- Email: [email protected]
Immunohistochemical Characterization of the Mouse Subcutaneous Perimuscular Fascial Plexus
ARTICLE IN PRESS Journal of Bodywork and Movement Therapies (2009) 13, 269–271
Journal of Bodywork and Movement Therapies www.intl.elsevierhealth.com/journals/jbmt
ABSTRACTS Tissue stretch decreases procollagen-1 and TGF-b1 in mouse subcutaneous fascia Nicole A. Bouffarda, Kenneth R. Cutroneob, Debbie Stevens-Tuttlea, Gary J. Badgerc, Helene M. Langevina,d a
Departments of Neurology, University of Vermont, Burlington, VT 05405, USA b Departments of Biochemistry, University of Vermont, Burlington, VT 05405, USA c Departments of Medical Biostatistics, University of Vermont, Burlington, VT 05405, USA d Departments of Orthopedics, University of Vermont, Burlington, VT 05405, USA
1.5 cm 1.5 cm area on one side of the back lateral to the midline, with the other side of the back serving as the control. Mice where then randomized to either stretch or no stretch. In the stretch group mice underwent stretching (approximately 30% strain) of the trunk for 10 min (mice suspended by the tail with the forelimbs touching a slightly inclined surface) twice a day for 7 days. All mice were sacrificed by decapitation at day 7. Subcutaneous fascia of the back was dissected, fixed and immunohistochemically stained for procollagen-1 (a marker for newly formed collagen). (2) Mouse subcutaneous fascia explant model: tissue was excised and kept in organ culture (DMEM culture media) for 4 days. One day after excision, fascia samples were either stretched (20% strain) for 10 min or not stretched. Aliquots of DMEM culture media were assayed by ELISA for TGF-b1 protein on days 0, 1 and 3 post stretch (or no stretch).
Background
Results
Increased collagen and transforming growth factor beta 1 (TGFb1) production are known to play an important role in connective tissue remodeling responses to injury, and in the production of excessive scarring and fibrosis. Although mechanical forces are known to influence connective tissue remodeling in vivo, their effect on TGF-b1 and collagen production in injured tissues are not well understood. We hypothesize that tissue stretch will attenuate TGF-b1-mediated collagen deposition in response to injury which may have implications for the therapeutic mechanisms of treatments that utilize tissue stretch.
In the in vivo microinjury model a significant difference in fascia procollagen-1 between injury and no-injury was observed in the absence of stretch (po 0.008). However, when injury was followed by tissue stretch, no significant difference was found between injury and no-injury (p ¼ 0.27). In the ex vivo model, both stretched and unstretched fascia samples showed increased TGF-b1 protein levels in the media from day 0 to day 3. However, at day 3, TGF-b1 protein levels were significantly lower in stretched vs. unstretched fascia (repeated measures ANOVA, po0.01).
Conclusion Methods Two different models were used to examine the effect of stretch on procollagen-1 and TGF-b1 protein levels in injured tissue:
(1) in vivo mouse microinjury model: a 5 mm incision was performed under anesthesia in the middle of the back at the level of the scapula. A microsurgery blade was introduced along the subcutaneous fascia plane and the attachments between the fascia and subcutaneous muscle were cut over a
These experiments suggest that short-term tissue stretch modulates TGF-b1-mediated tissue remodeling by attenuating procollagen-1 production. These results may be potentially relevant to the therapeutic mechanisms of treatments applying tissue stretch (e.g. massage, Rolfing, yoga, acupuncture).
Disclosure This study was funded by NCCAM Research Grant RO1 AT01121.
10.1016/j.jbmt.2008.04.017
Immunohistochemical characterization of the mouse subcutaneous perimuscular fascial plexus Sarah Coreya, Nicole Bouffarda, Helene Langevina,b a
Department of Neurology, The University of Vermont, Burlington, VT 05405, USA b Department of Orthopedics and Rehabilitation, The University of Vermont, Burlington, VT 05405, USA E-mail address: [email protected] (N.A. Bouffard). 1360-8592/$ - see front matter & 2008 Published by Elsevier Ltd. doi:10.1016/j.jbmt.2008.04.017
Purpose The innervartion of the deep fascia has not been fully described but may play an important role in musculoskeletal chronic pain. The pathophysiology of chronic musculoskeletal pain is poorly understood and may be related to nociceptive and/or mechanoreceptive sensory input originating from the perimuscular fascia. The goal of this study is to characterize the type and structure of the nerves and nerve terminals located within the subcutaneous perimuscular fascia of mouse tissue.
ARTICLE IN PRESS 270 Methods Post-mortem mouse subcutaneous tissue including the subcutaneous (panicular) muscle was excised from the backs of C57BL/6 mice and fixed in 3% paraformaldehyde. The perimuscular fascia was dissected from the panicular muscle and placed on slides as whole mounts. Immunohistochemistry and immunofluroescence was carried out with protein gene product 9.5 (PGP 9.5; marker of all neurons) and alpha-bungarotoxin (labels motor end plates). Imaging techniques included florescent and confocal microscopy.
