Interstitial Cells of Cajal (ICCs) and Smooth Muscle Actin (SMA) Activity After Non-Ablative Radiofrequency Energy Application to the Internal Anal Sphincter (IAS): An Animal Study

Poster presented at DDW 2013, submitted for publication

BACKGROUND: Interstitial cells of Cajal (ICCs) are pacemaker cells in the smooth muscles of the gut. ICCs are densely distributed throughout the muscularis of the IAS. However, the contribution of these cells to membrane potentials–Em and electrical rhythmicity in the IAS is unknown. Contraction in the IAS and the changes elicited by inhibitory motor nerves is primarily regu­lated by Em, and ICCs play an important role by modulating Em in adjacent SMCs. ICCs have been shown to be involved in neurotransmission of the lower oesophageal sphincter, pylorus, and Internal Anal Sphincter. ICCs deterioration has been confirmed in IAS Achalasia (IASA) infantile hypertrophic pyloric stenosis and Hirshprung’s disease. The contents and activity of smooth muscle actin (SMA) within the muscle represents its potential contractile properties.

Non-ablative radiofrequency (RF) smooth muscle remodeling has been successful in the treatment of GERD in the lower esophageal sphincter (Stretta) and fecal incontinence in the IAS (Secca). This unique RF therapy has demonstrated significant improvements in barrier function without evidence of stricture. The precise mechanism of non-ablative, RF-induced, IAS smooth muscle remodeling has not been studied yet. RF induced Heat Shock Proteins (HSP 27) may be involved in actin phosphorylation and its polimerysation.

In the first part of this animal tissue study** some of the mechanisms of nonablative radiofrequency (RF) induced tissue remodeling have been elucidated (see abstract below).

THE AIM OF THE STUDY: To define RF induced changes in IAS Interstitial Cells of Cajal (ICCs) distribution and Smooth Muscle Actin (SMA) contents following non-ablative RF therapy utilizing an animal model.

MATERIALS: A group of 20 large white pigs, mean age 181 days, weight 160 lbs. were included in the study. All animals were housed singly in cages for the duration of the study and received pig chow and water (standard protocol). Study was performed in three groups of age and weight matched pigs - FI group (6 animals underwent fecal incontinence model) - FI+RF (8 pigs with FI treated with RF spincter remodeling): Secca protocol - CONTROL (6 pigs): no treatment The animal experimental protocol was approved by the animal research committee Agriculture University Krakow/Poland and carried out at the same site.

METHODS OF STUDY: Experimental model of FI was created by pudendal nerve destruction (PND) (1.0 ml of 50% ethanol injection to pudendal nerve area, under PN stimulation guidance) and lateral sphincterotomy (LS) of external (EAS) and internal anal sphincter (IAS). FI was confirmed by anorectal manometry (BAP, SAP) (Pro-Medis Sphincterometer).

  • 6 weeks after FI model creation the 8 animals underwent non-nablative RF application using standard Secca protocol (64 points of delivery)
  • 10 weeks after RF treatment, animals were euthanized, and anal tissues harvested for pathologic evaluation. A control group of 6 age and weight matched pigs served as control.

RESULTS: Microscopic evaluation of the IAS in the control animals revealed characteristic circular muscle bundles separated by connective tissue septae. The basic architecture of the IAS was maintained following RF treatment. In all groups the circular muscle bundles greatly varied although the cross section area occupied by smooth muscle within the bundles increased after RF. There was strong peripherin immunoreactivity in the ganglia cells and nerve fibers in the IAS of the control animals. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. The number of peripherin-positive nerve fibers was markedly reduced in the IAS in sub­jects after RF treatment. There was a complete lack of peripherin immunoreactivity in the IAS, but hypertrophic nerve trunks stained strongly. Many c-Kit-positive ICCs were present among the muscle fibers and between the muscle bundles in the control IAS. In the FI group after RF, ICCs were absent or markedly reduced. Significant increase of smooth muscle actin within the IAS and fibroblasts have been observed suggesting phenotype switch of fibroblasts into myofibroblasts (reactive).

CONCLUSION: Non-ablative RF application to the IAS significantly influences the structural arrangement of IAS smooth muscle and IAS Interstitial Cell of Cajal distribution. Deterioration of the ICCs network within the IAS may be responsible for increased contraction or lack of relaxa­tion similar to IAS achalasia. Besides the increase of the smooth muscle fibers size and its number per muscle bundles as well as the collagen I content within IAS septa revealed in first part of our study, we also observed the increase of IAS smooth muscle actin (SMA) and reactive myofibrob­last contents within septa. All these features are potentially responsible for IAS remodeling after radiofrequency application to IAS (Secca procedure).