Published online Jan 26, 2018. doi: 10.4252/wjsc.v10.i1.1
Peer-review started: August 14, 2017
First decision: September 25, 2017
Revised: October 26, 2017
Accepted: November 27, 2017
Article in press: November 27, 2017
Published online: January 26, 2018
Fecal incontinence is a very prevalent (11% to 15% of adults), nonfatal illness associated with devastating consequences mainly in the psychosocial sphere and quality of life. Although in the last years sacral neuromodulation has been improving the poor results of available treatments, surgery remains the choice for the most severe or refractory cases, mainly if sphincter lesions are present (the most frequently observed anatomic alteration). Sphincter repair is the most successful technique used for traumatic fecal incontinence, but its results are not very satisfactory, mainly in the long term. Stem cells (SCs) and adipose-derived stem cells (ASCs) have demonstrated promising results in a wide variety of clinical settings, including particularly unfavorable environments for wound healing, such as anal fistulas and Crohn´s disease.
To test if SC therapy could improve postoperative healing mechanisms in patients with fecal incontinence. If this hypothesis is correct, surgical outcomes could be improved and more patients would benefit from surgery in the short and long term.
The first objective was obtaining an in-depth knowledge of rat anal region anatomy so as to design an easy and reproducible model for fecal incontinence or sphincter injury. The second objective was to establish a method for studying rat anal sphincter function, defining the best anesthetic method and physiological test to be used with low morbidity. Finally, the main objective was to study the feasibility and safety of ASC administration to rat anal sphincters via different methods. All of the previously mentioned objectives were accomplished. This furnishes future investigations with the proposed animal model for study of potential SC efficacy; and, if the expected results are obtained, they will support trying this therapy on humans.
Rat anal region normal anatomy was studied on BDIX and Wistar female rats. Once anatomy was well known, the authors studied a system capable of detecting the low-pressure waves supposed to be created in this area, and the capability of this system to detect sphincter lesions. Simultaneously, the authors needed to select a model for fecal incontinence. Since simulated childbirth injury models are complex, associate morbidity in animals and generate highly variable injuries, the authors decided to create a simple model of sphincter injury. The authors tested different injury models and finally selected an anterior extra-mucosal longitudinal myotomy of 1 cm. Researchers studied its morphology and physiology to verify if this procedure injured both sphincters in a constant way. Going further, to decide the best system to detect sphincter pressures, the authors tested different anesthetic drugs (inhaled isoflurane, intraperitoneal ketamine, intraperitoneal ketamine plus xylazine) and different systems to detect pressure, including an endorectal balloon that was retired slowly (similar to human manometry), a normal saline infusion through a mini-laparotomy in the rectum (until anus became opened by rectal increasing pressure), and basal anal manometry with a stationary endoanal balloon. Preliminary studies led the authors to select intraperitoneal ketamine plus xylazine (provided the most stable functional registries) and basal anal manometry with a stationary endoanal balloon as the best options. ASCs were obtained from subcutaneous fat from two BDIX rat males and later were marked with eGFP. Later, preparations of 106 ASCs in 50 μL of normal saline or biosutures of polyglactin 910 6/0 suture covered with 1.5 × 106 ASCs were prepared. Finally, those preparations were applied to 36 BDIX virgin female adult rats that underwent functional studies prior to and after the surgery. Then, safety and cell tracking studies were performed during a follow-up period lasting 7 d. Animals were distributed among three groups: biosuture repair; cell injection without repair; and conventional suture repair and cell injection. Moreover, some quality studies were performed with biosutures, trying to establish the real cell dose administered during their use.
A dose of 820000 to 860000 ASCs adhered to the suture, but not all of them remained on the animal tissue. The described lesion produced a constant injury to both anal sphincters. Rat anal sphincter spontaneous function is composed of heterogeneous, spontaneous, consistent, and rhythmic contractions in the form of “plateaus”, with multiple fasciculations that disappeared consistently with the described surgical damage. ASCs are able to survive in this potentially septic area for at least 7 d. We were able to identify them in 84% of the studied animals, mainly in the muscular section area or in the tissue that appeared between both muscular endings. ASCs form a kind of “conglomerate” in rats treated with injections, while they wrapped around the suture in the biosuture group. ASCs were also able to migrate to the damaged zone, and the most frequent disposition was formation of small groupings. No relevant adverse events or mortality could be related to the SCs in our study. We also did not find unexpected tissue growths. So, this cell therapy was deemed as safe, at least in the short term. As unresolved issues, safety must be studied in the long term, ASC survival must be confirmed in longer follow-ups, systems to improve SC function and survival could be tested, and biosuture cell dosing must be studied thoroughly.
The authors propose an easy, reproducible and safe method for rat sphincter injury that could represent a model for posttraumatic fecal incontinence. ASC administration through cell injections or biosutures is feasible and safe. Both systems are suitable for cell delivery, and biosutures do not change the surgical technique or suture manageability. Applied ASCs are able to survive in this complex area. By this approach, ASCs could offer a benefit for postoperative healing in fecal incontinence.
The model for rat anal sphincter injury can be used in future experiments for testing potential SC efficacy and obtaining long-term results. Anesthetic and functional study methodologies may need some minor changes. Areas prone to be studied deeply are: obtaining knowledge about the potential mechanism of action of ASCs; improving SC delivery, survival and function in the receiving animals (cytokine or molecule addition, etc.); and, supplying SCs through minimally invasive methods. This study allows continuing studies on animal models prior to human use, and, if the expected results are obtained, support trying this therapy on humans (only in empirical and highly controlled settings).