International Urogynecology Journal (2022) 33:3283–3289

https://doi.org/10.1007/s00192-022-05195-5

ORIGINAL ARTICLE

Chemokine therapy for anal sphincter injury in a rat model: a pilot

study
Amr S. El Haraki1 · S. Lankford2 · Wencheng Li3 · Koudy J. Williams2 · Catherine A. Matthews1 · Gopal H. Badlani1

Received: 18 January 2022 / Accepted: 20 March 2022 / Published online: 21 April 2022
© The International Urogynecological Association 2022

Abstract
Introduction and hypothesis To determine whether delayed administration of CXCL12 alters anorectal manometric pres-
sures and histology in rats following anal sphincterotomy compared to primary surgical repair alone.
Methods Adult female rats were divided into three groups: A, a control group that did not undergo surgery; B, anal sphinc-
terotomy with primary surgical repair; C, anal sphincterotomy with primary surgical repair and intra-sphincteric injection
of CXCL12 at 6 weeks post-injury. All rats underwent anal manometry measurements at baseline and at 6 and 12 weeks
post-injury. Histologic analysis of the anal sphincters was also performed.
Results At baseline and 6 weeks, there were no statistically significant differences among D, ­Tmax and P∆ of Groups A, B
and C. At 12-week manometry, the total duration of contractions on anal manometry was significantly less in Group C com-
pared to Groups A and B (3.65, 5.5, 5.3 p < 0.01) as was time to peak of contraction at 12 weeks (1.6, 2.1, 3.1, p < 0.01);
however, group C had a significantly higher P∆ at 12 weeks compared to Groups A and B (2.25, 1.4, 0.34, p < 0.01). There
were no statistically significant differences in the ratio of muscle to collagen at the site of injury; however, muscle fibers
were significantly smaller in group C and less per bundle than the other groups.
Conclusions Administration of chemokine therapy at 6 weeks post-repair using CXCL12 enhanced the magnitude of anal
sphincter contractions in a rat model of anal sphincter injury but decreased overall duration of contraction. Increased anal
sphincter contraction magnitude was not explained by histologic differences in explanted specimens.

Keywords CXCL12 · chemokine · sphincterotomy · manometry

Abbreviations Background
Group A No intervention group
Group B Sphincterotomy and primary repair Fecal incontinence is a quality-of-life condition, which has a
Group C Sphincterotomy, primary repair and CXCL12 complex etiology. One major identified risk factor in women
D Duration of contraction is mechanical trauma, i.e., anal sphincter tear that occurs
Tmax Time to peak of contraction during childbirth [1–3]. Most patients present with fecal
PΔ Change in contraction pressure from baseline incontinence later in life rather than immediately following
the inciting factor [4].
The anal sphincter functions through a voluntarily regu-
lated external anal striated muscle (EAS) and the smooth
muscle of the internal anal sphincter (IAS) [5, 6]. The EAS
is innervated by the pudendal nerve, which is also vulnerable
* Amr S. El Haraki
aelharak@wakehealth.edu to injury during childbirth [7, 8]. Other factors that influence
continence include local cytokines that may help modulate
1
Department of Urology, Wake Forest Baptist Medical basal IAS tone as well as the puborectalis muscle [9].
Center, 1 Medical Center Blvd, Winston‑Salem, NC 27157, Fecal incontinence can be treated medically and surgi-
USA
cally but results are suboptimal [10, 11]. Adjunctive thera-
2
Wake Forest Institute for Regenerative Medicine, pies such as regenerative cell therapies and chemokines have
Winston‑Salem, NC, USA
also been researched for the treatment of fecal incontinence.
3
Department of Pathology, Wake Forest Baptist Medical Studies using injected cell therapy for anal sphincter injuries
Center, Winston‑Salem, NC, USA

