Name: transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time
Uploaded: 2013-03-10T14:00:00
Description: Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

We acknowledge Drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier and Daniel Nyqvist for fruitful discussions. We also thank Eleut Hernandez and Diego Espinosa-Heidmann for technical assistance, and Mike Valdes and Margaret Formoso for help with video recording. Byron Maldonado recorded, edited, and produced the final video. Research support was provided by the Diabetes Research Institute Foundation (<a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a>), the NIH/NIDDK/NIAID (F32DK083226 to M.H.A.; NIH RO3DK075487 to A.C.; U01DK089538 to P-O.B.). Additional research support to P-O.B was provided through funds from the Karolinska Institutet, the Swedish Research Council, the Swedish Diabetes Foundation, the Family Erling-Persson Foundation, the Family Knut and Alice Wallenberg Foundation, the Skandia Insurance Company Ltd., VIBRANT (FP7-228933-2), Strategic Research Program in Diabetes at Karolinska Institutet, the Novo Nordisk Foundation, and the Berth von Kantzow&#x2019;s Foundation.

The following procedure is performed under the stereoscope in 2 steps, the first step involves loading the islets into the cannula and the second step is the actual transplantation into the ACE. All procedures performed on animals were approved by the institutional animal care and use committee (IACUC) of the University of Miami.
1. Loading Islets in Cannula for Transplantation
<ol>
 <li>Center the islets in culture dish by spinning the dish in narrowing circles.</li>
 <li>Disconnect th...

<ol>
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Murine pancreatic islets were isolated using collagenase digestion followed by purification on density gradients, as described previously 33. Isolated islets were cultured overnight before transplantation. While this may not be required, it is recommended to allow the islets to recover from the isolation procedure. This is critical when transplantation is performed in diabetic recipients as it will ensure transplantation of surviving/robust islets. 
Transplantation is performed ...

Advances in intravital microscopy have revealed physiological phenomena not predicted by in vitro studies1. This highlights the challenge in translating findings obtained by conventional in vitro methods into the living animal. In the last decade, visualization of tissues in living animals was considerably improved by technological advances in imaging modalities2, 3, 4, 5, 6. This has spurred a need for in vivo imaging approaches with feasible application in experimental animal models to enable longitudinal visualization of target tissues non-invasively. 
Imaging techniques such as magnetic resonance imaging and positron emission tomography or bioluminescence have enabled non-invasive imaging of organs/tissues deep within the body7-8, 9. But these techniques cannot achieve single cell-resolution due to high background signals and low spatial resolution, despite the use of high contrast materials or tissue-specific luminescence4. This was addressed with the advent of two-photon fluorescence confocal microscopy10. Two-photon microscopy enabled intravital imaging studies to visualize and quantify cellular events with unprecedented details11, 12. This has led to the characterization of key biological processes in health and disease13, 14, 15, 16. While pioneering intravital imaging studies have primarily "mimicked" in vivo conditions in excised tissue (e.g. lymph nodes), other studies have used invasive approaches to image exposed target tissues in situ17, 18, 19, 20, 21. Other studies have also used "window chamber models" to circumvent limitations associated with invasive approaches and limited imaging resolution in vivo22, 23, 24, 25. In the window chamber model, a chamber with a transparent window is surgically implanted into the skin at different locations (dorsal or ear skin, mammary fat pad, liver, etc) on the animal (e.g. mouse, rat, rabbit). While this approach clearly enables high-resolution in vivo imaging, it requires an invasive surgery to implant the chamber and may not be able to accommodate longitudinal imaging studies over several weeks or months22.
It was recently demonstrated that combining high-resolution confocal microscopy with a minimally invasive procedure, namely transplantation into the anterior chamber of the eye (ACE) provides a "natural body window" as a powerful and versatile in vivo imaging platform26, 27. Transplantation into the ACE has been used in the last several decades to study biological aspects of a variety of tissues 28, 29, 30; and its recent combination with high-resolution imaging enabled studying the physiology of pancreatic islets with single cell-resolution non-invasively and longitudinally26, 27. This approach was used to study autoimmune responses during development of type 1 diabetes in animal models (unpublished data). It was also used to study pancreatic development, as well as, in studies of kidney function by transplanting into the ACE pancreatic buds or individual renal glomeruli, respectively (unpublished data). A recent report using this approach further demonstrated its application to study immune responses after pancreatic islet transplantation31. Importantly, this study showed that transplantation into the anterior chamber of the eye provides a natural body window to perform: (1) longitudinal, non-invasive imaging of transplanted tissues in vivo; (2) in vivo cytolabeling to assess cellular phenotype and viability in situ; (3) real-time tracking of infiltrating immune cells in the target tissue; and (4) local intervention by topical application or intraocular injection.
Here, we demonstrate how to perform transplantation into the anterior chamber of the eye using pancreatic islets.

<table border="1">
 <tbody>
 <tr>
 <td>Name 				of reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Description/Comments</td>
 </tr>
 <tr>
 <td>IsoTHESIA 				(Isoflurane)</td>
 <td>Buttler 				Animal Health Supply</td>
 <td>11695-6775-2</td>
 <td>99.9% 				Isoflurane/ml</td>
 </tr>
 <tr>
 <td>Ketaset (Ketamine 				HCL)</td>
 <td>Fort 				dodge Animal Health</td>
 <td>0856-2013-01</td>
 <td>Alternative 				injectable anesthesia</td>
 </tr>
 <tr>
 <td>Beprenex (Buprenorphine 				HCL)</td>
 <td>Reckitt 				Benckiser Health Care (UK) Ltd.</td>
 <td>12496-075-7-1</td>
 <td>0.3 				mg/ml</td>
 </tr>
 <tr>
 <td>Erythromycin 				Ophthalmic Ointment USP, 0.5%</td>
 <td>Akron</td>
 <td>17478-070-35</td>
 <td>Applied 				prophylactically to transplanted eye </td>
 </tr>
 <tr>
 <td>0.9% 				Sodium Chloride (Saline)</td>
 <td>Hospira 				Inc.</td>
 <td>0409-7983-03</td>
 <td>For iv 				injection. Sterile</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>10010-023</td>
 <td>1X. 				Sterile</td>
 </tr>
 <tr>
 <td>CMRL 				medium 1066</td>
 <td>Cellgro</td>
 <td>98-304-CV</td>
 <td>Supplemented, 				CIT modification. Preferred media for islets </td>
 </tr>
 </tbody>
 </table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

There are a few parameters that define a "good" transplantation. A good transplantation is one that proceeds without bleeding when making the incision as can be seen in the video. Bleeding is prevented/minimized by penetrating only the tip of the scalpel (needle) into the ACE (Figure 3a). This will also help prevent contact and puncture of the iris. It will also ensure a small incision which will heal very well without causing cloudiness of the cornea over time (Figure 3c, d). Anoth...

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

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