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Endoscopic suturing promotes healing of mucosal defects after gastric endoscopic submucosal dissection: endoscopic and histologic analyses in in vivo porcine models (with video)
ORIGINAL ARTICLE: Experimental Endoscopy
Endoscopic suturing promotes healing of mucosal defects after gastric endoscopic submucosal dissection: endoscopic and histologic analyses in in vivo porcine models (with video) Teppei Akimoto, MD,1,2 Osamu Goto, MD, PhD,1,2 Motoki Sasaki,1 Mari Mizutani, MD,1 Koshiro Tsutsumi, MD,1 Yoshiyuki Kiguchi, MD,1 Atsushi Nakayama, MD, PhD,1 Motohiko Kato, MD, PhD,1 Ai Fujimoto, MD, PhD,1 Yasutoshi Ochiai, MD, PhD,1 Tadateru Maehata, MD, PhD,1 Mitsuru Kaise, MD, PhD,2 Katsuhiko Iwakiri, MD, PhD,2 Naohisa Yahagi, MD, PhD1 Tokyo, Japan
GRAPHICAL ABSTRACT
Background and Aims: Endoscopic suturing of mucosal defects after endoscopic submucosal dissection (ESD) is expected to prevent postoperative adverse events. We aimed to endoscopically and histologically evaluate the healing process of post-ESD mucosal defects closed with endoscopic hand suturing (EHS) in in vivo porcine models. Methods: Twelve mucosal defects (2 cm in size) were created in 2 pigs (6 defects per pig). Initially, 2 defects were created: one was closed with EHS (sutured group) and the other was kept open (control group). On postoperative days (PODs) 7 and 14, 2 additional defects were created in each session, and they were treated in the same manner as in the initial procedure. On POD 21, the entire stomach, with the 6 lesion sites, was extracted for histologic evaluation after endoscopic observation. Results: Endoscopically, all sutured sites remained closed in all sessions (PODs 7, 14, and 21). Histologically, on POD 14, the epithelium and muscularis mucosae were appropriately connected. The mucosae were covered with the epithelium without inversion of the mucosal edge in the sutured group, whereas the ulcer bed was exposed in the control group. Furthermore, the degree of neovascularity and fibroblasts in the submucosa was smaller in the sutured group than that in the control group. Conclusions: Our findings suggest that endoscopic suturing promotes healing of post-ESD mucosal defects histologically in in vivo porcine models. Thus, endoscopic mucosal closure after ESD might be clinically useful for the prevention of delayed perforation/bleeding if secure suturing is performed endoscopically. (Gastrointest Endosc 2020;91:1172-82.)
(footnotes appear on last page of article)
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INTRODUCTION Endoscopic submucosal dissection (ESD) is gaining acceptance as one of the curative treatment options for early gastric neoplasms because of its minimal invasiveness and high possibility of complete resection.1-3 However, critical postoperative adverse events associated with ESD, which primarily include delayed bleeding and delayed perforation, sometimes occur even with proper management strategies, such as administration of proton-pump inhibitors and prophylactic hemostasis of vessels in mucosal defects immediately after ESD using hemostatic forceps.4,5 A possible reason for these postoperative adverse events is that post-ESD large mucosal defects remain open.6 Therefore, several researchers have endoscopically closed post-ESD gastric mucosal defects using endoclips, endoloops, or over-the-scope clips.7-10 Furthermore, endoscopic suturing techniques involving a needle and suture thread, such as Overstitch (Apollo Endosurgery Inc, Austin, Tex, USA), have been developed to achieve robust closure.11 We have also developed a suturing technique involving a through-the-scope needle holder and absorbable suture thread attached to a curved needle, which is called endoscopic hand suturing (EHS).12,13 However, it has not been fully investigated whether these suturing techniques promote the healing of postESD mucosal defects and contribute to the prevention of delayed adverse events. In this study, we assessed the healing speed and histologic healing process of endoscopically sutured post-ESD mucosal defects in in vivo porcine models and compared the findings with those of natural healing of post-ESD mucosal defects.
METHODS Preparation of animal models This study was approved by the institutional review board of our animal laboratory (approval no. 17060-(0)). Endoscopic procedures were performed with the animals under general anesthesia in 2 female pigs (Göttingen minipig; Oriental Yeast Co, Ltd, Tokyo, Japan; body weights of 20.6 and 23.8 kg). After environmental acclimation for 7 days, the pigs were fasted for 24 hours before the endoscopic procedures. Initially, 0.02 mg/kg medetomidine and 0.1 mg/kg midazolam were injected intramuscularly, and then an intravenous line was placed in an ear vein. Subsequently, a tracheal intubation tube was inserted and inhalation anesthesia was performed with 1.5% to 2.5% isoflurane. Then, an overtube was placed and the endoscopic procedures were performed. The pigs were allowed to drink water and eat soft feed a day after the endoscopic procedures.
Endoscopic submucosal dissection According to the experiment protocol described later in this section, a double-channel endoscope with www.giejournal.org
Endoscopic suturing promotes healing of mucosal defects
multi-bending function and a high-vision endoscope (GIF-2TQ260M and GIF-H260Z; Olympus Co, Ltd, Tokyo, Japan) were used for endoscopic treatment and endoscopic observation, respectively. The settings of the highfrequency power unit (ESG-100; Olympus) during ESD were the pulse cut first mode (30 W) for mucosal incision and the forced coagulation 2 mode (30 W) for mucosal marking and submucosal dissection. For marking the experiment sites on the stomach, a plastic disciform plate (2 cm in diameter) was placed at the target areas and marks were made around the plate using the tip of a dual knife (KD650-L; Olympus). ESD was performed conventionally using the dual knife, an injection needle (NM-610L-0425; Olympus), and a solution of 10% glycerin with 5% fructose plus 0.9% saline solution (Glyceol; Chugai Pharmaceutical Co, Ltd, Tokyo, Japan).
Endoscopic hand suturing Mucosal closure by EHS was performed for the mucosal defect as described previously.12,13 Briefly, a prototype of the through-the-scope needle holder (FG-Y0015; Olympus) and a curved needle with absorbable barbed suture (VLOCL0604; Covidien, Dublin, Ireland) were used as follows (Video 1, available online at www.giejournal.org). The curved needle was delivered to the stomach, grasping the suture at approximately 5 mm from the tail of the needle with the needle holder. In the stomach, the suture was released and the curved needle was grasped at one-third from the tail. The first stitch was placed on the distal mucosa outside the mucosal defect. Then, the needle was passed through a ring attached to the end of the suture, which acted as an anchor. Subsequently, the mucosal rim of the defect was sutured with the running suture technique from the distal to proximal end with an interval of less than 5 mm between 2 adjacent stitches and a width of approximately 7 to 8 mm for the suture bites.13 During EHS, grasping forceps (FG-21L-1; Olympus) advanced through another working channel were used to regrasp the needle and tighten the suture. After complete closure, a hemoclip (HX-610-090S; Olympus) was placed with the suture on the mucosa near the final stitch. Eventually, the suture was cut with a loop cutter (FS-5L-1; Olympus) and was extracted outside the stomach along with the needle.