13
Vol.:(0123456789)
3284
have been conducted in various animal models, including
rat, rabbit and canine models [12–15]. Due to the difficulty
and expense of cellular therapy, cytokine therapy has been
researched for the treatment of various disorders, including
fecal incontinence, as cytokines are believed to be important
mediators in modulation of basal IAS tone and for healing
following injury.
Stromal-derived factor 1 (SDF-1), also known as
CXCL12, is one such cytokine. It is a chemokine that has
the ability to mobilize mesenchymal stem cells and stimu-
late cellular regeneration [16, 17]. CXCL12 has been shown
to have beneficial effects on cardiac and urethral muscle
repair [16, 17]. Animal models of urethral sphincter injury
have actually demonstrated better treatment efficacy with
CXCL12 therapy than with progenitor cell therapy. In a
nonhuman primate model of chronic intrinsic sphincter
deficiency (ISD), both local intramuscular CXCL12 and
intramuscular progenitor cell therapy partially increased
urethral sphincter contraction pressure during pudendal
nerve stimulation; however, only CXCL12 therapy improved
resting pressures and decreased collagen while increasing
muscle content on urethral sphincter histologic quantitative
analysis post-treatment [18]. While local administrations of
both progenitor cell therapy and CXCL12 therapy 30 days
post-injury restored sphincter complex muscle content,
only CXCL12 restored urethral leak point pressures back to
baseline values [19]. These findings support the hypothesis
that CXCL12 may be important not only for localization
of muscle repair mediators to sites of injury but also for
subsequent tissue regeneration. Due to the innovative con-
cept of CXCL12 as a direct treatment for fecal incontinence,
an animal model is suitable for this study and may provide
evidence that is sufficient to implement human trials in the
future.
Anal sphincter injury, therefore, may be a similarly good
target for chemokine augmentation therapy. Our pilot study
aims to test the administration of CXCL12 as a modula-
tor of anal sphincter function following injury in a rat. We
hypothesize that rats which receive delayed administration
of CXCL12 in addition to primary surgical repair will have
improved anal sphincteric physiology and histologic mor-
phology when compared to rats that receive primary surgical
repair alone.
Methods
This study was approved by the Institutional Animal Care
and Use Committee (IACUC) of the Wake Forest Institute
for Regenerative Medicine. Our sample size was based on
the number of rats we could acquire from our institution
for this pilot study. Sixteen adult virgin female Sprague-
Dawley rats weighing 187–232 (mean: 205) g were selected
13
International Urogynecology Journal (2022) 33:3283–3289
and arbitrarily placed into three groups: Group A (control),
Group B (anal sphincterotomy with primary surgical repair)
and Group C (anal sphincterotomy with primary surgical
repair and injection of CXCL12 6 weeks post-operatively).
It has been shown that CXCL12 dissipates at the site of
injury at 21 days following the repair [20]. For this reason,
the timeline of 6 weeks post-injury was chosen to study a
separate repair response as opposed to an augmented pri-
mary repair. The animals were anesthetized using a SurgiVet
Anesthetic machine and vaporizer using 1–3% isoflurane,
and the animal was maintained for the duration of the pro-
cedure by nose cone.
Surgical materials
For surgery, Metzenbaum scissors were used for the sphinc-
terotomy. For suturing, 4-0 Vicryl with P-1 3/8 circle reverse
cutting needle was used. First, the animal was placed over
a warm water circulating blanket, in a prone position. Per-
ineal preparation of the anal tissues with 2% chlorhexidine
and betadine was performed. The anal canal was emptied
with abdominopelvic massage. A 5-mm sphincterotomy was
made in the right posterolateral aspect of the anal sphincter
beginning at the anal verge. This was chosen to simulate
incidental laceration during childbirth.
Sphincter repair and cell therapy
Immediate repair was performed with three to four inter-
rupted stitches. The animal was given subcutaneous flu-
ids and Meloxicam, removed from the anesthetic gas and
allowed to recover on a warm water-circulating blanket until
ready to return to the home cage.
For group C, 0.05 ml of 1000 ng/ml solution of CXCL12
in normal saline was administered at 6 weeks post-injury at
the 12, 3, 6 and 9 o’clock positions on the anal sphincter for
a total of 200 ng of CXCL12.
Anal manometry measurement
For anal manometry, an AD Instruments blood pressure
transducer connected to an AD Instruments Power Lab sys-
tem was used. The system was purged to avoid air bubbles,
and the latex balloon was filled to 10–25 mmHg (to ensure
reading of anorectal pressures). The rectum was manually
evacuated, and the balloon was inserted to the level of the
anal sphincter approximately 4–6 mm past the anal verge.
Once stable activity was reached on the manometry, 10
min of activity was recorded. The system was configured
to detect ten pulses per second, registered as mmHg. All
International Urogynecology Journal (2022) 33:3283–3289
Fig. 1  Example of typical
contraction as seen on anal
manometry with examples of
parameters measured
animals underwent manometry at baseline (prior to inter-
vention), 6 weeks post-injury and 12 weeks post-injury. For
each recording, the three strongest contractions as deter-
mined by operator’s assessment were chosen. For these
contractions, we calculated the mean values of the follow-
ing: total duration (D), time to peak (Tmax) and difference
between maximal and minimal pressure value (P∆) (Fig. 1).
Histopathologic analysis
After killiing the rats after the 12-week assessment, the
anal canals were harvested and fixed in formalin. They
were then embedded in paraffin and sectioned at 5 μm.
Masson-Trichrome staining was performed on the cross-
sections to quantify new tissue at the site of the defect
as well as corresponding site in control rats. The sec-
tions were viewed under a bright-field microscope at a
magnification of 10. The sections were scanned using
Olympus BX63 Automated Fluorescence Microscope.
The site of injury was identified in the injured rats at the
right posterolateral section of the sphincter. Slides were
sectioned at 20-μm intervals, and the most anatomically
distal complete slide was used for analysis in each animal.
Masson-Trichrome stains muscle fiber red, collagen blue
and nuclei black. We used Olympus cellSens incorporating
red and blue colors for volumetric analysis of each section
to assess the percentage of muscle and connective tissue
(collagen-rich fibrosis) in the area of injury compared with
the muscle and connective tissue in the uninjured area in
3285
control rats. Digital identification of each of the stained
tissues was performed, and the results were compared to
complete the volumetric analysis. A blinded pathologist
then reviewed the muscle fiber configuration on slides
where both external and internal sphincter fibers were pre-
sent and contained intact muscle bundle structures. The
number of muscle fibers in each bundle was counted, and
the diameters of each fiber were measured and compared.
Statistical analysis
Anal manometry data were analyzed using Kruskal-Wallis
for nonparametric distributions and one-way ANOVA for
parametric distributions. The analysis was used to test for
relationships between the anal manometry results recorded,
and the group the rat was placed in as well for the histo-
logic data. A p value < 0.05 was considered statistically
significant.
Results
Between February 2021 and April 2021, a total of 16 female
adult (3 months old) Sprague-Dawley rats (5 in Group A,
5 in Group B and 6 in Group C) underwent the planned
anal manometry testing and respectively assigned interven-
tions. For each rat, three contractions were used from each
anal manometry session for a total data set consisting of 45
contractions for group A, 45 contractions for group B and a
13
3286 International Urogynecology Journal (2022) 33:3283–3289

