Research to Prevent Blindness; Experimental assistance from Takayuki Nagasaki; This research complies with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. KJW is supported by NIH grants 5T32EY013933 and 5T32DK007647-20. VBM is supported by NIH grant K08EY020530.

1. Assemble Devices for the Subretinal Injection
<ol>
 <li>Purchase or make a 100 &mu;m diameter needle from a glass capillary tube. This can be done manually by using a Sutter P-97 pipet puller or other similar equipment. The end of the capillary tube will be heated and pulled until it reaches the desired diameter (100 &mu;m). A smaller diameter needle can be used for gene therapy vectors; however, this is the recommended diameter for cell injection without damage to the cells or the eye. Compared to steel needles, the glass capillary needle has a very fine, but blunt tip, to allow for visualization of the injection fluid and access to the subretinal space without causing retinal breakage. Clean these pulled needles if needed using 70% ethanol, and store in a sterile container. A 15 cm sterile tissue culture dish with tape to hold the needles in place is one way in which to keep the injection needles covered and in a sterile environment. At this point, complete the set-up within the surgical room where the procedure will take place. It is easiest to keep all equipment in the surgical room at all times in order for it to remain sterile, especially if this procedure is done within a barrier facility.</li>
 <li>Aspirate silicone into the injection needle and eject the silicone to leave a coating along the inner walls of the needle. The silicone coating maximizes the passage of cells and viruses through the needle bore, which would otherwise stick to the inner glass capillary walls.</li>
 <li>Obtain an approximately 8 cm length of sterile plastic tubing by cutting a 25 &frac34;-gauge blood collection set with luer-lock at the point directly after the needle (removing the needle from the tubing).</li>
 <li>Fill a 1 ml Sub-Q 26 5/8-gauge slip-tip syringe with sterile saline and remove the needle from the syringe. </li>
 <li>Attach the injection needle to the cut end of the plastic tubing and attach the syringe to the luer-lock at the other end. </li>
 <li>Aspirate sterile saline to fill the dead space in the tubing and capillary needle, ejecting some saline through the needle to ensure that it is correctly attached without any leakage. Next, aspirate a small, approximately 1 &mu;l amount of air, into the tip of the injection needle. This will separate the saline from the injection fluid and provide a visual cue indicating the end of the injection fluid during the surgical procedure. The injection apparatus should be kept on a clean surface wiped down with 70% ethanol during the surgical procedure. Additionally, all equipment used during the surgical procedure should be cleaned both before and after with 70% ethanol, for the exception of the injection needles which should already be kept sterile and ready for use. These needles should be disposed of after surgery, and not re-used.</li>
 <li>Finally, aspirate injection solution containing the desired packaged gene therapy viral vector or stem cells. This can be done by taking up the injection solution into a sterile pipet tip, then pipetting the solution onto the surface of a sterile cell culture dish. The solution can now be easily aspirated into the injection needle using the syringe. The amount needed will vary depending on the age of the mouse. For our purposes we use post-natal day 5 mice, which can be injected with 0.5-0.8 &mu;l of injection fluid. Smaller amounts, 0.5 &mu;l or less, can be used for younger mice and larger amounts, such as 1 &mu;l, can be used for adult mice. The viral titer or number of cells needs to be determined based on the virus/cells being injected and their desired target tissue, as too many viral particles or cells in a small volume can lead to toxicity for the retinal cells. In the video, for visualization purposes, we have used 1.5 &mu;l, a greater amount of dye than necessary to inject into the eye. </li>
</ol>
2. Accessing the Subretinal Space
<ol>
 <li>Within the sterile surgical room, stabilize or anesthetize the mouse according to the locally approved animal handling protocol, which will vary by age of the mouse. Common anesthesia for adult mice includes the use of isoflurane gas or an intra-peritoneal injection of 0.1 ml/10g body weight of ketamine (100 mg/ml) and xylazine (20 mg/ml) in sterile saline. For perinatal mice, common anesthesia techniques include the use of isoflurane gas or cryoanesthesia (hypothermia). All anesthesia techniques must be approved by your local IACUC committee before use. We typically use post-natal day (P) 5 mice, although similar techniques can be applied to mice as young as P0 or to adult mice. The surgical procedure cannot begin until the mouse has ceased moving the limbs and no longer responds to toe pinches. For perinatal mice, they will become noticeably lighter in color since the blood flow will slow down, another key to knowing that they are fully under anesthesia.</li>
 <li>In perinatal mice, use Vannas straight scissors to make an approximate 1.5 mm incision along the closed lid fissure. Be careful to cut at the indentation, where the eye will naturally open in the future. This ensures that the lids will heal and open properly during the development of the mouse. A surgical blade can be used for this purpose, although we prefer Vannas scissors since they reduce the risk of puncturing the eye during this procedure. In the video, the scissors were used to directly puncture and cut along the eyelid margin. When learning this technique, forceps should be used to lift the eyelid before cutting and reduce the risk of damaging the eye.</li>
 <li>Pull apart the eyelids with curved dressing forceps to proptose the eye and pinch the eyelids slightly underneath the eye to hold it proptosed for injection. If the lid incision made in step two is too large, the eye will slip back under the lids and prevent adequate exposure during the injection procedure. For adult mice, applying slight pressure around the eye with dressing forceps can hold the eye proptosed for injection.</li>
 <li>Make a small scleral incision at or posterior to the globe's equator by scratching the surface with a 15-degree microsurgery blade. This incision should be only large enough to pass the tip of the injection needle, but no larger than that. If preferred, a small, sharp needle such as an acupuncture needle can be used to make this incision into the subretinal space instead of the microsurgery blade. In darkly pigmented animals, a dark brown-pigmented tissue (the choroid-retinal pigment epithelium) will become visible at the point of incision. In lighter pigmented or albino mice, a surgical marking pen can be applied to the tip of the microsurgery blade to leave an ink spot indicating the incision site. If clear (vitreous) fluid refluxes from the incision site, the blade was placed too deep and entered the vitreous cavity and the therapeutic virus or cells may enter the intravitreal space during injection. For visualization purposes, we have made the incision to this point in the video. It is still possible to complete a subretinal injection in this case, however the retinal detachment caused by the presence of the injection fluid may not fully heal in this mouse. Therefore, care must be taken to cut only the outer surface of the eye and not through the retina before injection. </li>
 <li>Carefully insert the injection needle into the incision site and advance it parallel to the outer eye wall to enter the subretinal space. Advancing too close to the outer wall or too deep within the eye will direct the injection into the wrong location. The best method to understand whether the needle is in the correct location is to practice the technique. Practicing on lightly pigmented or albino mice can help to make precise and accurate injections into the subretinal space. This is because the needle and injection fluid is visible within these eyes.</li>
 <li>Apply light pressure to the syringe plunger and begin injection of the desired fluid. If the needle is correctly placed within the subretinal space, moderate backpressure will be felt. If the needle is within the vitreous, there will be significantly less backpressure and potentially some fluid reflux during the injection. If there is significant backpressure, hold the needle in place for at least an additional 15 seconds before removing the needle slowly. This will allow the high intraocular pressure to equalize instead of refluxing the injected virus or cells back out the incision site. Some of the air and saline may be injected into the subretinal space due to the increased pressure required during the injection procedure. Care should be taken not to over-inject air or saline, since it can flush out the injected virus or cells from the eye. It is also possible to cause permanent retinal detachment from the presence of too much fluid injected into the subretinal space. For visualization purposes, excess dye and air was injected into the subretinal space in the video. It can be clearly seen how some of the dye will be flushed back out of the subretinal space if too much is injected, but also how the air bubble remains within the subretinal space and does not disappear into the vitreous. This was used to emphasize that these injections were all within the correct location of the eye, so one can visualize how the needle should enter the incision site. All injections into the subretinal space cause a temporary retinal detachment due to the presence of the injection fluid. If the injection procedure is completed correctly, this temporary retinal detachment will heal within 24 hr and mice can be used for all further experiments. The eye may remain soft for approximately one week due to the surgical procedure, so a second injection should be avoided during this period of time.</li>
 <li>For neonatal mice, use the curved dressing forceps to gently push the eye back behind the lids and into the orbit. It may be useful to wait for a minute in order to allow the virus to settle within the subretinal space and not cause it to be flushed out of the incision site when gently pushing the eye back into its orbit. After surgery, one drop of 0.5% bupivacaine (marcaine) diluted to 0.25% in sterile saline from a 25-gauge needle can be placed at the injection site. Mice do not show overt signs of distress after subretinal surgery and the use of bupivacaine as a long-acting topical analgesic has been ample for the previous three years. Mice should always be monitored after the surgical procedure to look for any signs of distress; such as infection, swelling, or a decrease in the usual daily activities of the mouse. If these occur, the veterinarian should be notified and further pain medications such as buprenorphine (0.05 mg/kg) can be given as an intraperitoneal injection.</li>
 <li> Wake the mouse from anesthesia according to locally approved animal handling protocol. The mouse should be kept on a heating pad to maintain normal body temperature as it wakes from the anesthetic. The surgical procedure should take less than five minutes to complete, not counting the time for the mouse to undergo anesthesia. Therefore, a heating pad is not necessary during the actual procedure, but may be needed while one is learning the technique as the procedure could take a longer duration of time. The mouse should not be placed back into a clean cage until it has begun to move around on its own. For neonates, gentle toe pinches will help for them to fully recover from the anesthesia, and they should regain their pink color and movement before being placed back with the mother. Mice should be monitored over time for any signs of distress as listed previously.</li>
</ol>

