pulsed high intensity laser versus low intensity laser ...

Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 Original Research Article

Physical Therapy

International Journal of Pharma and Bio Sciences

ISSN 0975-6299

PULSED HIGH INTENSITY LASER VERSUS LOW INTENSITY LASER ON HEALING OF FULL THICKNESS WOUND IN DIABETIC RATS (HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY) HESHAM GALAL MAHRAN1, EMAN MOHAMED FARUK2, NASER A. ELSAWY3, WARDAHABDULLAH MOHAMMED ALASMARI4 AND ABDULLAH GLILRADA ALKUSHI4 1

Physical Therapy Department for Surgery, Faculty of Physical Therapy, Cairo University, Egypt. Department Histology and Cell Biology, Faculty of Medicine, Benha University, Egypt. 3 Department of Anatomy and Embryology Faculty of Medicine Zagazig University, Egypt. 4 Department of Human Anatomy Faculty of Medicine, Umm al Qura University, Makkah, Saudi Arabia. 2

ABSTRACT To evaluate and compare the effects of pulsed high-intensity and low-intensity laser on wound healing in diabetic rats, which was represented by transforming in wound surface areas, rate of healing and immunohistochemical study of Desmin phenotype characteristics of myofibroblasts. One hundred and twenty mature male albino rats had been divided into two groups: (G1) was used like a control to study the conventional skin structure. (G2) had been intraperitoneal injected by streptozotocin (STZ) to provide diabetic rat model. After anesthesia, full thickness wounds (2×2. 5cm) were created in all rats’ nape, and then divided directly into 3 equal groups; Group A) received pulsed high-intensity laser (Nd: YAG laser light, 1064nm), Group B) received low intensity laser (GAlAs Laser, 830 nm) with 5 j/cm2 and overall energy equal to 25 joules using a scanning technique, 3times/week for 3 weeks for both laser techniques. Group C) (control +ve) received no treatment. Measurements of wounded area, rate of epithelialization, histological and immunohistochemical evaluation also were conducted. In both HILT group and LILT group, there were better significant reductions of WSA along with better epithelization rate than those in the control group, and there was clearly no significant difference between each laser's effect on wound recovery. Histological results revealed fast reepithelization of epidermal level with the increase in dermal thickness, which characterized by thick collagen bundles and cellular infiltrates in both laser treated groupings, and immunohistochemical examination showed an increase in the number of desmin-positive immunoreactivity compared with that in the control group. Both HILT and LILT lasers at intensity of 5j/cm2 have facilitating effect on wound healing inform of shrinkage of WSA with no significant difference between both effects at that intensity level. KEYWORDS: Wound, Diabetic Rats Pulsed High Intensity Laser, and Low Intensity Laser.

WARDAHABDULLAH MOHAMMED ALASMARI* Department of Human Anatomy Faculty of Medicine, Umm al Qura University, Makkah, Saudi Arabia.

Received on: 19-01-2017 Revised and Accepted on: 20-04-2017 DOI: http://dx.doi.org/10.22376/ijpbs.2017.8.2.b874-888

This article can be downloaded from www.ijpbs.net

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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888

INTRODUCTION Wound healing impairment is an extremely common problem associated with diabetes plus it is a severe 1 clinical problem in practice . Up to15% of individuals with diabetes will develop ulcerated and wounded foot 2 .Healing of the wound is a complicated process with in traditional stages: inflammatory stage, proliferative stage and maturation stage. During inflammatory stage; injured tissues are debrided by a clot produced phagocytic cells. While during the proliferative stage; epithelialization, fibroplasias, plus angiogenesis are occurred. Meanwhile; formation of mature granulation 3 tissues as well as contraction of wound begins .Lastly, the maturation stage; tight links among collagens and with protein are formed that increasing scar tissue 4 strength .Desmin is class-III, advanced filaments present in muscle tissues. They type the fibrous system linking myofibrils to one another and to the plasma membrane layer in the periphery from the Z-line 5 constructions .Desmin-immunoreactivity has been seen in cells associated with subepidermal area, within the myoepithelial cellular material encircling sweat plus perspiration intrigue, within the fibroblast, soft muscles, endothelial cellular material associated with ships and a 6 few follicles of hair . It is popular that; will myofibroblasts within pores and skin lesions can communicate advanced cytoskeletons, like vimentin, 7 desmin, plus a-smooth muscle actin (a-SMA)] . Curiosity about the efficacy of lasers as a noninvasive tool intended for wound healing has created. These types of lasers include helium-neon, gallium-aluminum-arsenide (GaAlAs), gallium-arsenide, Nd: YAG, ruby, carbon 8&9 dioxide plus argon (ar) dye lasers . Laser therapy is useful in treatment as it has maximum absorption rate which lead to stimulation of the biological 10&11 processes.. .Various studies used many types of lasers with different wavelengths to boost the wound 12 healing such as helium neon 632nm , Argon laser 48813 14 514, krypton laser , Copper vapor laser 578nm diode 15lasers (685, 904, 830, 670, 980, 815 and 809 nm) 18 19 .Nd-YAG 1046nm .Cameron and colleagues documented that the frequency of the laser beam light, as well as the type of cells being irradiated, determines the depth to which light permeates. Laser produced by a gallium aluminum arsenide (GaAlAs) laser beam penetrates deeper, whereas laser beam light with shorter wavelength, such as the red light created by a 20 He-Ne laser beam, permeates less deep penetration . The neodymium: yttrium aluminum garnet (Nd: YAG) laser is utilized to provide high intensity laser beam therapy (HILT) and releases light at 1064nm. This wavelength is highly consumed in blood than in the nearby tissue. This leads to deep transmission and wide scattering within the tissue, thereby allowing the main 21 effect of theNd: YAG laser to occur . Over the last few years, HILT offers gained attention for its exceptional 22-23 results in traumatology and discomfort therapy . Unlike the extensive amount of literature confirming the effects of low level laser beam therapy (LLLT) on injury healing, no data are already reported to date on the associated with pulsed Nd: YAG laser beam therapy on the healing various types of pathological wounds. The problem and challenge in remedying of diabetic wound, lead to look for new therapeutic protocols, which means

this study aimed to investigate plus compare the effects of two sorts of lasers; (Nd: YAG and GaAlAs) with various wavelengths; (1064 nm and 830nm) respectively with energy of 5 J/cm2 on the healing of injury in diabetic rats, calculating the WSA changes, examining histological and immunohistochemal modifications and identifying the best laser beam therapy in this type of injury

