- Email: [email protected]
Effects of Therapeutic Touch on Healing of the Skin in Rats
Author’s Accepted Manuscript EFFECTS OF THERAPEUTIC TOUCH ON HEALING OF THE SKIN IN RATS André Luiz Thomaz de Souza, David Patrick Carvalho Rosa, Bruno Anjos Blanco, Patrícia Passaglia, Angelita Maria Stabile www.elsevier.com/locate/jsch
PII: DOI: Reference:
S1550-8307(17)30240-9 http://dx.doi.org/10.1016/j.explore.2017.06.006 JSCH2216
To appear in: Explore: The Journal of Science and Healing Cite this article as: André Luiz Thomaz de Souza, David Patrick Carvalho Rosa, Bruno Anjos Blanco, Patrícia Passaglia and Angelita Maria Stabile, EFFECTS OF THERAPEUTIC TOUCH ON HEALING OF THE SKIN IN RATS, Explore: The Journal of Science and Healing, http://dx.doi.org/10.1016/j.explore.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECTS OF THERAPEUTIC TOUCH ON HEALING OF THE SKIN IN RATS
André Luiz Thomaz de Souza1*, David Patrick Carvalho Rosa2, Bruno Anjos Blanco3, Patrícia Passaglia4, Angelita Maria Stabile5. 1
Department of Nursing, School of Nursing, Integrated College of the Ribeira Valley,
Registro, SP, Brazil. E-mail: [email protected] 2
School of Nursing of the Integrated College of the Ribeira Valley, Registro, SP, Brazil. E-
mail: [email protected] 3
Department of Nursing, School of Nursing, Integrated College of the Ribeira Valley,
Registro, SP, Brazil. E-mail: [email protected]
Department of Medicine, School of Medicine, University of São Paulo. Ribeirão Preto, SP,
4
Brazil. E-mail: [email protected] 5
Departmentt of Nursing, School of Nursing, University of São Paulo, Ribeirão Preto, SP,
Brazil. E-mail: [email protected]
*Correspondence to:
André Luiz Thomaz de Souza, Integrated College of the Ribeira Valley Street: Oscar Yoshiaki Magário, 185, Jardim das Palmeiras Registro-SP, Brazil. E-mail: [email protected] Contact: (16) 98137-3251
Abstract Context: Therapeutic touch is a complementary treatment directed toward the balance of the energy field surrounding living beings. Objective: This study’s aim was to investigate the effect of therapeutic touch on wound area contraction and fibroblast proliferation in rat skin. Design: This study was conducted using 24 male Wistar rats with dorsal wounds of diameter 8 mm. The rats were divided into two groups: a control group: in this, the wounds were sanitized with filtered water and neutral-pH soap; a treatment group: in this, the wounds were sanitized as in the control group but the rats also underwent to daily sessions of therapeutic touch. Wound area was measured on days 1, 4, and 7 using imagelab software, version 2.4 R.C. On days 4 and 7, six animals in each group were euthanized so that the lesioned tissue could be collected for fibroblast counts and histological evaluations. Results: On days 1 and 4, wound areas were similar in both groups. Moreover, no significant differences in fibroblast counts were observed on day 4. On day 7, however, fibroblast counts were significantly higher in the treated group than in the control group, with a subsequent wound shrinkage Conclusion: These data indicate that therapeutic touch may accelerate wound repair, possibly by increasing fibroblast activity. Key words: Therapeutic Touch; Fibroblasts; Healing.
INTRODUCTION
Wound healing is a complex process in which injured tissues are replaced with healthy tissues1 by means of a continuous sequence of events divided into three phases: inflammatory, proliferative, and maturation.2,3The initial inflammatory phase develops within 24 h of injury, and it lasts for an average of 2–3 days1,4; it is characterized by hemostasis and inflammation.2 The proliferative phase sets in following the inflammatory phase and it lasts for an average of 2–3 weeks.5,6 The features of this phase are epithelialization, angiogenesis, granulation tissue formation, and collagen-fiber deposition.2 The final phase is maturation, which lasts the entire lifetime of the wound, considering that in approximately 1 year, 70%– 80% of the skin will be intact.6 The primary feature of this process is the transition from granulation tissue to scar formation, with increased collagen deposition and wound contraction.2 Different therapeutic alternatives can be used in clinical practice to treat acute and chronic tissue wounds, such as topical medication, dressings, and debridement.7,8 In addition to traditional methods, the use of complementary therapies, specifically energy therapies, appears as a potential alternative to accelerate skin healing; however, such methods require further studies to confirm their effectiveness. One of the earliest studies on energy therapies, also called curative energies, was conducted in the 1960s by Dr. Bernard Grad of McGill University in Montreal, Canada. After recognizing the therapeutic influences of the so-called spiritual and psychic healers, Dr. Grad investigated the effects of laying on of hands on cellular physiology, and conducted studies in animals and plants.9 During his studies, Dr. Grad had the assistance of one healer named Oscar Estebany, a Hungarian colonel known to have healing powers with his hand touch. While investigating
