematoxylin-Eosin, Sirius red
and immunohistochemistry (MMP-2, MMP-9 and 8-OHdG) analysis. Mdx showed focal lesions with intense inflammation and fibrosis related to immunoexpression of MMP-2 and MMP-9, proving the hypothesis that these MMPs are linked to muscular tissue degeneration, which can be regenerated by their inhibition, improving the treatment of DMD carriers. Histopathologicalfindings related to centralized nuclei increase were related to higher 8-OHdG immunomarked nuclei in Mdx, which signalizes oxidative stress associated with DNA damage provoked by DMD. Such result shows that the evaluation of 8-OHdG during the evolution of the disease could be a method to evaluate DMD disease progression.
1. Introduction
Duchenne muscular dystrophy (DMD) is a neuromuscular degen-erative progressive recessive X-linked disease (Lott et al., 2014), more precisely to the gene of dystrophin, a protein capable of maintaining the musclefiber integrity by its association with the transmembrane gly-coprotein complex and cytoskeleton (Petrof, 1998). DMD is estimated to affect one in 3.500 male births (Henderson et al., 2014).
To investigate the pathophysiology of DMD, the Mdx mice, which have no dystrophin detectable, are the most commonly employed models in DMD research. As observed in humans carriers of DMD, DMDmdxmice have mechanical instability on their muscles due to the absence of dystrophin, which after repetitive contraction-relaxation process, is capable of making the skeletal muscle suffer of necrosis process (Grounds et al., 2008;Nakagaki and Camilli, 2012).
Inflammation and oxidative stress have increasingly appeared as evidence to explain the process of this disease (Chabouni et al., 2010). The damaged muscular tissue counts on inflammatory cells, which gather grow factors and pro inflammatory cytokines (Abdel-Salam et al., 2009). Therefore, the accumulation of components of
extracellular matrix, such as the collagen, is responsible for thefibrosis (Zanotti et al., 2011).
Matrix Metalloproteinases (MMPs) are capable of breaking the ex-tracellular matrix, since they are proteolytic enzymes (Birkedal-Hansen et al., 1993). During the degradation of the extracellular matrix, MMP-2 breaks the collagen IV and helps with the formation of new myofibers. On the other hand, the MMP-9 is associated with the inflammatory process of the tissue and, unlike MMP-2, it participates in the muscular regeneration (Serrano and Muñoz-Cánoves, 2010).
Free radical reactions are caused by oxidative stress. One of the most important forms of free radicals in lesions that causes oxidative stress is the 8-hydroxy-2′-deoxyguanosine (8-OHdG), and, because of that, this free radical has been chosen as a biomarker to oxidative stress (Valavanidis and Fiotakis, 2009) and represents a biomarker of DNA damage (Cao et al., 2016).
The relationship between a panel of biomarkers and the DMD out-come is necessary to indicate disease progression and the response to rehabilitation programs. Thus, the aim of this study was to analyze the connective tissue of the skeletal muscle of DMDmdxmice and its relation with the immunoexpression of MMPs 2 and 9, as well as to investigate
https://doi.org/10.1016/j.acthis.2019.151458
Received 21 May 2019; Received in revised form 13 September 2019; Accepted 7 October 2019
⁎Corresponding author at: Departamento de Biociências, Universidade Federal de São Paulo– UNIFESP, Rua Silva Jardim, 136 – Lab 328, CEP: 11015-020, Santos,
SP, Brazil.
E-mail address:flavia.oliveira@unifesp.br(F. de Oliveira).
Acta Histochemica 122 (2020) 151458
0065-1281/ © 2019 Elsevier GmbH. All rights reserved.
the immunoexpression of a biomarker for oxidative stress associated with DNA damage (8-OHdG) in skeletal muscle.
2. Materials and methods
2.1. Experimental groups and obtaining tissue
Sixteen-week-old C57BL/10 (n = 05) and C57BL/10-Dmdmdx(n = 05) male mice were distributed in control and Mdx groups, respectively. All procedures were approved by the Animal Ethics Committee in the Use of Animal of Federal University of Sao Paulo, Brazil (8165240614).
The animals of different groups were euthanized with a lethal IP injection of Ketamin (150 mg/ml) and Xylasin (30 mg/ml). Biceps brachii muscles were dissected andfixed in 10% formalin phosphate buffer for 24 h for histological analysis.
2.2. Histopathological, connective tissue and immunohistochemistry analysis
The specimens were routinely embedded in paraffin blocks and cut in transversal sections (4μm). The slides were stained with hematoxylin and eosin (H&E) for histopathological analysis and Sirius red to eval-uate connective tissue.