Experiment protocol According to a previous study,14 to obtain multiple histologic specimens at different observation periods from a small number of pigs, this animal study included 4 sessions. The first session involved the initial operation. The second, third, and fourth sessions were performed on postoperative days (PODs) 7, 14, and 21, respectively. In one pig, experiments were performed as described here and in Figure 1A. In the first session, 2 mucosal defects of 2 cm were created using the ESD technique at Volume 91, No. 5 : 2020 GASTROINTESTINAL ENDOSCOPY 1173
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Observation Observation 3. ESD 4. ESD+EHS
Observation 5. ESD 6. ESD+EHS Sacrifice
Day 0
Day 7
Day 14
Day 21
Anterior wall
group, and the open mucosal defects were included in a control group. In the fourth session, all 6 lesions (3 in the sutured group and 3 in the control group) were observed and measured. Subsequently, the pig was killed and the stomach was extracted. Chronologic endoscopic views (Fig. 2A) and tissue specimens of the sutured and control groups were obtained from this pig. Conversely, in the other pig, open mucosal defects (control group) and sutured mucosal defects (sutured group) were created on the PW and AW, respectively.
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Evaluation of outcomes
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the lower body of the stomach (one on the anterior wall [AW] and another on the posterior wall [PW]). The mucosal defect on the PW was closed by EHS (Fig. 1B), whereas the mucosal defect on the AW was kept open (Fig. 1B). In the second and third sessions, the suture site and mucosal defects that were created in the previous session were observed, and the defect size was measured using the 2-cm plastic disciform plate. Subsequently, a sutured mucosal defect and an open defect were newly created on the PW and AW, respectively, of both the upper body (second session) and middle body (third session), similar to the approach in the first session. The sutured mucosal defects were included in a sutured
With regard to the technical outcomes of EHS, the following parameters were evaluated: the success rate of complete closure using EHS immediately after ESD, procedure time of EHS (time from the first stitch to complete closure), bites per suture applied for a mucosal defect, suturing speed (minutes per stitch), and proportion of complete closure without dehiscence on POD 7. With regard to endoscopic evaluation, the main outcome measures were the healing speed of mucosal defects after ESD and that of endoscopically sutured mucosal defects. To evaluate the macroscopic healing speed, the endoscopic widths of the mucosal defects at all sessions were measured. When the mucosal defect was replaced with scar tissue or was completely closed without dehiscence, the endoscopic width of the mucosal defect was recorded as zero. When the sutured mucosal defect exhibited dehiscence, the maximum distance between the mucosal rims was recorded as the endoscopic width of the mucosal defect. The mean endoscopic widths of the mucosal defects on PODs 7, 14, and 21 were compared between the sutured and control groups. For histologic and immunohistochemical evaluations, the lesion sites in the sutured and control groups were fixed in 10% neutral buffered formalin. Then, slices obtained from the center of the tissues were embedded in paraffin, cut into 4-mm-thick sections, and stained with hematoxylin and eosin (HE) and alpha-smooth muscle actin (aSMA) antibody. To evaluate histologic and immunohistochemical healing, the width of absent epithelium between mucosal rims in HE-stained sections and the width of absent muscularis mucosae between mucosal rims in aSMA-stained sections were measured. In addition, the neovascular and fibroblast area was assessed and defined as the area of positive aSMA in the submucosa at the lesion site. The area of positive aSMA was measured in 3 randomly selected fields (original magnification 400) in the middle layer of the submucosa under the lesion site for each section, using ImageJ software (National Institutes of Health, Bethesda, Md, USA) with the immunohistochemistry profiler plugin.15 In addition, the area of positive aSMA in normal submucosa (nonlesion sites) was assessed and measured at the marginal site in specimens from the sutured group. The widths of absent epithelium and absent muscularis mucosae on PODs 7, 14, and 21 were
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Oral side
Anal side 2
4 6
A
Posterior wall
B Figure 1. Study protocol and endoscopic images of the lesions. A, Time course of the study and schema of the lesion sites in the first pig. Pairs of mucosal defects (anterior wall) and sutured mucosal defects (posterior wall) are created at the lower body (1 and 2), upper body (3 and 4), and middle body (5 and 6) of the stomach on the first day, postoperative day (POD) 7, and POD 14, respectively. On POD 21, the 3 mucosal defects and 3 sutured defects are observed and extracted for histology. Conversely, in the other pig, mucosal defects and sutured mucosal defects are created on the posterior wall and anterior wall, respectively. B, An endoscopic view of a mucosal defect and a sutured defect immediately after each procedure. ESD, Endoscopic submucosal dissection; EHS, endoscopic hand suturing.
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Figure 2. Endoscopic evaluation. A, Changes in endoscopic images in both groups (lower body of the anterior wall). The mucosal defect is open in the control group on postoperative day (POD) 21, whereas it has closed without dehiscence in the sutured group at the same time point. B, Comparison of endoscopically measured widths of the mucosal defects. The mean widths are significantly shorter in the sutured group than in the control group on PODs 7 and 14.
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Endoscopic widths of mucosal defects (mm)
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25 Sutured group Control group 20
P < .05
15
15.0 2.6 mm, 6.3 2.8 mm, and 2.0 2.0 mm, respectively, in the control group. There were significant differences between the 2 groups on PODs 7 and 14 (P Z .0027 and P Z .020, respectively). All sutured mucosal defects remained closed during the observation period from POD 7 to POD 21.
Histologic and immunohistologic evaluations
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The chronologic endoscopic widths of the mucosal defects in the sutured and control groups are presented in Figure 2B. The mean endoscopic widths of the mucosal defects on PODs 7, 14, and 21 (n Z 6, 4, and 2, respectively, in each group) were 0 0 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and
The histologic findings regarding the epithelium are presented in Figure 3. The mean widths of absent epithelium on PODs 7, 14, and 21 (n Z 2, 2, and 2, respectively, in each group) were 0.88 0.30 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and 10.7 2.2 mm, 4.2 3.5 mm, and 1.1 1.1 mm, respectively, in the control group. On POD 7, the mean width of absent epithelium was significantly shorter in the sutured group than in the control group (P Z .023). However, there was no significant difference in the mean width between the 2 groups on PODs 14 and 21 (P Z .18 and P Z .21, respectively). With regard to the morphology of the sutured sites, the muscularis mucosae fused to each other, and the fused area was appropriately covered by connected epithelium without inversion on PODs 14 and 21. The immunohistochemical findings regarding the muscularis mucosae and neovascular and fibroblast area are presented in Figure 4. The mean widths of absent muscularis mucosae on PODs 7, 14, and 21 (n Z 2, 2, and 2, respectively, in each group) were 2.8 0.7 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and 13.2 2.6 mm, 8.2 2.9 mm, and 7.5 1.0 mm, respectively, in the control group (Fig. 4B). All the widths of absent muscularis mucosae were shorter in the sutured group than in the control group with significant differences on PODs 7 and 21 (P Z .030 and P Z .0093, respectively) and without a significant difference on POD 14 (P Z .055). The mean neovascular and fibroblast areas on PODs 7, 14, and 21 (n Z 6, 6, and 6, respectively, in each group) were 4573 1881 mm2, 3600 1875 mm2, and 2319 841 mm2, respectively, at the lesion sites in the sutured group; 4672 2289 mm2, 11722 1694 mm2, and 13831 4504 mm2, respectively, at the lesion sites in the control group; and 1860 448 mm2, 2219 856 mm2, and 2012 637 mm2, respectively, at the nonlesion sites (Fig. 4D). On POD 7, the mean neovascular and fibroblast area was significantly larger at the lesion sites in the sutured and control groups than at the nonlesion sites (P Z .021 and P Z .026, respectively). On PODs 14 and 21, the mean neovascular and fibroblast areas were significantly smaller at the lesion sites in the sutured group than at the lesion sites in the control group (P < .0001 and P < .0001, respectively). Furthermore, there were no significant differences in the mean neovascular and fibroblast area between the lesion sites in the sutured group and the nonlesion sites on PODs 14 and 21 (P Z .18 and P Z .86, respectively).
