The authors would like to thank Chiyoko Kobayashi and Kiyokazu Agata for helpful advice on planarian transplantation as well as past and present members of the S&aacute;nchez lab for invaluable discussions during the development of these techniques. This work was supported by NIH Training Grant (5T32 HD0791) to OCG and NIH R37GM057260 to ASA. ASA is a Howard Hughes Medical Institute Investigator.

Note: this protocol suggests the use of potentially hazardous materials (lead and chloretone). Acquire, read, and follow MSDS for all potentially hazardous materials.
1. Animal Culture, Selection, and Preparation
<ol>
 <li>For animal culture and handling use planarian water (1X Montju&iuml;c salts9) and plastic transfer pipettes.</li>
 <li>Sexual biotype Schmidtea mediterranea can be used when raised in the laboratory under normal culture conditions10. To produce asexual specimens of requisite size, S. mediterranea raised at room temperature under normal conditions9 should be fed at double to triple normal frequency (2-3 times per week) for one to two months prior to use. Alternatively, asexual animals fed at normal frequency may be kept at 10 degrees Celsius indefinitely in order to increase their average size.</li>
 <li>Select animals that are between 1 to 2 cm in length and wider than 2 mm then starve animals 3-7 days prior to use.</li>
 <li>If performing any pharmacological or radiological treatments on intended hosts, donors, or partially irradiated animals, perform treatment(s) at this point.</li>
 <li>If pharmacological treatments were performed that required feeding the animals, starve animals an additional 3 to 7 days prior to use.</li>
</ol>
2. Preparation of Solutions and Materials
<ol>
 <li>Prepare chloretone solution, a mild local anesthetic, by dissolving 0.1-0.2% w/v chloretone in planarian water and chilling the solution on ice.</li>
 <li>If performing partial irradiation only, proceed to step 2.7. For tissue transplantation continue on to step 2.3.</li>
 <li>Using a Bunsen burner, bend 0.75 mm interior diameter, used for cutting the graft tissue, and 0.7 mm exterior diameter, used for creating a hole in the host which will receive the graft, capillary tubes to a 90&deg; angle at 1-2 cm from the end of each tube. To save materials, bend both ends of each capillary tube and break them in half to produce two tools. Take care not to flame the very ends of the tubes.</li>
 <li>Cut the following papers to the indicated sizes:
 <ul>
 <li>Black filter paper (cut into rectangles approx. 2.5 cm x 1.5 cm)</li>
 <li>Whatman #3 filter paper (cut into rectangles approx. 2 cm x 0.5 cm)</li>
 <li>Kimwipe (folded and cut into wads approx. 3 cm x 0.5 cm x 4 ply)</li>
 <li>Cigarette rolling paper (remove gum strip and cut into rectangles approx. 3 cm x 2 cm)</li>
 </ul>
 </li>
 <li>Prepare modified Holtfreter's solution (3.5 g/L NaCl, 0.2 g/L NaHCO3, 0.05 g/L KCl, 0.2 g/L MgSO4, 0.1 g/L CaCl2, pH 7.0-7.5) and casein saturated Holtfreter's solution and chill both to 4 &deg;C.</li>
 <li>Attach a folded Kimwipe to a square of Parafilm and place on Peltier cooler plate or other cooling device situated under a dissecting microscope. Saturate the Kimwipe with chilled Holtfreter's solution and place two black filter paper rectangles on the Kimwipe.</li>
 <li>Line Petri dishes with Whatman #2 filter paper. Moisten the filter paper with Holtfreter's solution and chill the dishes on ice. For partial irradiation a larger dish and filter paper liner may be used.</li>
</ol>
3. Anesthetization and Immobilization
<ol>
 <li>Fill a Petri dish with chilled chloretone solution and pipette worms into the dish. For transplantation only anesthetize one host and one donor at a time. For partial irradiation many (n &gt; 10) animals can be anesthetized at once.</li>
 <li>Allow worms to soak in the chloretone solution until they become motionless (5-10 min).</li>
 <li>Rinse worms by pipetting them into a dish filled with chilled Holtfreter's solution.</li>
 <li>Immobilize animals by pipetting them onto black filter paper saturated with chilled Holtfreter's solution and orient them ventral side down with forceps. If animals are able to locomote, soak up excess Holtfreter's solution, slightly decrease the temperature of your Peltier or cold plate, or increase the length of chloretone treatment.</li>
</ol>
4. Partial Irradiation
Note: Follow these steps to prepare animals for partial irradiation. If performing transplantation instead, proceed to section 5.
<ol>
 <li>Place a chilled Petri dish from step 2.7 on ice in an ice bucket that will fit inside a top source X-ray irradiator.</li>
 <li>Arrange anesthetized animals in a Petri dish by moving the black filter paper on which they are immobilized. Using forceps to move anesthetized worms directly may injure them.</li>
