J.A.P. and J.M.J. jointly designed the experimental approach for evaluating Nanoemulsions in the Chronic Constriction Injury rat model for effects on neuropathic pain. J.M.J. conceived and designed the overall macrophage-targeted drug delivery approach with nanoemulsions, the nanoemulsion composition, and processes for fabrication. J.M.J. produced the nanoemulsion, which was further fabricated by L.L. under the guidance of J.M.J. The stability of the nanoemulsion was assessed by J.M.J., L.L., and S.P. Animal care, surgery, behavior, tail vein injections, and NIRF imaging were carried out jointly by M.S. and A.M.S. under the guidance of J.A.P. The manuscript was written and prepared by M.S., and the protocol was written by A.M.S.
NIR optical imaging was performed on the Small Animal Imaging System at Duquesne University (supported by the Pittsburgh Tissue Engineering Initiative Seed Grant). J.M.J. acknowledges support from the DOD award number FA8650-17-2-6836, NIDA award number 1R21DA039621-01, NIBIB award number R21EB023104-02 and AFMSA Award number FA8650-17-2-6836. J.A.P. and J.M.J acknowledge support from the Pittsburgh Tissue Engineering Initiative Seed Grant. J.A.P. also acknowledges the Hunkele Dreaded Disease Award, the Samuel and Emma Winters Foundation, the Charles Henry Leach II Fund, and the Commonwealth Universal Research Enhancement Award. J.A.P. and J.M.J. acknowledge support from the Duquesne University Inaugural Provost&#8217;s Interdisciplinary Research Consortia Grant, which supports the Chronic Pain Research Consortium.