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Postoperative period (days) Figure 2. Continued.
compared between the sutured and control groups. The neovascular and fibroblast areas on PODs 7, 14, and 21 were compared among the lesion sites in the sutured and control groups and the nonlesion sites. Furthermore, the histologic morphology of the mucosal layer in the sutured group was assessed.
Statistical analysis Data regarding the technical outcomes of EHS are expressed as medians or frequencies (proportions), and other data are expressed as means. The data were compared between 2 groups using the Student t test, and all analyses were performed using JMP version 14.0 (SAS Institute Inc, Cary, NC, USA). Statistical significance was set at a P value <.05.
RESULTS Technical outcomes of EHS In the sutured group, the success rate of complete closure immediately after ESD and the rate of complete closure without dehiscence on POD 7 were 100% (6 of 6) and 100% (6 of 6), respectively. The median procedure time of EHS was 26.5 minutes (range, 24-32 minutes), the median bites per suture applied to a lesion was 6 (range, 6-7), and the median time per stitch was 4.4 minutes (range, 3.6-5.3 minutes).
Endoscopic evaluation
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A
Histological widths of absent epithetlium (mm)
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C
15 Sutured group Control group P < .05 10
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7
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Postoperative period (days)
Figure 3. Histologic evaluation of hematoxylin and eosin-stained sections. A, Chronologic variation of the lesion areas in the sutured and control groups. Arrows indicate the width of absent epithelium between the rims of each mucosal defect. The width of absent epithelium is measured in each section. B, On postoperative day (POD) 7 (left), there is a narrow section of absent epithelium between the inverted mucosal rims in the sutured group. On POD 14 (right), regenerated epithelium completely covers the mucosal defect. C, Comparison of the width of absent epithelium between the sutured and control groups. On POD 7, the mean width of absent epithelium is significantly shorter in the sutured group than in the control group. On POD 21, the surface of the mucosal defect is not completely covered with epithelium in the control group.
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Widths of absent muscularis mucosae (mm)
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Figure 4. Immunohistochemical evaluation of alpha-smooth muscle actin (aSMA)-stained sections. A, Chronologic variation of the lesion areas in the sutured and control groups. Arrows indicate the width of absent muscularis mucosae between the rims of each mucosal defect. B, Comparison of the width of absent muscularis mucosae between the sutured and control groups. On postoperative day (POD) 14, the muscularis mucosae in the sutured group appear to fuse. C, Magnified views of the middle submucosal layer at the lesion sites in the 2 groups and at the nonlesion site on POD 14. The mean neovascular and fibroblast area (defined as positive aSMA tissue) is larger at the lesion sites in the control group than at the lesion sites in the sutured group and at the nonlesion sites. The red areas in each square (field orig. mag. 400) indicate positive aSMA tissue. The mean value of the red areas in 3 fields randomly selected from each section is measured using ImageJ software to determine the neovascular and fibroblast area. D, Comparison of the neovascular and fibroblast area. On POD 7, the mean area is significantly larger at the lesion sites in the sutured and control groups than at the nonlesion sites. On PODs 14 and 21, the mean areas are significantly larger at the lesion sites in the control group than at the lesion sites in the sutured group and at the nonlesion sites; however, there are no significant differences in the mean areas between the lesion sites in the sutured group and the nonlesion sites.
DISCUSSION This in vivo porcine study demonstrated that endoscopic suturing increased the healing speed of post-ESD
gastric mucosal defects compared with the speed during the natural healing process. In the histologic and immunohistochemical evaluations, the sutured mucosal defects were completely covered with regenerated epithelium
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C Neovascular and fibroblast areas (1000 μm2)
Sutured group Non-lesion site Control group 20
P < .05
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Postoperative period (days) Figure 4. Continued
within 14 days after suturing, and the muscularis mucosae fused to each other at the same time. Furthermore, histologically, layer-to-layer healing was noted. These findings provide novel information regarding the benefit of endoscopic suturing with regard to healing at the post-ESD site and the possible prevention of postoperative adverse events. The healing process at the sutured site can be summarized as follows. Macroscopically, the sutured site remained closed without dehiscence until scar formation. Histologically, the widths of absent epithelium and absent muscularis mucosae between the mucosal rims remained narrow until POD 7. In addition, the muscularis mucosae fused to each other, and the fused area was appropriately covered by connected epithelium without inversion by POD 14. Moreover, the neovascular and fibroblast area of the submucosal layer, which plays an important role in www.giejournal.org
gastric ulcer healing, especially in the late active stage and healing stage,16-19 stabilized to the same degree as that in normal submucosa. Considering these results, we expect that a securely sutured post-ESD gastric mucosal defect will heal faster histologically compared with the healing of an intact defect, even in the clinical setting. Post-ESD gastric mucosal defects in clinical practice were reported to remain open as ulcers at 4 weeks after ESD.20,21 However, Fujimoto et al22 reported that postESD gastric mucosal defects in in vivo porcine models closed within 21 days after ESD. Therefore, the observation period in this study was set to 21 days, which was considered sufficient for the investigation of the healing process of iatrogenic mucosal defects in a porcine model. Although it is ideal to prepare 1 lesion per pig to exclude influences from other lesions, this approach would require a considerable number of animals; however, this should be avoided Volume 91, No. 5 : 2020 GASTROINTESTINAL ENDOSCOPY 1179
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as much as possible from an ethical point of view. In terms of animal protection and cost-effectiveness, we consider that our protocol is acceptable for investigating individual histologic changes in sutured mucosal defects. Kobayashi et al23 performed an in vivo animal study in which post-ESD gastric mucosal defects were sutured using OverStitch. In this previous study, the success rate of complete closure was 86% (12 of 14), median defect size was 45 mm, median procedure time of closure was 15.5 minutes, and median bites per suture applied for a lesion was 2.23 However, all 12 lesions with complete closure exhibited dehiscence on POD 7.23 Because we know that mucosal suturing for a porcine mucosal defect easily shows dehiscence according to previous findings23 and our experience,13 the mucosal defect was sutured with a broad bite (approximately 7–8 mm) in the present study, which resulted in complete closure without dehiscence. The intervals between stitches in the previous study using OverStitch and the present study using EHS were estimated as 22.5 mm (2 stitches for a 45-mm mucosal defect) and 4 mm (5 stitches for a 20-mm mucosal defect, excluding the first stitch, which acts as an anchor outside the defect), respectively. The relatively narrow intervals with EHS might have contributed to the maintenance of complete closure on POD 7. Because most endoscopic centers do not use EHS, the results of the study might not be generalizable, especially considering the fact that the type of suturing done in this study is significantly different from the currently available suturing device. During histologic evaluation, we demonstrated that the endoscopically approximated mucosal defects by EHS healed without dehiscence during the