Table 1  Total duration (D), Control (Group A) Injury without Injury with P value
time to peak (­ Tmax) and CXCL12 (Group CXCL12 (Group
pressure changes (P Δ) in anal B) C)
manometry in all rat models at
baseline, 6 weeks post-injury Baseline D (s) 9.80 (7.80-16.0) 7.00 (5.60-9.00) 8.95 (5.00-15.4) 0.06
and 12 weeks post-injury
Tmax (s) 5.00 (3.70-9.00) 3.10 (3.00-3.70) 5.55 (2.50-7.50) 0.12
P Δ (mmHg) 9.90 (3.50-16.8) 4.6 (3.10-5.40) 4.30 (2.70-10.7) 0.17
6 weeks post-injury D (s) 6.00 (5.00-6.30) 5.00 (4.00-6.50) 5.00 (4.00-6.00) 0.51
Tmax (s) 3.00 (2.40-3.00) 2.60 (2.20-3.00) 2.80 (2.20-4.00) 0.82
P Δ (mmHg) 2.40 (0.80-3.10) 0.70 (0.56-3.00) 2.10 (0.80-6.10) 0.10
12 weeks post-injury D (s) 5.50 (4.00-7.00) 5.30 (4.80-11.0) 3.65 (2.50-4.40) <0.01
Tmax (s) 2.10 (1.90-3.20) 3.10 (2.70-4.70) 1.60 (1.20-2.40) <0.01
P Δ (mmHg) 1.40 (0.50-1.90) 0.34 (0.27-0.60), 2.25 (0.80-4.15) <0.01

