Name: a mouse model of intestinal partial obstruction
Uploaded: 2018-03-05T14:30:00
Description: Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

The authors would like to thank Benjamin J Weigler, D.V.M., Ph.D. and Walt Mandeville, D.V.M. (Animal Resources &#38; Campus Attending Veterinarian, University of Nevada, Reno) for their excellent animal services provided to the mice as well as their counsel on surgical procedures.

The following protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nevada-Reno (UNR) Animal Resources and complies with all institutional ethical guidelines regarding the use of research animals.
1. Animals.
<ol>
	<li>Obtain mature (4-6 weeks old) C57BL/6 mice weighing between 20-30 g. House the colony of laboratory mice in a centralized animal facility at UNR Animal Resources.</li>
</ol>
2. Partial Obstruction Surge...

<ol>
	<li>Millat, B., Guillon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiopathology+and+principles+of+intensive+care+in+intestinal+obstructions.">Physiopathology and principles of intensive care in intestinal obstructions.</a> Rev Prat. 43, 667-672 (1993).</li><li>Tonelli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+Crohn's+disease:+solution+of+doctrinal+mysteries+and+reinstatement+as+a+surgically+treatable+disease.+1.+The+process+is+not+a+form+of+enteritis+but+lymphedema+contaminated+by+intestinal+contents.">New developments in Crohn's disease: solution of doctrinal mysteries and reinstatement as a surgically treatable disease. 1. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+Brunner's+glade+adenoma+causing+chronic+small+intestinal+obstruction+in+a+dog.">Duodenal Brunner's glade adenoma causing chronic small intestinal obstruction in a dog.</a> J Small Anim Pract. 53, 136-139 (2012).</li><li>Bettini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophy+of+intestinal+smooth+muscle+in+cats.">Hypertrophy of intestinal smooth muscle in cats.</a> Res Vet Sci. 75, 43-53 (2003).</li><li>Macdonald, J. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+PDGFR&#945;++Cells+identifies+T-types+Ca2++channel+CACNA1G+as+a+new+pathological+marker+for+PDGFR&#945;++cell+hyperplasia.">Transcriptome analysis of PDGFR&#945;+ Cells identifies T-types Ca2+ channel CACNA1G as a new pathological marker for PDGFR&#945;+ cell hyperplasia.</a> PLoS One. 12, e0182265 (2017).</li><li>Park, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+response+factor+is+essential+for+prenatal+gastrointestinal+smooth+muscle+development+and+maintenance+of+differentiated+phenotype.">Serum response factor is essential for prenatal gastrointestinal smooth muscle development and maintenance of differentiated phenotype.</a> J Neurogastroenterol Motil. 21, 589-602 (2015).</li><li>Spencer, N. J., Sanders, K. M., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+motor+complexes+do+not+require+electrical+slow+waves+in+the+mouse+small+intestine.">Migrating motor complexes do not require electrical slow waves in the mouse small intestine.</a> J Physiol. 553, 881-893 (2003).</li><li>Langford, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nat Methods. 7, 447-449 (2010).</li><li>Terez, S. D., Notari, L., Sun, R., Zhao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+smooth+muscle+responses+to+inflammation.">Mechanisms of smooth muscle responses to inflammation.</a> Neurogastroenterol Motil. 24, 802-811 (2012).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+hyperplasia/hypertrophy+is+the+most+prominent+histological+change+in+Crohn's+fibrostenosing+bowel+strictures:+A+semiquantitative+analysis+by+using+a+novel+histological+grading+scheme.">Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: A semiquantitative analysis by using a novel histological grading scheme.</a> J Crohns Colitis. 11, 92-104 (2017).</li><li>Huizinga, J. D., Chen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+Cells+of+Cajal:+Update+on+Basic+and+Clinical+Science.">Interstitial Cells of Cajal: Update on Basic and Clinical Science.</a> Curr Gastroenterol Rep. 16, 363 (2014).</li><li>Jirkof, P., Touvieille, A., Cinelli, P., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buprenorphine+for+pain+relief+in+mice:+repeated+injections+vs+sustained-release+depot+formulation.">Buprenorphine for pain relief in mice: repeated injections vs sustained-release depot formulation.</a> Lab Animal. 49, 177-187 (2015).</li><li>Spencer, N. J., Dinning, P. J., Brookes, S. J., Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+mechanisms+underlying+colonic+motor+patterns.">Insights into the mechanisms underlying colonic motor patterns.</a> J Physiol. 594, 4099-4116 (2016).</li></ol>

We demonstrated that mice receiving the intestinal PO surgery consistently and reproducibly develop intestinal smooth muscle hypertrophy, which mimics human intestinal obstruction. Intestinal obstruction surgeries have been developed for different animals including mice7, rats10, guinea pigs11, dog12 and cats13. The mouse model of intestinal obstruction has time, cost, size, and phenotypic advantages ov...