MATERIALS AND METHODS Animals A hundred and twenty adult male albino rats approximately four months of age at research initiation, and weighed 150-250 g. were obtained from house of animal in Faculty of Medicine, Umm Al Qura University. Rats were looked after in accordance with the Guide for that Care and Use of Experimental Animals of Umm AlQura University, KSA, and the whole experiment was carried out according to the International Principles associated with Laboratory Animal Research. Rats were individually housed within stainless steel cages with wire-mesh flooring in a controlled atmosphere at 2325°C and 50 percent humidity with artificial light cycle on the 12: 12-h dark-light period (07. 00-19. 00 lamps on). Food (#5322 Purina Licensed Rat Ration, and obtainable ad libitum for all. Rats were housed in strong bottomed cages; food and drinking water were maintained on a pellet diet and tap water advertisement libitum during the entire amount of the study (3 weeks). Procedures Chemical induction of diabetes Permission of ethical committee: the study was started after obtaining permission in the ethical committee of the organization. Induction of type two diabetes Mellitus in experimental animals The animals were fed on rich fatty diet (RFD) for the initial 2 weeks ad libitum. A couple weeks after of dietary supplements, the animals were injected with streptozotocin 35 mg/kg intraperitoneally STZ-injected rats had blood glucose of 16. five mmol/L. Animals were examined regarding blood glucose level 7 days after STZ injection. After overnight fasting animals was administered glucose (3 g/kg) via gastric gavage. After that, 2 hours later blood sample through a lateral tail 24 . vein were collected for serum blood sugar estimation Level of blood glucose Before surgery and prior to scarification, level of blood glucose of each rat was examined. Blood glucose levels were tested using a glucometer and check the strips (One Touch Super; IifeScan Co., USA), blood samples collection from the rat tail before the experiments had been performed. 3 days after STZ injection, Average blood sugar was presented as indicated blood glucose, animals having serum glucose ≥200 mg/dL was diabetic and selected for the study. The rats were permitted to 25 remain to become fed on their own diet routine . Wound surgery model Before surgical treatment, the blood glucose level of every rat was checked once again. For ninety rats, the


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 full-thickness excisional wound has been performed as the animals had been anesthetized with prior to the development of the wounds. The skin has been shaved by electric clipper, disinfected with 70% alcoholic beverages. An area of uniform injury 5 cm2 in size (2. 5x2 cm) has been excised from the nape of the dorsal neck of all rats. Research design The study was randomized controlled study as rats were randomly assigned straight into 2groups. Treatment intervention Group 1 (Control -ve group). Group 2(Experimental group): Diabetic rats were divided into three equal groups (30 rats each). Group(2A): Rats received GaAlAs laser beam therapy, (830 nm, BTL 5000, UK) with the subsequent parameters; 5J/cm2, 3times/week/3weeks, making use of scanning method. Group (2B): Rats received Nd: Yag laser beam therapy (1064 nm, design: HIRO 3. 0, ASA srl company, Italy) using the following parameters; 5J/cm2, 3times/week/3weeks, using scanning method. Group (2C):Control (+ve) (received no treatment). Measurement and evaluation Wound area and healing rate measurement The injured area was measured simply by placing a transparent doing a trace for the paper over the wound. The tracing paper was put on 1 mm2chart sheet, and traced away. The squares were measured and the area was recorded. Additionally, digital camera was used to catch wound surface area. -Rate associated with epithelialization was calculated utilizing the following 26 : [(Area of 1stday− Area of X days)/Area formula associated with 1stday] × 100. -In each group, wound area plus the rate of epithelialization for each rat had been measured three times; after 7 days, 14 days, and twenty-one days of treatment. -In every group and at each time span of measurement (7&14&21days after treatment), 10 rats had been measured for their wound then were sacrificed intended for isolation of skin for examination histologically and Immunohistochemically. Histological technique The animals had been sacrificed by an overdose of anesthesia (phenobarbital sixty mg intraperitoneal); skin individuals were taken from the wound area of dorsum with the encircling normal skin. Skin examples were cut into areas (4∼5 µm thickness), appropriate fixation were done by utilizing 10% neutral buffered chemical. Then tissues had been dried out, cleared in xylol, and then embedded in paraffin polish. Finally, skin sections had 27 been stained with H&E spot and examined below light microscope. Immunohistochemical procedures An antibody is intended for using in vitro diagnostic (IVD). Ventana Medical Techniques, Inc. (Ventana) confirm anti-Desmin (DE-R-11) Primary Antibody is a mouse monoclonal antibody (IgG1, light chain kappa)

instructed against desmin. This antibody is intended for use to qualitatively identify desmin by gentle microscopy in skin parts of formalin fixed, paraffin inlaid tissue on a Ventana automatic slide stainer. Endogenous peroxidase activity was blocked along with 5% H2O2 in overall methanol for 10min with room temperature. Then, the sections were rinsed within phosphate buffered saline (PBS) and were incubated along with primary antibodies against anti- Desmin. Degree of Protein expression was evaluated by the streptavidin biotin peroxide kit. Areas were stained with diaminobenzidine (DAB) as chromogen intended for Desmin detection and areas were then lightly 28 . counterstained with hematoxylin Morphometric study Image analysis had been used to assay the number of desmin-positive cells in 10 fields in the dermis and in the epidermis in each slide from each group at ×400 using Image-Pro Plus system version six.Media Cybernetics Incorporation., Bethesda, Baltimore, USA). Epidermal thickness also was measured in five nonoverlapping fields from each group at ×400 using a Leica image analyzer (Leica) atThe National Institute for Research in Cairo.

