VOLUMEolume 3 NOo. 1 JANUuARY 2012 • pages 39-47 MALAYSIAN JOURNAL OF VETERINARY RESEARCH THE USE OF MODIFIED MASSON’S TRICHROME STAINING IN COLLAGEN EVALUATION IN WOUND HEALING STUDY SUVIK A. AND EFFENDY A.W.M Bio-Toxico Analysis Laboratory, Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Mengabang Telipot, Kuala Terengganu,Terengganu. [email protected] (corresponding author) & [email protected] Tel No: 09 668 3104/3661 Fax No: 09 668 3105 ABSTRACT. A number of studies have a clear view of collagen fibers deposition measured collagen fibers and collagen and re-organisation compared to H&E deposition in wound healing process with staining. This finding could validate the advances imaging techniques. However, using of modified MT staining which leads these are performed by complicated to accurate histopathological analysis and methods and need specific tools. In search of observation in wound healing study. the easier ways in routine histopathological Keywords: wound healing, laboratory, collagen measurement and hematoxylin and eosin staining, Masson’s staining pattern of wound healing process trichrome staining, collagen were observed in wounded skin of Sprague Dawley’s rat by using two different stains INTRODUCTION which are standard haematoxylin and eosin (H&E) and modified Masson’s Histopathological study of wound healing trichrome staining (MT). The comparison process is normally used for evaluating between these staining in wounded tissues the efficacy of pharmacological products was made to evaluate the advantages which promote and accelerate dermal skin and disadvantages of both staining in substitutes. The study is usually related to wound healing study for 21 days post- phases of cutaneous wound repairing which wounding. Tissues which stained with can be categorised into four phases such as MT staining was then evaluated its homeostasis, inflammation (early and late), collagen re-organization and density by proliferation and remodeling phases. In using polarized light microscope with the histopathological study of wound healing, aid of image analyzer software. Results a number of criteria are considered to showed that tissues stained with standard determine the level of histopathological H&E could not be used to measure and change such as the depth and length of differentiate the collagen deposition which healed wound, epithelial stratification, is contradictory to MT staining. Wounded leucocytes and macrophage infiltration, tissue stained with MT staining has showed fibroblast, extent of elastin formation and 39 MALAYSIAN JOURNAL OF VETERINARY RESEARCH VOLUME 3 NO. 1 JANUARY 2012 the most important is collagen fiber as it used in the histopathological study of wound plays a dominant role in maintaining the healing. Differing from H&E staining structural integrity of wound healing the MT staining is able to differentiate (Ukong et al., 2008). clearly the important morphological keys However, by using a conventional for wound healing assessment such as staining method such as haematoxylin keratin, haemoglobin, and muscle fiber and eosin (H&E), the study of wound (red colour), cytoplasm and adipose cells healing becomes more challenging as the (light red or pink), cell nuclei (dark brown stain is not able to differentiate important to black) and collagen fiber which stained histopathological change in the wound blue in colour and later could be measured healing process such as collagen deposition by using imaging analysis software. and scab formation which could later lead Clear differentiation of morphological to misinterpretation in histopathological and anatomical structure in the stained observations. Many studies have attempted skin tissue are advantageous and provide to quantify the amount of collagen change further understanding in histopathological and orientation in any stage of wound study of wound healing in future. healing such as by using the epipolarisation microscope with picrosirius red-stained MATERIALS AND METHODS (Noorlander et al., 2002), computer vision analysis of collagen fiber bundles Animals (Elbischger et al., 2005), Fourier transform infrared (FTIR) spectral imaging (Potter Eighteen clinically healthy Sprague et al., 2001), and laser scanning confocal Dawley’s female rats weighing between microscopy (Taylor et al., 2002). There 200 to 250 g were obtained from the was also a study made by Dallon et Animal Laboratory of Universiti Sains al., (2006) which used a mathematical Malaysia, Kubang Kerian, Kelantan approach and equation model to evaluate for wound healing study. All animals the alignment and arrangement of collagen were housed in standard environmental fibers in wound healing process; however conditions with temperature of 25 ± 1°C this method is difficult to be understood with 12 hours light and 12 hours dark and interpreted by non-mathematicians. cycle. They were acclimatised to a hygienic Conversely, all these methods and protocols laboratory condition for 7 days before the are complicated steps which require some start of experiment and observed for any special technicians or equipment in