Healing Incisional Surgical Wounds Using Rose Hip Oil in Rats *

Methods: Twenty-one days after oophorectomy procedure, twentyseven female, adult, Wistar rats were distributed into three groups: Control group (wound treatment with distilled water); Collagenase group (treatment with collagenase ointment); and Rose Hip group (wound treatment with Rose Hip oil). Each group was distributed according to date of euthanasia: 7th, 14th and 21st postoperative day. The wound was evaluated considering macroscopic and microscopic parameters.


Introduction
The healing process is common to all wounds, regardless of the agent that caused it, is systemic and dynamic and relates directly to the general conditions of the body [1,2].The topical use of substances for improving the healing process has been widely studied [3].
Brazil has the largest diversified forest reserve on the planet.Many of these species used for medicinal purposes are consumed with little or no evidence of their pharmacological properties.The use of medicinal plants is not restricted to rural areas or regions devoid of medical and pharmaceutical care.They are also used extensively in urban areas, as an alternative or complement to allopathic medicines [4].
The Rose Hip (Rosa rubiginosa L.) belongs to the genus Rosa, Rosaceae family, and has approximately 70 different species worldwide [5].Native to the Mediterranean region and Central Europe, was brought to South America by Spanish colonizers, and grows in south and central Chile [6] as a wild plant, beneath dry soils agricultural value [7].
The objective of this study was to investigate the effects of Rose Hip (Rosa rubiginosa L.) oil on the healing of incisional surgical wounds in rats.

Methods
Twenty-seven (15-20 weeks) female Wistar rats (Rattus norvegicus) weighting 250-350g were used in this study.The animals were kept in a vivarium of the Experimental Research Group at the Centro Universitário do Estado do Pará (GPE-CESUPA) with a controlled temperature, light, humidity, and noise; water and food was provided ad libitum.The project was previously approved by the Animal Use and Care Committee of University Center of the Pará State (Protocol 02/2014) and followed the rules of Brazilian National Law for Animal Care (Law: 11.794/08).

Experimental protocol
Three groups of study comprised this study: -Control Group (CG): distilled water were applied everyday and once in a day until euthanasia, a volume of 0.3 ml.-Collagenase Group (CLG): collagenase ointment (0.6 U/g) were applied everyday and once in a day, a volume of 0.3 ml until euthanasia.-Rose Hip Group (RG): Rose Hip oil were applied everyday and once in a day until euthanasia, a volume of 0.3 ml.Animals were then randomly assigned into three subgroups (N= 3 each): Distilled Water Group seven, 14 and 21 days (CG7, CG14 and CG21), Collagenase Group seven, 14 and 21 days (CLG7, CLG14 and CLG21) and Rose Hip Group seven, 14 and 21 days (RG7, RG14 and RG21) according to the time elapsed from surgery to animal euthanasia.

Oophorectomy
All animals were anesthetized with ketamine hydrochloride (70 mg/kg) and xylazine hydrochloride (10 mg/kg), submitted to trichotomy and then a 2cm laparotomy was performed to reach the ovaries, which were switched both proximal and distal at 1cm distance from the cervix and then sectioned.The laparorrhaphy was performed with 4-0 nylon.After 21 days, the female rats were subjected to exfoliative vaginal cytology, then, the content was deposited on the surface of a glass dish, which was immersed into absolute alcohol for fixation.The glass dishes were stained by Shorr (1941) [8] method and then analyzed using an optical microscope in order to characterize the estral cycle, to confirm anoestrus [9].

Surgical procedures
21 days after oophorectomy, all surgical procedures were performed newly under anesthesia.Dorsal region was shaved and then antisepsis of region was done.Next, a full-thickness skin surgical incision measuring 20 mm in length and 2 mm in depth was made in the skin of the back of the animal in the caudocranial direction and incision was not sutured [10].During postoperative days, animals remained in individual cages and isolated from their bedding through a metallic net to avoid wound infection.The rats were euthanized by anesthetic overdose.

Macroscopic analysis
To determine the largest diameter of the wound and its macroscopic used a magnifying glass with 2.5x.The wound was measured with a pachymeter graduated in millimeters to calculate the area.

Histological analysis
After euthanasia, wounds were harvested, fixed in 10% formaldehyde, embedded in paraffin, stained using Hematoxyline-Eosine (HE).In HE stained tissues, multiple sections were analyzed with regard to the presence of re-epithelialization, intensity of local inflammatory reaction, type of inflammatory response as the predominant cell, the presence of granulation tissue, neovascularization and fibrosis [11].

Statistics
Analysis of variance (ANOVA), followed by Tukey posthoc test correction, was performed for the macroscopic analysis and proportion of collagen fibers.Kruskal-Wallis test was used for the histological parameters.Statistical significance was assumed at p<0.05.