STATISTICAL ANALYSIS The outcomes were collected and examined using the SPSS program sixth as means plus standard deviations were determined in addition, as parametric procedures, one way ANOVA in addition to repeated measures were utilized to compare mean values among groups as well as within a group respectively. The differences had been considered statistically significant once the p value < 0. 29 05 .

RESULTS Wound Surface Area (WSA) and rate of healing The results displayed in table 1 and in figure 1 show a highly significant sequential reduction in wound surface within all groups (p value < 0.0001) for all measures, as data were analyzed by repeated measures ANOVA. After1stweek, the results in table 1 and figure 2 show that the mean value of the WSA in HILT group was (2.76±0.64) with a healing rate mean about (44.7% ±12.83), andit was (2.56±0.75) for LLLT group and with a healing rate means about (48.66%±15) while it was (2.88±0.71) for control group with a healing rate means about (42.34±14.26), analysis of outcomes by one-way ANOVA, exposed that there were no statistically significant differences between groups as (p value equal to 0. 41). In addition, the Post Hoc test exposed that there were no substantial differences between; the mean of W.S.A or healing rate in HILT group and in the LLLT group, the mean of W.S.A or healing rate in HILT group and in the control group, and the mean of W.S.A or healing rate in the LLLT group and in the control group after 1stweekas p value (0.4), (0.62), (0.19) respectively.


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 Table 1 Comparison of wound surface area (WSA) and healing rate mean values after (1st, 2nd and 3rd weeks) within group and between groups. Mean ± SD

HILT G. LLLT G. Control G. P value Post Hoc test F value

1st week Healing Rate (%) WSA (cm2) 2.76±0.64 44.7±12.83 2.56±0.75 48.66±15 2.88±0.71 42.34±14.26 0.41 1vs2=(0.4)2vs3=(0.19) 1vs3=(0.62) 0.90

2nd week WSA Healing Rate (%) (cm2) 0.72±0.43 85.4±8.6 0.82±0.61 83.50±12.2 1.32±0.65 73.6±13 0.04 1vs2=(0.68)2vs3=(0.046) 1vs3=(0.018) 3.5

3rd week WSA (cm2)

P value Healing Rate (%)

0.09±.07 98.1±1.43 0.12±0.11 97.5±2.26 0.29±0.18 94.±3.56 0.046 1vs2=(0.60)2vs3=(0.042) 1vs3=(0.021) 3.8

0.0001 0.0001 0.0001

(1): HILT group, (2): LLLT group, (3): Control group. (1) vs. (2): Comparison between HILT group and LLLT group., (2) vs. (3): Comparison between LLLT group and control group. (1) vs. (3): Comparison between HILT group and control group.

After the 2nd week, table 1 and figure 2 show that; 2 mean of the WSA in HILT group was (0.72±0.43) cm , with a healing rate mean (85.4%±8.6) while the mean of the WSA was (0.82±0.61) cm2 with a healing rate mean (83.50%±12.26)in LLLT group and it was (1.32±0.65) cm2 and healing rate mean (73.6%±13.03)in the control group; the analysis of these data revealed that there was a significant difference between the groups (p value = (0.04). Detailed analysis of the data revealed that; there were no significant differences between the mean of W.S.A or rate of healing in HILT group and that in LLLT group as p value =(0.68), while there were significant differences between; the mean of W.S.A or rate of healing in HILT group and that in the control group and the mean of W.S.A or rate of healing in LILT group and that in control group as p values (0.018), (0.046) respectively. After 3rd week table 1 and figure 2

show that; mean of the WSA in HILT group was 2 (0.09±.07) cm , with a healing rate mean (98.1±1.43), the mean of the WSA was (0.12±0.11) cm2 with a healing rate mean about (97.5±2.26) in LLLT group and it was (0.29±0.18) cm2 and healing rate mean about (94. ±3.56) in control group; the evaluation of these data revealed there was a significant difference between groups (p value = (0. 046). Detailed analysis of the data revealed that; there were no significant differences between the mean of W.S.A or rate of healing inHILT group and that in LLLT group (B) as p value = (0.60), while there were significant differences between; the mean of W.S.A or rate of healing in HILT group and that in control group and the mean of W.S.A or rate of healing in LILT group and that in control group as p values (0.021), (0.042) respectively.

Figure 1 Comparison of wound surface area (WSA) means between groups at different treatment phases.


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Figure 2 Comparison of wound surface area Means within each group at different treatment phases

Figure 3 Shows examples of captured images by digital camera for wound surface area (WSA) providing comparative st nd rd images for wounds within each group and between groups pretreatment and after (1 ), (2 ), and (3 ) weeks. Blood Glucose Level The results displayed in table 2 show a highly significant increase in blood glucose level within diabetic groups (p value < 0.001) for all measures during the study, as data were analyzed by repeated measures ANOVA. st After1 week, the results in table 2 and show that the mean value of blood glucose in HILT group was (394.58±19.98) and it was (396.23±27.20) for LLLT group while it was (420.15±9.33) for control + group, analysis of results by one-way ANOVA, revealed that therewere no statistically significant differences between treated groups and control positive group.After 2nd week, table 2 show that; mean of the blood glucose level in HILT group was (381.05±8.03), while the mean

of the blood glucose level was (382.34±6.362) in LLLT group and blood glucose level mean was (404.59±6.80)in control positive group ; the analysis of outcomes by one-way ANOVA, exposed that there were no statistically significant differences between treated groups and control positive group, After 3rd week table 2 show that; mean of the blood glucose level in HILT group was (344.04±11.37), while the mean of the blood glucose level was (328.01±13.67) in LLLT group and blood glucose level mean was (367.40±9.04)in control positive group ; the analysis associated with results by one-way ANOVA, revealed that there were simply no statistically significant differences among treated groups and control positive group.


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 Table 2 Comparison of fasting serum glucose (mg/dl) mean values after (1st, 2nd and 3rd weeks) within group and between groups. Mean ± SD Control –ve G. HILT G. LLLT G. Control + G. P value F value

1st week 97.72±4.08 394.58±19.98*** 396.23±27.20*** 420.15±9.33*** < 0.001 378.78

2nd week 96.11±3.03 381.05±8.03*** 382.34±6.362*** 404.59±6.80*** < 0.001 539.24

3rd week 91.37±3.27 344.04±11.37*** 328.01±13.67*** 367.40±9.04*** < 0.001 809.37

***

(1): HILT group, (2): LLLT group (3):Control group. Significant difference compared to control –ve group. (1) vs. (2): Comparison between HILT group and LLLT group. (2) vs. (3): Comparison between LLLT group and control group. (1) vs. (3): Comparison between HILT group and control group.