 <li>Transport arranged animals to a top-source X-ray irradiator and situate the ice bucket such that the distance from the cathode tube to the animals is minimized, thus maximizing the effective dose rate.</li>
 <li>Position lead shield(s) (Figure 1) between the animals and the cathode tube as desired. Shields should be 4.5 to 6 mm thick to allow for 97 to 99% attenuation of a 325kV X-ray beam11.</li>
 <li>Deliver X-ray dose. If complete stem cell ablation from non-shielded regions is desired, deliver 30 Gy or greater using an X-ray irradiator. For reference, 30 Gy is equivalent to 3.6 minutes at 320 kilovolts and 10 milliamps in a Precision X-Ray Inc. XRAD320 with a field-to-source distance of 30 centimeters.</li>
 <li>Immediately after dosing is complete, handling worms by the black filter paper, transfer animals into chilled planarian water. Allow the planarian water to warm to room temperature and the animals to dislodge themselves from the black filter paper. The partial irradiation procedure is now complete.</li>
</ol>
5. Tissue Transplantation
<ol>
 <li>Using a transfer pipette and forceps, arrange anesthetized host and donor worms on separate rectangles of black filter paper on the Kimwipe which has been cooled on the Peltier or cooler plate under the dissecting microscope.</li>
 <li>Using a 0.75 mm inner diameter capillary tube cut out the graft plug from the donor and, using forceps, place it on an out of the way portion of the host. If graft material gets stuck in the capillary tube, dislodge with forceps.</li>
 <li>Using a 0.7 mm outer diameter capillary tube remove a plug from the host and using forceps position the graft into the hole that is left behind.</li>
 <li>Transfer the transplanted host on its black filter paper rectangle into the Petri dish prepared in step 2.7.</li>
 <li>Wet a piece of rolling paper with casein saturated Holtfreter's solution and place it on top of the transplanted host as diagramed in Figure 2A.</li>
 <li>Soak four pieces of filter paper in casein saturated Holtfreter's solution and encase the transplanted host as diagramed in Figure 2B.</li>
 <li>Soak four wads of cut Kimwipe in casein saturated Holtfreter's solution and lay them over the filter paper from step 5.6 (Figure 2B). Replace the lid and put the Petri dish on ice.</li>
 <li>Transfer the donor worm into planarian water to recover, heal, and regenerate.</li>
 <li>When all transplants are completed, place the transplanted worms into a 10 &deg;C incubator overnight.</li>
 <li>The following morning, taking care not to disturb the graft, uncover the worm and transfer it (on its black filter paper) to a Petri dish filled with planaria water.</li>
 <li>Either allow the worm to dislodge itself from the filter paper or gently remove it with forceps.</li>
 <li>Change the planarian water once every 2-3 days.</li>
</ol>
6. Representative Results
Immediately following partial irradiation planaria will appear normal and unaffected. Depending on the delivered dose and the geometry of the shield used, irradiated tissue may regress and even disintegrate7. Shielded tissue should remain intact. Following tissue regression and a loss of tissue integrity, a blastema will form and missing structures will be regenerated (Figure 3A). If an amputation is made in the partially irradiated region, the irradiated tissue will be rescued (i.e. prevented from regressing or disintegrating) (Figure 3B). In both the uninjured and the amputated case regeneration will be delayed as compared to an amputated non-irradiated planaria (Figure 3C). If an X-ray dose of 30 Gy was delivered and the partially irradiated animal was uninjured, successful stem cell ablation in a pattern corresponding with the lead shield used can be confirmed 2 to 3 days following partial irradiation by in situ hybridization for the stem cell marker Smed-piwi-112 (a.k.a. smedwi-1) (Figure 4).
The morning following transplantation, a successful graft of the transplanted tissue should be obvious within the host tissue, having adhered to both the dorsal and ventral surfaces of the host (Figure 5A). Occasionally, the graft will adhere to only the ventral or dorsal surface. If the transplantation was completely unsuccessful, no sign of the graft will be visible from either the dorsal or ventral surface of the host (Figure 5B). Shortly following a successful graft of non-irradiated tissue into a host that had been ablated of stem cells by lethal irradiation13,14 , in situ hybridization for Smed-piwi-1 will reveal that stem cells are present primarily within the graft (Figure 5C). Additionally, successful grafts of non-irradiated tissue into lethally irradiated hosts will result in rescue of host tissue and long-term survival of the host15.
<img alt="Figure 1" src="/files/ftp_upload/4015/4015fig1.jpg" /> 
