Name: a new ex vivo model for the evaluation of endoscopic submucosal injection material performance
Uploaded: 2018-10-19T13:30:00.000+00:00
Duration: 3 min 54 s
Description: Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

This work was supported by Kyoto Innovative Medical Technology Research &amp; Development Support System, and by the Translational Research program; Strategic PRomotion for practical application of INnovative medical Technology (TR-SPRINT) from Japan Agency for Medical Research and Development (AMED).

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.
<ol>
	<li>Cut&#160;gastric specimens into squares with approximate dimensions of 6&#160;&#215; 6&#160;cm.</li>
	<li>Store the gastric specimens immediately at a temperature of -30 &#176;C.</li>
	<li>Thaw frozen gastric specimens right before the measurement procedure to ensure uniform measurement conditions.</li>
</ol>
2. Detailed Setup Methodology of a New Ex Vivo Model
NOTE: Stretch out the thawed specimen&#160;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):
<ol>
	<li>Connect the stainless-steel clip and&#160;the key wire and the S shaped hook (Figure 3A).</li>
	<li>Connect the wire and&#160;the S shaped hook and the weight (Figure 3A).</li>
	<li>Connect the hook to the other end of the wire. A traction device is completed in the above process (Figure 3B).</li>
	<li>Fix the pulleys (Figure 2b) at both ends of the base (Figure 3C).</li>
	<li>Place the rubber plate (6&#160;x 6&#160;cm) on the center of the base (Figure 3C).</li>
	<li>Place the gastric specimen on the rubber plate and pinch the specimen ends with the clip of the traction device.</li>
	<li>Hang the weight through the pulley (both side). Thereby, constant tension can be applied to the specimen (Figure 4).</li>
	<li>Start the measurement of SEH, because&#160;after the setup of the new model is completely finished (See Step 3 below).</li>
</ol>
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&#39;s t-test, and the magnitudes with P &#60; 0.05 were considered significant. The measurement of SEH is as follows (Figure 5).
<ol>
	<li>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.</li>
	<li>Inject 2.0 mL of each solution horizontally into the submucosa from the specimen margins using a 2.5-mL syringe and 23-gauge&#160;needle, to perform a submucosal elevation procedure (Figure 5A-C).</li>
	<li>Measure&#160;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.</li>
	<li>Perform three independent measurements, and express the obtained results as the mean and standard deviation.</li>
	<li>Analyze the obtained data using appropriate statistical software and evaluate the performance of SIMs (The performance can be compared between each SIM.)</li>
</ol>

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The authors have nothing to disclose.

The porcine stomach used for the new model should be stored in a freezer immediately after&#160;resection, and be used within a few months after freezing, since the freshness of the swine stomach is essential for SEH measurement. (We measured&#160;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 recommended to exclude obviously thick specimens or specimens with many folds before&#160;measurement. Furthermore, some specimens may be inappropriate specimens for SEH measurement due to fibrosis. It is recommended to exclude the inappropriate specimens where submucosal elevation is not found due to fibrosis.
Since the digestive tract is expanded by endoscopic treatment, some tension is applied to the gastrointestinal mucosa. It was revealed that SIM performance (evaluated by measuring the values of SEH) decreased with increasing the values of tension applied to the specimens. Therefore, the tension was an important factor affecting the SIM performance (i.e., the values of SEH)23. The application of&#160;tension of 1.5-3.0 N&#160;can reproduce an environment closer to the human gastrointestinal mucosa. However, a limitation of this method is that the optimal tension may depend on the difference of the specimen used for analysis.
In the conventional model, since the tension applied to each specimen varies depending on the degree of specimen fixation, the variations of measured SEH are large (which correspond to the high standard deviations of SEH). Therefore, these high standard deviations make it difficult to compare each SEH in detail and perform statistical analysis. On the other hand, owing to small variations of SEH measured in the new model, SIM performance can be compared accurately ex vivo and precise statistical analysis is performed.
In conclusion, the new ex vivo model enables accurate SEH measurement and detailed comparison of SIM performance. Descriptions of detailed setup methodology will contribute to the dissemination of the new model and the development of high-performance materials.

Both endoscopic submucosal dissection (ESD) and endoscopic mucosal resection (EMR) are currently common treatments for early-stage gastrointestinal cancer1,2. Injecting a submucosal injection material (SIM) into the submucosa is one of the most important steps for both the EMR and ESD procedures2,3. 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 therapy4,5, sodium hyaluronate (HA) was introduced as a treatment in recent years6,7. HA became widely used in endoscopic treatments as a superior SIM due to its high performance8,9,10,11. Currently, a performance comparison between the existing SIMs was conducted, and high-performance SIMs were developed to identify another superior SIM5,12,13,14,15,16,17,18.
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 difficult19,20,21,22. 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&#39;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&#160;new ex vivo model consists of parts that are&#160;easily available and can be quickly set up. Descriptions of detailed setup methodology will contribute to the dissemination of the new model.

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

a new ex vivo model for the evaluation of endoscopic submucosal injection material performance

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&#39;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.