survival period. The histologic healing process noted in this study can be generalized to other closure techniques if the sutured sites are kept closed without dehiscence; however, the results might fluctuate owing to differences in depth and suture density per unit area. Some previous studies have reported that endoscopic closure for a mucosal defect after endoscopic resection reduces the risk of postoperative bleeding.6,7,24,25 However, it is relatively difficult to close gastric mucosal defects completely and keep them closed, because the thickness of the gastric wall is greater than that of other GI areas.6 Recently, the number of patients receiving antithrombotic therapy, who are considered high-risk patients with regard to postoperative bleeding after endoscopic resection,26-30 has been increasing because of the aging population.31 Discontinuation of antithrombotic agents might lead to life-threatening events, such as cerebrovascular embolism and myocardial infarction. On the other hand, acute onset anemia due to GI bleeding associated with continuation of antithrombotic use can worsen the prognosis of heart failure.32,33 Therefore, secure measures to deal with postoperative bleeding are urgently required.6 Reliable closure of post-ESD gastric mucosal defects is clinically desirable, and endoscopic suturing approaches, such as
EHS, might be promising techniques for the prevention of postoperative adverse events. Furthermore, in this study, immunohistochemical evaluations revealed that endoscopic suturing reduced the neovascular and fibroblast area compared with natural healing of post-ESD mucosal defects, suggesting that endoscopic suturing can reduce the risk of delayed bleeding directly. In the case of a positive horizontal resection margin clinically, the residual tumor is transiently inverted between the sutured rims immediately after EHS. If the epithelium of the mucosal rims after ESD followed by EHS is completely buried under the suture line during the healing process, recurrence might involve a subepithelial tumor, which can be difficult to detect and remove endoscopically. On histologic evaluation of the sutured sites in this study, layer-to-layer healing without inversion of the epithelium was confirmed. Therefore, submucosal involvement in the case of local recurrence after suturing might be prevented according to the histologic healing process of this experiment performed on healthy gastric tissues of animal models; however, this should be addressed by evaluating actual clinical outcomes. Unlike in this well-designed in vivo study, in clinical practice, it is not rare to extend dissection into the muscular layer. For further examination, it would be informative to replicate this scenario in mucosal defects with muscular damage. The present study has several limitations. First, this animal study included a small number of pigs. However, the number of lesions prepared for histologic assessment was considered sufficient. Second, this study was performed on porcine healthy gastric tissues in both the sutured and control groups. The results of this study might not be reproducible in clinical practice owing to the presence of various tissue conditions and pathologies. Third, the target sites in this study were limited to the AW and PW of the stomach. It is unclear whether the results of this study are reproducible in other parts of the stomach. Furthermore, creating multiple lesions step-by-step in a single pig might influence the results. Fourth, aSMA is specific for not only neovascularity and fibroblasts but also muscularis mucosae and all vessels related to neovascularity. The positive aSMA area defined as the neovascular and fibroblast area was measured in the middle layer of the submucosa to avoid including the muscularis mucosae. In addition, the neovascular and fibroblast areas at the lesion sites in the sutured and control groups were compared with those in nonlesion sites to adjust for the original vessels. Fifth, the operators included an endoscopist (A.T.) and an assistant (M.S.), who had experience with a large number of ESD procedures and several cases of EHS in clinical practice. In conclusion, the findings of our in vivo porcine study suggest that successful endoscopic suturing might promote the histologic healing of post-ESD gastric mucosal defects and therefore might decrease the risk of
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postoperative adverse events. The clinical usefulness of endoscopic suturing for the prevention of post-ESD bleeding should be further investigated in future studies.
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17. 18.
ACKNOWLEDGMENTS
19.
We thank Mr Isamu Shinohara and Mr Kazunori Honjo for creating the graphical abstract.
20.
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gastric fibroblasts: a new angiogenic factor for gastric ulcer healing. Biochem Biophys Res Commun 1997;234:493-8. Tarnawski A. Molecular mechanisms of ulcer healing. Drug News Perspect 2000;13:158-68. Szabo S, Vincze A. Growth factors in ulcer healing: lessons from recent studies. J Physiol Paris 2000;94:77-81. Sanchez-Fidalgo S, Martin-Lacave I, Illanes M, et al. Angiogenesis, cell proliferation and apoptosis in gastric ulcer healing. Effect of a selective cox-2 inhibitor. Eur J Pharmacol 2004;505:187-94. Yoshizawa Y, Sugimoto M, Sato Y, et al. Factors associated with healing of artificial ulcer after endoscopic submucosal dissection with reference to Helicobacter pylori infection, CYP2C19 genotype, and tumor location: multicenter randomized trial. Dig Endosc 2016;28:162-72. Otsuka T, Sugimoto M, Ban H, et al. Severity of gastric mucosal atrophy affects the healing speed of post-endoscopic submucosal dissection ulcers. World J Gastrointest Endosc 2018;10:83-92. Fujimoto A, Uraoka T, Nishizawa T, et al. Rebamipide solution: a novel submucosal injection material to promote healing speed and healing quality of ulcers induced by endoscopic submucosal dissection. Gastrointest Endosc 2018;87:1114-20. Kobayashi M, Sumiyama K, Ban Y, et al. Closure of iatrogenic large mucosal and full-thickness defects of the stomach with endoscopic interrupted sutures in in vivo porcine models: are they durable enough? BMC Gastroenterol 2015;15:5. Liaquat H, Rohn E, Rex DK. Prophylactic clip closure reduced the risk of delayed postpolypectomy hemorrhage: experience in 277 clipped large sessile or flat colorectal lesions and 247 control lesions. Gastrointest Endosc 2013;77:401-7. Zhang QS, Han B, Xu JH, et al. Clip closure of defect after endoscopic resection in patients with larger colorectal tumors decreased the adverse events. Gastrointest Endosc 2015;82:904-9. Koh R, Hirasawa K, Yahara S, et al. Antithrombotic drugs are risk factors for delayed postoperative bleeding after endoscopic submucosal dissection for gastric neoplasms. Gastrointest Endosc 2013;78:476-83. Ono S, Fujishiro M, Yoshida N, et al. Thienopyridine derivatives as risk factors for bleeding following high risk endoscopic treatments: Safe Treatment on Antiplatelets (STRAP) study. Endoscopy 2015;47:632-7. Tounou S, Morita Y, Hosono T. Continuous aspirin use does not increase post-endoscopic dissection bleeding risk for gastric neoplasms in patients on antiplatelet therapy. Endosc Int Open 2015;3:E31-8. Ueki N, Futagami S, Akimoto T, et al. Effect of antithrombotic therapy and long endoscopic submucosal dissection procedure time on early and delayed postoperative bleeding. Digestion 2017;96:21-8. So S, Ahn JY, Kim N, et al. Comparison of the effects of antithrombotic therapy on delayed bleeding after gastric endoscopic resection: a propensity score-matched case-control study. Gastrointest Endosc 2019;89:277-85 e2. Hallas J, Dall M, Andries A, et al. Use of single and combined antithrombotic therapy and risk of serious upper gastrointestinal bleeding: population based case-control study. BMJ 2006;333:726. Yamauchi T, Sakata Y, Takada T, et al. Prognostic impact of anemia in patients with chronic heart failure- with special reference to clinical background: report from the CHART-2 study. Circ J 2015;79:1984-93. Kajimoto K, Sato N, Takano T. investigators of the Acute Decompensated Heart Failure Syndromes (ATTEND) registry. Association between anemia, clinical features and outcome in patients hospitalized for acute heart failure syndromes. Eur Heart J Acute Cardiovasc Care 2015;4:568-76.