For immunohistochemistry analysis, specimens were incubated with primary antibodies: mouse monoclonal anti-MMP-2, (dilution of 1:100, clone 8B4, catalogue number sc-13595, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-MMP-9 (dilution of 1:100, clone 2C3, catalogue number sc-21733, Santa Cruz Biotechnology, Dallas, TX, USA) and mouse monoclonal anti-8-OHdG (dilution of 1:100, clone 15A3, catalogue number sc-66036, Santa Cruz Biotechnology, Dallas, TX, USA). Then they were washed in PBS and incubated with biotin-conjugated anti-rabbit secondary antibody IgG
Fig. 1. Biceps barchii muscle of Control (a, d) and Mdx mice (b, c, e, f). Observe in (a) that the Control animals present homogeneous muscle fascicles and in higher magnification (d) muscle fibers with equidistant spacing, homogeneous size and peripheral nuclei. Conversely in (b) Mdx mice present heterogeneity muscle fascicles with focal lesions with intense inflammatory infiltrate highlighted in dotted rectangle. This highlighted area was shown in (c) with higher magnificence and numerous inflammatory cells and rounded muscle fibers with centralized nuclei and necrotic fragments of muscle fibers (head arrows). (e) and (f) represent general characteristics of dystrophic muscle cells distant from focal lesions. In (e), there is a large space between thefibers when compared with control muscle fibers, heterogeneity in muscularfibers size (small arrows), centralized nuclei and fibers at splitting process (prominence image). In addition, in (f), there are rounded fibers with acidophilic aspect (big arrows) and presence of inflammatory infiltrate between muscle cells (star). Hematoxylin & Eosin.
Fig. 2. Biceps brachii muscle of Control (C) and Mdx mice. In C, animals presentedfibers with equidistant spacing and muscular wrapping (endomysium and perymisium). In a focal lesion region of Mdx, we observed differentiated spacing between the muscle fibers and intense process of fibrosis, represented in red (stars). Sirius Red (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article).
(Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:200 in PBS. Then specimens were counterstained with Harris hematoxylin. Negative controls were analyzed by omitting primary antibody from the staining procedure. The analyses were carried out using a computerized imaging system (software Axio Visio 4.5, Carl Zeiss, Oberkochen, Germany) attached to a binocular light microscope (Axio Observer D1, Carl Zeiss, Oberkochen, Germany).
3. Results
3.1. Histopathological and connective tissue analysis
Histopathological evaluation presented Mdx Group with hetero-geneous muscle fascicles fibers and focal lesions with intense in-flammatory infiltrate with necrotic fragments of muscle fibers (Fig. 1). Musclefibers of Mdx groups distant from focal lesions showed more centralized phagocytic cells and cells nuclei and there was a large amount of splitting in muscular tissue in addition to rounding cells.
The connective tissues analysis in Fig. 2illustrates that Control animals showed a normal standard of presentation of the wrapping of connective tissue (endomysium and perimysium). Nevertheless, in the areas with focal lesions, Mdx animals showed large intercellular spaces filled with connective tissue.
3.2. Immunohistochemistry analysis (MMP-2, MMP-9 e 8-OHdG) The analysis for MMP-2 (Fig. 3) revealed that the Mdx animals presented immunolabeling in the areas of connective tissue thickening
and in the areas of focal lesions with intense inflammatory infiltrate. In the same way, the immunolabeling of MMP-9 (Fig. 3) occurred in areas of connective tissue thickening and focal lesions with inflammatory infiltrate. Regarding 8-OHdG (Fig. 4), the Mdx group showed more 8-OHdG immunoreactive nuclei when compared with the Control group. 4. Discussion
Inflammation and unregulated accumulation of collagen (fibrosis) in the muscular tissue are characteristics DMD changes. Furthermore, oxidative stress, along with intense inflammatory process is capable of changing physiological and morphological characteristics of the ske-letal muscle, rendering relevant to know the expression of a biomarker for oxidative stress related to DNA damage, since its function in the skeletal muscle in Mdx mice is still not unknown. This study aimed to analyze the histopathology, connective tissue and immunoexpression of MMP-2, MMP-9 and 8-OHdG of biceps brachii muscle of Mdx mice. Main results revealed the presence of focal lesions with inflammatory infiltrate and connective tissue thickening related to the increase of immunolabeling of MMP-2 and MMP-9. In addition, we noticed that the histopathological changes are related to the increase in the im-munolabeling of 8-OHdG.
During the immunohistochemistry analysis, we determined im-munolabeling of MMP-2 in the connective tissue of the muscle. This result enhances thefindings ofFukushima et al. (2007), who showed that the immunomarking for MMP-2 was increased in dystrophic ani-mals. In our study, we observed that this immunomarking was present mainly in cellular perimysium. The deficiency of dystrophin takes to a
Fig. 3. MMP-2 immunohistochemistry of biceps brachii muscle of Control (a) and Mdx mice (b,c); MMP-9 immunohistochemistry of biceps brachii muscle of Control (d) and Mdx mice (e,f); Immunohistochemistry negative control of Control group (g) and Mdx group (h). In MMP-2, black arrows in (b) represent immunolabeling at thickened connective tissue in the Mdx group. White arrows in (c) represent immunolabeling for MMP-2 in focal lesions with intense inflammatory infiltrate. In MMP-9, a“cord” of connective tissue with immunolabeling (white arrows) appears in (e). Black arrows in (f) show the area of intense inflammatory infiltrate immunomarked.
constant cycle of degeneration-regeneration of the extracellular matrix components, occurring thus a replacement of musclefibers by collagen fibers and consequent fibrosis, decreasing the muscular functionality of the disease carrier. In these tissues, MMP-2 has increased, making us believe that the larger the immunomarking in this tissue by MMP-2, the larger the degradation of extracellular matrix (Serrano and Muñoz-Cánoves, 2010). In the same way, we analyzed the expression of MMP-9 in loose connective tissue between musclefibers of Mdx animals, this tissue, with intense inflammatory infiltrate; while in Control animals, which does not present these modifications; immunolabeling was not visible, which means that MMP-9 is also associated withfibrosis and muscular tissue regeneration. Besides the marking by MMP-9 in Mdx animals’ tissue, numerous fibers were observed in the regeneration process, in other words, with central nuclei.
