Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD’s complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mdx mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics. NEW & NOTEWORTHY This study uses novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.

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Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice

AJP Cell Physiology ◽

10.1152/ajpcell.00369.2002 ◽

2003 ◽

Vol 285 (3) ◽

pp. C522-C528 ◽

Cited By ~ 21

Author(s):

David R. Plant ◽

Gordon S. Lynch

Keyword(s):

Muscle Fibers ◽

Voltage Regulation ◽

Mdx Mouse ◽

Mdx Mice ◽

Channel Activity ◽

Release Channel ◽

Transverse Tubular System ◽

Skinned Muscle ◽

Dystrophin Deficiency ◽

And Control

Dystrophin is absent in muscle fibers of patients with Duchenne muscular dystrophy (DMD) and in muscle fibers from the mdx mouse, an animal model of DMD. Disrupted excitation-contraction (E-C) coupling has been postulated to be a functional consequence of the lack of dystrophin, although the evidence for this is not entirely clear. We used mechanically skinned fibers (with a sealed transverse tubular system) prepared from fast extensor digitorum longus muscles of wild-type control and dystrophic mdx mice to test the hypothesis that dystrophin deficiency would affect the depolarization-induced contractile response (DICR) and sarcoplasmic reticulum (SR) function. DICR was similar in muscle fibers from mdx and control mice, indicating normal voltage regulation of Ca2+ release. Nevertheless, rundown of DICR (<50% of initial) was reached more rapidly in fibers from mdx than control mice [control: 32 ± 5 depolarizations ( n = 14 fibers) vs. mdx: 18 ± 1 depolarizations ( n = 7) before rundown, P < 0.05]. The repriming rate for DICRs was decreased in fibers from mdx mice, with lower submaximal DICR observed after 5, 10, and 20 s of repriming compared with fibers from control mice ( P < 0.05). SR Ca2+ reloading was not different in fibers from control and mdx mice, and no difference was observed in SR Ca2+ leak. Caffeine (2–7 mM)-induced contraction was diminished in fibers from mdx mice compared with control ( P < 0.05), indicating depressed SR Ca2+ release channel activity. Our findings indicate that fast fibers from mdx mice exhibit some impairment in the events mediating E-C coupling and SR Ca2+ release channel activity.

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β-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation

Biomolecules ◽

10.3390/biom11111742 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1742

Author(s):

Paola Mantuano ◽

Brigida Boccanegra ◽

Elena Conte ◽

Michela De Bellis ◽

Santa Cirmi ◽

...

Keyword(s):

Mechanism Of Action ◽

Ex Vivo ◽

Mdx Mouse ◽

Mdx Mice ◽

Partial Recovery ◽

Glycoprotein Complex ◽

Promising Therapeutic Target ◽

Potential Synergy ◽

Dystrophin Glycoprotein Complex

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of β-dystroglycan (β-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle β-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-β-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of β-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-β-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-β-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing β-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing β-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

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Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

Biomolecules ◽

10.3390/biom10030416 ◽

2020 ◽

Vol 10 (3) ◽

pp. 416 ◽

Cited By ~ 3

Author(s):

Alex Addinsall ◽

Leonard Forgan ◽

Natasha McRae ◽

Rhys Kelly ◽

Penny McDonald ◽

...

Keyword(s):

Monoclonal Antibody ◽

Ex Vivo ◽

Fiber Type ◽

Mdx Mouse ◽

Mdx Mice ◽

Protein Levels ◽

Positive Effects ◽

Hindlimb Muscles ◽

Fiber Size ◽

Fast Twitch

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensor digitorum longus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect markers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

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Transcriptional Heterogeneity ofCryptococcus gattiiVGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways

mSphere ◽

10.1128/msphere.00445-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 7

Author(s):

Rhys A. Farrer ◽

Christopher B. Ford ◽

Johanna Rhodes ◽

Toni Delorey ◽

Robin C. May ◽

...

Keyword(s):

Stress Response ◽

Differential Expression ◽

Ex Vivo ◽

Early Time ◽

Regulatory Proteins ◽

Differentially Expressed ◽

Content Type ◽

Transcriptional Profiles ◽

Time Points ◽

Mouse Macrophages

ABSTRACTCryptococcus gattiiis a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages ofC. gattii(lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons ofin vitrotoex vivogene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related geneCAP2and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule geneCAS3in VGIV, which have lost theCAS31gene. Differential expression of one in fiveC. gattiigenes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.IMPORTANCEThe transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccaseLAC1to expression ofLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.