the effects of laying on of hands on rats in which experimental goiter was induced, Dr. Grad revealed that after 40 days of contact Oscar Estebany’s hands, the animals had a significantly lower goiter incidence than animals that received no intervention.9 Dr. Grad also investigated the effects of laying on of hands on experimental wounds in rats, and revealed that the wound healing process was significantly improved in the group receiving the intervention.9 After disseminating his results to the scientific community, many scientists speculated on the mechanisms that could accelerate wound healing. A proposed theory was that the hand placement of the healers intensified healing by accelerating the cellular and enzymatic activities that are involved in the scarring process.9 After becoming aware of Dr. Grad's studies, the biochemist Dr. Justa Smith, with the assistance of Oscar Estebany, conducted a study that evaluated the effects of hand laying on the digestive enzyme trypsin. Dr. Justa Smith discovered that Estebany's laying on of hands resulted in increased enzymatic reaction speed and confirmed Dr. Grad's Theory.10 From these findings regarding the healing energy through laying on of hands, in the 1970s, the nurse Dolores Krieger and the farsighted Dora Kunz proposed that to perform the laying hands is not necessary to be a healer, but rather to have knowledge about the stages involving treatment. According to Krieger and Kunz, every human being by means of intentionality in “doing good” is able to intervene on the energy field (EF) surrounding living beings, and assist in the rehabilitation process within pathological frameworks.11 It should be emphasized that there is no exact definition for EF, nor established ways to measure it.12 In this context, Krieger and Kunz refer to laying on of hands as therapeutic touch (TT), which is considered a contemporary version of ancient healing practices.11 The Krieger– Kunz method was then developed, which involves four steps for TT: (a) centralization of consciousness; (B) EF evaluation; (C) rebalancing or resynchronization of EF; (D) the EF reassessment.11
Among complementary treatments, TT has attracted the interest of the scientific community in the last decades because of its potential effects on several experimental and clinical conditions, especially in relation to fibroblast proliferation13, inflammatory response14, acceleration of healing response15, effects on pain thresholds 14,16,17, and increased hematocrit and hemoglobin levels.18 However, its use in the treatment for skin healing has not been well understood until now, and there is no strong evidence to support its use in clinical practice19, which reinforces the need for developing animal model experimental studies, followed by clinical trials. Hence, the present study’s objective was to investigate the effect of TT on wound area contraction and fibroblast proliferation during skin healing in rats.
MATERIAL AND METHODS
Animal and study design
This laboratory experimental study was conducted using 24 male Wistar rats, which were 90 days old and weighed 270 ± 30g. The animals were housed individually in PVC plastic boxes, in an environment with temperature controlled at 25°C ± 2°C, a 12/12-h light/dark cycle, ad libitum access to water, and balanced commercial solid food. Before lesion induction on the skin, the animals were randomly divided into two experimental groups and individuals were assignee numbers of 1–12: Control (n = 12) and Treated (n = 12). All of the experiments were performed in agreement with the Guide for the Care and Use of Laboratory Animals of the International Association for Assessment and Accreditation of Laboratory Animal Care (1996), and according to the ethical recommendations by Vale do Ribeira Integrated Colleges, Registro, São Paulo, Brazil (protocol number: 2015/02-02).
Lesion induction of the skin
To create skin lesions, animals were anesthetized with a solution of 2% xylazine hydrochloride (20 mg/ml) and 10% Ketamine Hydrochloride (50 mg/ml). The dosage was set at 10 mg/kg and 70 mg/kg, respectively, and the dose was administrated in a single dose via intraperitoneal (IP) injection. Afterward, antisepsis with povidone–iodine was initiated and a trichotomy carried out on the dorsal-cervical region. A circular wound was made in that region using an 8-mm trephine punch, and the skin was then removed. At the end of the procedure, animals received analgesia in the form of sodium dipyrone 500 mg/ml in a single dose of 875 mg/kg diluted in water20; they were also allowed to have water ad libitum and were kept in their individual boxes until the end of the experiment.
Treatment protocol
Standard treatment for the wound area (sanitization) in both groups was performed daily by sanitizing with filtered water and vegetable glycerin soap with a neutral pH (HIDRADERM, Farmax®). In the treated group, apart from lesion sanitization, complementary treatment was delivered in the form of TT sessions once a day. The sessions were conducted by a nurse with 3 years of experience in the use of this intervention, and with training in the basic module in a TT course. The reference was the method by Krieger– Kunz.11 During TT, the animals were maintained on a bench, having as TT sessions performed on each animal at a time. The nurse held her hand at a distance of 5 cm from the animal (Figure 1) with no physical contact. Each TT session lasted 2 min and always began at
2:00 p.m. so that circadian variations could be prevented. To aid the interventionist’s concentration, all sessions were conducted in a secluded, peaceful, and quiet environment. Seven TT sessions were conducted altogether.
Figure 1. Positioning of both animal and interventionist’s hand during the therapeutic touch session.
