Topical Misoprostol and Wound Healing in Rats

Disclosure: Funds for this study were provided by a grant from the IOER Research and Grant Fund #3122 at Des Moines University.

Cutaneous ulceration is a common clinical problem encountered by nearly all disciplines and specialties in medicine. These ulcerations are commonly found among patients that have diabetes, spinal cord injury, stroke, myelodysplasia, or peripheral neuropathy.¹ A myriad of treatments exists; however, none routinely utilize the healing properties of prostaglandins.
This preliminary study seeks to determine if topical misoprostol can speed the process of healing in surgically created full thickness wounds in healthy adult rats. By definition, an acute wound was created, ie, one that heals in an orderly and predictable manner that results in the normal restoration of dermatologic anatomy and function.²
Upon tissue disruption, the body responds by isolating the injury and decreasing blood loss by initiating hemostasis.³ This process includes the activation of platelets.⁴ Within the platelet membrane, phospholipase A2 releases arachidonic acid from various membrane phospholipids; cyclooxygenase and 5-lipooxygenase then act upon arachidonic acid. Cyclooxygenase produces prostaglandins (D, E, F, G, H, I), prostacyclin, and thromboxane A2.⁵
A prostaglandin is a 20 carbon-long fatty acid that contains a 5-membered carbon ring.⁶ Prostaglandins are arbitrarily assigned to 1 of 9 groups with the letter designations A through I. These designations are further divided into 3 subgroups,with the exception of prostaglandin I by a subscript 1, 2, 3.⁶ These numbers represent the number of double bonds present in 2 side chains of the prostaglandin molecule.⁶
Prostaglandins have been shown to have many effects on wound healing. Prostaglandin E2 (PGE2) potentiates the inflammatory response by increasing the effects of other mediators.⁷ PGE2 also stimulates vasodilation and promotes the development of edema.⁸ This net result is to increase capillary permeability in order to facilitate the extravasation of serum proteins (cytokines, chemokines, growth factors) into the wound,which help orchestrate the healing process.⁹ Silverman et al¹⁰ noted an increase in nutritive perfusion after the topical administration of 120 mg of PGE2 analog. The observed local circulatory changes observed by were not accompanied by systemic effects as measured by vital signs attributing local perfusion to the local application of the analog itself.¹⁰
Misoprostol is a synthetic prostaglandin E1 analog (PGE1) and has both antisecretory and mucosal cytoprotective properties.¹¹ Its use is currently approved for the prevention of NSAID-induced gastric ulcers at 200 mg 4 times daily.¹² In addition, duodenal ulcer treatment may include misoprostol use at 100 mg 4 times daily. Other unapproved uses for misoprostol can be found in the literature. Olivery and Mueller¹³ found that misoprostol is a treatment option for canine atopic dermatitis. It has been found to have cytoprotectant effects against mucosal radiation^14,15 and to reduce bacterial translocation and modulate bacterial clearance in mice following a burn injury.^16,17 There are numerous references to its use as a wound healing agent for diabetic foot lesions or decubitus ulcers when used in various combinations with lidocaine, phenytoin, metronidazole, pentoxyphylline, and gentamicin.^18–20,21 When administered intragastrically following colonic anastomoses in rats, misoprostol was found to increase tissue collagen levels by day 14 without influencing inflammation.²² For years the School of Dentistry at the University of Iowa has used misoprostol to heal mucosal ulcers (personal communication with Rex McKee, PharmD, October 2005). Other clinical trials in progress are investigating the use of prostaglandins in wound healing. It is used as an abortifacient in the second trimester of pregnancy when administered intravaginally or extra-amniotically via a transcervical catheter.²³
The effects on skin suggest that a once daily application of misoprostol may increase the speed in which a surface wound heals. This hypothesis formed the basis for the present study.

Materials and Methods
A Des Moines University statistician performed a power analysis before the study began to ensure statistical significance. Per the findings of this analysis, a minimum subject number of 22 would be capable of showing a difference between the 2 groups tested. As such, 26, 300-g, 11-week-old, male Sprague-Dawley rats were used in this study. Thirteen rats served as the control group (saline application), while the remaining 13 rats were assigned to the treatment group (topical misoprostol application). Each rat was randomly identified and assigned to 1 of the 2 treatment groups by number draw. Each animal was housed separately in a stainless steel cage in a climate-controlled room. Care was administered in accordance with the Des Moines University Institutional Animal Care and Utilization Committee guidelines.
The rats were allowed 1 week to adjust to their environment and handling by the research team.Each rat was handled on a daily basis simulating dressing changes. After 1 week, anesthesia was induced with isoflurane and following induction, each rat was administered an intraperitoneal (IP) injection of pentobarbital (50 mg/kg). The dorsal surface of the back was prepped for aseptic dissection. A 2.5-cm x 2.5-cm, full-thickness section of skin was removed from the upper back (caudal to the scapula) down to muscle, midline, utilizing a combination of sharp and blunt dissection (Figure 1).

