A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Summary

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.  The detailed setup methodology of this new model is explained.

Abstract

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen's ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

Introduction

Both endoscopic submucosal dissection (ESD) and endoscopic mucosal resection (EMR) are currently common treatments for early-stage gastrointestinal cancer^1,2. Injecting a submucosal injection material (SIM) into the submucosa is one of the most important steps for both the EMR and ESD procedures^2,3. High submucosal elevation and maintenance of submucosal elevation are critical criteria for safely conducting EMR/ESD.

Although normal saline (NS) has been used as a SIM since the invention of endoscopic therapy^4,5, sodium hyaluronate (HA) was introduced as a treatment in recent years^6,7. HA became widely used in endoscopic treatments as a superior SIM due to its high performance^8,9,10,11. Currently, a performance comparison between the existing SIMs was conducted, and high-performance SIMs were developed to identify another superior SIM^{5,12,13,14,15,16,17,18}.

The ex vivo model using a porcine stomach specimen has been used to evaluate SIM performance, because the estimation of SIM performance in the human gastrointestinal tract is very difficult^19,20,21,22. However, this conventional ex vivo model is extremely simple, and has the scope for improvement. Reproducing an environment closer to the human gastrointestinal mucosa will enable accurate evaluation of SIM performance.

In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen's ends. We also confirmed that the proposed new ex vivo model, allows accurate SHE measurement under uniform conditions and a detailed comparison of the performances of various types of SIMs²³.

In this study, we present a complete appearance of the new ex vivo model, and the detailed setup methodology of the new ex vivo model is explained in detail using videos and figures. The new ex vivo model consists of parts that are easily available and can be quickly set up. Descriptions of detailed setup methodology will contribute to the dissemination of the new model.

Protocol

The following protocol follows the animal care guidelines of the Kyoto Prefectural University of Medicine.

1. Preparation of Specimens Using a Porcine Stomach

NOTE: The first step is to prepare specimens to be used in the ex vivo model (Figure 1). The thickness of the porcine gastric wall varies in different areas of the stomach. Use the upper third of the porcine stomach, which is relatively similar to the human stomach. Exclude inappropriate specimens where submucosal elevation is not found due to fibrosis.

1. Cut gastric specimens into squares with approximate dimensions of 6 × 6 cm.
2. Store the gastric specimens immediately at a temperature of -30 °C.
3. Thaw frozen gastric specimens right before the measurement procedure to ensure uniform measurement conditions.

2. Detailed Setup Methodology of a New Ex Vivo Model

NOTE: Stretch out the thawed specimen on a board in two different ways. In the conventional ex vivo model, fix the specimen with pins (Figure 1A)^19,20,21,22. On the other hand, in the new ex vivo model, fix or stretch both ends of the specimen with clips to produce a constant tension (Figure 1B, C). All parts of the new model are easy to obtain, and the setup of the new model can be completed quickly (Figure 2). The procedure of the new model is as follows (Figure 3):

1. Connect the stainless-steel clip and the key wire and the S shaped hook (Figure 3A).
2. Connect the wire and the S shaped hook and the weight (Figure 3A).
3. Connect the hook to the other end of the wire. A traction device is completed in the above process (Figure 3B).
4. Fix the pulleys (Figure 2b) at both ends of the base (Figure 3C).
5. Place the rubber plate (6 x 6 cm) on the center of the base (Figure 3C).
6. Place the gastric specimen on the rubber plate and pinch the specimen ends with the clip of the traction device.
7. Hang the weight through the pulley (both side). Thereby, constant tension can be applied to the specimen (Figure 4).
8. Start the measurement of SEH, because after the setup of the new model is completely finished (See Step 3 below).

3. Evaluation of SIM Performance

NOTE: In this study, we used normal saline (NS) and 0.4% sodium hyaluronate (HA) as SIMs to be tested, and measure SEH of the two SIMs. Three independent measurements are performed. The obtained data are expressed as the mean and standard deviation (S.D.). Statistical analysis was performed by using the statistical analysis software (GraphPad Prism 7). We analyzed continuous variables (SEH) with the Student's t-test, and the magnitudes with P < 0.05 were considered significant. The measurement of SEH is as follows (Figure 5).

1. Perform zero-point adjustment of the height gauge, based on the height of mucosa before a submucosal elevation procedure. In detail, perform zero-point adjustment by pushing the PRESET button after fixing the scriber at the height of the mucosal surface.
2. Inject 2.0 mL of each solution horizontally into the submucosa from the specimen margins using a 2.5-mL syringe and 23-gauge needle, to perform a submucosal elevation procedure (Figure 5A-C).
3. Measure SEH promptly using a digital height gage at 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 30, 45, and 60 min after the injection (Figure 5D). In detail, record the height displayed on the height gauge when fixing the scriber to the top of the submucosal elevation.
4. Perform three independent measurements, and express the obtained results as the mean and standard deviation.
5. Analyze the obtained data using appropriate statistical software and evaluate the performance of SIMs (The performance can be compared between each SIM.)

Results

SEH was measured over time in the new ex vivo model or conventional ex vivo model. The values of SEH (NS) measured using the conventional model [NS was injected into the submucosa of the specimen fixed with pins (0.0 N)] were 5.7 mm (0 min), 3.6 mm (5 min), 3.0 mm (10 min), and 2.2 mm (30 min).In this way, the values of SEH decreased with increasing post injection time.A similar analysis was performed using 0.4% HA instead of NS. The values of SEH (0.4% HA) were 6.5 mm (...

Discussion

The porcine stomach used for the new model should be stored in a freezer immediately after resection, and be used within a few months after freezing, since the freshness of the swine stomach is essential for SEH measurement. (We measured SEH using both frozen and unfrozen gastric specimens, and confirmed that there was no difference in the result of SEH measurement.)

The quality of gastric specimens is greatly influenced by the individual differences of porcine stomachs. Hence, it is...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
weight (153.1 g)
fixed type pulley	H.H.H. MANUFACTURING	VS25
stainless steel wire with a diameter of 0.45 mm	Nissa Chain	Cut wire Y-5
stainless steel clip of width 147 mm	KOKUYO	none
stainless steel key wire with a length of 12 cm	Nissa Chain	P-702
stainless steel S shaped hook	TRUSCO NAKAYAMA	TCS1.2
lockable stainless steel S-shaped hook	Mizumoto Machine Mfg	B2054
rectangular wooden base (45 x 60 cm)	none	none
rubber plate (5 x 5 cm)	none	none
digital height gage	Mitutoyo	HDS-20C
2.5-mL syringe	Terumo	SS-02SZ
23-gauge needle	Terumo	NN-2332R
MucoUp	Boston Scientific	none	0.4% sodium hyaluronate (HA)
saline (20 mL)	Otsuka Pharmaceutical	none	normal saline (NS)
GraphPad Prism 7 software	GraphPad Inc	none