total of 54 contractions in group C. During anal manometry Discussion

performed at baseline, there were no statistically significant
differences between D, Tmax and P∆ between groups. At
6-week post-injury, on anal manometry, there were no sta-
tistically significant differences among D, Tmax and P∆ of
Groups A, B and C as well (Table 1). At 12-week manom-
etry, the total duration of contractions on anal manometry
was significantly less in Group C compared to Groups A and
B (3.65, 5.5, 5.3 p = 0.000432). The time to peak of con-
traction at 12 weeks was also significantly less in Group C
compared to Groups A and B (1.6, 2.1, 3.1, p = 0.0007167);
however, group C had a significantly higher P∆ at 12 weeks
compared to Groups A and B (2.25, 1.4, 0.34, p = 0.001345).
Histology performed after euthanasia showed that there
was no significant difference among groups in the ratio of
muscle to collagen in the area of injury (A: 2.56 ± 2.11; B:
1.40 ± 1.04; C: 1.57 ± 1.12; p = 0.177). On reviewing mus-
cle fiber configuration, we did not observe any difference
in the configuration of internal sphincter muscle bundles
among the three groups. Interestingly, we noticed that the
architecture of the external sphincter muscle bundles was
disrupted in the group C (Fig. 2). To confirm the morpho-
logic impression, we measured the number of muscle fibers
in each bundle and the diameter of each fiber. The portion
of the fiber that was thickest was measured. Only longitudi-
nal fibers were measured for standardization. For each fiber,
three measurements were taken and averaged. The diameters
of the external sphincter muscle fibers were statistically dif-
ferent among the three groups. A total of 20 muscle bundles
were evaluated in groups A and B with 39 muscle bundles
in group C. Group A had the thickest muscle fibers 24.3 ±
4.6 μm followed by group B at 19.3 ± 4.2 μm with group
C showing the smallest muscle fiber diameter at 12.5 ± 3.2
μm. Additionally, group A had the most number of fibers per
muscle bundle at 6 fibers/bundle (IQ range: 5-6) followed by
group B at 5 fibers/bundle (IQ range: 4-5) and group C at 2
fibers/bundle (IQ range : 2-3) (Table 2)
Fecal incontinence is a common complication of anal
sphincter injury despite surgical attempts at repair [21].
Investigation of augmenting cytokine and cellular therapies,
therefore, is a rational avenue for research [22]. In our pilot
study, we showed that the rats that received delayed admin-
istration of CXCL12 demonstrated higher pressure changes
in shorter bursts on anorectal manometry. Qualitative and
quantitative analysis of the microscopic sections of the dif-
ferent anal sphincters in the groups did show a significant
difference. It seems that injection of CXCL12 did decrease
the number of fibers per muscle bundle in addition to caus-
ing a decrease in the individual muscle fiber diameters. This
may explain the shorter duration of the contractions as seen
on anal manometry. The smaller diameter fibers may be
explained by the emergence of new muscle fibers, but we
could not explain why there were fewer fibers in the area of
the injury following CXCL12 injection.
One other study has demonstrated a beneficial effect of
CXCL12 in a rat model of chronic anal sphincter injury [23]
in which CXCL12 plasmid injection therapy was injected 3
weeks post-sphincterotomy. They found that all sphincters
that were treated with CXCL12 had resting pressures that
returned to near normal levels by 8 weeks after treatment.
We could not replicate this in our study, and the timing and
dose of injection could potentially have been one factor in
this difference. Overall, their study demonstrated CXCL12
administration alone to be a potential therapy for chronic
anal sphincter injury without the need for additional stem
cell therapy.
Using anorectal manometry in rats as a proxy for anal
sphincter function and recovery after injury is challenging as
anal pressures may continue to be generated after sphincter-
otomy by undamaged muscle fibers continuously generating
force and pressure [24]. In our study, surgical incision of the
rat external anal sphincter did not decrease manometric pres-
sures in either repair group compared to controls at 6 weeks.