All protocols were performed in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) at Duquesne University.
1. Preparation and Anesthesia
NOTE: Aseptic techniques are used for the entirety of the procedure. Only new sterile materials and autoclaved sterile instruments are to be used. Personal protective equipment (sterile gloves, hair bonnet, surgical mask, scrubs) needs to be worn to avoid contamination.
<ol>
	<li>Adult male Sprague-Dawley rats weighing 250&#8211;300 g were used in this protocol. Acclimate the rats to standard living conditions, keep them on a 12 h light/12 h dark cycle, and provide food and water ad libitum. House the animal socially, keep them on paper bedding, and provide a special diet (see Table of Materials) to avoid autofluorescence during imaging.</li>
	<li>With the use of a properly placed heating pad, anesthetize the animal under an initial 5% isoflurane in 20% oxygen, followed by a maintenance level of not less than 1.5% isoflurane and not more than 3%, unless the animal wakes up or retains feeling.</li>
	<li>Confirm proper anesthesia via a lack of response to tail pinches. Monitor the blood flow as well via vital signs throughout the procedure.</li>
</ol>
2. Pre-injection Image Aquisition
<ol>
	<li>Image the animal in a preclinical NIR fluorescence imager by positioning the animal laterally to expose the injection site on the lateral tail to establish a baseline of fluorescence in the tail (Figure 1C,E).&#160;Perform a simultaneous image acquisition of both a white light (body view) and near infrared channels using the NIRF imager and associated software, with linked lookup tables (LUT).</li>
	<li>Following imaging, move the animal back to the surgical table, and place it under anesthesia for the tail vein injection. 
	NOTE: Continue monitoring the animal&#8217;s vital signs and recheck proper anesthetization via tail pinch.</li>
</ol>
3. Tail Vein Injection with NIRF-containing Agent
<ol>
	<li>With the animal in the prone position, orient the tail with the dorsal side facing up. Dilate the tail vasculature in warm water for a minimum of 1 min. Orient the tail vein so the lateral side (either right or left) is turned 30&#176; (clockwise or counterclockwise) to expose the right or left tail vein (Figure 1B).
	<ol>
		<li>Once a lateral tail vein has been located (which appears dark-colored upon dilation), disinfect&#160;the entire tail with alcohol pads, repeating 2x.</li>
		<li>At an appropriate dosage based on the study design, begin injections in the distal coccygeal vertebrae region of the tail and moving more proximal if proper needle placement fails.</li>
	</ol>
	</li>
	<li>Insert a 25&#8211;27 G sterile needle, bevel up, into the lateral tail vein, with the tail at a 180&#176; angle, inserting the needle parallel to the lifted tail. Observe blood flashback in the rim of the needle to ensure correct placement. If no flashback is apparent, slowly move the needle tip (without removing it from the tail) to find vein insertion. If placed subcutaneously, no blood flashback will occur.</li>
	<li>Insert the syringe with the injectable materials into the rim of the needle. When proper placement is achieved, the injectable fluid will not incur resistance upon injection. The injection will advance smoothly and easily. Once injected, remove the needle and the syringe, apply pressure with sterile gauze for at least 1 min to ensure clotting, and mark the spot of injection with a pen on the tail, ensuring it is visible on the white light image. 
	NOTE: No hematoma or lesion will be visible at the site of injection.</li>
	<li>If the needle tip moves during the syringe insertion, remove the needle and retry the needle entry procedure more proximal on the ipsilateral tail vein. Do not reuse the same needle if a different reentry point is tried. 
	NOTE: Alternatively, the injection can be performed with an IV catheter with a blood-flow indicator (see Table of Materials). This has the benefit of visual confirmation of the catheter during venipuncture. Insert the catheter, bevel side up, at the angle previously described. Observe prompt flashback in the entire length of the blood-flow indicator to ensure correct placement. Slight back pressure can be used to pull blood into the syringe to confirm proper placement in the vessel before injecting. Again, no resistance will be felt.</li>
</ol>
4. Post-injection Image Acquisition
<ol>
	<li>Perform quality assessment following tail vein injection in a preclinical NIR fluorescence imager in the same orientation as the baseline pre-injection image. Ensure the animal is still properly anesthetized &#8212; and will be so for the duration of imaging &#8212; prior to placing it in the imager. 
	NOTE: An imaging system containing a drawer housingwith anesthesia connections and a mask for the animal should be used if available.&#160;&#160;</li>
	<li>Orient the animal on its lateral side to expose the injection site (as marked) on the lateral tail. Check to see if a NIRF signal is present only at the site of injection. This indicates&#160;a successful tail vein injection (Figure 1D). 
	NOTE: If the signal is dispersed throughout the entire tail, it is considered to be subcutaneous and, hence unsuccessful (Figure 1F). Figure 2 shows additional examples of failed injections.</li>
</ol>
5. Image Quantification
NOTE: Image quantification can be performed with the imaging software that accompanies the NIR imager, if this&#160;function is available. Alternatively, other commercially available image analysis&#160;software may be used24.
<ol>
	<li>In the post-injection image, draw a region-of-interest around the area of fluorescence at the injection site2,6.&#160;</li>
	<li>Measure the area and relative fluorescence intensity and record as area/intensity. Compare post-injection and baseline pre-injection images either qualitatively or quantitively by using appropriate statistical analysis(dependent on study groups and conditions). 
	NOTE: The researcher can decide on thresholds that discriminate good from bad injections or assign a percentage of quality to the injection.</li>
</ol>

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The authors have nothing to disclose.