Data collection
Data collection and treatment protocol were initiated immediately after wounds were created. With a digital camera (Kodak Easy Share C813®) coupled to a tripod at a fixed height (22 cm), images were obtained of the wounds on days 1 (baseline data), 4, and 7 after injury. From the images obtained, the lesion bed area was calculated using the software imagelab version 2.4 R.C. Area calculation was performed by multiplying the anteroposterior measurement times the laterolateral measurement of the injured bed, with values being expressed in mm2. Statistical analyses to compare the evolution of the cicatrization of the wound healing was performed through the subtraction of the areas from days 4 and 7 from the initial area. The wound area contraction demonstrates the reduction of the injury in mm2.. The larger the contraction area, the smaller wound size. On day 4, the animals numbered 1–6 in each group and on day 7, the animals numbered 7–12 in each group were euthanized by anesthetic overdose. Tissue samples were
collected from the lesioned region and fixed in 10% formalin solution until histological processing. The samples were then embedded in paraffin, and 5-μm histological sections were obtained, which were laid onto slides and colored with hematoxylin–eosin for morphological assessment and fibroblast count.21,22 Histological slides were analyzed with an optical microscope at a magnification of 400×, and the images were captured using a digital camera, Moticam 2000® with 2.0 megapixels coupled to the microscope. Six fields of each histological section of the specimens of each animal were analyzed for the fibroblast count, and the average of these scores was calculated. To preserve the integrity of analyses, the images obtained from the injured bed, the geometric measurements of the injured bed and the quantification of fibroblasts were performed by the same researcher, who was not aware of the distribution of the experimental groups.
Statistical analysis
Data collected were analyzed with the use of statistical software SPSS: Statistical Package for the Social Sciences, version 23.0. Initially, data were submitted to Shapiro–Wilk test for normalization. After normality was established an unpaired two-tailed Student’s t-test, with a freedom degree equal to n − 2, where n is the total number of animals examined on, day 1 (baseline), day 4, and day 7 was conducted. The null hypothesis of the test is that there is no difference between control and treated groups. The alternative hypothesis is that the groups have differences in the averages. The significance level was established at 5%, and the results were presented as averages and standard errors.
RESULTS
Results showed that on days 1 and 4 the wound area in both groups was equivalent (Table 1). Furthermore, no significant differences were observed in the fibroblast counts between the groups on day 4. Nevertheless, on day 7, the average fibroblast count in the TT group was significantly higher than that in the control group (Table 2 and Figure 3). Along with this increase, on day 7, the wound area of the treated group was significantly lower than that of the control group, as observed by the increased contraction (Table 1 and Figure 2).
Table 1. Intergroup analysis of the wound contraction area on day 1 (n = 24), day 4 (n = 12), and day 7 (n = 12). Values expressed in average ± SEM, Registro-SP, Brazil, 2016. 2
Wound contraction area (mm ) Groups Day 1
t
P value
Day 4
T
P value
Day 7
t
P value
121.07 ± 44.74 ± 69.71 ± a a 5.10 8.45 11.28a 0.569 +0.575 0.244 0.828 0.03 115.45 ± 41.74 ± 101.14 ± 2.400 Treated a a b 8.44 8.94 6.64 Note: Averages followed by the same letter in the column for intergroup comparison are statistically equivalent in the unpaired Student’s t-test. Significance level is 5%. The area of wound contraction is related to the reduction of the lesion in mm2, thus, the larger the contraction area, the smaller the wound size. Control
Table 2. Amount of fibroblasts identified per specimen collected on day 4 (n = 12) and on day 7 (n = 12). Values expressed in average ± SEM, Registro-SP, Brazil, 2016. Fibroblasts (counts) Groups Day 4 Control
50.83 ± 3.03a
Treated
50.47 ± 6.67a
T
P value
Day 7
t
P value
−2.616
0.026
40.31 ± 3.96a 0.049
0.962 54.42 ± 3.65b
Note: Averages followed by the same letter in the column for intergroup comparison are statistically equivalent in the unpaired Student’s t-test. Significance level is 5%.
Surgical wound (Day 1) CT
Day 4 CT
Day 7 CT
Surgical wound (Day 1) TT
Day 4 TT
Day 7 TT
Figure 2. Representation of the clinical evolution of the wounds during treatment. CT: Control; TT: Treated.
Day 4 CT
Day 7 CT
Day 4 TT
Day 7 TT
Figure 3. Photomicrograph with objective lens of 400×. Skin wound area colored with hematoxylin–eosin. Fibroblast identification on days 4 and 7. CT: Control; TT: Treated.
DISCUSSION
This study shows that TT influences skin healing, accelerates tissue repair by increasing fibroblast counts on day 7, followed by a reduction in wound area (Tables 1 and 2; Figures 2 and 3). However, the lack of evidence on the mechanisms modulated by TT imposes limitations on understanding how it interacts with the body. The effects observed in this study mirror the discoveries of Dr. Bernard Grad and Dr. Justa Smith that TT increases the speed of enzymatic reactions, such as those involving proteases10, which may result in the enhancement of cellular activities that are part of the healing process and/or other organic reactions.9 In tissue repair, proteases, specifically the matrix metalloproteinases (MMPs), have an essential role in protein breakdown. This facilitates the immune response, elimination of foreign elements, remodeling of the extracellular matrix (MEC) damaged by injury, cell motility, release of growth factors, cytokines, and cell proliferation. This process favors new tissue formation and skin healing progression.23,24 However, the imbalance in protease activity during skin healing can cause damage to MEC, leading to destruction of healthy tissue because of the delay in the healing process, and, consequently, to lesion chronicity.24 According to the experiments of Dr. Justa Smith, the changes in the enzymatic activity triggered by laying on of hands are always related to the improvement of cellular activity. Consequently, the organic response is improved, independent of the enzyme that is under the influence of TT.10 In light of Dr. Justa Smith findings on the effects of TT and the evidence available in the literature on the role of proteases in skin healing10,2, we believe the effects of TT in this study may be related to the improved response of MMPs that participate in skin healing. However, this should be investigated in future studies.