13
International Urogynecology Journal (2022) 33:3283–3289 3287

Additionally, there was no measurable or qualitative change

A in the muscle structure between the control group and the
B was performed since this was a pilot study. Since the tissue
C need to be performed to be able to detect if there indeed
Fig. 2  Masson-Trichrome stain of right posterolateral section of anal
sphincter at 10x magnification
primary surgical repair group (group B). It is interesting that
differences in manometric parameters emerged at 12 weeks,
suggesting some effect of CXCL on the site of injury. We do
not know if the observed manometry findings in the CXCL
group of a higher amplitude contraction of shorter duration
translates into better or worse anal sphincter function. Our
results, however, prompt consideration of future studies to
compare different dosing and administration schedules. Our
study has several important limitations. First, our sample
size used for the experiments was small but we limited our
aims to standardization of our injury model and performance
of reliable and reproducible manometric values. This sample
was also a convenience sample, and no power calculation
is friable and tissue yield is low, a bigger sample would have
further strengthened our comparisons. There is no standard-
ized method for anal manometry, in rats and we relied on
previous studies which looked at similar parameters [24–29].
Each group that studied anal manometry in rats, however,
performed their measurements slightly differently. We can-
not correlate the clinical significance of the anal manom-
etry measurements. While group C showed higher pressure
changes with shorter time to peak and shorter duration of
contractions, we can only postulate this actually impacts anal
sphincter function. Adding EMG analysis to this study may
further shed light on the function of the anal sphincter based
on its contractility and innervation. Larger animal studies
is a difference with CXCL12 as rat self-healing might be
too rapid. This may explain the similarity in anal manom-
etry measurements between group A and groups B and C at
6 weeks post-injury. Another explanation may be that the
sphincterotomy performed was not extensive enough. In a
larger animal, we plan to obtain data regarding the normal
anal sphincter function before and after injury. Additionally,
this study mimics anal sphincter tears, which is only one
potential cause of fecal incontinence during childbirth. In
reality, there are other possible confounding causes such as
concomitant nerve injury [28].
In conclusion, we have established replacement of col-
lagen with host-derived cells in urethral sphincter injury
and a renal ischemic model, but in this pilot study, we have
gained initial experience with the use of local injection

Table 2  Histologic data Control (Group A) Injury without Injury with P value
between different rat groups CXCL12 (Group B) CXCL12 (Group
C)

Muscle-collagen ratio 2.56± 2.11 1.40 ± 1.04 1.57 ± 1.12 0.177

Muscle fibers per bundle 6 (5-6) 5 (4-5) 2 (2-3) <0.01
Muscle fiber diameter (μm) 24.3 ± 4.6 19.3 ± 4.2 12.5 ± 3.2 <0.01