Results Within the perimuscular fascia, we observed an extensive plexus of nerve fibers. Immunofluorescent labeling of tissue whole mounts with PGP 9.5 demonstrated an extensive and diverse network of nerve fibers in the perimuscular fascia. To gain a better understanding of the types of nerves present in this tissue
G. Voyer we used alpha-bungarotoxin to identify motor end plates. Double label immunohistochemistry showed that fibers from the same bundle of nerves would terminate in varying fashion. Some groups of fibers terminated at motor end plates. Other groups of fibers appeared to terminate at free nerve endings. Further study to characterize the composition and function of the free endings is ongoing.
Conclusions An extensive plexus of nerve fibers was observed within the mouse perimuscular fascia associated with the subcutaneous (panicular) muscle. Preliminary data using alpha-bungarotoxin suggests that a large number of these fibers are sensory. Currently we are working to identify characteristics of these sensory nerves that may play an important role in conveying the sensation originating from deep fascial tissue.
10.1016/j.jbmt.2008.04.015
Importance of evaluation of the foot muscular tone in type 1 diabetes mellitus patients Ricardas Kerpe, MD,PhD, Aleksandras Krisciunas, MD,PhD Department of Rehabilitation, Kaunas University of Medicine, Mickeviciaus Str. 9 LT- 44307, Kaunas, Lithuania
Background In 2003, Ardic et al. established that Dupuytren’s contracture was the most frequent complication of the musculoskeletal system in the diabetics they had examined. In these cases, Dupuytren’s contracture most often involved palmar aponeuroses. In 2005, D’Ambrogi et al. determined that diabetics have thickened Achilles’ tendons and plantar fascias and, according to them, this could play a significant role in the alteration of the biomechanics of the foot–ankle complex. The aim of this study was to evaluate muscular tone of the feet in type 1 diabetes mellitus patients.
Method The study involved 70 at random selected subjects with type 1 diabetes mellitus: 31 men (44.3%) and 39 women (55.7%) at the age of 32.4710.3 years. The duration of diabetes was 18.778.3 years. The study also involved 31 healthy subjects as controls—12 men (38.7%) and 19 women (61.3%) at the age of 33.3710.0 years. The study of the selected patients was performed using a standardized questionnaire and examination. Myotonometry equipment and a pedobarography device were used in this study.
Results It was found that the tone of the tibialis anterior muscle in the patients with type 1 diabetes mellitus was decreased, compared with the healthy controls. It was also determined that the tone of
the flexor digitorum brevis muscle in the patients with type 1 diabetes mellitus was increased, compared with healthy controls. A Pearson correlation was observed between the muscular tone of the right tibialis anterior muscle and the right body side weight pressure distribution (index P) in the patients with type 1 diabetes mellitus (r ¼ 0.54; pp0.05). A linear regression was observed between the muscular tone of the right tibialis anterior muscle and the body weight pressure distribution (index P) in the patients with type 1 diabetes mellitus: (R2 ¼ 0.29; pp0.05).
Conclusion We have concluded that the increased tone of the flexor digitorum brevis muscle in the patients with type 1 diabetes mellitus could result in the changes not only in the muscle itself but also in the plantar aponeurosis and plantar fascia. These changes are similar to those found in Dupuytren’s contracture and may lead to the deformation typical of the diabetic foot. The foot becomes more convex which leads to increased pressure on the inferior part of metatarsal heads. Therefore rehabilitation of the patients with type 1 diabetes mellitus should include approaches influencing myofibroblast activity in order to decrease tension in the flexor digitorum brevis muscle and ensure a less convex shape of the foot and an equalizing weight pressure’s distribution in the feet.
References Ardic, F., Soyupek, F., Kahraman, Y., Yorgancioglu, R., 2003. The musculoskeletal complications seen in type II diabetes: predominance of hand involvement. Clinical Rheumatology 22 (3), 229–233. D’Ambrogi, E., Giacomozzi, C., Macellari, V., Uccioli, L., 2005. Abnormal foot function in diabetic patients: the altered onset of Windlass mechanism. Diabetic Medicine 22 (12), 1713–1719.
10.1016/j.jbmt.2008.04.039
Treatment of Be ´raud’s Vertebro-pericardial Fascia Guy Voyer, D.O. Sutherland Academy of Osteopathy, Montreal, Toronto, Marseille E-mail address: [email protected]
Background Be´raud’s Vertebro-pericardial Fascia extends from C6-C7-T1 to the base of the posterior aspect of the pericardium. In this sense, it is a link between the cervico-thoracic articulation and the diaphragm. This raises the question as to whether it would be hard to treat the vestibule of the mediastinum without treating this very important fascia.