Histological results Microscopic examination of sections of control unwounded skin (-ve control G) revealed the normal histological structure of the thin skin which is formed of two principal layers: the epidermis and the dermis. The epidermis was formed of stratified squamous keratinized epithelium resting on a wavy basement membrane with four distinct cell layers: stratum basale, stratumspinosum, stratum granulosum, plus stratum corneum. The epidermal-dermal junction showed many skin ridges and dermal papillae.The dermal layer contained connective tissue with collagen fibers and epidermal appendages (hair follicles & sebaceous gland). The hypodermis was present deep to the dermis that contained adipose connective tissue. (Fig.4; A&B and C). While examination of skin of (+ve control G.) one week postwounding, showed separation of the two edges of the wound with loss of superficial layer of the epidermis and destructed collagen fibers in the demial layer . .. (Fig. 5), while sections stained two-weeks post wounding showed that the two wounds margin were covered by skin crust which is formed of degenerated inflammatory cells, RBCs and keratin with irregularly arranged epithelial cells of the wound edges with increased thickness of collagen fibers if compared with control group (Fig.6).Sections of control +vegroup (3 weeks post wounding) showed an apparent thinning out of the epidermis and loss of epidermal ridges and dermal papillae as compared with the (–ve)control rats. Less dense connective tissue fibers were also seen in the dermis (Fig.7).H&E-stained sections one week post wounding of LILT and HILT groups one week postwounding were almost similar together were showed newly formed epithelium with irregularly arranged keratinocytes with vacuolated cytoplasm and darkly stained small nuclei and mononuclear cells infiltration (Fig.8; A&B).Skin sections two-weeks post wounding of LILT and HILT groups showed closure of the wound with a newly formed layer of epithelium. The keratinocytes appeared less normal. The dermo-epidermal junction was flattened with loss of the dermal papillae. The underlying granulation tissue was replaced by new

dermal tissue. This dermis contained dense, interrupted and irregular newly formed collagen fibers (Fig.9; A&B). Whereas the appearance of the epidermis and the dermis of group LILT and HILT groups were almost similar to that of the control group three-weeks post wounding (Fig.10-A&B). Immunohistochemicaland results Desmin expression appeared as dark brown cytoplasmic stain in positive reaction. Severe desmin expression was detected in the normal rat skin. It was mainly detected in the hair follicle cells and around blood vessels in the dermis (Fig.11). In control +ve group there was a moderate expression of desminoneweek post wounding sections, while two and three-weeks post wounding there were mild cytoplasmic immunoreactions in specimens (Fig.12&13 and 14 respectively). In both LILT and HILTgroups showed moderate to severe desminimmunoreactivity in one, two and three-weeks post wounding (Fig.15&16 and 17 respectively). Morphometricresults See Table 3 As regards desminimmunoreactivity, it was measured as 12.988±0.669 control – vegroup .There was a highly significant decrease (P < 0.001) in the desmin in the skin cells of diabetic untreated group (6.184±1.779, 8.241±0.725 and 5.692±0.462)in 1st,2nd and 3rd week post wound respectivelycompared with the control group. However, there was a highly significant increase (P < 0.001) in the treated diabetic laser groups when compared with they compared with controlgroup. While, the epidermal thickness of control group was measured as 21.34 ± 0.4 urn. There was a highly significant increase (P < 0.001) in the mean thickness of the epidermis in group 2 B (20.93 ± 0.65,21.05 ± 0.11 and 22.53 ± 0.85)in 1st,2nd and 3rd week respectively when compared with the control group. While, a significant decrease (P > 0.05) in the mean thickness of the epidermis was observed in +ve control group (17.22 ± 0.65,18.87 ± 0.34and 20.89 ± 0.34 ) when compared with the control group (Table 4).


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Figure 4 A photomicrograph of the section in adult man albino rat thin skin (control-vegroup.),showing a normal histological structure of the thin skin which formed of thin epidermis (E) which is formed of stratified squamous epithelium resting on a wavy basement membrane and covered by scales of keratin (K) and the dermis with its upper papillary (P) and lower reticular (R) layers. Notice the hair follicles (H) and associated sebaceous glands (S) can be seen. [H&Ex100].Small window shows magnified layers of the epidermis: basal layer (B) and keratin of horny (C) H&E, 100; inset, 1000

Figure 5 A photomicrograph of a section in adult male albino rat thin skin (control+vegroup) one weekpost wounding showing, that widely separation of the two edges of wound with retraction (arrows). Also, hair follicles (H) and sebaceous glands(S) can be observed. [H&Ex100].

Figure 6 A photomicrograph of a section in adult male albino rat thin skin (control+vegroup) two weeks post wounding , showing one margin of wound which covered by crust (S) which is formed of degenerated inflammatory cells, RBCs and keratin. Notice dermal collagen fibers and hair follicles (arrows) were distorted. [H&Ex100]. This article can be downloaded from www.ijpbs.net

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Figure 7 A photomicrograph of a section in adult male albino rat thin skin (control+vegroup) three-weeks post wounding, showing thinning out of the epidermis with loss of epidermal ridges(arrow).[H&Ex100].

A

B

Figure 8 Fig.(8-A) photomicrograph of a section in adult male albino rat thin skin (LILT G.) one -week post wounding, showing margin of the wound with granulation tissues (G) and irregularly arranged epithelial cells of the wound edges with vacuolated cytoplasm (V). Fig.(8-B) A photomicrograph of a section in adult male albino rat thin skin (HILT G) ) one- week post wounding, showing margin of the wound with granulation tissues (G) with mononuclear inflammatory cells infiltration (F) and irregularly arranged epithelial cells of the wound edges (arrow). [H&Ex200].