<ol>
	<li>Abe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+retina+using+pigment+epithelial+cell+transplantation.">Regeneration of the retina using pigment epithelial cell transplantation.</a> Nihon Ganka Gakkai Zasshi. 106, 778-803 (2002).</li><li>Jacobson, S. G., Cideciyan, A. V., Ratnakaram, R., Heon, E., Schwartz, S. B., Roman, A. J., Peden, M. C., Aleman, T. S., Boye, S. L., Sumaroka, A., 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=Gene+therapy+for+Leber+congenital+amaurosis+caused+by+RPE65+mutations:+safety+and+efficacy+in+15+children+and+adults+followed+up+to+3+years.">Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.</a> Arch. Ophthalmol. 130, 9-24 (2011).</li><li>Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379, 713-720 (2012).</li><li>Wang, N. K., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+reprogrammed+embryonic+stem+cells+improves+visual+function+in+a+mouse+model+for+retinitis+pigmentosa.">Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.</a> Transplantation. 89, 911-919 (2010).</li><li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Tosi, J., Sancho-Pelluz, J., Davis, R. J., Hsu, C. W., Wolpert, K. V., Sengillo, J. D., Lin, C. S., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-mediated+expression+of+cDNA+and+shRNA+slows+degeneration+in+retinitis+pigmentosa.">Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa.</a> Exp. Biol. Med. 236, 1211-1217 (2011).</li><li>Tucker, B. A., Park, I. H., Qi, S. D., Klassen, H. J., Jiang, C., Yao, J., Redenti, S., Daley, G. Q., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+adult+mouse+iPS+cell-derived+photoreceptor+precursors+restores+retinal+structure+and+function+in+degenerative+mice.">Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.</a> PLoS One. 6, e189992 (2011).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A high-throughput Mouse Eye Phenomics System. , (2010).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384 (2011).</li></ol>

