Effects of Copaiba oil in the healing process of urinary bladder in rats

ABSTRACT Introduction The appropriate closure of the urinary bladder is important to many urologic procedures to avoid the formation of fistulas and strictures by excessive fibrosis. This paper presents the alterations in the bladder healing process of rats after the topical use of Copaiba oil (Copaifera reticulata). Material and Methods Ten male Wistar rats were used and randomly divided into two groups: Control group (CG): injected 1ml/kg of saline solution on the suture line; and Copaiba group (CpG): 0.63ml/kg of copaiba oil applied to the suture line. Euthanasia was performed on the seventh day after surgery. The criteria observed were adherences formation, histopathological modifications and stereology for collagen. Results Both groups showed adhesions to the bladder, with no statistically significant difference (p=0.1481). The microscopic evaluation revealed a trend to more severe acute inflammation process on the CpG, but there was statistical difference only in the giant cells reaction (p=0.0472) and vascular proliferation (p=0.0472). The stereology showed no difference. Conclusion The copaiba oil modified the healing process, improving the quantity of giant cells and vascular proliferation, but not interfered in the collagen physiology.


INTRODUCTION
The appropriate closure of the urinary bladder is important to many urologic procedures to avoid the formation of fistulas and strictures by excessive fibrosis (1). Although healing occurs similarly in different tissues, some organs have their peculiarities. In the case of the urinary bladder, scarring may be impaired by the presence of urine (2). Although when compared with healing of the intestines, the bladder has a faster healing rate and tensile strength gain (3).
Regenerative medicine has been trying alternatives to common suture threads to impro-ve the rapid regeneration of the bladder, such as adhesives and barbed sutures (4). In addition, the use of medicinal plants in bladder healing has shown interesting results in experimental research (5,6).
The copaiba oil (Copaifera sp.) is a native Amazonic herb and has many proprieties described such as anti-inflammatory, antibiotic, wound healing and anti-neoplastic (7). The majority of these were already scientifically described in experimental and clinical research, but none of that research tested this oil on the bladder healing process. To develop knowledge about the potential benefits of this plant, we evaluated the use of copaiba oil in the healing process of the urinary bladder in rats by macro-and microscopic analysis, regarding the formation of adherences, histopathological characteristics and stereology for collagen. This paper presents the alterations on the infl ammatory response and collagens physiology in the bladder healing process of rats after the topical use of copaiba oil (Copaifera reticulata).

Animals
Ten male Wistar rats (Rattus norvegicus), weighing between 200g and 250g, were used in this study. The animals were housed in steel cages, cleaned twice a week, and put in a controlled environment with a temperature of 22±2ºC, with adequate humidity and artifi cial light in a photoperiod of 12/12 hours. Water and food were offered ad libitum throughout the study. The experimental procedures started only after the approval of this research by the Ethics Committee in Animal's Use from the Pará State University by the protocol 25/2015.

Experimental design
Rats were randomly divided into two groups, with fi ve animals each. Groups were established as follows: Control group (cystotomy was performed followed by suture and the injection of 1mg/kg of saline solution directly on the suture. N: 5) and Copaiba group (cystotomy was performed followed by suture and the injection of 1mg/ kg of copaiba oil directly on the suture. N: 5).

Copaiba oil
The Copaiba Oil was obtained commercially from Amazon Oil © . The oil was extracted in the city of Itaituba, Brazil and was certifi ed by a chemical engineer. Its composition was 45% of β-caryophyllene.

Surgery procedure
The animals were anesthetized by intraperitoneal injection of ketamine (70mg/kg) and xylazine (10mg/kg). Then, the rats were put in supine position on a surgical board. The trichotomy and antisepsis of the lower third of the abdominal region were performed and a median incision of 2cm made to expose the bladder (Figure-1A). Then, an 1cm cystotomy was performed and sutured with 4-0 Polydioxanone -PDS© ( Figure-1B). For the cystotomy instead of incising the serosa, muscular and mucosa at one time, we divided this step into two stages, reserving an incision only for the mucosa. Immediately following, either the saline solution or copaiba oil were injected, depen- ding on the group. Then, all the animals had their abdominal wall closed with 4-0 nylon in continuous suture for the muscle-aponeurotic plane and separated for the skin.

Macroscopic analysis
On the seventh day after surgery, the rats were euthanized by an overdose of halothane by inhalation. Then, the macroscopic analysis began with inspection of the abdominal scar and verification of presence or absence of infection in the cavity. With the abdominal wall opened, we proceeded to evaluate adhesion formation, using the Nair score (8) ( Table-1).

