Adults Wistar rats weighing between 170 – 200 g, were used in this study to investigate the effect of the assayed natural products on burn wound healing. They were obtained from the Pasteur Institute of Algiers (Algeria) and were kept in standard stainless-steel cages and maintained in the animal house under controlled laboratory conditions with free access to water and food ad libitum (Principles of Laboratory Animal Care NIH publication no, 85-23, revised 1985).
Burn wound healing study
The experimental protocol used in this study complies with the ARRIVE (Animals in Research: reporting in vivo experiments) guidelines and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). All care was taken to minimize the suffering of the animals. The experimental protocol was approved by the National Ethics Committee.
The wound healing activity was tested on males and females Wistar rats according to Pirbalouti et al. Animals from each sex were divided into four groups in which the healing effect of the mixture was compared to that of Euphorbia honey and two conventional treatments (betadine solution and silver sulfadiazine ointment); the first group received Euphorbia honey, the second group betadine solution, the third one silver sulfadiazine while the fourth receives the mixture Euphorbia honey-A. sativum juice. The rats were anesthetized with an intramuscular injection of ketamine (85 mg/kg) and xylazine (10 mg/kg) and then shaved on the dorsum. Square metals of 1 cm 2 were used to induce burn wounds. The metals were heated in boiling water during 10 min and then applied 20 sec on the dorsal part of the rats. Each piece of the induced burn wound received a specific treatment that was renewed daily the first week then every other day until complete healing of the wound.
Histological and morphometrical analyses
In order to reduce as much as possible animal testing, histological analyze was limited to the study of samples that have already demonstrated enough interest in this experiment. The tissue specimens of scarred skin were collected at the end of the epithelialization process and fixed in a 5% formaldehyde solution and underwent paraffin inclusion. Histological sections of 5 ?m width were performed with the microtome. The paraffin sections were stained by Hematoxylin-Eosin stain for morphological changes and Masson’s trichrome protocol for demonstration of skin collagen. The microphotographs were taken from the generated set of slides with a microscope Leica DMI 3000 (Leica Microsystems, Wetzlar, Germany) and subsequently analyzed using Image-Pro Plus 7.0 (Media Cybernetics, Silver Spring, MD, USA).
Quantitative morphometric studies were performed on the obtained microphotographs in order to evaluate epidermis and dermis thickness, interdigitation index and collagen organization using Fourier transform. At least 5 captions were taken for each case and each parameter, following a method of semi-randomization which excluded altered zones. The magnification of the microphotographs was adjusted individually for each parameter. The mean for each image was calculated and, later, the mean of the case from the values of the 5 images as described by Marcos-Garces et al.
The interdigitation index was investigated with the above-mentioned computerized image analyzing system. The mouse cursor was led along the interdigitation border, the computer measured its length and calculated the distance between the starting point and end point. From these data the interdigitation index was calculated as described by Timár et al. . Briefly, the length of the line following the interdigitation between two points on the border between the epidermis and the dermis was divided by the length of a straight line between the same two points. Results demonstrating an elevation in interdigitation index might indicate some rejuvenation effect the treatment. Collagen orientation in the dermis was measured in 20x magnification micrographs following the methodology validated by van Zuiljen et al. . This technique applies the Fourier transform to the image and, over the resulting 2D power plot the length and the width of the central figure are measured. The relation between both data is representative of the orientation of the fibres: values which tend to 0 indicate higher orientation, whilst values tending to 1 define disorientation of the collagen fibres.
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