Intestinal obstructions are a partial or complete blockage in the small or large intestine which prevents digested food, fluids, and gas from moving through the intestines1. Due to the obstruction, the blockage induces the intestinal walls to become thickened, narrowing the lumen2. Intestinal obstruction can occur as a result of abdominal or pelvic surgeries that cause abdominal adhesion tissue formation or from GI disorders such as inflammatory bowel diseases (Crohn&#39;s disease), diverticulitis, hernias, volvulus, stricture, intussusception, constipation, fecal impaction, pseudo-obstruction, cancers and tumors3,4,5. Intestinal obstructions in these cases often lead to the hypertrophy of the tunica muscularis of the intestine6.
PO of the lumen induces intestinal distention, and increases smooth muscle layer thickness around the obstruction in response to the need to continue functional peristalsis7,8,9,10,11,12,13. Animal models of intestinal PO have been developed to study smooth muscle hypertrophy in mice7, rats10, guinea pigs11, dogs12, and cats13 that consistently develop similar hypertrophy within the intestinal muscle layers.
A mouse model of intestinal PO is the most cost effective way to generate and study intestinal obstructions in vivo. Small intestine obstructions are carried out in mice by using a silicone ring surgically placed surrounding the ileum. The PO mice showed an early increase in the number of cells (hyperplasia), and an increase in muscle layer thickness (hypertrophy) after PO surgery8,15. SMC are the primary plastic cells that are growing within smooth muscle layers in response to the hypertrophic conditions14, but other cells such as ICC and PDGFR&#945;+ cells that are closely associated with SMC, are also repopulated. We have previously reported that the PO mice develop hypertrophy in the small intestine, in which SMC are dedifferentiated into PDGFR&#945;+ cells that are highly proliferative7,15,16. Conversely, ICC are degenerated and lost within the hypertrophied smooth muscle layers during the development of intestinal obsruction7. Another major benefit of the PO model is its capacity to induce changes in the enteric nervous system and propagating neurogenic motor patterns. The major propagating neurogenic motor pattern in the mouse small bowel is the migrating motor complex (MMC), which is neurogenic and does not require ICC or electrical slow waves17. The PO model can provide clear insights into how MMCs and enteric nerves are remodeled by partial obstruction.
Here, we propose a murine protocol for intestinal PO surgery using a silicone ring. Mice receiving PO surgery reliably produce hypertrophy in the tunica muscularis of the small intestine. Within hypertrophic muscle, SMC, ICC, PDGFR&#945;+, and neuronal cells are dramatically remodeled.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical drape</td>
			<td>Medical and veterinary supplies</td>
			<td>SMS40</td>
			<td>40&#8221;X100 yards</td>
		</tr>
		<tr>
			<td>Underpad, econ, pro plus</td>
			<td>Medical and veterinary supplies</td>
			<td>MSC281224</td>
			<td>17x24&#8221;</td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Braintree scientific, Inc</td>
			<td>SC-i-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Vantage</td>
			<td>V95-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont electronic &#38; jeweler tweezers</td>
			<td>Dumont</td>
			<td>98-180-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided absorbable suture</td>
			<td>Covidien polysorb</td>
			<td>SL-5687G</td>
			<td>5-0, polyglactin</td>
		</tr>
		<tr>
			<td>Nylon non-absorbable mono filament</td>
			<td>AD surgical</td>
			<td>S-N618R13</td>
			<td>6-0, nylon</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Dynarex</td>
			<td></td>
			<td>No.15</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Jacobson microvascular</td>
			<td>36-1342TC</td>
			<td>8.5 inch</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Flinn scientific</td>
			<td>AB1049</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>WPI</td>
			<td>503305</td>
			<td></td>
		</tr>
		<tr>
			<td>Petrolatum ophthalmic ointment</td>
			<td>Puralube VET</td>
			<td></td>
			<td>3.5 g</td>
		</tr>
		<tr>
			<td>Fluriso (isoflurane)</td>
			<td>Vetone</td>
			<td>V1 502017</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Steri-strip reinforced skin closure</td>
			<td>3M</td>
			<td>R1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Medline</td>
			<td>MSG2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear loop face mask</td>
			<td>The safety zone</td>
			<td>RS700</td>
			<td></td>
		</tr>
		<tr>
			<td>Avant gauze non-woven sponges</td>
			<td>Caring</td>
			<td>PRM25444</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical cup</td>
			<td>Admiral&#160; craft OYC-2</td>
			<td>725-A42</td>
			<td>2.5 oz</td>
		</tr>
		<tr>
			<td>Swabstick</td>
			<td>ChloraPrep</td>
			<td>260103</td>
			<td>2% w/v Chlorhexidine&#160; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Zoo pharm</td>
			<td>BZ8069317</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Vetone</td>
			<td>G-6336-04</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>Fast acting gel cream remover</td>
			<td>Veet</td>
			<td>8111002</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>AHS</td>
			<td>AH01T2516</td>
			<td>1 ml with needle</td>
		</tr>
		<tr>
			<td>Silicon ring</td>
			<td>VWR</td>
			<td>60985-720</td>
			<td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>4-6 weeks old</td>
		</tr>
	</tbody>
</table>

a mouse model of intestinal partial obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Partial obstruction (PO) was surgically induced in one month old mice by placing a silicone ring around the ileum close to the ileocecal sphincter. This ring created a partial blockage in the ileum. Sham operations (SO) were also performed without a ring on age/sex matched mice and these mice did not show any symptoms similar to those found in PO mice. Mice quickly recovered from PO surgery within a few hours. They showed no obvious behavioral changes or weakness within the first week, bu...