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Expression of the glucose transporter GLUT4 in the muscular dystrophic mdx mouse

Biochemical Journal ◽

10.1042/bj2910257 ◽

1993 ◽

Vol 291 (1) ◽

pp. 257-261 ◽

Cited By ~ 12

Author(s):

C Olichon-Berthe ◽

N Gautier ◽

E Van Obberghen ◽

Y Le Marchand-Brustel

Keyword(s):

Skeletal Muscle ◽

Plasma Membranes ◽

Glucose Transporter ◽

Mrna Level ◽

Mdx Mouse ◽

Mdx Mice ◽

Mrna Levels ◽

Skeletal Tissue ◽

Muscle Tissues ◽

And Control

Glucose transporter protein levels have been investigated in mdx and control (C57Bl/10) mice. Crude membrane fractions (microsomes plus plasma membranes) were prepared from skeletal muscle, heart, diaphragm and brain of 5-6-week-old and 6-7-month-old control and mdx mice. Using Western blot analysis with C-terminal-specific anti-peptide antibodies, we investigated the glucose transporters GLUT4 in the different muscle tissues and GLUT1 in brain. In skeletal tissue from the hindlegs, GLUT4 was increased by approximately 55% in mdx mice compared with control mice at both ages studied. In the diaphragm, the amount of GLUT4 protein was unchanged in young mdx mice, and was decreased by 37.4 +/- 4.7% in older mice compared with age-matched control mice. No difference was observed between mdx and control mice in the amounts of GLUT4 and GLUT1 in heart and brain preparations respectively. To determine whether the change in GLUT4 protein observed in the diaphragm and skeletal muscle of mdx mice was regulated through changes at the level of glucose transporter mRNA, Northern blot analyses were performed. In skeletal muscle, GLUT4 mRNA level per tissue was not different between the two groups of mice at both ages studied. In contrast, the decrease in the amount of GLUT4 protein observed in the diaphragm of 6-7-month-old mdx mice was accompanied by a decrease in the GLUT4 mRNA level. In conclusion, the levels of GLUT4 protein were modified in muscle tissues from mdx compared with control mice, and these modifications were different depending on the muscle involved and the age of the mice. An increase in the amount of GLUT4 protein in the skeletal muscle of mdx mice was not due to changes at the mRNA level. The diaphragms of 6-7-month-old mdx mice exhibited decreases in GLUT4 protein and mRNA levels that were not detected in young animals (5-6 weeks old).

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Transcriptional heterogeneity ofCryptococcus gattiiVGII compared with non-VGII lineages underpins key pathogenicity pathways

10.1101/396796 ◽

2018 ◽

Cited By ~ 1

Author(s):

Rhys A. Farrer ◽

Christopher B. Ford ◽

Johanna Rhodes ◽

Toni Delorey ◽

Robin C. May ◽

...

Keyword(s):

Stress Response ◽

Differential Expression ◽

Ex Vivo ◽

Early Time ◽

Regulatory Proteins ◽

Differentially Expressed ◽

Transcriptional Profiles ◽

Over Expression ◽

Time Points ◽

Mouse Macrophages

AbstractCryptococcus gattiiis a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages ofC. gattii(VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison ofin vitrotoex vivogene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related geneCAP2, and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule geneCAS3in VGIV having lostCAS31. Differential expression of one in fiveC. gattiigenes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.ImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccaseLAC1toLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.

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Investigating the Potential for Sulforaphane to Attenuate Gastrointestinal Dysfunction in mdx Dystrophic Mice

Nutrients ◽

10.3390/nu13124559 ◽

2021 ◽

Vol 13 (12) ◽

pp. 4559

Author(s):

Kristy Swiderski ◽

Suzannah J. Read ◽

Audrey S. Chan ◽

Jin D. Chung ◽

Jennifer Trieu ◽

...

Keyword(s):

Muscular Dystrophy ◽

Corn Oil ◽

Therapeutic Potential ◽

Ex Vivo ◽

In Vivo Studies ◽

Muscle Pathology ◽

Mdx Mouse ◽

Mdx Mice ◽

Dystrophic Mice

Gastrointestinal (GI) dysfunction is an important, yet understudied condition associated with Duchenne muscular dystrophy (DMD), with patients reporting bloating, diarrhea, and general discomfort, contributing to a reduced quality of life. In the mdx mouse, the most commonly used mouse model of DMD, studies have confirmed GI dysfunction (reported as altered contractility and GI transit through the small and large intestine), associated with increased local and systemic inflammation. Sulforaphane (SFN) is a natural isothiocyanate with anti-inflammatory and anti-oxidative properties via its activation of Nrf2 signalling that has been shown to improve aspects of the skeletal muscle pathology in dystrophic mice. Whether SFN can similarly improve GI function in muscular dystrophy was unknown. Video imaging and spatiotemporal mapping to assess gastrointestinal contractions in isolated colon preparations from mdx and C57BL/10 mice revealed that SFN reduced contraction frequency when administered ex vivo, demonstrating its therapeutic potential to improve GI function in DMD. To confirm this in vivo, four-week-old male C57BL/10 and mdx mice received vehicle (2% DMSO/corn oil) or SFN (2 mg/kg in 2% DMSO/corn oil) via daily oral gavage five days/week for 4 weeks. SFN administration reduced fibrosis in the diaphragm of mdx mice but did not affect other pathological markers. Gene and protein analysis revealed no change in Nrf2 protein expression or activation of Nrf2 signalling after SFN administration and oral SFN supplementation did not improve GI function in mdx mice. Although ex vivo studies demonstrate SFN’s therapeutic potential for reducing colon contractions, in vivo studies should investigate higher doses and/or alternate routes of administration to confirm SFN’s potential to improve GI function in DMD.