According to the precursor of the Kriger–Kunz method11, TT is able to intensify the recovery and/or healing processes in different clinical situations, with response of the autonomic nervous, lymphatic, circulatory, peripheral vascular, and musculoskeletal systems. Moreover, some other systems such as the collagen and endocrine systems may also react to TT in specific situations. One of the features of the proliferative phase during skin healing is the formation of granulation tissue, which consists of fibroblasts, inflammatory cells, and neovascularization that has invaded MEC with the presence of collagen, fibronectin, and hyaluronic acid.3,26 In this study, we opted to evaluate the effects of TT on fibroblast proliferation between the fourth and the seventh day, because it is a period in which they reach a higher production peak and can be visualized in significant numbers.27 The primary function of fibroblasts during healing is to stimulate collagen synthesis, and this occurs early during the inflammatory phase.5,6 The effects of TT observed in our study also reveal its action on fibroblast proliferation (Table 2 and Figure 2) during the proliferative phase. This favors the shortening of the healing time, because it initiates a faster deposition of collagen fibers, and consequently, favors the wound contraction. Importantly, fibroblast activity also involves the participation of MMPs, which are responsible for their migration and proliferation, which later favors scar tissue formation.24 Apart from stimulating collagen-fiber synthesis, fibroblasts form the framework for newly formed blood vessels, which directly influence tissue nutrition. As collagen is responsible for tissue support, its participation in the healing process is fundamental for the lesion bed contraction to occur.5,6 Studies about the use of TT in both in-vitro and in-vivo experimental models show significant results in fibroblast proliferation increase13, in pain reduction14, and in the acceleration of healing response.7,28,29 These results corroborate the
findings of the present study. Nevertheless, further research is necessary to validate its use in the clinical practice. Because it is a low cost, easy-to-use therapy, with potential effects on the organic system, TT seems to be a useful treatment for tissue repair. However, so far its effect on skin healing has not been investigated. As a study limitation, there was no investigation of other factors associated with skin healing, such as macrophage counts, neovascularization, collagen-fiber distribution, and the absence of a simulated TT group (placebo). In addition, it is essential to investigate the effects of TT on all phases of skin healing and its effects on MMP activity in association with the reduction of the lesion bed and the improvement of the scarring response.
CONCLUSION
The data from this study suggest that seven sessions of TT performed daily for a period of 2 min were enough to induce acceleration in wound repair in Wistar rats. This result was observed by means of the reduction in the wound area and by the increase in fibroblast counts on day 7.
REFERENCES
1. Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res. 2010;89:219-29. 2. Sinno H, Prakash S. Complements and the Wound Healing Cascade: An Updated Review. Plastic Surgery International. 2013;ID 146764:1-7. 3. Li B, Wang JH. Fibroblasts and myofibroblasts in wound healing: Force generation and measurement. J Tissue Viability. 2011;20:108-20. 4. Busti AJ, Hooper JS, Amaya CJ, Kazi S. Effects of perioperative antiinflammatory and immunomodulating therapy on surgical wound healing. Pharmacotherapy. 2005;25:1566-91. 5. Tazima MFGS, Vicente YAMVA, Moriya T. Wound healing and biology. Medicina (Ribeirão Preto). 2008;41:259-64. 6. Isaac C, Ladeira PRS, Rêgo FMP, Aldunate JCB, Ferreira MC. Physiological wound healing. Rev. Med. (São Paulo). 2010;89:125-31. 7. Powers JG, Higham C, Broussard K, Phillips TJ. Wound healing and treating wounds: Chronic wound care and management. J Am Acad Dermatol. 2016;74:607-25. 8. Mandelbaum SH, Di Santis, EP, Mandelbaum MHS. Cicatrization: current concepts and auxiliary resources - Part II. An. Bras. Dermatol. 2003;78:525-542. 9. Gerber R. Vibrational medicine: A medicine for the future. São Paulo: Cultrix; 2007. 10. Smith J. The Influence on Enzyme Growth by the “Laying on of Hands”, in The Dimensions of Healing: A Symposium. Los Altos, CA: The Academy of Parapsychology and Medicine, 1972. 11. Krieger D. Therapeutic touch: modern version of the ancient technique of laying on of hands. São Paulo: Cultrix; 1995. 12. Souza ALT, Prado BO, Silva RP, Chaves ECL, Iunes DH. Clinical research with Therapeutic Touch: a systematic review. Sci Med. 2014.24:404-10. 13. Gronowicz GA, Jhaveri A, Clarke LW, Aronow MS, Smith TH. Therapeutic Touch Stimulates the Proliferation of Human Cells in Culture. J Altern Complement Med. 2008;14:233-39. 14. Santos DS, Marta IER, Cárnio EC, Quadros AU, Cunha TM, Carvalho EC. Using an experimental model for the study of therapeutic touch. Rev. Lat-Am. Enfermagem. 2013;21:18. 15. Savieto RM, Silva MJP, Pozzi DHB, Ferreira-Neto PA. Scarring effects of water energized with therapeutci touch on mice skin. Rev. Enferm. UERJ. 2007;15:423-29.
16. Marta IER, Baldan SS, Berton AF, Pavam M, Silva MJP. The effectiveness of Therapeutic Touch on pain, depression and sleep in patients with chronic pain: clinical trial. Rev Esc Enferm USP. 2010;44:1100-06. 17. Lu DF, Hart LK, Lutgendorf SK, Perkhounkova Y. The effect of healing touch on the pain and mobility of persons with osteoarthritis: a feasibility study. Geriatr Nurs. 2013;34:314-22. 18. Movaffaghi Z, Hasanpoor M, Farsi M, Hooshmand P, Abrishami F. Effects of therapeutic touch on blood hemoglobin and hematocrit level. J Holist Nurs, 2006;24:41-48. 19. O´Mathuna DP. Therapeutic touch for healing acute wounds. Cochrane Database Syst Rev. 2016;(5):CD002766. 20. Pereira VA, Ávila MA, Loyola YCS, Nakagaki WR, Camilli JA, Garcia JAD. et al. Effects of phenobarbital on bone repair and biomechanics in rats. Acta Paul. Enferm. 2011;24:794-98. 21. Junqueira LC, Carneiro J. Basic Histology: text and atlas. 10ª ed. Rio de Janeiro: Guanabara Koogan; 2004. 22. Paixão DR, Flausino PA, Reis NG, Lima CC, Bernardes MTCP, Santos L.. et al. Propolis effects on proliferation of fibroblasts in skin lesions in rats. Rev Ciênc Farm Básica Apl. 2014;35:413-17. 23. Löffek S, Schilling O, Franzke CW. . Biological role of matrix metalloproteinases: a critical balance. European Respiratory Journal. 2011,38(1):191-08. 24. McCarty SM; Percival SL. Proteases and delayed wound healing. Adv Wound Care (New Rochelle). 2013,2(8):438-47. 25. Rohani MG, Parks WC. Matrix remodeling by MMPs during wound repair. Matrix Biol. 2015,44-46:113-21. 26. Midwood KS, Williams LV, Schwarzbauer JE. Tissue repair and the dynamics of the extracellular matrix. Int J Biochem Cell Biol. 2004;36:1031-37. 27. Willoughby DA. Some views on the pathogenesis of inflammation. In: Montagna W, Bently JP, Dobson R (eds) The dermis. Advances in the biology of skin. Appelton-CenturyCrofts, Norwalk.; 1970:221-230. 28. Savieto RM, Silva MJ. Effects of therapeutic touch on the healing of skin lesions of guinea pigs. Acta Paul. Enferm. 2004;17:377-82. 29. Savieto RM, Silva MJ. Therapeutic touch for the healing of skin injuries in guinea pigs. Rev Bras Enferm. 2004;57:340-43.
PII: DOI: Reference:
S1550-8307(17)30240-9 http://dx.doi.org/10.1016/j.explore.2017.06.006 JSCH2216
To appear in: Explore: The Journal of Science and Healing Cite this article as: André Luiz Thomaz de Souza, David Patrick Carvalho Rosa, Bruno Anjos Blanco, Patrícia Passaglia and Angelita Maria Stabile, EFFECTS OF THERAPEUTIC TOUCH ON HEALING OF THE SKIN IN RATS, Explore: The Journal of Science and Healing, http://dx.doi.org/10.1016/j.explore.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECTS OF THERAPEUTIC TOUCH ON HEALING OF THE SKIN IN RATS
André Luiz Thomaz de Souza1*, David Patrick Carvalho Rosa2, Bruno Anjos Blanco3, Patrícia Passaglia4, Angelita Maria Stabile5. 1
Department of Nursing, School of Nursing, Integrated College of the Ribeira Valley,
Registro, SP, Brazil. E-mail: [email protected] 2
School of Nursing of the Integrated College of the Ribeira Valley, Registro, SP, Brazil. E-
mail: [email protected] 3
Department of Nursing, School of Nursing, Integrated College of the Ribeira Valley,
Registro, SP, Brazil. E-mail: [email protected]
Department of Medicine, School of Medicine, University of São Paulo. Ribeirão Preto, SP,
4
Brazil. E-mail: [email protected] 5
Departmentt of Nursing, School of Nursing, University of São Paulo, Ribeirão Preto, SP,
Brazil. E-mail: [email protected]
*Correspondence to:
André Luiz Thomaz de Souza, Integrated College of the Ribeira Valley Street: Oscar Yoshiaki Magário, 185, Jardim das Palmeiras Registro-SP, Brazil. E-mail: [email protected] Contact: (16) 98137-3251
Abstract Context: Therapeutic touch is a complementary treatment directed toward the balance of the energy field surrounding living beings. Objective: This study’s aim was to investigate the effect of therapeutic touch on wound area contraction and fibroblast proliferation in rat skin. Design: This study was conducted using 24 male Wistar rats with dorsal wounds of diameter 8 mm. The rats were divided into two groups: a control group: in this, the wounds were sanitized with filtered water and neutral-pH soap; a treatment group: in this, the wounds were sanitized as in the control group but the rats also underwent to daily sessions of therapeutic touch. Wound area was measured on days 1, 4, and 7 using imagelab software, version 2.4 R.C. On days 4 and 7, six animals in each group were euthanized so that the lesioned tissue could be collected for fibroblast counts and histological evaluations. Results: On days 1 and 4, wound areas were similar in both groups. Moreover, no significant differences in fibroblast counts were observed on day 4. On day 7, however, fibroblast counts were significantly higher in the treated group than in the control group, with a subsequent wound shrinkage Conclusion: These data indicate that therapeutic touch may accelerate wound repair, possibly by increasing fibroblast activity. Key words: Therapeutic Touch; Fibroblasts; Healing.