13
3288
of CXCL12 for anal sphincter injury. CXCL2 produces a
measurable effect on anorectal manometric parameters and
an effect on the local muscle morphology post-injection.
In our study, CXCL12 increased magnitude of contractions
of the anal sphincter while decreasing duration. On histol-
ogy, there were fewer fibers at the area of CXCL12 injec-
tion that had smaller fiber diameters. These results could
not be correlated. Our data in this study lead us to refine our
hypothesis regarding the potential for CXCL to enhance anal
sphincter healing to concentrate on optimization of dose and
timing of administration. Future directions include investiga-
tion of regeneration and repair with more extensive use of
EMG studies. Additionally, we will aim to study CXCL12
administration in larger animals with concomitant neural
injury. Using larger animal models may provide more insight
as well in the use of CXCL12 in anal sphincter injury. We
hope, after more extensive preclinical testing, CXCL12 can
be used as an adjunctive treatment for anal sphincter injury.
Author contributions AS El Haraki: Rat measurements and surgery,
data analysis and manuscript writing
S Lankford: Rat measurements, data collection and surgery
Wencheng Li: qualitative histologic analysis
JK Williams: Project development, manuscript editing
C Matthews: Manuscript writing
GH Badlani: Principal investigator, project development, manu-
script editing
Declarations
Conflicts of interest None.
References
1. Snooks SJ, Henry MM, Swash M. Faecal incontinence due to
external anal sphincter division in childbirth is associated with
damage to the innervation of the pelvic floor musculature: a dou-
ble pathology. Br J Obstet Gynaecol. 1985;92(8):824–8 5.
2. Allen RE, et al. Pelvic floor damage and childbirth: a neurophysi-
ological study. Br J Obstet Gynaecol. 1990;97(9):770–9 6.
3. Sandridge DA, et al. Vaginal delivery is associated with occult
disruption of the anal sphincter mechanism. Am J Perinatol.
1997;14(9):527–33 7.
4. Sultan AH, Kamm MA, Hudson CN, et al. Third Degree Obstetric
Anal Sphincter Tears. British Med J. 1994;308(6933):887–91.
5. Gearhart S, Hull T, Floruta C, et al. Anal manometric parameters:
predictors of outcome following anal sphincter repair? J Gastroin-
testinal Surg. 2005;9(1):115–20. https://​doi.​org/​10.​1016/j.​gassur.​
2004.​04.​001.
6. Sangwan YP, Solla JA. Internal anal sphincter: advances and
insights. Dis Colon Rectum. 1998;41(10):1297–311. https://​doi.​
org/​10.​1007/​bf022​58232.
7. Chaliha C, Sultan A, Bland J, et al. Anal function: Effect of preg-
nancy and delivery. Am J Obstet Gynecol. 2001;185(2):427–32.
13
International Urogynecology Journal (2022) 33:3283–3289
8. Hamdy S, Enck P, Aziz Q, et al. Laterality effects of human
pudendal nerve stimulation on corticoanal pathways: evidence
for functional asymmetry. Gut. 1999;45(1):58–63.
9. Azpiroz F, Fernandez-Fraga X, Merletti R, et al. The puborec-
talis muscle. Neurogastroenterol Motil. 2005;17:68–72.
10. Alavi K, Chan S, Wise P, et al. Fecal Incontinence: Eti-
ology, Diagnosis, and Management. J Gastrointest
Surg. 2015;19(10):1910–21. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 007/​
s11605-​015-​2905-1.
11. Saldana Ruiz N, Kaiser AM. Fecal incontinence - Challenges and
solutions. World J Gastroenterol. 2017;23(1):11–24. https://​doi.​
org/​10.​3748/​wjg.​v23.​i1.​11.
12. Lane FL, et al. In vivo recovery of the injured anal sphincter after
repair and injection of myogenic stem cells: an experimental
model. Dis Colon Rectum. 2013;56(11):1290–7 27.
13. Aghaee-Afshar M, et al. Potential of human umbilical cord matrix
and rabbit bone marrow-derived mesenchymal stem cells in repair
of surgically incised rabbit external anal sphincter. Dis Colon Rec-
tum. 2009;52(10):1753–61 28.
14. Kajbafzadeh AM, et al. Functional external anal sphincter recon-
struction for treatment of anal incontinence using muscle progeni-
tor cell auto grafting. Dis Colon Rectum. 2010;53(10):1415–21
29.
15. Oh HK, et al. Functional and histological evidence for the targeted
therapy using biocompatible polycaprolactone beads and autolo-
gous myoblasts in a dog model of fecal incontinence. Dis Colon
Rectum. 2015;58(5):517–25.
16. Cottler-Fox MH, et al. Stem cell mobilization. Hematology Am
Soc Hematol Educ Program. 2003;2003:419–37 34.
17. Lapidot T, Petit I. Current understanding of stem cell mobi-
lization: the roles of chemokines, proteolytic enzymes, adhe-
sion molecules, cytokines, and stromal cells. Exp Hematol.
2002;30(9):973–81.
18. Williams JK, et al. Cell versus chemokine therapy in a nonhuman
primate model of chronic intrinsic urinary sphincter deficiency. J
Urol. 2016;196(6):1809–15.
19. Koudy Williams J, et al. Efficacy and initial safety profile of
CXCL12 treatment in a rodent model of urinary sphincter defi-
ciency. Stem Cells Transl Med. 2017;6(8):1740–6.
20. Salcedo L, et al. Chemokine upregulation in response to anal
sphincter and pudendal nerve injury: potential signals for stem
cell homing. Int J Color Dis. 2011;26(12):1577–81.
21. Sideris M, McCaughey T, Hanrahan JG, et al. Risk of obstetric
anal sphincter injuries (OASIS) and anal incontinence: A meta-
analysis. Eur J Obstet Gynecol Reprod Biol. 2020;252:303–12.
https://​doi.​org/​10.​1016/j.​ejogrb.​2020.​06.​048.
22. Sun L, Xie Z, Kuang M, et al. Regenerating the Anal Sphinc-
ter: Cytokines, Stem Cells, or Both? Dis Colon Rectum.
2017;60(4):416–25. https://​doi.​org/​10.​1097/​dcr.​00000​00000​
000783.
23. Sun L, Kuang M, Penn M et al. 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(12). Retrieved from https://j​ ourna​ ls.l​ ww.c​ om/d​ crjou​ rnal/​
Fullt​ext/​2017/​12000/​Strom​al_​Cell_​Deriv​ed_​Factor_​1_​Plasm​id_​
Regen​erates.​16.​aspx
24. Salcedo L, Damaser M, Butler R, et al. Long-term effects on pres-
sure and electromyography in a rat model of anal sphincter injury.
Dis Colon Rectum. 2010;53(8):1209–17. https://​doi.​org/​10.​1007/​
DCR.​0b013​e3181​de7fe0.
25. Zutshi M, Salcedo LB, Zaszczurynski PJ, et al. Effects of sphinc-
terotomy and pudendal nerve transection on the anal sphincter in
a rat model. Dis Colon Rectum. 2009;52(7):1321–9. https://​doi.​
org/​10.​1007/​DCR.​0b013​e3181​9f746d.
International Urogynecology Journal (2022) 33:3283–3289 3289