Once MMPs are related to inflammatory process and fibrosis, Zimowska et al. (2012) used MMP-2 and 9 inhibitors in tissue in re-generation stage of Wistar rats. They found that the tissue treated with the inhibitor improved its regeneration capacity, and the fibrosis showed a decrease in comparison with untreated muscles. Studies with inhibitors of inflammatory process and even inhibitors of MMPs, could also have positive effects in DMD.Hindi et al. (2013), using an ex-perimental model Mdx, showed their study that satellite cells have a better activation when MMP-9 is inhibited and, in that way, there is an improvement in the regeneration of the tissue. Thereby, we suggest that from the relation obtained in this study on histopathological and
connective tissue changes with MMP-2 and 9, as mensuration of these MMPs as their control could be related to therapy and DMD disease staging.
The immunohistochemistry analysis of 8-OHdG showed marking in both groups; however, the Mdx group presented more immunomarked nuclei than the Control group. Control group presented marking due to the presence of reactive oxygen species original by cellular metabolism and not by a pathologic condition, as we can infer by the study made by Gianni et al. (2004). Conversely, the Mdx group presented more im-munoreactive nuclei, indicating the presence of reactive oxygen species in large amount, which also generates oxidative stress in large amount, justifying, thereby, the large 8-OHdG immunolabeling. In a study about the oxidative stress from advanced age in mice,Ryan et al. (2010), found results similar to ours, highlighting that the more cells suffered from the oxidative stress presented, the more marking by 8-OHdG they presented. The 8-OHdG could also be evaluated by urine analysis which makes the evaluation of this component related to oxidative stress easy to be measured in patients (Rodriguez and Tarnopolsky, 2003). Al-though DMD is still incurable, recently, multiple drug development focus on preventing the progress of the disease. In addition to corti-costeroids, therapies such as stop codon readthrough agents, exon skipping agents, utrophin modulators and other new therapies to repair muscle (Reinig et al., 2017) aim to promote these patients quality of life and long life expectancy. Furthermore, exercise interventions in motor rehabilitation has a positive role in many diseases but the intensity,
Fig. 4. 8-OHdG immunohistochemistry of biceps brachii muscle of Control (a) and Mdx mice (b); Immunohistochemistry negative control of Control group (c) and Mdx group (d). In (a), we observed the presence of rare immunomarking nuclei (arrows), while in Mdx (b), the majority of the nuclei were immunomarked (arrows), including centralized nuclei.
type and frequency in DMD has controversial recommendations (Gianola et al., 2013;Spaulding and Selsby, 2018) because of the sus-ceptibility to muscle lesions by exercise in DMD. Meanwhile, bio-markers could help provide evidence of the efficacy of several treat-ments. Creatine kinase, for example, is very important for the diagnosis, but the higher activity of creatine kinase in the plasma of patients with dystrophy is not suitable to monitor disease progression and response to therapy because its concentration is easily influenced by muscular membrane disruption (Hathout et al., 2016). Consequently, the relation of 8-OHdG to cellular damage showed in this study makes us infer that a study about the control of 8-OHdG as a potential biomarker in pa-tients could be a good precursor of the level of oxidative stress asso-ciated with muscular damage caused by DMD.
In conclusion, we observed immunoexpression of MMP-2 and 9 in focal lesions withfibrosis and inflammation, these regions were found just in Mdx animals, proving the hypothesis that these MMPs are linked to muscular tissue degeneration, which can be regenerated by their inhibition, improving thus the treatment of DMD carriers. In addition, histopathological findings were related to the increase in 8-OHdG, which signalizes oxidative stress associated with DNA damage pro-voked by DMD. This result shows that the evaluation of 8-OHdG during the evolution of the disease could be a method to evaluate DMD pro-gression.
Author statement
I declare that all authors have made substantial contributions in the paper entitled“Presence of metalloproteinases 2 and 9 and 8-OHdG in thefibrotic process in skeletal muscle of Mdx mice”, as follow: L.B.S. and C.M. and F.O. participated in performance of the research, data analysis and writing the article. M.C.L. and H.T.Q. contribute to per-formance of research design and data analysis. T.C.T. and V.I.A.B. contribute in performance of research and analysis. All authors con-tribute to reviewing the article.
Declaration of Competing Interest
The authors declare that they have no conflict of interest. Acknowledgments
We would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) forfinancial grants (2014/22211-6). References
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