A

B

Figure 9 Fig.(9-A).A photomicrograph of a section in adult male albino rat thin skin (LILT G.) two-week post wounding, showing large skin crust (C) above the wound. Re epithelisation is formed with irregular epithelial layer (arrow). Granulation tissue (G) fills the wound space and under the epithelial layer. Fig.(9-B).A photomicrograph of a section in adult male albino rat thin skin (HILT G.) two-weeks post wounding, showing newly formed epithelium with variable thickness (E) and irregular epithelial cells with vacuolated cytoplasm (v). Notice thick irregular dermal connective tissue (arrow) [H&Ex100].


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A

B

Figure 10 Fig.(10-A).A photomicrograph of a section in adult male albino rat thin skin (LILT G.) three weeks post wounding, showing normal histological skin formed of epidermal (E) and dermal layers (D).Fig.(10-B).A photomicrograph of a section in adult male albino rat thin skin (HILT G.) three weeks post wounding , showing normal histological skin structure formed of epidermal (E) and dermal layers (D). Notice epidermaldermal junction showing many epidermal ridges (arrow). [H&Ex100].

Figure 11 Fig. (11).A photomicrograph of a section in adult male albino rat thin skin (unwounded control group G I), showing, moderate cytoplasmic immunoreaction for desmin in epidermal cells and hair follicles cells (arrows). [Antidesmin antibody x200].

Figure 12 A photomicrograph of a section in adult male albino rat thin skin (+ve control G) one week postwounding showing, mild cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H). [Antidesmin antibody x200].


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Figure 13 A photomicrograph of a section in adult male albino rat thin skin (+ve control G) two weeks post wounding , showing moderate cytoplasmic immunoreaction for desmin in epidermal cells (C) and hair follicles cells (H). [Antidesmin antibody x200].

Figure 14 A photomicrograph of a section in adult male albino rat thin skin (+ve control G) three-weeks post wounding, showing mild or absence cytoplasmic immunoreaction for desmin in epidermal cells (C) and hair follicles cells (H). [Antidesmin antibody x200].

A

B

Figure 15 Fig.(15-A). A photomicrograph of a section in adult male albino rat thin skin (LILT G.) one week post wounding, showing mild or absence cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H).Fig.(15-B). A photomicrograph of a section in adult male albino rat thin skin (HILT G.) three weeks post wounding, showing mild or absence cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H).[Antidesmin antibody x400].


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A

B

Figure 16 Fig. (16A). A photomicrograph of a section in adult male albino rat thin skin (LILT G.) two-weeks post wounding, showing moderate cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H). [Antidesmin antibody x200]. Fig. (16B). A photomicrograph of a section in adult male albino rat thin skin (HILT G.) two-weeks post wounding, showing moderate cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H). [Antidesmin antibody x400].

A

B

Figure 17 Fig. (17A). A photomicrograph of a section in adult male albino rat thin skin (LILT G.) three-weeks post wounding, showing severe cytoplasmic immunoreaction for desmin in epidermal cells(C) and hair follicles cells (H). Fig. (17B). A photomicrograph of a section in adult male albino rat thin skin (HILT G.) one week post wounding, showing severe cytoplasmic immunoreaction for desmin in epidermal cells (C)and hair follicles cells (H). [Antidesmin antibody x200]. Table 3 Showing the mean number of desmin-positive cells, SD and P value compared with control-vegroup. Groups

Control (-ve) group (no wound ) 12.988±0.669

1st week post wound 2nd week post wound 3rd week post week Significance SD = standard deviation S = Significant

Control (+ve) group

LLLT group

HILT group

P value

6.184±1.779 8.241±0.725 5.692±0.462 HS

10.482±.321 10.962±0.475 11.285±1.002 HS

11.065±.452 11.624±2.796 12.064±0.869 HS

0.0001 0.0001 0.0001

Table 4 showing the mean epidermal thickness (mean ± SD) in different groups and P value compared with control-ve group. Groups

Control (-ve) group 21.34 ± 0.4 urn

Control (+ve) group

1st week post wound 17.22 ± 0.65 2nd week post wound 18.87 ± 0.34 3rd week post week 20.89 ± 0.34 Significance S SD = standard deviation S = Significant

LLLT group

HILT group

P value

19.42±.39 20.52±0.42 20.99±.09 HS

20.93 ± 0.65 21.05 ± 0.11 21.13 ± 0.05 HS

0.0001 0.0001 0.0001


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DISCUSSION Pulsed Nd: YAG laser light has proved its flexibility and efficacy in the remedying of many different musculoskeletal diseases in fact it is believed to have 30 anti-inflammatory, anti-edema and analgesic effects .In recent studies, also regenerative effects have been 31-32 uncovered .Within our study an excisional diabetic wound model was employed to investigate and compare the result of Nd: YAG laserlight, 1064nm as pulsed highintensity and GaAlAs Laser, 830 nm as low-intensity 2 atthe same intensity level (5j/cm ). The results of this study provided proof that both pulsed high-intensity and low-intensity laser impact the wound healing process in diabetic rats. Notably, despite simply no significant differences, the evaluation between groups after the first week of treatment uncovered that the healing process in both groupings (A) and (B) by means of shrinking of wound surface area and healing rate had been better than in group (C), two weeks later after therapy, the healing process in both groupings (A) and (B) had been significantly better than in group C. as p value = (0. 04), plus post hoc analysis uncovered that no significant difference in between group (A) and group (B) as pvalue = (0.68) Lastly, and after three weeks associated with treatment, the previous results were verified in that; the healing process both in groups (A) and (B) was significantly better than within group C. as g value = (0. 046), and no significant difference between team (A) and group (B) as p value = (0. 60). These results were agreed with some prior published studies as stick to: The bio modulatory effects of laserlight therapy are based on the theory that photon energy is ingested by cellular photo acceptor elements, such as oxyhemoglobin, hemoglobin, cytochrome c oxidase and melanin then the energy is changed into chemical 33 energy within the cellular . Cytochrome m. oxidase receives photons plus promotes a change in the mitochondrial redox leading to moving of ions across the internal mitochondrial membrane as well as a boost in 34 ATP synthesis . There is also an increase within intracellular calcium (Ca2+), which usually stimulates DNA and RNA synthesis, thereby activating the cascade 35 of intracellular indicators . This eventually stimulates DNA duplication, raises protein synthesis, regulates oxidative stress, and modulates the availability of 36-37 different cytokines . These events result in the bio modulation of different cellular types involved in tissue 38 39 reconstruction also affect fibroblast division , 21 greater angiogenesis , changes in the activity of 40 cytokines , and assistance in the transformation of 38 . fibroblasts into myofibroblasts Many correctly reported the influence associated with LLLT with normal plus impaired wound healing procedures in animals and human beings. Indeed, enhanced closing of the wound, increased fibroblasts, increased growth of blood vessel endothelial cells, improved tensile strength, a lower inflammatory reaction, and the induction of collagen synthesis have already been reported for normal 39 41 cutaneous wounds, stress ulcers, superficial wounds 42 43 in healthy individuals, and uses up in rats .In comparison with other laser resources, the advantages of the pulsed Nd: YAG laser consists within high power using a wavelength emission (1064 nm) which is weakly absorbed simply by cell and tissue chromophores, thus