Immediately following wound creation, the first treatment, either saline or misoprostol was administered. Thirteen rats in the control group were administered normal saline via gooseneck spray bottle with 3 sprays directly onto the wound. The remaining 13 rats were treated with 3 consecutive sprays of a 1:1000 concentration of misoprostol powder/saline (300 μg of powder in 30 cc of normal saline). The misoprostol powder/saline solution was prepared by NuCara Pharmacy (Waterloo, Iowa).NuCara pharmacy estimated that 3 sprays (0.5 cc) delivered 5 μg of misoprostol. For both treatment groups, a 5-cm x 5-cm nonadherent sterile gauze pad was directly applied to the wound with 1- in Coban^™ (3M Healthcare, St. Paul, Minn) used to secure the dressing by encircling the thoracic region.The compression was such that the handler’s index finger could fit between the skin surface and the gauze. Respiratory pattern was also monitored to ensure each rat’s safety at the initial bandage change and every bandage application thereafter. Labored breathing or high-pitched vocal rat sounds would indicate that the bandage was tootight.The bandage would then be loosened appropriately.
Twelve rats in the treatment group and 11 in the control group survived the 3-week study. One of the 3 deceased rats did not survive the wound creation procedure and died within 12 h after anesthetization. Unlike the other rats, this subject showed evidence of intolerance to the anesthesia with labored breathing while recuperating after the procedure. The other 2 rats died within the first 96 h after the procedure. Their cause of death is undetermined. These rats exhibited no behavioral changes or evidence of infection.
The dressings were changed daily for 3 weeks utilizing the above protocol. High-pressure irrigation with 10 cc of normal saline was used daily prior to dressing change for mild debridement. In addition, on days 2, 5, 8, 11, 14, 17, and 20, a digital photograph was taken of each wound. Two connecting lines forming a 90-degree angle were marked on a small piece of paper measuring 1 cm x 3 cm. This paper was placed in all photographs, thereby acting as a standard.To standardize camera distance from the wound, the camera was attached to a fixed pedestal. The attachment point was nonvariable, and the distance from camera to wound was 12 in. UTHSCSA ImageTool software was used to calculate the area of each wound in the individual photographs.The area was measured 10 times per wound. An average of the area was then determined. Likewise, the area of the 1-cm x 3- cm marking was measured 10 times, and the average was determined. The software program gave wound area in pixels. Conversion from pixels to cm² was performed as follows: a ratio was calculated using the average area of the wound compared to the average area of the 1-cm x 3-cm marking. The ratio was then multiplied by 3 cm² (from the known control marking in every photo), giving the result of a wound in metric units. The resultant wound areas were then recorded and followed through time until wound healing occurred.Wound healing was defined as complete epithelialization at the skin interface (Figure 2). During the study, areas of eschar were noted at the healing site in several rats at multiple times in both the treatment and control groups with the same frequency. Any eschars were gently removed with a moist saline soaked sponge. The eschars were removed because it was thought that they might prevent microprostol or saline from reaching the wound surface, thus limiting their biological effects. Additionally, at each dressing change a standard form was filled out to monitor the rat for signs of infection, behavioral changes, bandage condition, and general wound characteristics, including presence or absence of granulation tissue.

On day 20, the rats were euthanized by the IP administration of pentobarbital (150 mg/kg) and cremated per institution protocol.
A Des Moines University statistician performed statistical analysis using nonparametric Mann-Whitney U test.

Results
The measurements taken during the study period were summarized by calculating the area under thecurve (AUC) for the healing progress for each laboratory animal. Table 1 shows the AUC measurements calculated for the placebo group.Table 2 shows the AUC measurements for the misoprostol group.
The mean AUC for the placebo group was 14.67 (SD ± 7.82). For the misoprostol group, the mean was 10.46 (SD ± 1.77). Hence the treatment group not only has a low average AUC measurement, but also has a lower standard deviation. The next step was to proceed with providing inferential evidence through statistical hypothesis testing.

