Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to absence or decrease of dystrophin protein generating progressive muscle degeneration. Cell therapy using mesenchymal stem cell (MSC) has been described as a treatment to DMD. In this work, MSC derived from deciduous teeth, called stem cells from human exfoliated deciduous teeth (SHED), were injected in acupoint as an alternative therapy to minimize muscle degeneration in twenty-two mdx mice. The treatment occurred three times with intervals of 21 days, and animals were analyzed four times: seven days prior treatment (T-7); 10 days after first treatment (T10); 10 days after second treatment (T31); and 10 days after third treatment (T52). Animals were evaluated by wire test for estimate strength and blood was collected to perform a creatinine phosphokinase analysis. After euthanasia, cranial tibial muscles were collected and submitted to histological and immunohistochemistry analyses. Treated groups presented improvement of strength and reduced creatinine phosphokinase levels. Also, a slight dystrophin increase was observed in tibial cranial muscle when aquapuncture was associated SHED. All therapies have minimized muscle degeneration, but the association of aquapuncture with SHED appears to have better effect, reducing muscle damage, suggesting a therapeutic value.
Muscular dystrophies are neuromuscular diseases genetically related that compromise progressively skeletal muscles. Regarding that, Duchenne muscular dystrophy (DMD) presents worldwide frequency of approximately 1 : 3500 male children. DMD is a recessive inheritance pattern, with the first symptoms starting at 3–6 years old [
Several studies tried some therapeutic strategies to treat DMD, such as antibiotics, glucocorticoids, and calcium channel blockers [
Stem cells from human exfoliated deciduous teeth (SHED) were first isolated as a population of multipotent stem cells from the pulp of exfoliated deciduous teeth [
The dystrophin deficient mice, mdx (C57BL10 ScSn-DMD-mdx/J) are widely used as an animal model to study DMD in order to investigate various aspects of this disease and for preclinical test or proof-of-concept studies. Mdx mice have a milder clinical behavior than human, presenting muscle degeneration in waves leading to necrosis, as well as presenting some serum biochemical parameters and histological changes, similar to human affected by DMD [
Acupuncture, a traditional oriental medicine is therapeutic approach that has been used to contribute to pain control, analgesia, inflammatory process, and others factors improving the treatment in several degenerative diseases like Parkinson’s, DMD, and spinal cord injury [
Regarding this possible beneficial effect, this study aimed to use acupoint injection with SHED in order to verify the benefits of this approach to treat mdx mice.
This work was approved by the ethics committee on animal use, from the School of Veterinary Medicine and Animal Science, São Paulo University, protocol number 2431/2011. Animal research was conducted in accordance with the Ethical Principles in Animal Experimentation, according to the National Institute of Health Guide for the Care and Use of Laboratory Animals, 1996. Twenty-two male mdx mice (14–20 g) between 4 and 6 weeks of age from IPEN animal facility (Nuclear and Energy Research Institute, Brazil) were used in this work. Animals were kept in cage (
SHED were obtained using a modified Miura’s protocol [
Four groups of animals were distributed to do the following treatments: (A) SHED injection in false acupoint (
Acupoint injections and timeline of treatments. (a) Acupoints used in this study in mdx mice: BL47, BL49, and BL52 (white arrows). (b) Experimental timeline considering days of injection and days for treatment evaluation. (T-7) seven days before treatment; (T10) ten days after first treatment (SHED or saline injection); (T31) twenty-one days after second treatment (SHED or saline injection); (T52) twenty-one days after third treatment (SHED or saline injection).
Animals were submitted to clinical evaluation by wire test, creatinine phosphokinase, and histological analyses.