INTRODUCTION
Wound healing is a complex process in which injured tissues are replaced with healthy tissues1 by means of a continuous sequence of events divided into three phases: inflammatory, proliferative, and maturation.2,3The initial inflammatory phase develops within 24 h of injury, and it lasts for an average of 2–3 days1,4; it is characterized by hemostasis and inflammation.2 The proliferative phase sets in following the inflammatory phase and it lasts for an average of 2–3 weeks.5,6 The features of this phase are epithelialization, angiogenesis, granulation tissue formation, and collagen-fiber deposition.2 The final phase is maturation, which lasts the entire lifetime of the wound, considering that in approximately 1 year, 70%– 80% of the skin will be intact.6 The primary feature of this process is the transition from granulation tissue to scar formation, with increased collagen deposition and wound contraction.2 Different therapeutic alternatives can be used in clinical practice to treat acute and chronic tissue wounds, such as topical medication, dressings, and debridement.7,8 In addition to traditional methods, the use of complementary therapies, specifically energy therapies, appears as a potential alternative to accelerate skin healing; however, such methods require further studies to confirm their effectiveness. One of the earliest studies on energy therapies, also called curative energies, was conducted in the 1960s by Dr. Bernard Grad of McGill University in Montreal, Canada. After recognizing the therapeutic influences of the so-called spiritual and psychic healers, Dr. Grad investigated the effects of laying on of hands on cellular physiology, and conducted studies in animals and plants.9 During his studies, Dr. Grad had the assistance of one healer named Oscar Estebany, a Hungarian colonel known to have healing powers with his hand touch. While investigating
the effects of laying on of hands on rats in which experimental goiter was induced, Dr. Grad revealed that after 40 days of contact Oscar Estebany’s hands, the animals had a significantly lower goiter incidence than animals that received no intervention.9 Dr. Grad also investigated the effects of laying on of hands on experimental wounds in rats, and revealed that the wound healing process was significantly improved in the group receiving the intervention.9 After disseminating his results to the scientific community, many scientists speculated on the mechanisms that could accelerate wound healing. A proposed theory was that the hand placement of the healers intensified healing by accelerating the cellular and enzymatic activities that are involved in the scarring process.9 After becoming aware of Dr. Grad's studies, the biochemist Dr. Justa Smith, with the assistance of Oscar Estebany, conducted a study that evaluated the effects of hand laying on the digestive enzyme trypsin. Dr. Justa Smith discovered that Estebany's laying on of hands resulted in increased enzymatic reaction speed and confirmed Dr. Grad's Theory.10 From these findings regarding the healing energy through laying on of hands, in the 1970s, the nurse Dolores Krieger and the farsighted Dora Kunz proposed that to perform the laying hands is not necessary to be a healer, but rather to have knowledge about the stages involving treatment. According to Krieger and Kunz, every human being by means of intentionality in “doing good” is able to intervene on the energy field (EF) surrounding living beings, and assist in the rehabilitation process within pathological frameworks.11 It should be emphasized that there is no exact definition for EF, nor established ways to measure it.12 In this context, Krieger and Kunz refer to laying on of hands as therapeutic touch (TT), which is considered a contemporary version of ancient healing practices.11 The Krieger– Kunz method was then developed, which involves four steps for TT: (a) centralization of consciousness; (B) EF evaluation; (C) rebalancing or resynchronization of EF; (D) the EF reassessment.11
Among complementary treatments, TT has attracted the interest of the scientific community in the last decades because of its potential effects on several experimental and clinical conditions, especially in relation to fibroblast proliferation13, inflammatory response14, acceleration of healing response15, effects on pain thresholds 14,16,17, and increased hematocrit and hemoglobin levels.18 However, its use in the treatment for skin healing has not been well understood until now, and there is no strong evidence to support its use in clinical practice19, which reinforces the need for developing animal model experimental studies, followed by clinical trials. Hence, the present study’s objective was to investigate the effect of TT on wound area contraction and fibroblast proliferation during skin healing in rats.
MATERIAL AND METHODS
Animal and study design
This laboratory experimental study was conducted using 24 male Wistar rats, which were 90 days old and weighed 270 ± 30g. The animals were housed individually in PVC plastic boxes, in an environment with temperature controlled at 25°C ± 2°C, a 12/12-h light/dark cycle, ad libitum access to water, and balanced commercial solid food. Before lesion induction on the skin, the animals were randomly divided into two experimental groups and individuals were assignee numbers of 1–12: Control (n = 12) and Treated (n = 12). All of the experiments were performed in agreement with the Guide for the Care and Use of Laboratory Animals of the International Association for Assessment and Accreditation of Laboratory Animal Care (1996), and according to the ethical recommendations by Vale do Ribeira Integrated Colleges, Registro, São Paulo, Brazil (protocol number: 2015/02-02).