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

The overall goal of this surgical procedure is to reliably and consistently produce intestinal obstructions in mice that facilitate the study of the molecular and phenotypic changes in gastrointestinal tissue that occur in response to the obstructions. This method can help answer key questions in the gastrointestinal field surrounding the cellular mechanisms underlying the phenotypic changes that result from intestinal obstruction. The main advantages of this technique is that it's reliable, replicable, and cost effective.The implication of this technique extend toward the therapy of intestinal obstruction as they mimic phenotypic conditions observed in human pathologies caused by bowel obstruction. Demonstrating this procedure will be Se Eun Ha, a post doc, and Lai Wei, a graduate student, both from our laboratory. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and place the mouse onto a warming pad.Remove the hair from the abdomen with depilatory cream and clean the exposed skin with 70%ethanol. Disinfect the surgical site with povidone-iodine solution and drape the animal with a 25 by 50 centimeter sterile paper with a 2.5 by 2.5 centimeter opening for the surgical area. Secure the drape with sterile strips at the boundaries of the opening and the skin and use a number 15 scalpel blade to make a three centimeter longitudinal skin incision.Using forceps and surgical scissors, carefully separate the skin from the musculoperitoneal layer without incising the tissue and locate the linea alba. Then use micro forceps and scissors to cut two centimeters along the linea alba to expose the intraperitoneal cavity and carefully locate the cecum. Use the micro forceps to slowly and gently move the cecum, proximal colon, and ileum from within the intraperitoneal cavity onto the sterile drape and immediately moisten the intestinal tissue with 0.9%sterile saline soaked gauze.Locate the mesentery between the ileum and proximal colon and make a one centimeter incision in the mesentery parallel to and just below the ileum taking care to avoid cutting any vasculature. Cut longitudinally to open the mesenteric vessel and use micro forceps to open an appropriately sized autoclaved sterilized silicone ring. Insert one end of the ring through the incision and bring the end of the ring into contact with the first end to return the instrument to its original shape.Confirm that the ring is completely surrounding the ileum and close the ring with a suture. The incisions in the mesentery should be large enough for the ring to fit, but small enough that no blood vessels are incised. Also make sure the ring is placed on the ileum, not on the colon.Carefully return the intestines to their original anatomical location and use an absorbable suture in a simple continuous stitch to close the musculoperitoneal layer along the linea alba. Clean any bleeding with 0.9%sterile saline soaked gauze and use a new nylon suture and a simple continuous stitch to close the skin. Then disinfect the wound site with povidone-iodine and inject antibiotics before placing the animal in a warming gate with monitoring until full recumbency.In this experiment, the small intestine was partially filled and distended at eight days post partial obstruction surgery and fully filled and distended at 13 days post partial obstruction surgery compared to control sham operated animals. Further, fecal pellet formation in the colon of day eight and 13 post partial obstruction surgery mice was reduced compared to that in control animals. The smooth muscle layer of the ileum just upstream of the ring was hypertrophied at eight days post partial obstruction surgery with further hypertrophy observed at day 13.The smooth muscle cells grew at progressively rapid rates in all three layers of tissue at eight and 13 days post partial obstruction surgery as assessed by immunochemical analysis while the interstitial cells of Cajal degenerated within the intra and intermuscular layers. Platelet-Derived Growth Factor Receptor alpha positive cell subsets also known as PDGFR alpha cells were dynamically remodeled within the intermuscular layers of partial obstruction mice with growth observed at eight days post surgery that had degenerated by day 13. In addition, neuronal cells were also significantly lost in the inter and intramuscular regions of partial obstruction mice at both days eight and 13 post surgery.Once mastered, this technique should be completed within 30 minutes to ensure the survival of the mouse. While attempting this procedure, it is important to treat the tissue quickly and delicately. Following this procedure, other methods like gastrointestinal trend testing can be performed to answer additional questions about how obstructions alter intestinal peristalsis.After its development, this technique paved the way for researchers in the field of gastrointestinal research to explore the consequences of intestinal obstruction in mice. After watching this video, you should have a good understanding of how to quickly and properly place a ring around the mouse ileum for the creation of intestinal partial obstruction. Don't forget that working with surgical tools and needles can be extremely hazardous so take the proper precautions.Also, be sure to wear sterile gloves and practice aseptic techniques while performing this procedure.

a-mouse-model-of-intestinal-partial-obstruction

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In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Aluminum foil was microsurgically inserted between the testes of Spodoptera litura to obstruct the fusion of testis. The procedure includes freezing, fixing, disinfection, incision, placing the barrier, suturing, postoperative feeding, and inspection. This approach provides a method to interfere with tissue formation.

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...