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 (0 min), 5.2 mm (5 min), 4.8 mm (10 min), and 4.1 mm (30 min).The resulting SEHs of 0.4% HA were higher than those of NS regardless of the post injection time.The SEHs (NS and 0.4% HA) obtained using the conventional model (in the absence of the applied tension) exhibited relatively large variations (in other words, their standard deviations were high) (Figure 6A).
Next, the values of SEH (NS) measured using the conventional model [NS was injected into the submucosa of the specimen stretched at a constant tension (1.5 N)] were 4.8 mm (0 min), 3.0 mm (5 min), 2.4 mm (10 min), and 1.8 mm (30 min). When the tension was increased to 3.0 N under the same conditions, the values of SEH (NS) were 4.5 mm (0 min), 2.3 mm (5 min), 1.5 mm (10 min), and 1.3 mm (30 min).The SEH measured at various post injection times decreased with increasing tension. The SEHs obtained using the new model exhibited small variations (in other words, their standard deviations were low) (Figure 6B, C).
For evaluating the relationship between SEH and tension applied to the specimen, we compared SEH measured at different tensions (0.0-3.0 N). In the analysis with the new model, the SEH obtained at a tension of 3.0 N was significantly lower than the SEH obtained at a tension of 1.5 N (in all cases, the condition P &#60; 0.001 was satisfied). In contrast, since standard deviations of SEHs obtained using the conventional model (0.0 N) were high, there was no significant difference between SEHs obtained using the conventional model (0.0 N) and the new model (1.5 N) (Figure 6D, E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58029/58029fig1.jpg" /> 
Figure 1. New ex vivo model and conventional ex vivo model. In the conventional ex vivo model, the porcine specimen was fixed with pins (A). On the other hand, in the new ex vivo model, both ends of the specimen were stretched with clips to produce a constant tension (B). This model can be tensioned uniformly by using a weight, and the tension can be arranged by changing the weight (C). Each SIM was injected into the submucosa of the specimen, leading to submucosal elevation (D). This figure has been modified from Hirose et al.23. <a href="https://www.jove.com/files/ftp_upload/58029/58029fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58029/58029fig2.jpg" /> 
Figure 2. All parts used for the new model. The new ex vivo model consists of parts that are easily available. All parts used for the new ex vivo model: (a) approximately 50-300 g of weights (the weight can be changed appropriately depending on the applied tension); (b) fixed type pulley with&#160;pulley diameter of 25 mm; (c) stainless steel wire with a diameter of 0.45 mm; (d) stainless steel clip of&#160;width&#160;147 mm; (e) stainless steel key wire with a length of 12 cm; (f) stainless steel S shaped hook; (g) lockable stainless steel S-shaped hook. (This figure has been modified from Hirose et al.23). <a href="https://www.jove.com/files/ftp_upload/58029/58029fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58029/58029fig3.jpg" /> 
Figure 3. The detailed setup method of the new ex vivo model. The new ex vivo model can be quickly set up. (A) Connect the stainless steel clip (Figure 2d) and&#160;the key wire (Figure 2e) and the S shaped hook (Figure 2g). Next, connect the wire (Figure 2c), the S shaped hook (Figure 2f) and the weight (Figure 2a). (B) Finally, connect the hook (Figure 2g) to the other end of the wire (Figure 2c). A traction device is completed in the above process. (C) Fix the pulleys (Figure 2b) at both ends of the base [rectangular wooden base (45 x 60 cm) for assembling the model]. Next, place the rubber plate (6&#160;x 6&#160;cm) on the center of the base. <a href="https://www.jove.com/files/ftp_upload/58029/58029fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58029/58029fig4.jpg" /> 
Figure 4. The complete appearance of the new ex vivo model. Accurate measurement of SEH can be performed. <a href="https://www.jove.com/files/ftp_upload/58029/58029fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58029/58029fig5.jpg" /> 
Figure 5. The measurement procedure by using the new ex vivo model. To evaluate SIM performance, the magnitude of SEH was measured by a digital height gage (A). Using a 2.5-mL syringe with a 23-gauge&#160;needle, 2.0 mL of each SIM was injected into the submucosa from the specimen margins to create a submucosal elevation (B, C). The digital height gage was used to measure of the height of the submucosal elevation (i.e., the values of SEH) (D). <a href="https://www.jove.com/files/ftp_upload/58029/58029fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58029/58029fig6.jpg" /> 
Figure 6. Measurement of SEH using either the new or conventional model. After the injection of NS or 0.4% HA into the submucosa of the specimen fixed with pins (0.0 N) (A) or stretched at a constant tension (1.5 N or 3.0 N) (B, C), SEH was measured using the height gage. Next, we compared the values of SEH measured at different tensions (0.0, 1.5, and 3.0 N) after the submucosal injection of NS (D) or 0.4% HA (E). Data are expressed as mean &#177; S.D. of more than three independent experiments. (This figure has been modified from Hirose et al.23)&#160;<a href="https://www.jove.com/files/ftp_upload/58029/58029fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

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

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.&#160; The detailed setup methodology of this new model is explained.

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

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.1K Views.  Kyoto Prefectural University of Medicine. 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.

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

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Cancer Research

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Medicine

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Immunology and Infection

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

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

Summary

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Chapters in this video

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Postoperative Ileus Murine Model