the clinical sign such as diarrhoea, food and routine of histopathological laboratory. water intake, behavior and blood in urine To overcome this problem, an (Tuffery, 1995). Animals were fed with alternative staining such as modified standard commercial pellet diet (10% of Masson’s trichrome staining (MT) can be 40 VOLUME 3 NO. 1 JANUARY 2012 MALAYSIAN JOURNAL OF VETERINARY RESEARCH their body weight) and distilled water ad Histopathological studies libitum. Skin specimens for Masson’s trichrome Experimental design for wound healing staining was fixed in Bouin’s solution, study meanwhile for standard haematoxylin and eosin staining the skin specimens was A total of 18 clinically healthy male fixed in normal 10% buffered formalin. white rats were used in the wound healing All the epithelial tissues which subjected study and divided into three groups with to these staining were assessed under six rats per group for different interval light microscope to evaluate fibroblast days of 7, 14 and 21 of post-wounding. All proliferation, collagen formation and animals in each group was anaesthetised re-ephithelization and wound healing with light ether prior to the wound creation processes (Reddy et al., 2007). and 70% of alcohol was applied as topical disinfection on a shaved area at the dorsal Haematoxylin and Eosin (H&E) thoracic region. An 8 mm diameter of Staining full thickness wound was created by using sterile wound biopsy punch. Each Slides were placed in staining jar and animal was wounded on the dorsal part deparaffinized by submerging into three individually to represent a duplicate. At series of absolute xylene for 4 minutes the end of the interval days, samples of followed by 100%, 100%, 95%, 90%, the skin were harvested and processed for and 70% of ethanol for 4 minutes of each histology examination with three samples percentage. Next, slides were washed in of wounded skin from each group was running tap water for 2 minutes. Then, slides subjected to Masson’s trichrome staining were submerged into Harris Hematoxylin and another three rats were subjected to (Sigma-Aldrich, GERMANY) for 2 standard haematoxylin and eosin staining. minutes and then washed in running tap The wounds were measured based on the water for 2 minutes. The slides then were percentage according to the healed wound submerged into 1% acid alcohol for 3 dips area. The epitelisation time was measured to decolorize it and washed in running from initial day (Singhai et al., 2006). tap water for 2 minutes. Next, slides were submerged into 2% potassium acetate for Percentage of wound contraction 3 minutes and again washed in running tap water for 2 minutes. After that, slides Healed area (mm2) = ––––––––––––––––––× 100 were submerged into Eosin for 2 minutes Total wound area (mm2) followed by washing in running tap water for 2 minutes. Stained slides were dried for 24 hours at 38°C. Before observation, 41 MALAYSIAN JOURNAL OF VETERINARY RESEARCH VOLUME 3 NO. 1 JANUARY 2012 slides were dipped into absolute xylene for each percentage. Before observation, slides 1 minute and finally mounted with cover were dipped into absolute xylene for 1 slip using DPX mounting. minute and finally mounted with cover slip using DPX mounting. Collagen Special Stain (Modified Masson’s Trichrome Staining) Collagen Density Evaluation Method was modified from Kiernan Method was modified from Elizabeth et al., (2008). Granulation skin tissue slides were (1995) and Ukong et al., (2008). The slides placed in staining jar and deparaffinised stained with Masson’s trichrome stain were by submerging into three series of absolute examined using polarised light microscope xylene for 4 minutes each followed by (Leica, Germany) and with the aid of a 100%, 95%, 90%, 80% and 70% of ethanol software image analyser (Video Test- for 4 minutes in each percentage. The Master 4.0 software), measurements were slides then were submerged in warmed made at the intensity of blue colour which Bouin’s solution at 60°C for 45 minutes. represent the collagen density. Collagen Next, the slides were washed in running density was measured under the wound tap water until yellow colour in samples area compared to normal dermis at 100× disappeared. To differentiate nuclei, slides magnification. The mean of the collagen then were immersed in modified Weigert’s values obtained for the normal dermis was haematoxylin for 8 minutes, after that accepted as the equivalent of 100. For each washed in running water for 2 minutes. In group, the mean of the collagen density order to stain cytoplasms and erythrocytes, under wound area was expressed in the slides were submerged in anionic dyes, acid ratio of percentage compared to collagen fuschin (C.I. 42590, Merck, Germany) for density of normal dermis during the post- 5 minutes; then again slides were washed wounding day. with running tap water for 2 minutes. Next, slides were treated with phosphomolybidic Ratio acid solution for another 10 minutes as Average collagen intensity a mordant and immediately slides were under wound = –––––––––––––––––– × 100 submerged into methyl blue (C.I.