Abbreviations: aSMA, alpha-smooth muscle actin; AW, anterior wall; ESD, endoscopic submucosal dissection; EHS, endoscopic hand suturing; HE, hematoxylin and eosin; POD, postoperative day; PW, posterior wall. DISCLOSURE: Dr Goto: Consultant and paid speaker for Olympus Co, Ltd; Dr Yahagi: consultant and royalty fee from Olympus Co, Ltd. All
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other authors disclosed no financial relationships. The flexible needle holder and the scissors forceps that were used in this study were provided by Olympus Co, Ltd.
Akimoto et al Copyright ª 2020 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 https://doi.org/10.1016/j.gie.2019.12.032 Received August 20, 2019. Accepted December 6, 2019.
This video can be viewed directly from the GIE website or by using the QR code and your mobile device. Download a free QR code scanner by searching “QR Scanner” in your mobile device’s app store.
Current affiliations: Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University, School of Medicine, Tokyo (1); Department of Gastroenterology, Nippon Medical School, Tokyo, Japan (2). Reprint requests: Osamu Goto, MD, PhD, Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
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Endoscopic suturing promotes healing of mucosal defects after gastric endoscopic submucosal dissection: endoscopic and histologic analyses in in vivo porcine models (with video) Teppei Akimoto, MD,1,2 Osamu Goto, MD, PhD,1,2 Motoki Sasaki,1 Mari Mizutani, MD,1 Koshiro Tsutsumi, MD,1 Yoshiyuki Kiguchi, MD,1 Atsushi Nakayama, MD, PhD,1 Motohiko Kato, MD, PhD,1 Ai Fujimoto, MD, PhD,1 Yasutoshi Ochiai, MD, PhD,1 Tadateru Maehata, MD, PhD,1 Mitsuru Kaise, MD, PhD,2 Katsuhiko Iwakiri, MD, PhD,2 Naohisa Yahagi, MD, PhD1 Tokyo, Japan
GRAPHICAL ABSTRACT
Background and Aims: Endoscopic suturing of mucosal defects after endoscopic submucosal dissection (ESD) is expected to prevent postoperative adverse events. We aimed to endoscopically and histologically evaluate the healing process of post-ESD mucosal defects closed with endoscopic hand suturing (EHS) in in vivo porcine models. Methods: Twelve mucosal defects (2 cm in size) were created in 2 pigs (6 defects per pig). Initially, 2 defects were created: one was closed with EHS (sutured group) and the other was kept open (control group). On postoperative days (PODs) 7 and 14, 2 additional defects were created in each session, and they were treated in the same manner as in the initial procedure. On POD 21, the entire stomach, with the 6 lesion sites, was extracted for histologic evaluation after endoscopic observation. Results: Endoscopically, all sutured sites remained closed in all sessions (PODs 7, 14, and 21). Histologically, on POD 14, the epithelium and muscularis mucosae were appropriately connected. The mucosae were covered with the epithelium without inversion of the mucosal edge in the sutured group, whereas the ulcer bed was exposed in the control group. Furthermore, the degree of neovascularity and fibroblasts in the submucosa was smaller in the sutured group than that in the control group. Conclusions: Our findings suggest that endoscopic suturing promotes healing of post-ESD mucosal defects histologically in in vivo porcine models. Thus, endoscopic mucosal closure after ESD might be clinically useful for the prevention of delayed perforation/bleeding if secure suturing is performed endoscopically. (Gastrointest Endosc 2020;91:1172-82.)
(footnotes appear on last page of article)
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INTRODUCTION Endoscopic submucosal dissection (ESD) is gaining acceptance as one of the curative treatment options for early gastric neoplasms because of its minimal invasiveness and high possibility of complete resection.1-3 However, critical postoperative adverse events associated with ESD, which primarily include delayed bleeding and delayed perforation, sometimes occur even with proper management strategies, such as administration of proton-pump inhibitors and prophylactic hemostasis of vessels in mucosal defects immediately after ESD using hemostatic forceps.4,5 A possible reason for these postoperative adverse events is that post-ESD large mucosal defects remain open.6 Therefore, several researchers have endoscopically closed post-ESD gastric mucosal defects using endoclips, endoloops, or over-the-scope clips.7-10 Furthermore, endoscopic suturing techniques involving a needle and suture thread, such as Overstitch (Apollo Endosurgery Inc, Austin, Tex, USA), have been developed to achieve robust closure.11 We have also developed a suturing technique involving a through-the-scope needle holder and absorbable suture thread attached to a curved needle, which is called endoscopic hand suturing (EHS).12,13 However, it has not been fully investigated whether these suturing techniques promote the healing of postESD mucosal defects and contribute to the prevention of delayed adverse events. In this study, we assessed the healing speed and histologic healing process of endoscopically sutured post-ESD mucosal defects in in vivo porcine models and compared the findings with those of natural healing of post-ESD mucosal defects.
METHODS Preparation of animal models This study was approved by the institutional review board of our animal laboratory (approval no. 17060-(0)). Endoscopic procedures were performed with the animals under general anesthesia in 2 female pigs (Göttingen minipig; Oriental Yeast Co, Ltd, Tokyo, Japan; body weights of 20.6 and 23.8 kg). After environmental acclimation for 7 days, the pigs were fasted for 24 hours before the endoscopic procedures. Initially, 0.02 mg/kg medetomidine and 0.1 mg/kg midazolam were injected intramuscularly, and then an intravenous line was placed in an ear vein. Subsequently, a tracheal intubation tube was inserted and inhalation anesthesia was performed with 1.5% to 2.5% isoflurane. Then, an overtube was placed and the endoscopic procedures were performed. The pigs were allowed to drink water and eat soft feed a day after the endoscopic procedures.