resulting in high penetration capacity and ability to deal with deep tissues. Moreover, the photothermal impact can be controlled in terms of affected person safety and 44 comfort bymodulating pulse intensity and regularity . The exposure to pulsed Nd: YAG laser triggered the synthesis of ECM molecules in connective tissue cells 45-46 .This kind of high intensity in such a limited time prevents the heat accumulation from the tissues as happens by using Nd: YAG laser along with continuous emission. These functions result in a greater propagation of the radiation in the tissues using a very low histolsesive risk, resulting in the possibility of 47 treating deep tissue and structures . Photostimulation promotes tissues repair by accelerating the availability of collagen and market overall connective tissue. Several studies have found that HILT seems to be more effective than LLLT because of its higher intensity and to 44. the increased depth arrived at by the laser Histological result in line with the laserlight result as, one week post wounding skin sections demonstrated retraction of the two sides of the wound. This might end up being occurred due to recoil associated with collagen and 48 elastic fibres. Al-Henhena et al., (2011) showed that will contractore of wound because of increasing in the fibroblasts phase (proliferative phase) and so wound 49 shrinkage. Ejaz et al (2009) said that woundnatural healing is regenerating skin and epidermal tissue within bodyand divided into (the inflammatory, proliferative, and redesigning phases). Two-weeks post wounding, the epithelium had the adjustable thickness and flattening associated with dermo-epidermal junction. The epithelial cells appeared irregularly organized, with vacuolated cytoplasm. Granulation tissue filled the wound which formed of mononuclear cellular infiltration. 22 Braiman et al., (2007) stated that seven to 10days post wounding, formation of scab along with the migration of the epidermal sides (re-epithelization), proliferation of the granulation cells and inflammatory reaction. Liang et 23 found that re-epithelizationis due to al., (2012) activation, migration plus proliferation of keratinocytes that migration from epidermis So, epithelialization is mainly done by keratinocytes, which have an important 50 role in this process. Gal et al., (2008) was with our finding asthey foundthat the proliferative phase is containing angiogenesis, collagen deposition, granulation tissue formation and epithelization. Threeweeks post wounding, wound closure was including a newly formed layer of epithelium. The epithelial cells made an appearance more or less normal. The dermoepidermal junction was flattened along with the loss of the dermal papillae. The underlying granulation tissue had been replaced simply by dermal tissues. Alfars, 51 (2009) supported our findings; he stated that the thickness of the newly formed epidermis was not similar to intact epidermis throughout proliferative phase in diabetic wound healing. Desmin reflection was detected in the present research, one, two, three days, post wounding. The top of the expression was confirmed three-weeks post wounding. This increase in desmin reflection might be explained by the growth of myofibroblast during wound healing. Also, this reflection observed around hair follicles within the dermis of the 52 wound area. Hayashi et al., (2004) stated that the top of desmin expression takes place with skin wound process of recovery, corresponding to the proliferative stage which histologically evidenced by simply


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Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 granulation tissue formation in addition re epithelization. Myofibroblastscan end up being detected by immunoexpressions for vimentin (V), a-SMA (A) plus desmin (D); these might be variously expressed depending on building phases of myofibroblasts(Ide et 53 al., 2004) . In line with the expression levels of these more advanced filaments, myofibroblasts are split into the following types: cellular activating to vimentin plus aSMA (VA type), cellular activating to vimentin and desmin (VD type), and cellular activating to vimentin, a54 SMA plus desmin (VAD type) .Alenaet al., 55 (2003) supported the finding as proved that Laser therapy reduced the inflammatory reaction, increased collagen deposition and proliferation associated with myofibroblasts in experimental cutaneous wounds. 56 Alenaet al., (2008) stated the fact that laser photobiomodulationcould encourage many modifications in the healing process, and the newly-formed collagen fibers be better organized.

CONCLUSION Both HILT plus LILT lasers at regarding 5j/cm2 have facilitating effect on wound healing inform of shrinking of WSA and boosting of epithelization rate with no significant difference between both results at that intensity level.

ACKNOWLEDGMENT We acknowledge the resources and financial support for the study was provided by the Institute of Scientific Research and Revival Islamic Heritage at Umm Al-Qura University (project # 43509022) for research project finance and financial support.