Research laboratories incur significant costs as a result of the misadministration of testing agents. Tail vein injections are a difficult technique to master to attain consistent success rate, with the most experienced of technologists often incurring misadministration errors. There is no reliable way to confirm a successful injection. This protocol offers a solution to this problem by giving researchers a qualitative and quantitative method to validate the success of a murine tail vein injection. Here, a NIRF-labelled nanoemulsion7,8,25&#160;incorporates the agent of choice (in this case, a drug) and is imaged at the site of injection in a NIRF small-animal imager. There is also the option to develop a non-nanoemulsion-based agent and use the same principle of NIRF imaging by incorporating commercially available infra-red dyes. Additionally, ready-to-use imaging agents with a variety of applications, such as tumor imaging, metabolic imaging, cell trafficking, and apoptosis are also commercially available. An injection is performed either by using a sterile needle or, alternatively, an IV catheter; this depends on the preference of the researcher. In addition, automated tail vein injectors26&#160;have been used to assist in this process and are compatible with this methodology. However, this technology has not yet become commercially available.
There are important steps in the tail vein injection method that ensure a higher rate of correct agent administration. First, the tail should be cleaned with ethanol to remove any dirt or debris, allowing researchers to better visualize the vein. Dilating the vein by submerging the tail in warm water is also a very important step in the method, as it allows a greater surface area for injection. Injecting at a more distal point on the tail vein allows for some error, in the event that multiple attempts are required. Injection should be attempted at a more proximal position in the tail as the tail vein increases in size as the caudal aspect of the animal&#8217;s body is approached. In addition, the contralateral tail vein can be used if needle placement fails in more than three to five sites on the ipsilateral tail vein.
A successful administration of a test agent results in little to no NIRF signal at the point of injection. If no resistance is felt during the administration of the injection and there is little to no fluorescence at the tail, then the injection can be recorded as successful. If resistance is felt during injection and there is a trail of NIRF signal along some length of the tail, then the injection is recorded as unsuccessful and is likely partly subcutaneous. Fluorescence images are taken pre- and post-injection, and the quality of the injection is assessed by observing qualitatively or analyzing quantitatively the fluorescence signal at the site of injection. The software accompanying the NIR fluorescence imager is often capable of performing this analysis.
The method can be adapted in several ways. It is applicable to tail vein injection in both mice and rats. Most small-animal NIR fluorescence imagers will be capable of accommodating murine rodents. Levels of anesthesia need to be adjusted depending on the weight of the animal, in accordance with the research laboratory&#8217;s IACUC protocol. Another possible modification is the preparation of a non-nanoemulsion-based probe either by incorporating an infrared dye into the researcher&#8217;s formulated agent or by purchasing a ready-to-use imaging agent, tailored to a specific biological application.
If a rat is relatively large, it can often be difficult to position it in the small-animal imager. It is thus recommended that a test image is taken with the animal in the drawer before injecting, and a field of view ascertained where the tail is visible. It is helpful to tape the tail to the drawer of the imager, to ensure it does not move during imaging.
Alternative methods seeking to assess the quality of tail vein injections in small animals are limited to the utilization of labeling reagents that do not interfere with concurrent experimental procedures and require euthanasia of the animals postinjection12,13. Some reagents may impact study outcomes and the therapeutic assessment of the animals involved, so care in experimental design is recommended.
This method can, in the future, be refined with advances in small-animal imaging technology, as well as improvements in infrared fluorescent probes. Biological probes with an incorporated infrared dye, designed for a variety of different applications, can be used at the agent administration stage of a study design to validate the quality of an injection, as outlined in this method2,3,27,28,29,30,31,32.