Journal of Bodywork and Movement Therapies www.intl.elsevierhealth.com/journals/jbmt
ABSTRACTS Tissue stretch decreases procollagen-1 and TGF-b1 in mouse subcutaneous fascia Nicole A. Bouffarda, Kenneth R. Cutroneob, Debbie Stevens-Tuttlea, Gary J. Badgerc, Helene M. Langevina,d a
Departments of Neurology, University of Vermont, Burlington, VT 05405, USA b Departments of Biochemistry, University of Vermont, Burlington, VT 05405, USA c Departments of Medical Biostatistics, University of Vermont, Burlington, VT 05405, USA d Departments of Orthopedics, University of Vermont, Burlington, VT 05405, USA
1.5 cm 1.5 cm area on one side of the back lateral to the midline, with the other side of the back serving as the control. Mice where then randomized to either stretch or no stretch. In the stretch group mice underwent stretching (approximately 30% strain) of the trunk for 10 min (mice suspended by the tail with the forelimbs touching a slightly inclined surface) twice a day for 7 days. All mice were sacrificed by decapitation at day 7. Subcutaneous fascia of the back was dissected, fixed and immunohistochemically stained for procollagen-1 (a marker for newly formed collagen). (2) Mouse subcutaneous fascia explant model: tissue was excised and kept in organ culture (DMEM culture media) for 4 days. One day after excision, fascia samples were either stretched (20% strain) for 10 min or not stretched. Aliquots of DMEM culture media were assayed by ELISA for TGF-b1 protein on days 0, 1 and 3 post stretch (or no stretch).
Background
Results
Increased collagen and transforming growth factor beta 1 (TGFb1) production are known to play an important role in connective tissue remodeling responses to injury, and in the production of excessive scarring and fibrosis. Although mechanical forces are known to influence connective tissue remodeling in vivo, their effect on TGF-b1 and collagen production in injured tissues are not well understood. We hypothesize that tissue stretch will attenuate TGF-b1-mediated collagen deposition in response to injury which may have implications for the therapeutic mechanisms of treatments that utilize tissue stretch.
In the in vivo microinjury model a significant difference in fascia procollagen-1 between injury and no-injury was observed in the absence of stretch (po 0.008). However, when injury was followed by tissue stretch, no significant difference was found between injury and no-injury (p ¼ 0.27). In the ex vivo model, both stretched and unstretched fascia samples showed increased TGF-b1 protein levels in the media from day 0 to day 3. However, at day 3, TGF-b1 protein levels were significantly lower in stretched vs. unstretched fascia (repeated measures ANOVA, po0.01).
Conclusion Methods Two different models were used to examine the effect of stretch on procollagen-1 and TGF-b1 protein levels in injured tissue:
(1) in vivo mouse microinjury model: a 5 mm incision was performed under anesthesia in the middle of the back at the level of the scapula. A microsurgery blade was introduced along the subcutaneous fascia plane and the attachments between the fascia and subcutaneous muscle were cut over a
These experiments suggest that short-term tissue stretch modulates TGF-b1-mediated tissue remodeling by attenuating procollagen-1 production. These results may be potentially relevant to the therapeutic mechanisms of treatments applying tissue stretch (e.g. massage, Rolfing, yoga, acupuncture).
Disclosure This study was funded by NCCAM Research Grant RO1 AT01121.
10.1016/j.jbmt.2008.04.017
Immunohistochemical characterization of the mouse subcutaneous perimuscular fascial plexus Sarah Coreya, Nicole Bouffarda, Helene Langevina,b a
Department of Neurology, The University of Vermont, Burlington, VT 05405, USA b Department of Orthopedics and Rehabilitation, The University of Vermont, Burlington, VT 05405, USA E-mail address: [email protected] (N.A. Bouffard). 1360-8592/$ - see front matter & 2008 Published by Elsevier Ltd. doi:10.1016/j.jbmt.2008.04.017
Purpose The innervartion of the deep fascia has not been fully described but may play an important role in musculoskeletal chronic pain. The pathophysiology of chronic musculoskeletal pain is poorly understood and may be related to nociceptive and/or mechanoreceptive sensory input originating from the perimuscular fascia. The goal of this study is to characterize the type and structure of the nerves and nerve terminals located within the subcutaneous perimuscular fascia of mouse tissue.
ARTICLE IN PRESS 270 Methods Post-mortem mouse subcutaneous tissue including the subcutaneous (panicular) muscle was excised from the backs of C57BL/6 mice and fixed in 3% paraformaldehyde. The perimuscular fascia was dissected from the panicular muscle and placed on slides as whole mounts. Immunohistochemistry and immunofluroescence was carried out with protein gene product 9.5 (PGP 9.5; marker of all neurons) and alpha-bungarotoxin (labels motor end plates). Imaging techniques included florescent and confocal microscopy.