Figure 1. General arrangement of basic components for partial irradiation. In an X-ray irradiator with a top positioned X-ray source (cathode tube) the anesthetized planarian is positioned directly below the X-ray source within the irradiation field. In order to maximize the X-ray dose rate, the distance between the planarian and the X-ray source should be minimized. A lead shield should be positioned between the cathode tube and the anesthetized worm, as close to the worm as practical. The lead shield should be designed, manufactured, and positioned so that it shields the desired tissue but completely exposes the rest of the worm. Many commercial manufactures will produce custom lead shields from your simple design diagram; we have successfully used Alpha Systems Corp. (Bluffdale, UT). The lead should be sufficiently thick to allow for the desired amount of shielding. For example, if near complete shielding of a 320 kV X-ray beam is desired, the lead should be 4.5 to 6 mm thick. The planarian and shield are positioned on a Holtfreter's soaked filter paper lined Petri dish which rests in an ice bucket. Given a sufficiently large X-ray irradiation field and a number of identical lead shields, many specimens may be partially irradiated at one time (not pictured).
<img alt="Figure 2" src="/files/ftp_upload/4015/4015fig2.jpg" /> 
Figure 2. Building the recovery chamber. (A) An exploded view of the tissue transplantation recovery chamber, showing all of the components that are layered on top of one another after being soaked in casein saturated Holtfreter's Solution. (B) A nearly completed recovery chamber, illustrating the interlocking placement of the Whatman #3 filter paper rectangles which snugly encase the anesthetized planarian, preventing movement during healing. The careful construction of the recovery chamber prevents animal movement and desiccation, promoting rapid healing and greater efficacy of tissue transplantation.
<img alt="Figure 3" src="/files/ftp_upload/4015/4015fig3.jpg" /> 
Figure 3. Representative outcomes of simple posterior partial irradiation. As Dubois described7, (A) when the posterior half of planarians were shielded with lead and then exposed to x-ray irradiation the anterior tissue was observed to regress back to the boundary between the irradiated and shielded tissue at which point unpigmented blastemas formed and the animals began to regenerate. (B) On the other hand, when animals were decapitated following the same partial irradiation performed in (A), tissue regression was not observed and the remaining irradiated anterior tissue was rescued. The decapitated partially irradiated animals regenerated heads (B), however, regeneration was significantly delayed as compared to unirradiated decapitated controls (C).
<img alt="Figure 4" src="/files/ftp_upload/4015/4015fig4.jpg" /> 
Figure 4. Representative outcome of partial stem cell ablation following partial irradiation. Whole-mount In situ hybridization (WISH) for the stem cell marker Smed-piwi-1 reveals that wild type planaria have stem cells distributed throughout their bodies with the exception of the tissue anterior to the photoreceptors (arrowhead) and the pharynx proper (asterisk)14. (A) WISH for Smed-piwi-1 in irradiated but fully shielded control planaria fixed three days following irradiation shows a stem cell distribution that is indistinguishable from that of wild type planaria. (B) On the other hand, WISH for Smed-piwi-1 in animals that were only partially shielded, leaving the anterior and posterior exposed, but were also fixed three days following irradiation shows that stem cells are ablated from the non-shielded regions. Scale bars are 500 microns.
<img alt="Figure 5" src="/files/ftp_upload/4015/4015fig5.jpg" /> 
Figure 5. Examples of successful and unsuccessful tissue transplantation. (A) Live images of dorsal and ventral views of a successfully transplanted planarian three days following transplantation. The graft (indicated) is clearly visible on both dorsal and ventral surfaces and is surrounded with characteristic unpigmented tissue at the graft-host interface. (B) Correspondingly, unsuccessful transplantations display no visible graft tissue at the transplantation site (indicated) and instead show a healed, unpigmented, lateral wound from the failed transplantation. A graft that adheres to only the dorsal or the ventral surface can resemble a successful transplantation (A) when viewed from one side and an unsuccessful transplantation (B) when viewed from the other. (C) When wild type (wt) tissue is grafted into an irradiated host that has been ablated of resident stem cells and the transplanted stem cells are later revealed by WISH for Smed-piwi-1 two days following transplantation, the success of the transplantation is clearly displayed by the specific presence of stem cells only in or around the graft location (arrowheads). Scale bars are 500 microns.