Endoscopic submucosal dissection According to the experiment protocol described later in this section, a double-channel endoscope with www.giejournal.org
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multi-bending function and a high-vision endoscope (GIF-2TQ260M and GIF-H260Z; Olympus Co, Ltd, Tokyo, Japan) were used for endoscopic treatment and endoscopic observation, respectively. The settings of the highfrequency power unit (ESG-100; Olympus) during ESD were the pulse cut first mode (30 W) for mucosal incision and the forced coagulation 2 mode (30 W) for mucosal marking and submucosal dissection. For marking the experiment sites on the stomach, a plastic disciform plate (2 cm in diameter) was placed at the target areas and marks were made around the plate using the tip of a dual knife (KD650-L; Olympus). ESD was performed conventionally using the dual knife, an injection needle (NM-610L-0425; Olympus), and a solution of 10% glycerin with 5% fructose plus 0.9% saline solution (Glyceol; Chugai Pharmaceutical Co, Ltd, Tokyo, Japan).
Endoscopic hand suturing Mucosal closure by EHS was performed for the mucosal defect as described previously.12,13 Briefly, a prototype of the through-the-scope needle holder (FG-Y0015; Olympus) and a curved needle with absorbable barbed suture (VLOCL0604; Covidien, Dublin, Ireland) were used as follows (Video 1, available online at www.giejournal.org). The curved needle was delivered to the stomach, grasping the suture at approximately 5 mm from the tail of the needle with the needle holder. In the stomach, the suture was released and the curved needle was grasped at one-third from the tail. The first stitch was placed on the distal mucosa outside the mucosal defect. Then, the needle was passed through a ring attached to the end of the suture, which acted as an anchor. Subsequently, the mucosal rim of the defect was sutured with the running suture technique from the distal to proximal end with an interval of less than 5 mm between 2 adjacent stitches and a width of approximately 7 to 8 mm for the suture bites.13 During EHS, grasping forceps (FG-21L-1; Olympus) advanced through another working channel were used to regrasp the needle and tighten the suture. After complete closure, a hemoclip (HX-610-090S; Olympus) was placed with the suture on the mucosa near the final stitch. Eventually, the suture was cut with a loop cutter (FS-5L-1; Olympus) and was extracted outside the stomach along with the needle.
Experiment protocol According to a previous study,14 to obtain multiple histologic specimens at different observation periods from a small number of pigs, this animal study included 4 sessions. The first session involved the initial operation. The second, third, and fourth sessions were performed on postoperative days (PODs) 7, 14, and 21, respectively. In one pig, experiments were performed as described here and in Figure 1A. In the first session, 2 mucosal defects of 2 cm were created using the ESD technique at Volume 91, No. 5 : 2020 GASTROINTESTINAL ENDOSCOPY 1173
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Observation Observation 3. ESD 4. ESD+EHS
Observation 5. ESD 6. ESD+EHS Sacrifice
Day 0
Day 7
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Anterior wall
group, and the open mucosal defects were included in a control group. In the fourth session, all 6 lesions (3 in the sutured group and 3 in the control group) were observed and measured. Subsequently, the pig was killed and the stomach was extracted. Chronologic endoscopic views (Fig. 2A) and tissue specimens of the sutured and control groups were obtained from this pig. Conversely, in the other pig, open mucosal defects (control group) and sutured mucosal defects (sutured group) were created on the PW and AW, respectively.
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1
the lower body of the stomach (one on the anterior wall [AW] and another on the posterior wall [PW]). The mucosal defect on the PW was closed by EHS (Fig. 1B), whereas the mucosal defect on the AW was kept open (Fig. 1B). In the second and third sessions, the suture site and mucosal defects that were created in the previous session were observed, and the defect size was measured using the 2-cm plastic disciform plate. Subsequently, a sutured mucosal defect and an open defect were newly created on the PW and AW, respectively, of both the upper body (second session) and middle body (third session), similar to the approach in the first session. The sutured mucosal defects were included in a sutured
With regard to the technical outcomes of EHS, the following parameters were evaluated: the success rate of complete closure using EHS immediately after ESD, procedure time of EHS (time from the first stitch to complete closure), bites per suture applied for a mucosal defect, suturing speed (minutes per stitch), and proportion of complete closure without dehiscence on POD 7. With regard to endoscopic evaluation, the main outcome measures were the healing speed of mucosal defects after ESD and that of endoscopically sutured mucosal defects. To evaluate the macroscopic healing speed, the endoscopic widths of the mucosal defects at all sessions were measured. When the mucosal defect was replaced with scar tissue or was completely closed without dehiscence, the endoscopic width of the mucosal defect was recorded as zero. When the sutured mucosal defect exhibited dehiscence, the maximum distance between the mucosal rims was recorded as the endoscopic width of the mucosal defect. The mean endoscopic widths of the mucosal defects on PODs 7, 14, and 21 were compared between the sutured and control groups. For histologic and immunohistochemical evaluations, the lesion sites in the sutured and control groups were fixed in 10% neutral buffered formalin. Then, slices obtained from the center of the tissues were embedded in paraffin, cut into 4-mm-thick sections, and stained with hematoxylin and eosin (HE) and alpha-smooth muscle actin (aSMA) antibody. To evaluate histologic and immunohistochemical healing, the width of absent epithelium between mucosal rims in HE-stained sections and the width of absent muscularis mucosae between mucosal rims in aSMA-stained sections were measured. In addition, the neovascular and fibroblast area was assessed and defined as the area of positive aSMA in the submucosa at the lesion site. The area of positive aSMA was measured in 3 randomly selected fields (original magnification 400) in the middle layer of the submucosa under the lesion site for each section, using ImageJ software (National Institutes of Health, Bethesda, Md, USA) with the immunohistochemistry profiler plugin.15 In addition, the area of positive aSMA in normal submucosa (nonlesion sites) was assessed and measured at the marginal site in specimens from the sutured group. The widths of absent epithelium and absent muscularis mucosae on PODs 7, 14, and 21 were
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Oral side
Anal side 2
4 6
A
Posterior wall
B Figure 1. Study protocol and endoscopic images of the lesions. A, Time course of the study and schema of the lesion sites in the first pig. Pairs of mucosal defects (anterior wall) and sutured mucosal defects (posterior wall) are created at the lower body (1 and 2), upper body (3 and 4), and middle body (5 and 6) of the stomach on the first day, postoperative day (POD) 7, and POD 14, respectively. On POD 21, the 3 mucosal defects and 3 sutured defects are observed and extracted for histology. Conversely, in the other pig, mucosal defects and sutured mucosal defects are created on the posterior wall and anterior wall, respectively. B, An endoscopic view of a mucosal defect and a sutured defect immediately after each procedure. ESD, Endoscopic submucosal dissection; EHS, endoscopic hand suturing.
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Figure 2. Endoscopic evaluation. A, Changes in endoscopic images in both groups (lower body of the anterior wall). The mucosal defect is open in the control group on postoperative day (POD) 21, whereas it has closed without dehiscence in the sutured group at the same time point. B, Comparison of endoscopically measured widths of the mucosal defects. The mean widths are significantly shorter in the sutured group than in the control group on PODs 7 and 14.
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25 Sutured group Control group 20
P < .05
15
15.0 2.6 mm, 6.3 2.8 mm, and 2.0 2.0 mm, respectively, in the control group. There were significant differences between the 2 groups on PODs 7 and 14 (P Z .0027 and P Z .020, respectively). All sutured mucosal defects remained closed during the observation period from POD 7 to POD 21.
Histologic and immunohistologic evaluations
10
The chronologic endoscopic widths of the mucosal defects in the sutured and control groups are presented in Figure 2B. The mean endoscopic widths of the mucosal defects on PODs 7, 14, and 21 (n Z 6, 4, and 2, respectively, in each group) were 0 0 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and
The histologic findings regarding the epithelium are presented in Figure 3. The mean widths of absent epithelium on PODs 7, 14, and 21 (n Z 2, 2, and 2, respectively, in each group) were 0.88 0.30 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and 10.7 2.2 mm, 4.2 3.5 mm, and 1.1 1.1 mm, respectively, in the control group. On POD 7, the mean width of absent epithelium was significantly shorter in the sutured group than in the control group (P Z .023). However, there was no significant difference in the mean width between the 2 groups on PODs 14 and 21 (P Z .18 and P Z .21, respectively). With regard to the morphology of the sutured sites, the muscularis mucosae fused to each other, and the fused area was appropriately covered by connected epithelium without inversion on PODs 14 and 21. The immunohistochemical findings regarding the muscularis mucosae and neovascular and fibroblast area are presented in Figure 4. The mean widths of absent muscularis mucosae on PODs 7, 14, and 21 (n Z 2, 2, and 2, respectively, in each group) were 2.8 0.7 mm, 0 0 mm, and 0 0 mm, respectively, in the sutured group and 13.2 2.6 mm, 8.2 2.9 mm, and 7.5 1.0 mm, respectively, in the control group (Fig. 4B). All the widths of absent muscularis mucosae were shorter in the sutured group than in the control group with significant differences on PODs 7 and 21 (P Z .030 and P Z .0093, respectively) and without a significant difference on POD 14 (P Z .055). The mean neovascular and fibroblast areas on PODs 7, 14, and 21 (n Z 6, 6, and 6, respectively, in each group) were 4573 1881 mm2, 3600 1875 mm2, and 2319 841 mm2, respectively, at the lesion sites in the sutured group; 4672 2289 mm2, 11722 1694 mm2, and 13831 4504 mm2, respectively, at the lesion sites in the control group; and 1860 448 mm2, 2219 856 mm2, and 2012 637 mm2, respectively, at the nonlesion sites (Fig. 4D). On POD 7, the mean neovascular and fibroblast area was significantly larger at the lesion sites in the sutured and control groups than at the nonlesion sites (P Z .021 and P Z .026, respectively). On PODs 14 and 21, the mean neovascular and fibroblast areas were significantly smaller at the lesion sites in the sutured group than at the lesion sites in the control group (P < .0001 and P < .0001, respectively). Furthermore, there were no significant differences in the mean neovascular and fibroblast area between the lesion sites in the sutured group and the nonlesion sites on PODs 14 and 21 (P Z .18 and P Z .86, respectively).
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Postoperative period (days) Figure 2. Continued.
compared between the sutured and control groups. The neovascular and fibroblast areas on PODs 7, 14, and 21 were compared among the lesion sites in the sutured and control groups and the nonlesion sites. Furthermore, the histologic morphology of the mucosal layer in the sutured group was assessed.
Statistical analysis Data regarding the technical outcomes of EHS are expressed as medians or frequencies (proportions), and other data are expressed as means. The data were compared between 2 groups using the Student t test, and all analyses were performed using JMP version 14.0 (SAS Institute Inc, Cary, NC, USA). Statistical significance was set at a P value <.05.
RESULTS Technical outcomes of EHS In the sutured group, the success rate of complete closure immediately after ESD and the rate of complete closure without dehiscence on POD 7 were 100% (6 of 6) and 100% (6 of 6), respectively. The median procedure time of EHS was 26.5 minutes (range, 24-32 minutes), the median bites per suture applied to a lesion was 6 (range, 6-7), and the median time per stitch was 4.4 minutes (range, 3.6-5.3 minutes).
Endoscopic evaluation
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A
Histological widths of absent epithetlium (mm)
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C
15 Sutured group Control group P < .05 10
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Figure 3. Histologic evaluation of hematoxylin and eosin-stained sections. A, Chronologic variation of the lesion areas in the sutured and control groups. Arrows indicate the width of absent epithelium between the rims of each mucosal defect. The width of absent epithelium is measured in each section. B, On postoperative day (POD) 7 (left), there is a narrow section of absent epithelium between the inverted mucosal rims in the sutured group. On POD 14 (right), regenerated epithelium completely covers the mucosal defect. C, Comparison of the width of absent epithelium between the sutured and control groups. On POD 7, the mean width of absent epithelium is significantly shorter in the sutured group than in the control group. On POD 21, the surface of the mucosal defect is not completely covered with epithelium in the control group.
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Widths of absent muscularis mucosae (mm)
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Figure 4. Immunohistochemical evaluation of alpha-smooth muscle actin (aSMA)-stained sections. A, Chronologic variation of the lesion areas in the sutured and control groups. Arrows indicate the width of absent muscularis mucosae between the rims of each mucosal defect. B, Comparison of the width of absent muscularis mucosae between the sutured and control groups. On postoperative day (POD) 14, the muscularis mucosae in the sutured group appear to fuse. C, Magnified views of the middle submucosal layer at the lesion sites in the 2 groups and at the nonlesion site on POD 14. The mean neovascular and fibroblast area (defined as positive aSMA tissue) is larger at the lesion sites in the control group than at the lesion sites in the sutured group and at the nonlesion sites. The red areas in each square (field orig. mag. 400) indicate positive aSMA tissue. The mean value of the red areas in 3 fields randomly selected from each section is measured using ImageJ software to determine the neovascular and fibroblast area. D, Comparison of the neovascular and fibroblast area. On POD 7, the mean area is significantly larger at the lesion sites in the sutured and control groups than at the nonlesion sites. On PODs 14 and 21, the mean areas are significantly larger at the lesion sites in the control group than at the lesion sites in the sutured group and at the nonlesion sites; however, there are no significant differences in the mean areas between the lesion sites in the sutured group and the nonlesion sites.