Results Within the perimuscular fascia, we observed an extensive plexus of nerve fibers. Immunofluorescent labeling of tissue whole mounts with PGP 9.5 demonstrated an extensive and diverse network of nerve fibers in the perimuscular fascia. To gain a better understanding of the types of nerves present in this tissue
G. Voyer we used alpha-bungarotoxin to identify motor end plates. Double label immunohistochemistry showed that fibers from the same bundle of nerves would terminate in varying fashion. Some groups of fibers terminated at motor end plates. Other groups of fibers appeared to terminate at free nerve endings. Further study to characterize the composition and function of the free endings is ongoing.
Conclusions An extensive plexus of nerve fibers was observed within the mouse perimuscular fascia associated with the subcutaneous (panicular) muscle. Preliminary data using alpha-bungarotoxin suggests that a large number of these fibers are sensory. Currently we are working to identify characteristics of these sensory nerves that may play an important role in conveying the sensation originating from deep fascial tissue.
10.1016/j.jbmt.2008.04.015
Importance of evaluation of the foot muscular tone in type 1 diabetes mellitus patients Ricardas Kerpe, MD,PhD, Aleksandras Krisciunas, MD,PhD Department of Rehabilitation, Kaunas University of Medicine, Mickeviciaus Str. 9 LT- 44307, Kaunas, Lithuania
Background In 2003, Ardic et al. established that Dupuytren’s contracture was the most frequent complication of the musculoskeletal system in the diabetics they had examined. In these cases, Dupuytren’s contracture most often involved palmar aponeuroses. In 2005, D’Ambrogi et al. determined that diabetics have thickened Achilles’ tendons and plantar fascias and, according to them, this could play a significant role in the alteration of the biomechanics of the foot–ankle complex. The aim of this study was to evaluate muscular tone of the feet in type 1 diabetes mellitus patients.
Method The study involved 70 at random selected subjects with type 1 diabetes mellitus: 31 men (44.3%) and 39 women (55.7%) at the age of 32.4710.3 years. The duration of diabetes was 18.778.3 years. The study also involved 31 healthy subjects as controls—12 men (38.7%) and 19 women (61.3%) at the age of 33.3710.0 years. The study of the selected patients was performed using a standardized questionnaire and examination. Myotonometry equipment and a pedobarography device were used in this study.
Results It was found that the tone of the tibialis anterior muscle in the patients with type 1 diabetes mellitus was decreased, compared with the healthy controls. It was also determined that the tone of
the flexor digitorum brevis muscle in the patients with type 1 diabetes mellitus was increased, compared with healthy controls. A Pearson correlation was observed between the muscular tone of the right tibialis anterior muscle and the right body side weight pressure distribution (index P) in the patients with type 1 diabetes mellitus (r ¼ 0.54; pp0.05). A linear regression was observed between the muscular tone of the right tibialis anterior muscle and the body weight pressure distribution (index P) in the patients with type 1 diabetes mellitus: (R2 ¼ 0.29; pp0.05).
Conclusion We have concluded that the increased tone of the flexor digitorum brevis muscle in the patients with type 1 diabetes mellitus could result in the changes not only in the muscle itself but also in the plantar aponeurosis and plantar fascia. These changes are similar to those found in Dupuytren’s contracture and may lead to the deformation typical of the diabetic foot. The foot becomes more convex which leads to increased pressure on the inferior part of metatarsal heads. Therefore rehabilitation of the patients with type 1 diabetes mellitus should include approaches influencing myofibroblast activity in order to decrease tension in the flexor digitorum brevis muscle and ensure a less convex shape of the foot and an equalizing weight pressure’s distribution in the feet.
References Ardic, F., Soyupek, F., Kahraman, Y., Yorgancioglu, R., 2003. The musculoskeletal complications seen in type II diabetes: predominance of hand involvement. Clinical Rheumatology 22 (3), 229–233. D’Ambrogi, E., Giacomozzi, C., Macellari, V., Uccioli, L., 2005. Abnormal foot function in diabetic patients: the altered onset of Windlass mechanism. Diabetic Medicine 22 (12), 1713–1719.
10.1016/j.jbmt.2008.04.039
Treatment of Be ´raud’s Vertebro-pericardial Fascia Guy Voyer, D.O. Sutherland Academy of Osteopathy, Montreal, Toronto, Marseille E-mail address: [email protected]
Background Be´raud’s Vertebro-pericardial Fascia extends from C6-C7-T1 to the base of the posterior aspect of the pericardium. In this sense, it is a link between the cervico-thoracic articulation and the diaphragm. This raises the question as to whether it would be hard to treat the vestibule of the mediastinum without treating this very important fascia.