CONFLICT OF INTEREST Conflict of interest declared none.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

Rafehi H, A. El-Osta T. C. Karagiannis B. “Genetic and epigenetic events in diabetic wound healing,” International Wound Journal, 2011. 8 (1):12-21. Al-Watban F. A. H. Zhang XY. Andres B L. “Lowlevel laser therapy enhances wound healing in diabetic rats: a comparison of different lasers,” Photomedicine and Laser Surgery, 2007. 25(2): 72-77. Desmouliere A. Chaponnier C.Gabbiani G. Tissue repair, contraction and the myofibroblast. Wound Repair Regen,2005. 13(1):7–12. Ishida Y.Gao J. Murphy P.M. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function.J Immunol,2008. 180:569–579. Saha K.Saha A.Datta C.Chatterjee U. Ray S. Bera M. Does desmin immunohistochemistry have a role in assessing stage of urothelial carcinoma in transurethral resection of bladder tumor specimens?Clin Cancer Investig J;2014. 3(6):502-7.DOI: 10.4103/2278-0513.142634. Bär H.Strelkov SV.Sjöberg G.AebiU. Herrmann H. "The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?" J. Struct. Biol. 2004. 148 (2): 137– 52. doi:10.1016/j.jsb.04.003.PMID 15477095. Juniantito V. IzawaT. YuasaT. Ichikawa C. Yamamoto E.et al. Possibletransdifferentiation of blood vessel pericytes and perifollicular dermal sheath cells into myofibroblasts.HistolHistopathol,2012.27: 515527. Jenkins PA. Carroll JD .How to report low-level laser therapy (LLLT)/photomedicine dose and beam parameters in clinical andlaboratory studies Photomed Laser Surg,2011. 29:785–787. Bayat M. Vasheghani M.M. Razavi N. Effects of Low-level helium-neon laser therapy on the healing of third-degree burn in rats.J Photochem.Photobio.2006. 83(2), 87-93.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Pessoa E.S. Melhado R.M. Theodoro L.H.Garica V.G. histological assessment of influence of lowintensity laser therapy on wound healing in steroid-treated animals. J.Photomed Laser surg. 2004.22(3) Jun; 199- 204. Kreisler M. Chritoffers A.B. Al-Haj H. Willershausen B .Low level 809nm diode laserinduced in vitro stimulation of the proliferation of human gingival fibroblasts.Lasers surg. Med.2002. 30(5) 365-9. Capon A. Souil E. Gauthier B. Sumian C. Laser assisted skin closure (LASC) by using a 815 nm diode-laser system accelerates and improves wound healing .Lasers surg Med.2002 .(2) 16875. Gál P.Kilík R.Špaková T.Pataky F. Sabo J.Pomfy M et al. He-Ne laser irradiation accelerates inflammatory phase and epithelization of skin wound healing in rats. Biologia.2005.60: 691–696. Hawkins DH.Abrahamse H.The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation.Lasers Surg Med.2006. 38:74–83. doi:10.1002/lsm.20271. Houreld NN.Abrahamse H. Laser light influences cellular viability and proliferation in diabeticwounded fibroblast cells in a dose- and wavelength-dependent manner. Lasers Med Sci.2007.23:11–18. doi:10.1007/s10103–007– 0445-y. Wang CZ. Chen YJ. Wang YH.Yeh ML. et al. Low-level laser irradiation improves functional recovery and nerve regeneration in sciatic nerve crush rat injury model. PLoS One. 2014; 9: e103348. doi: 10.1371/journal.pone.0103348. pmid:25119457 Poetke M. Laser treatment in hemangiomas and vascular malformations. in: Applied Laser Medicine. H.P. Berlien, and J. Mueller (eds.). Berlin: Springer Verlag,2003. pp. 444–482. Lubich T. Mondardini P. Verardi L. Kanellopulu S.Zoratti M. Use of laser power in the early


B-886


19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

treatment and functional recovery of the injured.Sports Med.1997. 50, 71–83. ByrnesK.R.Barna K.Chenault M. et al., “Photobiomodulation improves cutaneous wound healing in an animal model of type II diabetes,” .Photomedicine and Laser Surgery,2004. 22(4):281-290. Al-Henhena N. Mahmood A. Al-magrami A. Nor Syuhada. A. B. Zahra, A. A.et al. Histological study of wound healing potential by ethanol leaf extract of Strobilanthescrispus in rats.Journal of Medicinal Plants Research,2011. 5 (16): 36603666. Ejaz S. Ashraf M. Nawaz M. Lim C.W. Total Particulate Matter and Wound Healing: An in vivo Study with Histological Insights. Biomedical and Environmental Sciences, 2009.22: 278-287. Braiman-Wiksman L. Solomonik I. Spira R. Tennenbaum T. Novel Insights into Wound Healing Sequence of Events. Toxicologic Pathology,2007. 35:767–779. Liang X. Bhattacharya S. Bajaj G. Guha G. Wang Z. et al. Delayed Cutaneous Wound Healing and Aberrant Expression of Hair Follicle Stem Cell Markers in Mice Selectively Lacking Ctip2 in Epidermis. PLoS ONE,2012. 7 (2): 1-15. Srinivasan K.Viswanand B.Asrat L.Kaul CL.Ramarao P. Combination of high-fat diet-fed and low dose of streptozotocin- treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol Res;2005.52:313-320. Reed MJ.Meszaros K.Entes LJ.Claypool MD, Pinkett JG, Gadbois TM, et al. A new rat model of type 2 diabetes, the fat-fed, streptozotocin-treated rat. Metabolism 2000;49:1390-1394. Alves AC. Vieira R. Leal-Junior E. dos Santos S.Ligeiro AP. et al. Effect of low-level laser therapy on the expression of inflammatory mediators and on neutrophils and macrophages in acute joint inflammation. Arthritis Res Ther.2013. 15: R116. pmid:24028507 doi: 10.1186/ar4296. Bancroft JD and Layton C: The Hematoxylin and eosin. In: Suvarna SK, Layton C and Bancroft JD editors. Theory & Practice of histological techniques.7th ed, Churchill Livingstone of El Sevier. Philadelphia. Ch. 10 and 11. 2013; 172 214. Jackson P and Blythe D: Immunohistochemical techniques. In: Suvarna SK, Layton C and Bancroft JD editors. Theory & Practice of histological techniques.7th ed., Churchill Livingstone of El Sevier. Philadelphia. Ch. 18. 2013; 381 - 434. RiedelF. Philipp K. Sadick H. Goessler U.et al.Immunohistochemical Analysis of Radiationinduced Non-healing Dermal Wounds of the Head and Neck. 2005. In vivo,2005. 19: 343-350. Zati A. Fortuna D.Valent A.FilippiMV.Bilotta TW. High Intensity Laser Therapy (HILT) versus TENS and NSAIDs in low back pain: clinical study. SPIE Proceedings, 2004, 5610: 277-283. Ninomiva T.Mivamoto Y. Ito T. Yamashita A. Wakita M.Nishisaka T. High-intensity pulsed laser irradiation accelerates bone formation

