Name: intraportal transplantation of pancreatic islets in mouse model
Uploaded: 2018-05-05T15:00:00
Description: Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

We would like to acknowledge Gundula Hertl for her support.

All procedures in this protocol have been approved by German Animal welfare law and guidelines. Consider using 10- to 12-week-old NMRI nude mice and C57Bl/6 mice (donor: old female; recipient: male) throughout the experiments. Use NMRI nude mice for islet isolation. Keep the mice under defined conditions according to local animal facility regulations.
1. Induction of Diabetes by Streptozotocin
<ol>
	<li>On day -4, measure the body weights of 10 to 12 week-old NMRI nude mice and C57Bl/6 mice ...

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. M., 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, 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=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. 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=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, O., 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=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, N., 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=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, 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=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, Z., 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=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

In this study, the intraportal route of islet transplantation is explored. This protocol is highly efficient in relieving diabetic stress and provides an in-depth opportunity to explore islet transplantation studies. This protocol confirms that about 350 murine islets are capable of reversing the hyperglycemic state. Moreover, none of the mice died during the procedure, and glycemic control was achieved in all recipients. Body weight and blood glucose levels were regularly recorded to reflect the competence, standard, an...

Islet transplantation is a promising approach to treat type 1 diabetes mellitus1. The first attempt to transfer a sheep pancreatic fragment into a diabetic patient was performed by Watson Williams in 1893. However, a major breakthrough in clinical islet transplantation was achieved with the Edmonton protocol, and thereafter, a series of national programs were developed2. In the past, several sites, such as the bone marrow, omental pouch, intramuscular region, gastric mucosal surface, spleen, and kidney capsule, were explored in preclinical models, but the portal venous system is considered as one of the suitable and effective site for clinical programs3,4,5,6.
Exogenous administration of insulin can be substituted by islet infusion into the portal vein. This is considered a preferred site because the oxygen supply is comparable to that in the native pancreas due to the location downstream of the confluence of the portal artery and vein. Moreover, there is a large surface area, so that the three-dimensional structure of the islets may be preserved, and vascularization could be facilitated5. In a recipient diabetic mouse, 2,000 human or porcine islet equivalents or 350 mouse islets are adequate to reverse the hyperglycemic state7,8. Euglycemic levels were reported for 15 days in the mouse recipient model of xenogeneic islets and in the allogeneic mouse-to-mouse model, and for more than 120 days in the syngeneic mouse-to-mouse transplantation.
Factors pertinent to the efficacy of islet transplantation via portal vein are appropriate anesthesia, method of puncture, and hemostasis9. Anesthesia can be induced by inhalation (5% isoflurane) or injection (ketamine, xylazine, or pentobarbital), and the substances are often combined. For control of the depth and time of anesthesia, attention should be paid to the state of the mouse, such as the color of the mucous membrane, eye lid, corneal reflex, respiratory rate, and body temperature. It is important to ensure that the animal is not struggling and that it survives the operation. The temperature can be maintained by different heating devices, such as heating pads, red light bulbs, etc. A warm thermal plate has been used to maintain a temperature of 25-30 &#176;C between the mouse and the operation table, which prevents the occurrence of hypothermia. The dosages of the anesthetics are important, as all of them are metabolized by the liver, and hepatic function may be transiently disordered by the infusion of the islet suspension. The ideal puncture point for the portal vein is the position between the first and second tributary vein.
The number of islets and the intended injection volume also influence the outcome of the transplant, as an excessive volume may increase the shear stress. A 0.2-mL volume is considered appropriate for islet transplantation into the portal vein. The puncturing wound (26-gauge needle) induces bleeding from the portal vein, which needs to be stopped in a timely and effective manner. Sterile gauze or a finger can be applied with minimal pressure at the site of the puncture for about 6 min to stop the bleeding. Taken together, portal islet injection is effective and provides regulation over blood glucose levels in chemically induced diabetic mouse models.

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		<tr height="20">
			<td>C57Bl/6 mice&#160;</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>BALB/c mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>NMRI nude mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Bela-pharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>CEVA TIERGESUNDHEIT GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe, 23G and 26G needle</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Falcon</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Carl Roth</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Fuhrmann</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking Water bath</td>
			<td>Kotterman</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Braun</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose Meter&#160;</td>
			<td>One Touch</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm Thermal Plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Braunol (povidone-iodide solution)</td>
			<td>B.Braun Melsungen AG</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liposic Edo - Sprinkled Ointment (eye ointment)</td>
			<td colspan="2">Dr. G Mann Chem.-Pharm.&#160; GmbH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Fa. Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flow Bench</td>
			<td>Herdus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Suture Vicryl</td>
			<td>Braun</td>
			<td></td>
			<td>(7.5 *1.75mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Michel Clamps (clips)</td>
			<td>Aesculap&#160;</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P/FCS Solution</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCM-199 (10x)</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1 M)</td>
			<td>Biochrom</td>
			<td>L 1613</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamycin</td>
			<td>Ratiopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ciprofloxacin</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#39;s Solution</td>
			<td>Gibco</td>
			<td>14060-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Roche</td>
			<td>11213865001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptozotocin</td>
			<td>Calbiochem</td>
			<td>572204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSITE-spray (wound healer)</td>
			<td>Smith and nephew</td>
			<td>66004978</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Reugular food</td>
			<td>Altromin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Head light LED 16042 (magnifying glass)</td>
			<td>Eschenbach</td>
			<td>16042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td>
			<td>Millipore, Chemicon</td>
			<td>CBL1337</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-rat secondary antibody</td>
			<td>Dianova</td>
			<td>712095153</td>
			<td>Dilution (1:400)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyclonal guinea pig anti-insulin primay antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-guinea pig secondary antibody</td>
			<td>Dianova</td>
			<td>706-259-148</td>
			<td>Dilution (1:400)</td>
		</tr>
	</tbody>
</table>