The route of administration of agents into small animals serves as a critical point of many experiments. It determines where the agent is to be delivered and, subsequently, what will happen to the agent thereafter. Although other routes can be used for agent administration1, the intravenous route of delivery is a preferred route for certain agents. IV injection allows agents to be directly injected into the bloodstream, bypassing first-pass tissue effects and the need for extraneous solute absorption1. This also allows for targeting cells in the bloodstream2,3 and direct delivery to all tissues within the circulatory system. In rodents, several veins can be considered, including the jugular, the saphenous, and the tail vein.
In this method, a NIRF dye containing a biological probe&#8212;in this case, a nanoemulsion (Figure 1A)3,4,5,6&#8212;is injected into the lateral tail vein of rats. This particular NIRF-containing nanoemulsion has been used previously to image and track neuroinflammation in vivo and ex vivo7,8 in a rat model9 of neuropathic pain2,3,4,5,10,11. Imaging is conducted before and after the injection with a preclinical NIR fluorescence imager (see Table of Materials). This serves as a tool to validate the quality of the agent administration. Imaging prior to the tail vein injection serves as a basis for obtaining a baseline image.
Increasingly in animal studies, intravenously administered nanoemulsions are being utilized as biological probes and targeting agents12,13,14,15. It is a proven challenge to administer an agent via the tail vein16,17&#8212;be it a drug, a viral vector, or another probe&#8212;and to ensure that the entire contents of the injection have successfully entered the bloodstream and not the surrounding tissues17. Therefore, a method of visualizing and evaluating the quality of a successful injection is beneficial.&#160;
Typically, a heat lamp or warm water is used to warm the tail, which causes dilation of the vein, permitting its visualization prior to injection. While this ensures easier entry into the vein, there is not a quantitative way to discern whether the compound has entered the bloodstream in its entirety18,19,20,21. This becomes more difficult still in strains of animals where the vein contrasts faintly with the skin, such as in black mice. Typically, the investigator can gauge a failed injection by experiencing resistance during the injection and, in some cases, visualizing a bulge on the tail, indicating a subcutaneous leakage of the agent22,23.&#160;
In this study, NIRF imaging of the nanoemulsion injected into the lateral tail vein of live rats is performed on a small-animal NIRF imaging system (see Table of Materials). Rats are fed a special purified diet (see Table of Materials) to reduce nonspecific gut fluorescence. Simultaneous image acquisition of white light and 800 nm fluorescence is captured using the NIRF imager and associated software. The relative fluorescence intensity is measured on the tail at the pre-injection and post-injection states. The fluorescence intensity for the region of interest (ROI) at the site of injection is recorded and divided by the area of the ROI. Qualitative assessments can be made on which injections are acceptable. Optionally, further quantitative analysis can be performed by setting thresholds for acceptable injections and assigning ROI measurements into groups, at which point statistical significance can be calculated.&#160;
By utilizing this validation strategy following tail vein injections, the standard of a research study improves due to increased consistency of agent administration. This method of assessing the quality of tail vein injection can be easily customized for different injectable agents to include infra-red fluorescent probes provided commercially by several companies.

<table border="0" cellpadding="0" cellspacing="0" style="width:1033px;" width="1032">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">100% Oxygen air tank</td>
			<td style="width:175px;">AirGas Heathcare</td>
			<td style="width:119px;">n/a</td>
			<td style="width:435px;">For ventilation of animal.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">70% Ethanol</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Alcohol Pads</td>
			<td style="width:175px;">Henry Schein</td>
			<td style="width:119px;">112-6131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Artificial Tears</td>
			<td style="width:175px;">Henry Schein</td>
			<td style="width:119px;">100-2634</td>
			<td>This protects the rats eyes while it is anesthetized.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Beaker</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td>This holds warm water to dilate the tail veins.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Distilled water</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Exhaust Fans</td>
			<td style="width:175px;">Hazard Technologies</td>
			<td style="width:119px;">n/a</td>
			<td>For ventilation of lab, if it is not built in.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Face Mask</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Gas Chamber with tubing and face mask</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Gauze Pads</td>
			<td style="width:175px;">Henry Schein</td>
			<td style="width:119px;">100-2634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Hair Bonnet</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Heating Lamp</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Heating Pad</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Isoflurane</td>
			<td style="width:175px;">Southmedic Inc.</td>
			<td style="width:119px;">ND66794-013-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Padded Bench Cloth</td>
			<td style="width:175px;">Box Board Products Inc.</td>
			<td style="width:119px;">026755100I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pearl Small Animal Imager</td>
			<td>Li-COR Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Pearl Trilogy Small Animal Imaging System</td>
			<td style="width:175px;">LI-COR Biosciences</td>
			<td style="width:119px;">n/a</td>
			<td>Quote available via manufacturers web site. Other manufacturers such as Perkin Elmer (VisEn Medical FMT) offer preclinical NIR fluoresence imagers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Scrubs, lab coat, shoe covers</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Sharps container</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">special diet</td>
			<td>Research Diets, Inc, New Brunswick, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sprague-Dawley rats</td>
			<td>Hilltop Animals, Springdale, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Sterile injection cap</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Sterile needle, 27G</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">SURFLO IV Catheter, 24G, yellow</td>
			<td style="width:175px;">TERUMO</td>
			<td style="width:119px;">SR+OX2419C1</td>
			<td>This is an alternative to using a sterile needle. It provides additional indication of correct venous insertion.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Surgical gloves</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Surgical Tape</td>
			<td style="width:175px;">Multiple sources</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