Results
No significant differences in age and body weight of the animals were found between the study before and after the intervention.

Macroscopic results
Comparing the groups according to the time of measurement, a statistical difference was observed between Control and Collagenase (Figure 1), in 7 th (p=0.0373) and 14 th day (p=0.0367).It was also found a difference between Control and Rose Hip oil (Figure 2) in 14 th (p=0.0275) and 21 st day (p=0.0008).In addition, when comparing collagenase and Rose Hip oil (Figure 3), there was difference only in 21 st day (p=0.0364).

Microscopic results
There was no statistical difference between the groups at different times regarding the degree of re-epithelialization and the presence of granulation tissue.However, predominance of chronic response type was noted in the groups at all wound evaluation times and was observed statistical difference regarding the intensity of the local inflammatory response with predominance of monomorphonuclear infiltrate at different times of collagenase and Rose Hip oil groups, when compared to the control group (p=0.0498 and p=0.0442, respectively).

Discussion
Several substances have been tested in wound healing process.However, the results are not always satisfactory when compared to the control group or even active compounds may slow the overall wound healing.Therefore, the study of incisional wound healing becomes of great value for scientific knowledge.
Throughout the experimental part of this article, all animals evolved well postoperatively, be it 7, 14 or 21 days without causing infection, death or replacement of animals to the study, proving that this model of incisional wound induction was efficient.
Proteolytic enzymes have been used in wound healing for many years and the most commonly used enzymes are fibrinolysin/DNAse and collagenase [12,13,14], where collagenases act by degrading native helical collagen fibrils [15].Recently, Tallis et al. (2013) showed that the collagenase ointment is tolerable and clinically effective in providing non-viable tissue removal and allow effective scar [16].
There was a significant difference in collagenase with control group on days 7 and 14, which demonstrates its mechanism of action, especially in the acute phase of incisional wound healing, not observed statistical difference on day 21 due to the lower effectiveness of its action against the chronicity of the incisional wound.
However, Rose Hip oil showed surprising results compared to the control group, obtaining a lower average of the wound area when compared to the other groups.Mainly in the macroscopy where a degree of almost total contraction of the incisional wound was observed on 21 day.
In a clinical study with a control group, the authors demonstrated an improvement in the time and quality of healing among patients using Rosa Mosqueta oil in comparison to the control group.Evaluated patients with varicose ulcers, post-traumatic ulcers and contact eczema.All showed positive results in relation to the comparison group, occurring a difference of up to 29 days of healing among the groups [22].
As noted, Rose Hip oil has proved to be a better treatment in the retraction of the wounds, especially for a late mechanism of action.Having more effective from the 14th day of incisional wound, compared to the control and collagenase group, maintaining a lower chronic inflammatory response, characterized by monomorphonuclear infiltration.
Essential fatty acids are required for many physiological processes, such as maintaining the integrity of the skin and the structure of cell membranes and the synthesis of biologically active compounds.Among the health benefits attributed to fatty acids are: anticarcinogenesis, antiatherosclerosis, inhibition of free radicals, alteration in the composition and metabolism of adipose tissue, immunomodulation, antibacterial and antidiabetic activity [24].
The essential fatty acids also have a structural function, forming part of the phospholipids of the cellular membranes of the tissues of the organism, being the precursors of prostaglandins and leukotrienes from the synthesis of arachidonic acid [24,25].Already phospholipids involved in various metabolic processes, such as the active phosphorylation process in mitosis and cell organization and ion exchange, which gives the Rose Hip oil a high potential in the treatment of wound healing [26].
We must consider that human skin differs from rat and the important difference is that human skin cures preferentially by re-epithelialization, whereas the skin of the rat heal mainly by contraction of the wound [27].We also recognize the limitations for translational relevance of our experimental study.However, skin lesions in animal models are relevant because they provide significant contributions to advances in the treatment of incisional wounds.
The experimental model demonstrated greater efficacy of topical application of Rose Hip oil in tissue formation, epithelialization, angiogenesis and collagen deposition in skin lesion compared to the other groups.

Conclusion
Rose Hip oil is able to reduce intensity of inflammatory response of the wound, maintaining the same type of inflammatory response when compared to the collagenase group, proven by macroscopic results, in which there was a greater retraction of wound in animals treated with oil and no evidence of incisional wound healing impaired.
International Archives of Medicine is an open access journal publishing articles encompassing all aspects of medical science and clinical practice.IAM is considered a megajournal with independent sections on all areas of medicine.IAM is a really international journal with authors and board members from all around the world.The journal is widely indexed and classified Q2 in category Medicine.