Name: 视频: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice
Uploaded: 2016-02-03T15:00:00

JCB and ELH-Y were supported by the US Department of Defense CDMRP Autism Research Program (AR11034). NHMRC (1047674) to ELH-Y.The May Stewart Bursary-University of Melbourne trust funded scholarship to MS. We thank Ali Taher, F&#225;tima Ramalhosa and Gracia Seger for technical contributions.

Animal handling and cervical dislocation of animals prior to all experiments were performed strictly according to protocols approved by the Animal Experimentation Committee for the University of Melbourne (Ethics ID: 1212494.7)
1. Tissue Collection and Dissection
<ol>
	<li>Euthanize adult mice by cervical dislocation. If possible avoid anesthesia to prevent influences on gut function via receptors located on neuronal populations of interest.</li>
	<li>Record the animal&#39;s total body weigh...

<ol>
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Using this video imaging technique, CMMC frequency was measured as an indication of colonic motility in wild type and NL3R451C mice, a mouse model of autism spectrum disorder17. Our results indicate a reduction in the number of CMMCs in mutant NL3R451C mice compared to wild type mice in the presence of the 5HT3/4 receptor antagonist Tropisetron suggesting that NL3R451C mice exhibit an increased sensitivity to Tropisetron. Accordingly, we propose that the neuroligin-3...

The enteric nervous system (ENS) is the intrinsic neuronal network of the gastrointestinal tract and modulates various functions such as digestion of intestinal content, absorption of nutrients and the secretion and reabsorption of fluid. Neurons of the ENS are located in the myenteric and submucosal plexuses. The myenteric plexus plays a major role in regulating gastrointestinal motility1 whereas the submucosal plexus is primarily involved in the control of secretion2,3. The myenteric plexus is situated between the longitudinal and circular muscle layers of the gastrointestinal wall. The contractile activity of the smooth muscle layers of the intestinal wall facilitates the primary functions of the gastrointestinal tract by mixing and propelling intestinal content along the length of the intestine3. Although the extrinsic nerve supply to the gastrointestinal tract from the CNS contributes to gastrointestinal function in vivo, the ENS is capable of regulating gastrointestinal function independently. This unique characteristic enables the functional investigation of enteric neuronal circuits and their contribution to gastrointestinal motility ex vivo.
Colonic migrating motor complexes (CMMCs) are spontaneous, neurogenic events that are the predominant motor pattern observed in isolated mouse colon in the absence of fecal pellets4-9. CMMCs are defined as rhythmic contractions that propagate along a horizontal distance that is at least half the total length of the colon (i.e.,&#160;from the cecum to the rectum)10. The relationship between CMMCs and the contractile patterns that propel fecal pellets is yet to be clearly established, however some pharmacological differences have been reported11. Nevertheless, the ability of the ENS to function independently of the CNS and the existence of neural-mediated motor patterns in the isolated colon provides an ideal assay system to examine disturbances in motility resulting from underlying ENS dysfunction. The spontaneity of gastrointestinal motor patterns allows functional changes in response to pharmacological stimuli to be evaluated.
The use of video imaging and spatiotemporal mapping was first developed to quantitatively examine small intestinal peristalsis in guinea pigs12. Here, an ex vivo technique is described that enables the study of mouse colonic motility patterns using video imaging and analysis of these recordings to construct high-resolution (~100 &#181;m, 33 msec) maps of colonic diameter as a function of position along the colon and of time (spatiotemporal maps). Using in-house edge detection software (Analyse2; available on request), data from full length colonic segments contracting in real time are processed to generate spatiotemporal maps for each experiment. In this step, video (AVI) files are summarized and converted to spatiotemporal maps using Analyse2. Spatiotemporal maps (Figure 2) depict contractility over time and enable the measurement of multiple parameters including propagation speed, magnitude, length and duration. Gut diameter is also recorded throughout the duration of the experiment as a measure of the overall contractility of the tissue segment. This method can be applied to identify differences in the point of initiation of contractile complexes which could indicate altered enteric neural connectivity.
A similar video imaging protocol designed to assess pellet propulsion in guinea pigs has been reported13 however here we outline the application of the video imaging approach for quantification of spontaneous colonic motility (i.e., in the absence of pellets). We also provide detailed information to assist in the dissection and preparation of gastrointestinal tissue for the video imaging approach. This protocol provides researchers with an accessible and easily replicated tool for analyzing enteric neural control of gastrointestinal function in animal models of disease including genetic mouse models.
The video imaging technique enables the analysis of colonic motility in response to various pharmacological agents. Drugs can be administered via the gut lumen or the organ bath external to the colonic preparation. Different regions of the mouse gastrointestinal tract exhibit specific motility patterns such as small intestinal segmentation and CMMCs in the colon.
This technique has been used to identify strain differences in small intestinal function; differential sensitivity to 5-HT3 and 5-HT4 antagonists were observed in the jejunum of Balb/c and C57/Bl6 mice due to the polymorphic nature of the tph2 gene expressed in the two strains6. The effect of 5-HT inhibition on motility remains controversial, as conflicting data has been reported on the importance of endogenous 5-HT on colonic peristalsis and CMMCs14,15. Alterations in motility pre- and postnatally during development7, and the effects of gene mutations on gastrointestinal motility in animal models of disease10 can also be examined by utilizing video imaging. Here we illustrate use of the method for a study of colonic motility in the NL3R451C mouse model of autism, which expresses a missense mutation in the Nlgn3 gene encoding the synaptic adhesion protein Neuroligin-316. This mutation was first identified in patients diagnosed with Autism spectrum disorder (ASD)17, which is strongly associated with GI dysfunction18-22. We investigated whether the NL3 R451C synaptic mutation affects neural outputs in the ENS using the video imaging technique. We present data characterizing CMMCs at baseline and in response to the serotonergic 5HT3/4 receptor antagonist tropisetron in the NL3R451C mouse model of autism.