intraportal transplantation of pancreatic islets in mouse model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

The competency of transplanted islets was studied in chemically induced diabetic mice. The islets were transplanted via the portal venous system in diabetic mice model. The donor-recipient ratio was 2:1. In the syngeneic mouse model, two donor mice were utilized to obtain 350 islets. The islets were then transplanted into diabetic mice. A reduction in blood glucose level was observed on day 1 after transplantation, suggesting a role of transplanted pancreatic islets in reducing the blood ...

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

I'm Thomas Linn, principle investigator of the Diabetes Research Group at Justus Liebig University, Giessen, Germany. Pancreatic islet transplantation is a treatment for type-1 diabetes mellitus. Experimental islet transplantation is performed to the kidney capsule, while clinical programs prefer the intra-portal route.Therefore, we believe that more research on intra-portal islet transplantation is needed in preclinical models. For example, islets sites exposed to blood, as the transplantation site is intravascular. Moreover, the microsurgery technique needs some experience.For example, after the injection of the portal vein, bleeding can occur, or there may be issues with anesthesia or with the maintenance of the bodyweight of the recipient mouse. Therefore, we think that visual explanation of the procedure is crucial. This video focuses on the intra-portal route of islet transplantation.To achieve normal glycemia in diabetic mice, it can be used for syngeneic and allogenic transplantation in black mice, but also in syngeneic models in nude mice recipients. Remove the isolated islets from the incubator, and place them under a flow bench. Center the islets with a circular swinging motion of the petri dish.Fill 100-microliter medium in one-ml syringe with the help of blue needle. Then add 350 islets in 300 microliter of Hank's Solution. Keep the syringe in a vertical position to allow the islets to settle.Decrease the volume to 50 microliter. Replace the blue needle with the brown needle. To the same syringe add 150 microliter of density-gradient medium to the top, to reach to the final volume of 200 microliter in the syringe.Avoid the air bubbles. Keep the syringe in vertical position. Rotate it slightly to avoid the clumping of islets.The islets will settle at the cone of syringe within two to three minutes. After three days of streptozotocin injection, check the blood-glucose level by puncturing the tail vein with the help of handheld glucose meter. Anesthetize the mice with dalzylozine and ketamine by intraperitoneal injection.Place the mice in supine position on warm thermal plate. Check the depth anesthesia by pinching the toe. Clean the skin with skinless infectant.To prevent the eyes from drying, keep them moist with eye ointment. Start with electrotomy by holding the skin with forcep and making a two-to-three-centimeter-long middle incision with scissor. Place a sterile wet gauze around the wound edges to keep it moist.Keep the intestine situs to the left and outside the abdominal cavity. Move the duodenum, and locate the pancreas. Use a finger and thumb to expose the portal vein.Inject the islets in 200 microliter slowly into the portal vein. Apply the pressure with the index finger at the puncture site for six minute to stop the bleeding. Carefully place the intestine situs, peritoneum, and abdominal-muscles layer back into the position.Using forcep, lift the skin, close the muscles incision, and upper-skin layer with absorbable silk suture. Apply a wound-healer. Maintain the body temperature at 37 degrees celsius on warm thermal plate.Observe the mouse until it regains sufficient consciousness. Provide tramadol along with fluid to manage pain. After transplantation of islets, a sudden drop in blood-glucose level was observed on day one, from 23 millimolar per liter to 4.6 millimolar per liter in strepto-induced diabetic mice.Normal glycemia was maintained for up to 121 days. The bodyweight and blood-glucose level roughly followed the same pattern. After the streptozotocin injection and surgery, the bodyweight decreased;from day two onwards it tend to rise towards normal, from 25.3 gram to 29.7 gram.In major-histocompatibility-mismatch mice after islet transplantation, the blood-glucose level dropped to 4.4 millimole per liter, and the normal glycemia observed for up to 10 days. Afterward, it starts to reject. The bodyweight of diabetic mice also follows the similar pattern.In successful engraftment of transplanted pancreatic islets into the liver. In this study, the intra-portal route of islet transplantation is explored. This protocol is highly efficient in relieving diabetic stress, and provide an in-depth opportunity to explore islet transplantation.This protocol confirms that about 350 islets are capable of reversing the hyperglycemic state. Moreover, none of the mice died during the procedure, and the glycemic control was maintained.

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A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...