Microscopic analysis
After total cystectomy, the bladder was fixed in 10% formalin by 48h and the suture threads were removed. The piece was cleaved into three parts, contemplating the border, center and incision of the bladder. The tissues were dehydrated, diaphanized in xylol and parafinized. Then, the tissues were cut into 5μm sections at 250μm intervals, mounted on slides and stained with hematoxylin/eosin and Masson's trichrome stains.
The criteria observed were acute inflammation, necrosis, foreign-body giant cell reaction, re-epithelialization and vascular proliferation in hematoxylin/eosin stain. Fibro-blast and collagen growth was estimated in the Masson's trichrome stain. Each item was graded from 1 to 4, as follows: 1-absent; 2-mild; 3-moderate; 4-intense.

Stereology
Five sections stained with Masson's trichrome were randomly analyzed from each bladder. In each section, five fields were analyzed, totaling 15 fields per bladder. The analyzed fields were digitized to a final magnification of x100 using a video camera coupled to a light microscope.
The volumetric density of the collagen fibers and muscle fibers in the muscular layer was analyzed by overlaying the M-42 grid system on the computed morphological image of the slides. The volumetric density was the relative density taken up by fibers in the tissue under examination. The stereological method determined quantitatively the parameters of the anatomical structural based on the two-dimensional thin sections, in three dimensions.
The equation Vv= ____ x100% Ps Pp was used to calculate the volume density of the collagen fibers, where: Vv=volumetric density, Ps=the number of structure points studied (collagen) and Pp=the number of possible test points (42 in this case).

Statistical analysis
The statistical analysis was made in the software Bioestat© 5.3. The Mann-Whitney test was used, with p <0.05 as the criterion for significance for all statistical comparisons.

Macroscopic analysis
There was no infection on the abdominal surgical wound in either group. In both the control and copaiba groups, we observed adhesion to the bladder with the omentum and the abdominal wall, with no statistically significant difference (p=0.1481). The categories of adhesions following the Nair Score found in each group are described in Table-2.

Microscopic analysis
From the histopathological parameters analyzed, the only differences identifi ed between the groups were the giant cells reaction (p=0.0472) and vascular proliferation (p=0.472) (Figure-2). The statistical analysis of histological parameters, as well as the p value for each variable, is summarized in Table-3.

Stereology
In the histological sections, we observed no differences between the groups (p=0.615) (Figure-3). The fi bers volumetric density in both groups is listed on Table-4.

DISCUSSION
The search for ways to improve the healing process and reduce its side effects dates to the Hippocratic era; at present, there is no therapeutic method to fully control the harmful effects of the wound healing process. The search for new drugs should be encouraged for better control of wound infl ammation, which in many cases causes surgical complications.
We know that the use of copaiba in the peritoneal cavity forms adherences because copaiba oil has a corrosive effect (9). However, there was no difference between the groups regarding the formation of adherences. This data disagree with other study that used copaiba oil directly on the peritoneal cavity, which caused the copaiba group to develop greater adherences (10). This probably occurred because the dosage of copaiba oil in our study was lower and concentrated in the bladder.
Regarding the microscopic analysis, the copaiba oil modifi ed the healing process when compared with saline solution. The copaiba group showed statistically different results in the quantity of giant cells and vascular proliferation. The giant cells are activated macrophages which play an important role in modulating the infl ammation process, performing down-regulation and protecting the tissue (11).
On the other hand, the higher vascular proliferation reinforces the idea that copaiba increases local infl ammation, since the factors for vascular proliferation are mainly released by activated macrophages (12). Those data suggest that the topical administration of copaiba oil may have     enhanced the inflammation due to its corrosive nature, which increased the inflammatory phase of healing and delayed the beginning of the proliferative phase.
The results might be different if the copaiba oil was administrated by gavage. Yasojima et al. (13) showed that the oral administration of copaiba modulates the inflammatory response and improves wound healing in abdominal wall defects.
In relation to the amount of collagen fibers, other studies showed that copaiba oil may improve the organization of those fibers in bone regeneration when topically applied, which was not observed in our results (14). Besides having a trending to have more collagen than the control group, there was not statistical difference in the total amount, as well as occurred in the quantity of fibroblasts. Then, in this study the copaiba oil did not enhance healing.

CONCLUSIONS
We conclude that the copaiba oil modified the healing process improving the quantity of giant cells and vascular proliferation, but not interfered in the collagen physiology.