DISCUSSION This in vivo porcine study demonstrated that endoscopic suturing increased the healing speed of post-ESD
gastric mucosal defects compared with the speed during the natural healing process. In the histologic and immunohistochemical evaluations, the sutured mucosal defects were completely covered with regenerated epithelium
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C Neovascular and fibroblast areas (1000 μm2)
Sutured group Non-lesion site Control group 20
P < .05
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Postoperative period (days) Figure 4. Continued
within 14 days after suturing, and the muscularis mucosae fused to each other at the same time. Furthermore, histologically, layer-to-layer healing was noted. These findings provide novel information regarding the benefit of endoscopic suturing with regard to healing at the post-ESD site and the possible prevention of postoperative adverse events. The healing process at the sutured site can be summarized as follows. Macroscopically, the sutured site remained closed without dehiscence until scar formation. Histologically, the widths of absent epithelium and absent muscularis mucosae between the mucosal rims remained narrow until POD 7. In addition, the muscularis mucosae fused to each other, and the fused area was appropriately covered by connected epithelium without inversion by POD 14. Moreover, the neovascular and fibroblast area of the submucosal layer, which plays an important role in www.giejournal.org
gastric ulcer healing, especially in the late active stage and healing stage,16-19 stabilized to the same degree as that in normal submucosa. Considering these results, we expect that a securely sutured post-ESD gastric mucosal defect will heal faster histologically compared with the healing of an intact defect, even in the clinical setting. Post-ESD gastric mucosal defects in clinical practice were reported to remain open as ulcers at 4 weeks after ESD.20,21 However, Fujimoto et al22 reported that postESD gastric mucosal defects in in vivo porcine models closed within 21 days after ESD. Therefore, the observation period in this study was set to 21 days, which was considered sufficient for the investigation of the healing process of iatrogenic mucosal defects in a porcine model. Although it is ideal to prepare 1 lesion per pig to exclude influences from other lesions, this approach would require a considerable number of animals; however, this should be avoided Volume 91, No. 5 : 2020 GASTROINTESTINAL ENDOSCOPY 1179
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as much as possible from an ethical point of view. In terms of animal protection and cost-effectiveness, we consider that our protocol is acceptable for investigating individual histologic changes in sutured mucosal defects. Kobayashi et al23 performed an in vivo animal study in which post-ESD gastric mucosal defects were sutured using OverStitch. In this previous study, the success rate of complete closure was 86% (12 of 14), median defect size was 45 mm, median procedure time of closure was 15.5 minutes, and median bites per suture applied for a lesion was 2.23 However, all 12 lesions with complete closure exhibited dehiscence on POD 7.23 Because we know that mucosal suturing for a porcine mucosal defect easily shows dehiscence according to previous findings23 and our experience,13 the mucosal defect was sutured with a broad bite (approximately 7–8 mm) in the present study, which resulted in complete closure without dehiscence. The intervals between stitches in the previous study using OverStitch and the present study using EHS were estimated as 22.5 mm (2 stitches for a 45-mm mucosal defect) and 4 mm (5 stitches for a 20-mm mucosal defect, excluding the first stitch, which acts as an anchor outside the defect), respectively. The relatively narrow intervals with EHS might have contributed to the maintenance of complete closure on POD 7. Because most endoscopic centers do not use EHS, the results of the study might not be generalizable, especially considering the fact that the type of suturing done in this study is significantly different from the currently available suturing device. During histologic evaluation, we demonstrated that the endoscopically approximated mucosal defects by EHS healed without dehiscence during the survival period. The histologic healing process noted in this study can be generalized to other closure techniques if the sutured sites are kept closed without dehiscence; however, the results might fluctuate owing to differences in depth and suture density per unit area. Some previous studies have reported that endoscopic closure for a mucosal defect after endoscopic resection reduces the risk of postoperative bleeding.6,7,24,25 However, it is relatively difficult to close gastric mucosal defects completely and keep them closed, because the thickness of the gastric wall is greater than that of other GI areas.6 Recently, the number of patients receiving antithrombotic therapy, who are considered high-risk patients with regard to postoperative bleeding after endoscopic resection,26-30 has been increasing because of the aging population.31 Discontinuation of antithrombotic agents might lead to life-threatening events, such as cerebrovascular embolism and myocardial infarction. On the other hand, acute onset anemia due to GI bleeding associated with continuation of antithrombotic use can worsen the prognosis of heart failure.32,33 Therefore, secure measures to deal with postoperative bleeding are urgently required.6 Reliable closure of post-ESD gastric mucosal defects is clinically desirable, and endoscopic suturing approaches, such as
EHS, might be promising techniques for the prevention of postoperative adverse events. Furthermore, in this study, immunohistochemical evaluations revealed that endoscopic suturing reduced the neovascular and fibroblast area compared with natural healing of post-ESD mucosal defects, suggesting that endoscopic suturing can reduce the risk of delayed bleeding directly. In the case of a positive horizontal resection margin clinically, the residual tumor is transiently inverted between the sutured rims immediately after EHS. If the epithelium of the mucosal rims after ESD followed by EHS is completely buried under the suture line during the healing process, recurrence might involve a subepithelial tumor, which can be difficult to detect and remove endoscopically. On histologic evaluation of the sutured sites in this study, layer-to-layer healing without inversion of the epithelium was confirmed. Therefore, submucosal involvement in the case of local recurrence after suturing might be prevented according to the histologic healing process of this experiment performed on healthy gastric tissues of animal models; however, this should be addressed by evaluating actual clinical outcomes. Unlike in this well-designed in vivo study, in clinical practice, it is not rare to extend dissection into the muscular layer. For further examination, it would be informative to replicate this scenario in mucosal defects with muscular damage. The present study has several limitations. First, this animal study included a small number of pigs. However, the number of lesions prepared for histologic assessment was considered sufficient. Second, this study was performed on porcine healthy gastric tissues in both the sutured and control groups. The results of this study might not be reproducible in clinical practice owing to the presence of various tissue conditions and pathologies. Third, the target sites in this study were limited to the AW and PW of the stomach. It is unclear whether the results of this study are reproducible in other parts of the stomach. Furthermore, creating multiple lesions step-by-step in a single pig might influence the results. Fourth, aSMA is specific for not only neovascularity and fibroblasts but also muscularis mucosae and all vessels related to neovascularity. The positive aSMA area defined as the neovascular and fibroblast area was measured in the middle layer of the submucosa to avoid including the muscularis mucosae. In addition, the neovascular and fibroblast areas at the lesion sites in the sutured and control groups were compared with those in nonlesion sites to adjust for the original vessels. Fifth, the operators included an endoscopist (A.T.) and an assistant (M.S.), who had experience with a large number of ESD procedures and several cases of EHS in clinical practice. In conclusion, the findings of our in vivo porcine study suggest that successful endoscopic suturing might promote the histologic healing of post-ESD gastric mucosal defects and therefore might decrease the risk of
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postoperative adverse events. The clinical usefulness of endoscopic suturing for the prevention of post-ESD bleeding should be further investigated in future studies.
Endoscopic suturing promotes healing of mucosal defects
17. 18.
ACKNOWLEDGMENTS
19.
We thank Mr Isamu Shinohara and Mr Kazunori Honjo for creating the graphical abstract.
20.
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Abbreviations: aSMA, alpha-smooth muscle actin; AW, anterior wall; ESD, endoscopic submucosal dissection; EHS, endoscopic hand suturing; HE, hematoxylin and eosin; POD, postoperative day; PW, posterior wall. DISCLOSURE: Dr Goto: Consultant and paid speaker for Olympus Co, Ltd; Dr Yahagi: consultant and royalty fee from Olympus Co, Ltd. All
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Endoscopic suturing promotes healing of mucosal defects
other authors disclosed no financial relationships. The flexible needle holder and the scissors forceps that were used in this study were provided by Olympus Co, Ltd.
Akimoto et al Copyright ª 2020 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 https://doi.org/10.1016/j.gie.2019.12.032 Received August 20, 2019. Accepted December 6, 2019.
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Current affiliations: Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University, School of Medicine, Tokyo (1); Department of Gastroenterology, Nippon Medical School, Tokyo, Japan (2). Reprint requests: Osamu Goto, MD, PhD, Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
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