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

metaphyseal trabecular bone in rat femur. J None Miner Metab.,2003. 21: 67-73. Arisu HD.Turkoz E.Bala O. Effect of Nd:YAG laser irradiation on osteoblast cell cultures. LasersMed. Sci., 2006, 21:175-180. 3 Houreld NN. Shedding light on a new treatment for diabetic wound healing: a review on phototherapy. ScientificWorldJournal:2014. 398412. doi: 10.1155/2014/398412. pmid: 24511283. Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J PhotochemPhotobiol B. 1999; 49: 1–17. pmid:10365442 doi: 10.1016/s10111344(98)00219-x. Silveira PC. Silva LA.Fraga DB.Et al. Evaluation of mitochondrial respiratory chain activity in muscle healing by low-level laser therapy.J Photochem Photobiol B.; 95: 89–92. doi: 10.1016/j.jphotobiol.2009.01.004. pmid:19232497. Alves AC. Vieira R. Leal-Junior E. dos Santos S. et al. Effect of low-level laser therapy on the expression of inflammatory mediators and on neutrophils and macrophages in acute joint inflammation. Arthritis Res Ther.2013. 15: R116. pmid:24028507 doi: 10.1186/ar4296 Ribeiro MA. Albuquerque RL .Ramalho LM.Pinheiro AL.et al. Immunohistochemical assessment of myofibroblasts and lymphoid cells during wound healing in rats subjected to laser photobiomodulation at 660 nm. Photomed Laser Surg. 2009; 27: 49–55.doi: 10.1089/pho.2007.2215. pmid:19250051. Bates–Jensen, B.M. The Pressure Sore Status Tool: a few thousand assessments later. Adv.Wound Care.1997. 10, 65–73. Bayat M. Azari.A.Golmohammadi M.G. Effects of 780-nm low-level laser therapy with a pulsed gallium aluminium arsenide laser on the healing of a surgically induced open skin wound of rat. Photomed. Laser Surg.2010. 28, 465–470. Fukuda TY. Tanji MM. Jesus JF. Sato MN.Duarte AJ.Plapler H. Single session to infrared low level diode laser on TNF-alpha and IL-6 cytokines release by mononuclear spleen cells in mice: a pilot study. Lasers Surg Med. 2010; 42: 584– 588.doi: 10.1002/lsm.20949. pmid:20662036. Schubert V. Effect of phototherapy on pressure ulcer healing in elderly patients after a falling trauma.Aprospective, randomized, controlled study. Photodermatol. Photoimmunol. Photomed. 2001. 17, 32–38. Hopkins J.T. Mcloda T.A. Seegmiller. J.C. Baxter. G.D. Low-level laser therapy facilitates superficialwound healing in humans: a triple-blind, sham controlled study. J. Athl. Train.2004. 39, 223–229. Monici M. Cialdai F. Fusi F. Romano G. Pratesi R. Effects of pulsed Nd:YAGleser at molecular and cellular level. A study on the basis of Hilterapia. Energy for Health, 2008, volume 03/2008. Sarasa-Renedo A.Chiquet M. Mechanical signals reglating extracellular matrix gene expression in fibroblasts. Scand J Med Sci Sports, 2005, 15: 223-230.


B-887

Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) 874-888 45.

46.

47.

48.

49.

50.

Chiquet M.Sarasa-Renedo A. Huber F.Flück M. How does fibroblast translate mechanical signals into changes in extracellular matrix production? Matrix Biology, 2003, 22: 73-80. Hawkins D.Abrahamse H. The role of fluence in cell viability, proliferation and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers in Surgery and Medicine, 2006a, 38(1): 74-83. AlGhamdi K.M. A. Kumar A.Moussa N.A. Lowlevel laser therapy: a useful technique for enhancing the proliferation of various cultured cells, Lasers Med.Sci. 27 (2012) 237–249. Al-Henhena N. Mahmood A. A. Al-magrami A. Nor Syuhada, A. B.et al .Histological study of wound healing potential by ethanol leaf extract of Strobilanthescrispus in rats.Journal of Medicinal Plants Research, 2011. 5 (16): 3660-3666. Ejaz S. Ashraf M. Nawaz M. Lim, C.W. Total Particulate Matter and Wound Healing: An in vivo Study with Histological Insights. Biomedical and Environmental Sciences,2009. 22: 278-287. Gal P. Kilik, R. Mokry M. Vidinsky B. Vasilenko T. Mozes, S.et al. Simple method of open skin wound healing model in corticosteroid-treated and diabetic rats: standardization of semi-quantitative and quantitative histological assessments. VeterinarniMedicina, 2008. 53, (12): 652–659.

51.

52.

53.

54.

55.

56.

Alfars A. A. Histological study of early events during wound healing in rabbits. Bas.J.Vet.Res., 2009. 8 (1):130-137. Hayashi T. Ishida Y. Kimura A. Takayasu T. Eisenmenger W. Kondo T. Forensic application of VEGF expression to skin wound age determination. Int J Legal Med, 2004. 118:320– 325. Ide M. Kuwamura M. Kotani T. Yamate J. Immunophenotypical changes of myofibroblasts appearing in thioacetamide-induced hepatic fibrosis in rats. J. Toxicol. Pathol.2004. 17, 231238. Chaurasia S.S. Kaur H. de Medeiros F.W. Smith S.D. Wilson S.E. Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury. Exp. Eye Res.2009. 89, 133-139. Alena P. Medrado BS, Lívia S. Pugliese BS SílviaR.A. Reis, Zilton A. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts, Lasers in Surgery and Medicine Volume 32, Issue 3 (2003) Pages 239–244. Alena P.MedradoB.S. Ana Prates S.Elisângela T. et al. Andrade: Influence of laser photobiomodulation upon connective tissue remodeling during wound healing. Journal of Photochemistry and Photobiology B: Biology 92 (2008) 144–152.


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