The strength of the animal’s forelimbs was assessed by wire test adapted from van Putten et al. (2012) [
Wire test score and the strength quantification. This table reveals the score attributed for each action using wire test. The total time used in this evaluation was 21 seconds. The number of times walking or holding on the wire was attributed considering 0 to 4 steps. For each animal, the points obtained from Table
Action | 0 step/holding until 9 s on the wire | 1 to 3 steps/holding 10–20 s on the wire | 4 or > turn/21 s |
---|---|---|---|
Raise the body above the wire | 0 point | 1 point | 2 points |
Walk on the wire | 0 point | 1 point | 2 points |
Put the pelvic member on the wire | 0 point | 1 point | 2 points |
Time on the wire (seconds only) | 0 point | 1 point | 2 points |
Animals’ blood was collected in the facial vein and plasma was diluted 1 : 100. The samples were analyzed kinetically by CK-NAC kit (Randox Laboratories, London, England) in Biotek Powerwave XS spectrophotometer by the KC4 software (v. 3.4) (Biotek, CA, USA). Results were plotted in GraphPad Prism 5 and analyzed using 2-way ANOVA.
After animal’s euthanasia, cranial tibial muscle and diaphragm were collected, frozen in liquid nitrogen, fixed in 4% paraformaldehyde, and subjected to routine histological procedures. Sections of 5
Here we proposed an alternative method to treat DMD using mdx as a model to test the benefits of acupuncture associated with stem cell therapy, a pioneer work in this field. Acupoints were selected bilaterally after an accurate assessment of DMD, associated with the benefits they could bring from Traditional Chinese Medicine. So, in this project the points used to apply acupuncture were Bladder 47 (B47), Bladder 49 (B49), and Bladder 52 (B52) based on the fact that DMD patients present a deficiency in liver, spleen-pancreas, and kidney meridians and the points chosen could nourish the first column of Shu points [
In this work, mdx mice were evaluated for the total time of 52 days (Figure
Concerning force using an adapted wire test, statistical differences were observed comparing treatments, with a strength improvement in animals submitted to SHED/true acupoints (
Analysis for the treatment using wire test and seric CPK, (a) scale force graphic using wire test, (b) seric CPK analysis.
Serum CPK analysis is a reliable measurement for the identification and quantification of muscle damage. This analysis infers animals’ improvement or worsening during the treatment [
The causes of the perpetuation of muscle degeneration are numerous such as free radicals release and lack of the dystrophin [
In addition, we observed a delay in disease progression in animals treated with SHED in false acupoints suggesting that cells could present a paracrine effect, which is already, known for SHED [
Histological analysis inferred that treatments did not interfere with anatomical structure of tissue and did not produce inflammatory reaction (Figures
Histological analyses of cranial tibial muscle using HE staining (a–d). In (c), inside the circle, centralized nuclei and increased heterogeneity in myofibers size, characteristics of mdx muscle degeneration. Dystrophin expression in tibial cranial muscles after 52 days of the treatment (e–i). Groups: SHED/false acupoints; saline/true acupoints; SHED/true acupoints; and control. Scale Bar—100
Immunohistochemistry using dystrophin protein analysis in cranial tibial muscle in mdx revealed that all treated groups presented a slight dystrophin improvement (Figures
Considering an acupuncture therapy for mdx and the results found here, acupuncture associated or not with SHED cells seems to increase dystrophin production and prevent the disease progression, being revealed as an alternative treatment for this untreated disease. An improvement in dystrophin expression was also observed in SHED/false acupoint suggesting the influence of cells in the treatment. Moreover, CPK decrease was observed in all treatments, but only SHED/true acupoint treatment was able to improve force, an important clinical parameter to DMD patients. In this work we can infer that acupuncture associated with SHED has a beneficial effect in muscle force as well as dystrophin expression and CPK decrease.
The use of acupuncture associated with SHED therapy appears to have a benefic effect in reducing disease progression in mdx mice and so far open new perspectives and opportunities to DMD treatment or could, at least, improve the quality of patient’s life.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Greyson Vitor Zanatta Esper and Graciela Conceição Pignatari contributed equally and are both first authors and the same to Patricia Cristina Baleeiro Beltrão-Braga and Maria Angelica Miglino, who are both last authors.
The authors are thankful to the Institute for Energy and Nuclear Research for the support regarding the obtaining of animals specially Maria Neide F. Mascarenhas, Cecília S. Ferreira, and Ismária S. Reis. They are also thankful to the Department of Surgery, Anatomy of Wild and Domestic Animals Service and National Council of Technological and Scientific Development (CNPq), and Sao Paulo Research Foundation (FAPESP) for the financial support.