No conflicts of interest declared.

This video technique provides instructions on completing the subretinal injection surgical procedure successfully, and ensuring that the gene therapy vector or stem cells are placed in the location necessary to efficiently treat the ophthalmic disease. This technique allows for the targeting of retinal cells such as the RPE or photoreceptors, since it places the gene therapy vectors or stem cell-derived tissues in the vicinity of these cells. Previous methods involved intravitreal injections, where the fluid is placed wi...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>0.8-1.10 x 100 mm Capillary Tube (glass)</td>
 <td>Kimble Glass, Inc.</td>
 <td>34502 99</td>
 <td></td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller </td>
 <td>Sutter Instrument</td>
 <td>P-97</td>
 <td>Narishige microforge can be used instead.&nbsp; Catalog #MF-900 </td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>Sigma Aldrich</td>
 <td>SL2-25ML</td>
 <td>Silicone</td>
 </tr>
 <tr>
 <td>Dubecco's Phosphate Buffered Saline with Calcium Chloride and Magnesium Chloride</td>
 <td>Gibco-Invitrogen</td>
 <td>14040-133</td>
 <td></td>
 </tr>
 <tr>
 <td>Safety-Lok 25 3/4G x 12"; Blood Collection Set</td>
 <td>B-D Vacutainer</td>
 <td>367298</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml Sub-Q 26 5/8G Slip-Tip Syringe</td>
 <td>Becton-Dickinson</td>
 <td>309597</td>
 <td></td>
 </tr>
 <tr>
 <td>0.5-10 &mu;l Finnpipette II Adjustable-Volume Pipetter</td>
 <td>Fisherbrand</td>
 <td>21-377-815</td>
 <td></td>
 </tr>
 <tr>
 <td>1-200 &mu;l Natural Beveled Tips</td>
 <td>USA Scientific, Inc.</td>
 <td>1111-1700</td>
 <td></td>
 </tr>
 <tr>
 <td>Discovery.V8 Stereo Microscope</td>
 <td>Zeiss</td>
 <td>MC1500</td>
 <td></td>
 </tr>
 <tr>
 <td>60 mm x 15 mm Style Treated Polystyrene Cell Culture Dish</td>
 <td>Corning Incorporated</td>
 <td>430166</td>
 <td></td>
 </tr>
 <tr>
 <td>Vannas Straight Scissors</td>
 <td>Storz Ophthalmics</td>
 <td>E3383 S</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved Dressing Forceps with Serrations Delicate</td>
 <td>Storz Ophthalmics</td>
 <td>E1408</td>
 <td></td>
 </tr>
 <tr>
 <td>15 Degree Microsurgery Knife</td>
 <td>Wilson Ophthalmic Corp.</td>
 <td>091204</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Ketaset III</td>
 <td>NADA #45-290</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd Laboratories</td>
 <td>NADA #139-236</td>
 <td></td>
 </tr>
 <tr>
 <td>Bupivacaine (Marcaine)</td>
 <td>AstraZeneca</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Buprenorphine </td>
 <td>Sigma Aldrich</td>
 <td>B9275</td>
 <td></td>
 </tr>
 </tbody>
</table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

A drawing of the mouse eye is shown with major structures labeled for reference, with arrows displaying the locations for both intravitreal and subretinal injection surgical procedures (arrowheads, Figure 1). Gene therapy vectors, such as the lacZ lentiviral vector (Figure 2), can be injected using these locations. Additionally, stem cells, such as mouse embryonic stem cells (Figure 3), can also be transplanted at these sites in the mouse eye. 
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Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

subretinal-injection-gene-therapy-vectors-stem-cells-perinatal-mouse

Research

JoVE Journal

Medicine

33.0K vistas.  Columbia University. Esta técnica quirúrgica ilustra la inyección de vectores de terapia génica y las células madre en el espacio subretiniano del ojo del ratón.

Video: Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...