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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