26. Vinograd I, Hanani M, Hadary A, et al. Animal model for Distention or Sphincter Transection in an Animal Model. Obstet
the study of internal anal sphincter activity. Eur Surg Res. Gynecol. 2008;111(6). Retrieved from https://​journ​als.​lww.​com/​
1985;17(4):259–63. https://​doi.​org/​10.​1159/​00012​8476. green​journ​al/​Fullt​ext/​2008/​06000/​Recov​ery_​of_​Exter​nal_​Anal_​
27. Trébol J, Georgiev-Hristov T, Vega-Clemente L, et al. Rat model Sphin​cter_​Contr​actile.​25.​aspx
of anal sphincter injury and two approaches for stem cell admin-
istration. World J Stem Cells. 2018;10(1):1–14. https://d​ oi.o​ rg/1​ 0.​ Publisher’s note Springer Nature remains neutral with regard to
4252/​wjsc.​v10.​i1.1. jurisdictional claims in published maps and institutional affiliations.
28. Gras S, Tolstrup CK, Lose G. Regenerative medicine provides
alternative strategies for the treatment of anal incontinence. Int
Urogynecol J. 2017;28(3):341–50.
29. Wai CY, Rahn DD, White AB et al. Recovery of External
Anal Sphincter Contractile Function After Prolonged Vaginal

13