Lesion induction of the skin
To create skin lesions, animals were anesthetized with a solution of 2% xylazine hydrochloride (20 mg/ml) and 10% Ketamine Hydrochloride (50 mg/ml). The dosage was set at 10 mg/kg and 70 mg/kg, respectively, and the dose was administrated in a single dose via intraperitoneal (IP) injection. Afterward, antisepsis with povidone–iodine was initiated and a trichotomy carried out on the dorsal-cervical region. A circular wound was made in that region using an 8-mm trephine punch, and the skin was then removed. At the end of the procedure, animals received analgesia in the form of sodium dipyrone 500 mg/ml in a single dose of 875 mg/kg diluted in water20; they were also allowed to have water ad libitum and were kept in their individual boxes until the end of the experiment.
Treatment protocol
Standard treatment for the wound area (sanitization) in both groups was performed daily by sanitizing with filtered water and vegetable glycerin soap with a neutral pH (HIDRADERM, Farmax®). In the treated group, apart from lesion sanitization, complementary treatment was delivered in the form of TT sessions once a day. The sessions were conducted by a nurse with 3 years of experience in the use of this intervention, and with training in the basic module in a TT course. The reference was the method by Krieger– Kunz.11 During TT, the animals were maintained on a bench, having as TT sessions performed on each animal at a time. The nurse held her hand at a distance of 5 cm from the animal (Figure 1) with no physical contact. Each TT session lasted 2 min and always began at
2:00 p.m. so that circadian variations could be prevented. To aid the interventionist’s concentration, all sessions were conducted in a secluded, peaceful, and quiet environment. Seven TT sessions were conducted altogether.
Figure 1. Positioning of both animal and interventionist’s hand during the therapeutic touch session.
Data collection
Data collection and treatment protocol were initiated immediately after wounds were created. With a digital camera (Kodak Easy Share C813®) coupled to a tripod at a fixed height (22 cm), images were obtained of the wounds on days 1 (baseline data), 4, and 7 after injury. From the images obtained, the lesion bed area was calculated using the software imagelab version 2.4 R.C. Area calculation was performed by multiplying the anteroposterior measurement times the laterolateral measurement of the injured bed, with values being expressed in mm2. Statistical analyses to compare the evolution of the cicatrization of the wound healing was performed through the subtraction of the areas from days 4 and 7 from the initial area. The wound area contraction demonstrates the reduction of the injury in mm2.. The larger the contraction area, the smaller wound size. On day 4, the animals numbered 1–6 in each group and on day 7, the animals numbered 7–12 in each group were euthanized by anesthetic overdose. Tissue samples were
collected from the lesioned region and fixed in 10% formalin solution until histological processing. The samples were then embedded in paraffin, and 5-μm histological sections were obtained, which were laid onto slides and colored with hematoxylin–eosin for morphological assessment and fibroblast count.21,22 Histological slides were analyzed with an optical microscope at a magnification of 400×, and the images were captured using a digital camera, Moticam 2000® with 2.0 megapixels coupled to the microscope. Six fields of each histological section of the specimens of each animal were analyzed for the fibroblast count, and the average of these scores was calculated. To preserve the integrity of analyses, the images obtained from the injured bed, the geometric measurements of the injured bed and the quantification of fibroblasts were performed by the same researcher, who was not aware of the distribution of the experimental groups.
Statistical analysis
Data collected were analyzed with the use of statistical software SPSS: Statistical Package for the Social Sciences, version 23.0. Initially, data were submitted to Shapiro–Wilk test for normalization. After normality was established an unpaired two-tailed Student’s t-test, with a freedom degree equal to n − 2, where n is the total number of animals examined on, day 1 (baseline), day 4, and day 7 was conducted. The null hypothesis of the test is that there is no difference between control and treated groups. The alternative hypothesis is that the groups have differences in the averages. The significance level was established at 5%, and the results were presented as averages and standard errors.
RESULTS
Results showed that on days 1 and 4 the wound area in both groups was equivalent (Table 1). Furthermore, no significant differences were observed in the fibroblast counts between the groups on day 4. Nevertheless, on day 7, the average fibroblast count in the TT group was significantly higher than that in the control group (Table 2 and Figure 3). Along with this increase, on day 7, the wound area of the treated group was significantly lower than that of the control group, as observed by the increased contraction (Table 1 and Figure 2).
Table 1. Intergroup analysis of the wound contraction area on day 1 (n = 24), day 4 (n = 12), and day 7 (n = 12). Values expressed in average ± SEM, Registro-SP, Brazil, 2016. 2
Wound contraction area (mm ) Groups Day 1
t
P value
Day 4
T
P value
Day 7
t
P value
121.07 ± 44.74 ± 69.71 ± a a 5.10 8.45 11.28a 0.569 +0.575 0.244 0.828 0.03 115.45 ± 41.74 ± 101.14 ± 2.400 Treated a a b 8.44 8.94 6.64 Note: Averages followed by the same letter in the column for intergroup comparison are statistically equivalent in the unpaired Student’s t-test. Significance level is 5%. The area of wound contraction is related to the reduction of the lesion in mm2, thus, the larger the contraction area, the smaller the wound size. Control
Table 2. Amount of fibroblasts identified per specimen collected on day 4 (n = 12) and on day 7 (n = 12). Values expressed in average ± SEM, Registro-SP, Brazil, 2016. Fibroblasts (counts) Groups Day 4 Control
50.83 ± 3.03a
Treated
50.47 ± 6.67a
T
P value
Day 7
t
P value
−2.616
0.026
40.31 ± 3.96a 0.049
0.962 54.42 ± 3.65b
Note: Averages followed by the same letter in the column for intergroup comparison are statistically equivalent in the unpaired Student’s t-test. Significance level is 5%.