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Novel assessment of leukocyte-rich platelet rich plasma on functional and patient reported outcomes in knee osteoarthritis: a pilot study

Regenerative Medicine ◽

10.2217/rme-2021-0032 ◽

2021 ◽

Author(s):

Prathap Jayaram ◽

Gu Eon Kang ◽

Brett L Heldt ◽

Olumide Sokunbi ◽

Bo Song ◽

...

Keyword(s):

Knee Osteoarthritis ◽

Patient Reported Outcomes ◽

Platelet Rich Plasma ◽

Early Time ◽

Therapeutic Effects ◽

Timed Up And Go ◽

End Points ◽

Time Points ◽

Gait Parameters ◽

Patient Reported

Background: Leukocyte-rich platelet-rich plasma (LR-PRP) has demonstrated to be beneficial for patient with knee osteoarthritis (KOA); however, reliable objective end points to accurately assess its therapeutic effects is lacking. Aim: To investigate the efficacy of LR-PRP as assessed by functional and patient-reported outcomes at early time-points (6 weeks). Materials & methods: We conducted a prospective cohort study in 12 patients with diagnosed KOA (Kellgren Lawrence score of II–III), who underwent a single ultrasound-guided LR-PRP injection. Results: There was significant improvement in timed up and go, pain and quality of life scales and balance parameters. There were nonsignificant improvements in range of motion and gait parameters. Conclusion: LR-PRP demonstrates efficacy in meaningful end points for functional and patient reported outcomes at early time points in patients with KOA.

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Fine structure of early degenerating myofibers in the tibialis anterior of young ‘MDX’ mouse

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010367x ◽

1988 ◽

Vol 46 ◽

pp. 322-323

Author(s):

H.D. Geissinger ◽

C.K. McDonald-Taylor

Keyword(s):

Fine Structure ◽

Muscle Regeneration ◽

Tibialis Anterior ◽

Genetic Defect ◽

Mdx Mouse ◽

Mdx Mice ◽

Histopathological Changes ◽

Electron Microscopic ◽

Dystrophic Mouse ◽

Preliminary Study

A new strain of mice, which had arisen by mutation from a dystrophic mouse colony was designated ‘mdx’, because the genetic defect, which manifests itself in brief periods of muscle destruction followed by episodes of muscle regeneration appears to be X-linked. Further studies of histopathological changes in muscle from ‘mdx’ mice at the light microscopic or electron microscopic levels have been published, but only one preliminary study has been on the tibialis anterior (TA) of ‘mdx’ mice less than four weeks old. Lesions in the ‘mdx’ mice vary between different muscles, and centronucleation of fibers in all muscles studied so far appears to be especially prominent in older mice. Lesions in young ‘mdx’ mice have not been studied extensively, and the results appear to be at variance with one another. The degenerative and regenerative aspects of the lesions in the TA of 23 to 26-day-old ‘mdx’ mice appear to vary quantitatively.

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Does transcranial direct current stimulation affect selective visual attention in children with left-sided infantile hemiplegia? A randomized, controlled pilot study

Brain Impairment ◽

10.1017/brimp.2020.20 ◽

2020 ◽

pp. 1-13

Author(s):

Raed A. Alharbi ◽

Saleh A. Aloyuni ◽

Faizan Kashoo ◽

Mohamed I. Waly ◽

Harpreet Singh ◽

...

Keyword(s):

Pilot Study ◽

Direct Current ◽

Transcranial Direct Current Stimulation ◽

Repeated Measures ◽

Sham Group ◽

Time Points ◽

Selective Visual Attention ◽

Subject Factor ◽

And Control

Abstract Objective: Infantile hemiplegia due to brain injury is associated with poor attention span, which critically affects the learning and acquisition of new skills, especially among children with left-sided infantile hemiplegia (LSIH). This study aimed to improve the selective visual attention (SVA) of children with LSIH through transcranial direct current stimulation (tDCS). Methods: A total of 15 children participated in this randomized, double-blinded, pilot study; of them, 10 experienced LSIH, and the remaining 5 were healthy age-matched controls. All the children performed the Computerized Stroop Color-Word Test (CSCWT) at baseline, during the 5th and 10th treatment sessions, and at follow-up. The experimental (n = 5) and control groups (n = 5) received tDCS, while the sham group (n = 5) received placebo tDCS. All three groups received cognitive training on alternate days, for 3 weeks, with the aim to improve SVA. Results: Two-way repeated measures analysis of variance (ANOVA) showed a statistically significant change in the mean scores of CSCWT between time points (baseline, 5th and 10th sessions, and follow-up) within-subject factor, group (experimental, sham) between-subject factor and interaction (time points X group) (p < 0.005). Furthermore, a one-way repeated measures ANOVA showed significant differences between time point (p < 0.005) for the experimental and control group but not the sham group. Conclusion: These pilot results suggest that future research should be conducted with adequate samples to enable conclusions to be drawn.

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