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			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>NaCl (MW: 58.44)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-250G</td>
			<td></td>
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		<tr>
			<td>KCl (MW: 74.55)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
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		<tr>
			<td>NaH2PO4.2H2O (MW: 156.01)</td>
			<td>Chem Supply</td>
			<td>471-500G</td>
			<td></td>
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		<tr>
			<td>MgSO4.7H20 (MW: 246.48)</td>
			<td>Chem Supply</td>
			<td>MA048</td>
			<td></td>
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			<td>CaCl2.2H2O (MW: 147.02)</td>
			<td>Chem Supply</td>
			<td>CA033</td>
			<td></td>
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			<td>D-Glucose anhydrous (MW: 180.16)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
			<td></td>
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		<tr>
			<td>NaHCO3 (MW: 84.01)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
			<td></td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Two chambered organ bath 
			Dimentions: 14 cm x 8 cm x 3 cm</td>
			<td>Custom Made</td>
			<td></td>
			<td>Contact Laboratory Directly&#160;</td>
		</tr>
		<tr>
			<td>&#160;732 MULTI -PURPOSE SEALANT CLEAR</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1890573</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT&#160;</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1064291</td>
			<td></td>
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		<tr>
			<td>STOPCOCK 3 WAY FEM-ML L/LOCK S</td>
			<td>Terumo Medical Corporation</td>
			<td>0912-2006</td>
			<td></td>
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		<tr>
			<td>SYRINGES with Luer Lock Tips 50mL, 20 mL, 10 mL</td>
			<td>Terumo Medical Corporation</td>
			<td>N/A</td>
			<td></td>
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			<td>1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
			<td>508-008</td>
			<td></td>
		</tr>
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			<td>1.02 mm (ID) x 2.16 mm (OD) - Silastic Tubing</td>
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			<td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
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			<td>1.60 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>4.40 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>3.10 mm (ID) - Platinum cured silicone tubing&#160;</td>
			<td>Masterflex</td>
			<td>96410 -16</td>
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			<td>Thermofisher Scientific&#160;</td>
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			<td>CHEMICAL RUBBER STOPPER 29 x 32mm</td>
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			<td>MATLAB R2012a&#160;</td>
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			<td>Logitech Webcam Software</td>
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video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

Up to 90% of patients with ASD experience an array of gastrointestinal disorders, including diarrhea and constipation18,24,25. However, the underlying causes of these gastrointestinal issues are unknown. Many mutations identified in patients with ASD are associated with synaptic proteins contributing to alterations and disturbances in synaptic transmission or function. One such mutation, in the gene coding for the cell adhesion molecule neuroligin-3 (NL3 R451C), was identified ...

Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

The overall goal of this technique is to identify changes in the neural regulation of colonic motility in mice using video imaging. This technique can help answer key questions related to the enteric neuroscience field, such as do subtle genetic mutations in neuronal genes affect colonic motility. The main advantage of this technique is that the tissue is a cyst in the absence of central nervous system input, and as a result the method allows investigation of the enteric nervous system's involvement in the control of gastrointestinal motility.This technique extends towards the therapy of neurodevelopmental diseases such as autism, as gene mutations altering synaptic function in the brain, might also alter gut motility. Begin by using dissecting forceps and scissors to make an incision through the epidermis overlying the lower abdominal muscle layers. Open the abdominal cavity along the midline to the sternum.Moisten the tissue with physiological saline and use the forceps to gently lift the cecum four to five centimeters above the abdomen. Then, using fine dissection scissors, trim the mesentery, taking care not to handle, stretch, or cut the gastrointestinal tissue. Remove the bladder, testes, and any other excess tissue.Then, using coarse dissecting scissors, make two vertical incisions approximately 0.5 centimeters from the midline into the pelvic bone on each side of the colon, and use the fine scissors to separate the rectum from the adjoining pelvic tissue. Trim the mesentery from the colon and rinse the full-length colon tissue in a beaker containing physiological saline. To remove the fecal pellets and other intestinal contents, first trim approximately one centimeter from the end of a 200 microliter pipette and attach the pipette to a five milliliter syringe filled with physiological saline.Then, after removing the cecum and the rectum, place an insect pin in the most proximal end of the colon where the striations in the mucosa are visible to orient the tissue. Now, gently grip the tissue at the proximal end, and insert the pipette tip into the lumen and flush the saline through the colon into a waste container. Next, with the same syringe and pipette tip, flush any debris from the tubing attached to a two-chambered organ bath with fresh physiological saline.Set the organ bath so that the chambers will be continuously superfused with physiological saline bubbled with carbogen and use a temperature probe to regularly confirm that the saline temperature remains between 33 and 37 degrees Celsius. Use a rubber stopper with a five millimeter inside diameter glass tube inserted through the center to maintain the pressure in the inflow reservoir. Then, fully submerge a five to seven centimeter piece of colon into the organ bath chamber and use forceps and standard cotton sewing thread to cannulate the proximal end of the colon to the inlet tube and the distal end of the colon to the outlet tube.After determining the baseline distance between the proximal and distal cannulations of the colon, equilibrate the tissue in physiological saline for 30 minutes. To record the intestinal movements, while the colon is acclimatizing, position a video camera 10 to 15 centimeters above the organ bath on a standard laboratory retort stand, and set up the appropriate video capture software. When the camera is ready, record four 15 minute video segments of the colon under control conditions.Then, superfuse the drug of interest into the organ bath for 30 to 60 minutes, capturing another four 15 minute video segments. Finally, replace the treatment solution with fresh physiological saline and record four more 15 minute video segments during the one-hour wash out period. Spatiotemporal maps of the colon, in response to the different treatment periods can then be generated.To assess whether synaptic mutations alter colonic migrating motor complexes, or CMMC's, when the enteric nervous system is pharmacologically perturbed, in this experiment, the five HT3 and 4 receptor antagonists, tropisetron, was added to the colon preparation organ bath chamber. In the presence of tropisetron, NL3R451C mice exhibited a decrease in CMMC frequency compared to their wild type litter mates. Although no significant differences were observed between the wild type and NL3R451C contractility during controlled conditions, tropisetron significantly reduced the CMMC frequency in both wild type and NL3R451C mice.Moreover, tropisetron imposed a larger effect on the frequency of CMMC's in NL3R451C mice compared to wild type animals. Once mastered, this technique can be completed in four hours if it's performed properly. While attempting this technique, make sure that the organ bath is maintained at a constant temperature, it has a continuous supply of carbogen, and overflow of physiological saline is prevented by a vacuum.Following this procedure, other techniques such as immunocytochemistry can be performed to answer additional questions such as are there changes in neuronal proportions or cell numbers in mice exhibiting altered colonic motility? Since its development by this method has allowed a variety of studies of intestinal motility in guinea pig and rabbit, rat, and other species, and most particularly in this case in the mouse. After watching this video, you should have a good understanding of how to identify changes in the neuronal regulation of colonic motility using video imaging.

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Research

JoVE Journal

Neuroscience

13.8K 次观看.  The University of Melbourne. The overall goal of this technique is to identify changes in the neural regulation of colonic motility in mice using video imaging. This technique can help answer key questions related to the enteric neuroscience field, such as do subtle genetic mutations in neuronal genes affect colonic motility. The main advantage of this technique is that the tissue is a cyst in the absence of central nervous system input, and as a result the method allows investigation of the enteric nervous system's invo...