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	<li>Morgan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+the+regeneration+of+Planaria+maculata.">Experimental studies of the regeneration of Planaria maculata.</a> Arch. Entw. Mech. Org. 7, 364-397 .</li><li>Reddien, P. W., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+planarian+regeneration.">Fundamentals of planarian regeneration.</a> Annu Rev Cell Dev Biol. 20, 725-757 (2004).</li><li>Randolph, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+the+tail+in+lumbriculus.">The regeneration of the tail in lumbriculus.</a> J. Morphol. 7, 317-344 .</li><li>Wagner, D. E., Wang, I. E., Reddien, P. 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No conflicts of interest declared.

Importance of immobilization
Immobilization is by far the most critical step for the successful completion of any of these processes. If planaria are improperly immobilized prior to partial irradiation, they may move beneath the lead shield, producing inconsistent and confounding results. Additionally, if animals are insufficiently immobilized following transplantation, the host worm will likely move away from the graft tissue, resulting in a failure of the graft to heal properly to the host. Pro...

<table><tbody><tr><td>General Purpose Transfer Pipette</td><td>Samco</td><td>691</td><td></td></tr><tr><td>Capillary tubes (ID 0.75 mm)</td><td>FHC</td><td>30-30-0</td><td></td></tr><tr><td>Capillary tubes (OD 0.7 mm)</td><td>FHC</td><td>30-50-08</td><td></td></tr><tr><td>Parafilm M</td><td>VWR</td><td>52858-076</td><td></td></tr><tr><td>Kimwipes 34155</td><td>VWR</td><td>500029-891</td><td></td></tr><tr><td>Black filter paper</td><td>Schleicher Schuell</td><td>10310809</td><td></td></tr><tr><td>Whatman #2 filter paper 1002-055</td><td>Fisher Scientific</td><td>09-810B</td><td></td></tr><tr><td>Whatman #3 filter paper 1003-185</td><td>Fisher Scientific</td><td>09-820E</td><td></td></tr><tr><td>Cigarette rolling paper</td><td>Zig-Zag, original</td><td>NA</td><td></td></tr><tr><td>Petri dishes</td><td>VWR</td><td>82050-544</td><td></td></tr><tr><td>Forceps DUMONT, INOX #5</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Chloretone</td><td>Sigma Aldrich</td><td>112054</td><td></td></tr><tr><td>Casein</td><td>Sigma Aldrich</td><td>C3400</td><td></td></tr><tr><td>Lead Shields</td><td>Alpha Systems Corp., Bluffdale, UT</td><td>Custom design</td><td></td></tr><tr><td>XRAD-320 Biological Irradiator</td><td>Precision X-Ray, North Branford, CT</td><td>NA</td><td></td></tr></tbody></table>

planarian immobilization, partial irradiation, and tissue transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

planarian-immobilization-partial-irradiation-tissue

Research

JoVE Journal

Biology

19.5K Views.  University of Utah School of Medicine. An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Video: Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Immunology and Infection

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengineering

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Cancer Research

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Developmental Biology

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Medicine

Partial Heterotopic Hindlimb Transplantation Model in Rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Summary

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Chapters in this video

Orthotopic Hind-Limb Transplantation in Rats

Use of Human Perivascular Stem Cells for Bone Regeneration

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

Partial Heterotopic Hindlimb Transplantation Model in Rats

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat