Evaluation of effect of local exogenous application of Myrrh oil on healing of wound incisions of facial skin (Histochemical, Histological and Histomorphometrical study in rabbits)

Aim of the study: Is to evaluate the effect of myrrh oil local application on the healing process of skin wounds histologically , histomorphometrically and , histochemically. Materials and methods:Twenty male white New Zealand rabbits were used in this study. An incisional wounds with full thickness depth and of 2 cm length were done on both sides of the cheek skin of each rabbit. The left sided incisions (the control group) were irrigated with distilled water (10μL). The right sided incisions (the experimental groups) were treated with myrrh oil (10μL). Each group was subdivided into 4 subgroups according to the healing interval into 1,3,7 and 14 days(5 rabbits for each group). Results: Histological findings of our current study showed a highly significant difference between the experimental and the control groups in count of the inflammatory cells which showed that mean values increased with time for the control and the experimental groups. The histomorphometrical findings had shown that the thickness of the epithelium was nearly completed at about 7 days for the experimental groups and at about 14 days for the control. The blood vessels count was recorded to have a highly significant difference between the groups at days 1 and 3 only. The histochemical findings had shown that the collagen fibers remodeling had recorded a highly significant difference between the control and the experimental groups at days 7 and 14. Conclusion: The current study had revealed that myrrh oil accelerates wounds healing in rabbits. Key word:Wound healing , Myrrh oil, Masson’s Trichrome staining. (Received: 25/11/2018; Accepted: 3/1/2019)

The skin can be defined as an interface or a barrier between the body and the external environment (1) . A wound can be described as a loss of the function and integrity of the body tissues. The injury can be usually caused by an external force (traumatic/accidental or surgical) and it can involve any type of organs or tissues (2) . The process of wound healing, which is considered a normal biological process of the body, can be achieved by four highly programmed, strict and precise phases. These phases include: "hemostasis, inflammation, proliferation, and remodeling". For a successful healing of any wound, the four phases must occur in the proper sequences and accepted time limit (3) . Myrrh oil is described as a hardened sap that oozes from the Commiphora molmol stem (Family Burseracea) tree. It is collected from the natural cracks or it can be from cuts made by human in the trees bark (4) . Myrrh had been used frequently in the recorded history and it had been concidered to be a fragrance and also a medical agent by "the ancient Chines and by the old Egyptians" (5) . "Caryophyllene C15H24" which is present in myrrh oil also has an anti inflammatory, antibacterial and anti tumor action (6) .

MARERIALS AND METHODS
Twenty New Zealand male rabbits, of (1.5-2 kg) body weight and (4-7) months of age were used in this study. All of the rabbits were housed with controlled ventilation conditions and temperature and were given a standard diet (barseem and pellet) with an easy accessed tap water. The animals were kept in a standard separated cages for 2 weeks in the same suitable environment before the surgical procedure. All of the animals underwent examination by "veterinarian staff in the animal house of Biotechnical Research Center at Alnahrain Unniversity" for the evaluation of their general condition and health in order to exclude any unhealthy animals. The animals then were fasted about 6-8 hours before the operation. All of the experimental procedures were done in accordance with "the animal experimentation ethical principles" (7) . A full skin thickness surgical incisional wounds and 2 cm length were done on both sides of the cheek skin for each rabbit (11) . The animals then were randomly divided in accordance to the healing intervals into 4 groups (1,3,7,14) (5 rabbits in each group) and each group was subdivided in to: 1-Control group: The left sided incisions were irrigated with distilled water. 2-Experimental group: The right sided incisions were treated with 10µL of a myrrh oil. All of the specimens were taken and prepared for the histological (H&E stain) and the histochemical examination with masson's trichrome stain for collagen fibers density assessment. Assessment of wound healing parameters clinical assessment -Wound contraction: At 3,7 and 14 days period interval the wounds were measured by ruler (8,9) . 2 Histological analysis 1. Inflammatory cells analysis : With power x40 lens, we counted the inflammatory cells number in five fields and then recorded the mean number of cells (10) .