The null hypothesis that the mean AUC for the 2 groups (drug and misoprostol) are equal was tested against the alternative that mean AUC for the misoprostol group is statistically significantly lower than that of the placebo group (1-tail test). To provide more robust evidence, the nonparametric Mann-Whitney U-test (rather than the parametric independent sample t-test) was conducted. The test resulted in the rejection of the null hypothesis H₀ (z = 1.79, P = 0.04); therefore, showing a difference in healing between the 2 groups with misoprostol treatment being beneficial.

Discussion
There is no ideal wound healing agent. Nor is there universal consensus on the best wound management protocol. With more than 2000 wound care products on the market, it is difficult to gauge which are the “best” to use. Selection is often guided by training preferences, local use patterns, and economic considerations (including supplier discounts for use of the specific products).
The authors were interested in investigating the use of topical prostaglandins in decreasing the healing time of acute wounds. The oral use of misoprostol, a PGE2 analog, to heal gastric and duodenal ulcerations is well known. As stated previously, the vasodilatory effects of topically applied prostaglandins may provide an easily administered vehicle to potentiate healing and decrease wound coverage time with less systemic effect.
It is reasonable to assume that prostaglandins exert their greatest effect in the inflammatory phase of wound healing. By attracting other mediators of healing, angiogenesis can proceed to form granulation tissue.This can begin to occur within 48 h of injury.²⁴ This study involved a protocol applying the prostaglandin analog immediately after creation of the acute wound. This application fell within the initial period where inflammatory factors are recruited. Whether this aided in faster healing of the misoprostol treatment group is unknown. However, further studies may shed light on this topic and help determine if the inflammatory effect is heightened with topical misoprostol use in wounds.
This study shows that topical misoprostol, when applied daily to an acute surgical wound, will decrease healing time. However, the most evident potential lies within the chronic wound.
Chronic wounds exhibit significant differences from acute wounds in immunohistochemistry and histopathology. These differences are noted throughout all 4 phases of wound healing, but it seems that it is the inflammatory and proliferative phases where the primary delay occurs.²⁵ This assumption is based upon the observation that as soon as a healthy wound bed has been formed, epithelialization can then occur normally.²⁵ There is prolonged expression of fibronectin, chondroitin sulfate, and tenascin in chronic diabetic and venous ulcers, compared to the acute wound healing model.²⁵ There is also a reduced amount of CD4 T-cells and high numbers of B-cells, plasma cells, and macrophages, without any evidence of increased autodigestion and phagocytosis in these chronic wound types.²⁵ While the inflammatory phase is of short duration (4 days) in acute wounds, these chronic wounds seem to be stagnant in this low-grade inflammatory state so that transition into the next phase of forming granulation tissue is not taking place.²⁵ Future experiments are needed to examine misoprostol and its effect on chronic wounds. One possible animal model is the diabetic rat.
Several limitations to this preliminary study exist. Firstly, one cannot compare the rate of wound healing in rats to humans. Indeed, some researchers have long held that incisions in rats may heal more slowly than wound models would predict.²⁶ Secondly, the acute wound healing animal model might be significantly different from the chronic wound healing model, making a direct correlation between the two not possible. Thirdly, rather than an exact determination of the amount of misoprostol on each wound, it was approximated from an amount sprayed from a powder/saline mixture. Future studies should employ another vehicle for drug delivery. If a gel were used, the total amount of drug delivered can be more accurately determined. Lastly, there were 4 occurrences of the dressing being removed overnight by a rat, so the amount of misoprostol exposed to the wound was probably decreased as a result. A learning curve was present with bandage application. As the study progressed the rats became more accustomed to being handled daily and were more tolerant of the bandage changes. It was found that placing the Coban more toward the midline thoracic area kept the dressing on better. By placing the Coban in this area, the rat could not reach or chew through the bandage. Statistical power for this study was maintained despite the death of 3 rats. However, future studies should include a larger subject number to ensure no difference statistically between the 2 groups.

Conclusion
This preliminary study demonstrated effectiveness of topical misoprostol powder in decreasing the healing time of acute wounds in rats. Further studies are needed to see if misoprostol powder can increase healing rates in chronic wounds.

Acknowledgment
Thanks to Dr. Simon Geletta, PhD, at Des Moines University for his invaluable assistance in providing statistical analysis.