Surgical wound (Day 1) CT
Day 4 CT
Day 7 CT
Surgical wound (Day 1) TT
Day 4 TT
Day 7 TT
Figure 2. Representation of the clinical evolution of the wounds during treatment. CT: Control; TT: Treated.
Day 4 CT
Day 7 CT
Day 4 TT
Day 7 TT
Figure 3. Photomicrograph with objective lens of 400×. Skin wound area colored with hematoxylin–eosin. Fibroblast identification on days 4 and 7. CT: Control; TT: Treated.
DISCUSSION
This study shows that TT influences skin healing, accelerates tissue repair by increasing fibroblast counts on day 7, followed by a reduction in wound area (Tables 1 and 2; Figures 2 and 3). However, the lack of evidence on the mechanisms modulated by TT imposes limitations on understanding how it interacts with the body. The effects observed in this study mirror the discoveries of Dr. Bernard Grad and Dr. Justa Smith that TT increases the speed of enzymatic reactions, such as those involving proteases10, which may result in the enhancement of cellular activities that are part of the healing process and/or other organic reactions.9 In tissue repair, proteases, specifically the matrix metalloproteinases (MMPs), have an essential role in protein breakdown. This facilitates the immune response, elimination of foreign elements, remodeling of the extracellular matrix (MEC) damaged by injury, cell motility, release of growth factors, cytokines, and cell proliferation. This process favors new tissue formation and skin healing progression.23,24 However, the imbalance in protease activity during skin healing can cause damage to MEC, leading to destruction of healthy tissue because of the delay in the healing process, and, consequently, to lesion chronicity.24 According to the experiments of Dr. Justa Smith, the changes in the enzymatic activity triggered by laying on of hands are always related to the improvement of cellular activity. Consequently, the organic response is improved, independent of the enzyme that is under the influence of TT.10 In light of Dr. Justa Smith findings on the effects of TT and the evidence available in the literature on the role of proteases in skin healing10,2, we believe the effects of TT in this study may be related to the improved response of MMPs that participate in skin healing. However, this should be investigated in future studies.
According to the precursor of the Kriger–Kunz method11, TT is able to intensify the recovery and/or healing processes in different clinical situations, with response of the autonomic nervous, lymphatic, circulatory, peripheral vascular, and musculoskeletal systems. Moreover, some other systems such as the collagen and endocrine systems may also react to TT in specific situations. One of the features of the proliferative phase during skin healing is the formation of granulation tissue, which consists of fibroblasts, inflammatory cells, and neovascularization that has invaded MEC with the presence of collagen, fibronectin, and hyaluronic acid.3,26 In this study, we opted to evaluate the effects of TT on fibroblast proliferation between the fourth and the seventh day, because it is a period in which they reach a higher production peak and can be visualized in significant numbers.27 The primary function of fibroblasts during healing is to stimulate collagen synthesis, and this occurs early during the inflammatory phase.5,6 The effects of TT observed in our study also reveal its action on fibroblast proliferation (Table 2 and Figure 2) during the proliferative phase. This favors the shortening of the healing time, because it initiates a faster deposition of collagen fibers, and consequently, favors the wound contraction. Importantly, fibroblast activity also involves the participation of MMPs, which are responsible for their migration and proliferation, which later favors scar tissue formation.24 Apart from stimulating collagen-fiber synthesis, fibroblasts form the framework for newly formed blood vessels, which directly influence tissue nutrition. As collagen is responsible for tissue support, its participation in the healing process is fundamental for the lesion bed contraction to occur.5,6 Studies about the use of TT in both in-vitro and in-vivo experimental models show significant results in fibroblast proliferation increase13, in pain reduction14, and in the acceleration of healing response.7,28,29 These results corroborate the
findings of the present study. Nevertheless, further research is necessary to validate its use in the clinical practice. Because it is a low cost, easy-to-use therapy, with potential effects on the organic system, TT seems to be a useful treatment for tissue repair. However, so far its effect on skin healing has not been investigated. As a study limitation, there was no investigation of other factors associated with skin healing, such as macrophage counts, neovascularization, collagen-fiber distribution, and the absence of a simulated TT group (placebo). In addition, it is essential to investigate the effects of TT on all phases of skin healing and its effects on MMP activity in association with the reduction of the lesion bed and the improvement of the scarring response.
CONCLUSION
The data from this study suggest that seven sessions of TT performed daily for a period of 2 min were enough to induce acceleration in wound repair in Wistar rats. This result was observed by means of the reduction in the wound area and by the increase in fibroblast counts on day 7.
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