Epithelial thickness assessment:
With power x40 lens, the measurement of the epithelial thickness was performed by measuring the distance from the outermost layer of the epidermal keratin to the inner most basal layer of it at the edges of the wound as a mean of two readings with the help of Image J computer software (11) .

Blood vessels analysis:
Blood vessels analysis was done using the Image J software. Using light microscope, power x40 lens (12) .

Collagen fiber density analysis:
The slides were stained with masson's trichrome stain and were examined under the light microscope, power x40, at 3,7 and 14 days period intervals with the help of image J software (13) .

RESULTS wound contraction estimation:
Table (1) is showing that with time there is a decrease in the recorded mean values, and that there is a high significant difference between the control and the experimental groups in all healing periods.

Inflammatory cell parameter
The results showed as in table (2) that the highest mean values were recorded for the experimental groups at day 3 and the lowest mean value was at 14 days.
The epithelial thickness parameter: results had revealed as shown in table (3) that the epithelial thickness highest mean value for the control and the experimental groups was recorded at day 14 and that the lowest mean value for both both groups wasrecorded at day 1, also it showed a highly significant differences between the control and the experimental groups at all healing period intervals (p<0.01).

DISCUSSION
Nowadays, medicinal herbals are considered to be one of the branches of the complementary and alternative medicine. Herbals use for caring of wounds and injuries had been known since the ancient civilizations (14,15) . Our present study had shown that wounds contraction was accelerated in the experimental groups as compared to the control groups. This acceleration in the wounds contraction can be explained be due to the increased proliferation and progression of the epidermal cells in the experimental groups and because of the antiinflammatory effect of this oil (16) . Histological and histomorphometrical evaluation: In our study the experimental groups had displayed variable degrees of the inflammatory reaction especially in the first 24 hours, in contrast to the control groups which had displayed a more prolonged and sever inflammatory reaction extending up to the 7th day attributed to the bacterial colonization and the lack of the immunomodulation and the antiinflammatory activities. This agrees with (Al-

Mobeeriek,2011) (17)
The intensity of the inflammatory cells in the wounds areas was found to be predominant and the density of the inflammatory reactions was more sever in experimental groups as compared to the control groups in the 1 st and 3 rd day, while in the 7 th and 14 th days the inflammatory cells had become mild because of the established and completed inflammatory stage and the start of the remolding stage in the experimental groups, while they recorded a high mean value in the control groups at day seven and a decrease in day fourteen. This may be because of the anti bacterial effects of the myrrh oil (18,19) and this had led to the promotion and the acceleration of the healing processes. Neovascularization is another very important event that should take place in the 2 nd stage of the wound healing (20) . In our study, in the 1 st day, the neovascularization in the control groups was found to be little or nearly absent, while in the experimental group, it was present but to a very little extent, High significant differences was recorded between the control groups and the experimental groups in day 1 and 3 but there was a none significant difference in the days 7 and 14. The early neovascularization in the experimental groups had led to promote the healing process because new vessels usually supplies oxygen and the required nutrients and remove the waste products (21) . The present study had shown that the reepithelialization had occurred faster in the experimental groups and it recorded a high significant difference as compared to the control groups because of the increased proliferation and the progression of the epidermal cells with an increment in the amount of the neovascularization, the fibroblast cells and the collagen fiber of the dermal layer in the experimental groups and this agrees with (Al-Mobeeriek,2011) (17) , The remodeling phase had started earlier in the experimental groups than in the control groups and this agrees with a previous study (Al-Mobeeriek,2011) (17) . "Masson's trichrome stain" was used in this study for assessment of the density of the collagen fiber, this agrees with previous study (Suvik, and Effendy, 2012) (13) The experimental groups had been found to have a higher collagen density than the control groups at day 3,7 and 14 and this agrees with a previous study (Enoch and Leaper, 2007;Reinke and Sorg, 2012) (22,23) CONCLUSION Myrrh oil has the ability to accelerate healing of the wounds because of the faster wounds contraction, early neovascularization, reepithelization and a higher collagen density than the control groups. Myrrh oil groups had shown an anti-inflammatory effects which is identified by a decrease in the count of the inflammatory cells with time.