SDF-1 plasmid to regenerate the anal sphincter: are we closer to translation?

Sun L1, Damaser M S1, Penn M1, Reitsch A1, Zutshi M1

Research Type

Basic Science / Translational

Abstract Category

Anorectal / Bowel Dysfunction

Abstract 659
Bowel Dysfunction and Sexual Function
Scientific Podium Short Oral Session 34
Friday 31st August 2018
15:05 - 15:12
Hall D
Anal Incontinence Basic Science Animal Study Pelvic Floor Stem Cells / Tissue Engineering
1. Cleveland Clinic , Cleveland , Ohio
Presenter
M

Massarat Zutshi

Links

Abstract

Hypothesis / aims of study
In a rat model, we have documented regeneration after a chronic large injury of the anal sphincter muscle at 4 and 8 weeks after treatment with a plasmid encoding stromal cell derived factor -1 (SDF-1) which is a cytokine that is chemotactic for stem cells and is responsible for aiding angiogenesis after injury

Hypothesis: SDF-1 plasmid regenerates muscle and increases pressures after a chronic large anal sphincter injury in a large animal model
Aim : To evaluate regeneration of a chronic large anal sphincter defect in a pig model after treatment with a plasmid encoding SDF-1.
Study design, materials and methods
Under an institution of animal care and use committee approved protocol 20 19 age and weight matched Sinclair minipigs were subjected to excision of the posterior half of the anal sphincter muscle and left to recover for 6 weeks. They were then randomly allocated to receive either saline treatment 1 ml (Saline, n=5), 1 injection of SDF-1 plasmid 2 mg/ml (1-SDF-1, n=109) or 2 injections of SDF-1, 2 mg/ml each at 2 week intervals (2-SDF-1, n=5) under anesthesia. Injections were made at the two ends of the incision in the region of the severed muscle.  Animals were euthanized 8 weeks after the last treatment and subjected to histopathology. 

Outcomes included anal manometry and anal ultrasound done pre-injury, at time of first injection and before euthanasia. Unpaired T-test followed by nonparametric testsOne way ANOVA followed Tukey test were used for data analysis (mean±SD), p<0.05 was regarded as significant.
Results
There were no treatment related complications. Two pigs in the control group died due to unrelated causes and were replaced. 
At pre-injury and post-treatment,  no significant difference was found among 3 groups. When comparing  the anal pressure among 3 time points within animal groups, there was no significant difference was found in saline group; in 1-SDF-1 group, at post-treatment had there was significantly higher pressure than both pre-injury (*p<0.001) and pre-treatment time point (#p<0.001); no significant difference was found within 2-SDF-1 group.

Single injection treatment: The pressures in the control group were higher at the pre-injury time point and statistically significant (p=0.002). At pre-treatment the pressure in the control group was significantly higher (p=0.002).  At 8 weeks after treatment, the difference in resting pressures in the control group was negative from pre-treatment pressures indicating that the pressures declined and did not return to pre-injury or pretreatment levels, while those in the  SDF-1 treatment group increased significantly from  pre –treatment (p=0.002) when compared with the control group.

Two injection treatment: After treatment the SDF-1 treatment group had significantly higher posterior anal pressures compared to saline group.  (p=0004) When comparing the mean  resting pressure between 3 time points within the animal group the pressure was not significantly different .

Ultrasound imaging at 8 weeks after treatment revealed disruption of the muscle posteriorly in the control group with more complete muscle in the SDF-1 group compared to pre-injury.  
 Histology qualitative analysis shows distortion of normal anatomy with patchy regeneration in the control group while muscle was more organized in the treatment group .Quantitative analysis is awaited.

Histological appearance of saline treated group on the left and SDF-1 1 injection on the right. A marks anterior while the defect is posterior
Interpretation of results
The pressures in the posterior channel at the site of the injury increased in both the SDF treated groups. The mean resting pressures in the one injection treatment were higher post treatment that at preinjury 

The two injection group although much improved did not shot a mean pressure increase that was significant. This may be due to the sample size. 

The pigs were also anesthetized and this may have had an effect on the anal manometry .

Histological regeneration however was qualitatively seen in both SDF-1 treated groups that  was more organized than the saline group
Concluding message
Eight weeks after both a single dose of SDF-1 and 2 doses injected 6 weeks after an excision of 50% of the circumference of the anal sphincter improved resting anal sphincter pressures, and regenerated muscle in the entire area of the defect. SDF-1 plasmid is safe and effective in treating chronic defects of the anal sphincter in a large animal and can be translated.
Figure 1
Figure 2
References
  1. Sun L, Kuang M, Penn M,Damaser MS, Zutshi M. Stromal Cell-Derived Factor 1 Plasmid Regenerates Both Smooth and Skeletal Muscle After Anal Sphincter Injury in the Long Term. Dis Colon Rectum. 2017; 60: 1320-1328.
Disclosures
Funding The Armed Forces Institute of Regenerative Medicine (AFIRM-2) W81XWH-13-AFIRMIIRP Clinical Trial No Subjects Animal Species Mini Pig Ethics Committee Cleveland Clinic IACUC