a new best practice for validating tail vein injections in rat with near-infrared-labeled agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Rats were injected with NIRF-containing nanoemulsion into the lateral tail vein, and pre- and post-injection images were taken with the small-animal imager (Table of Materials) as described in the protocol. Post-injection images are qualitatively assessed for injection quality and placed into &#8216;good injection&#8217; (n = 7) and &#8216;bad injection&#8217; (n = 4) groups. Qualitative assessment was carried out by observing the post-injection area fluorescence intensity. In an optimal injection, the NIRF signal is confined to the site of injection. No signal will be seen if the injection is successful because the agent has been fully displaced into the bloodstream. A bad-quality injection displays a NIRF signal that is dispersed along the length of the tail.
Images were analyzed with the accompanying NIRF imager software. Regions-of-interest&#160;were drawn at the site of pre-injection images (Figure 1C, E) and around the area of fluorescence in post-injection images (Figure 1D, F). Images where fluorescence was visible throughout the length of the tail were deemed unacceptable and removed from the analysis (Figure 2). Measurements of the area and fluorescence intensity were recorded. Values for area/fluorescence intensity were calculated and plotted (Figure 1G). A significant difference (unpaired t-test) in fluorescence intensity between pre- and post-injection images was observed in the &#8216;bad injection&#8217; group (Figure 1G) (p = 0.0024).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59295/59295fig1.jpg" /> 
Figure 1: NIRF based nanoemulsion and images of tail vein. (A) A nanoemulsion-based biological probe containing NIRF dye was injected into (B) the lateral tail vein and imaged in a NIRF imager. (C and D) Pre- and postinjection images of a good injection. (E and F) Pre- and postinjection images of a bad injection. White arrows indicate the point of injection. It is possible to qualitatively assess the success of a good injection compared to a bad injection by assessing the extent of the NIRF signal at the site of injection. Unacceptable injections display fluorescence throughout the length of the tail and were removed from the analysis (Figure 2). (G) The images can also be analyzed to reveal a quantitative measure of fluorescence intensity, with thresholds for injection quality assigned by the investigator. The error bars on the graph reflect the SEM. For the &#8216;good injection&#8217; group, n = 7. For the &#8216;bad injection&#8217; group, n = 4. There is a statistical difference in fluorescence intensity in the &#8216;bad injection&#8217; group when comparing pre- and postinjection images (unpaired t-test; p = 0.0024). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59295/59295fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59295/59295fig2.jpg" /> 
Figure 2: Examples of bad injections. (A) Fluorescent signal seen in part of the tail. (B) Fluorescent signal seen over the full length of the tail. (C) Fluorescent signal dispersed heavily in the entire tail and caudal area of the animal&#8217;s body. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59295/59295fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents at JoVE.com

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

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20.9K Views.  Duquesne University. Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Video: A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

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 ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative wound complications. This standardized protocol for antegrade endoscopic vein harvesting (EVH) from the lower leg has the potential to be introduced to routine coronary artery bypass grafting (CABG). Patients undergoing CABG surgery are positioned on a surgical table with two additional foam rollers below the extended legs, enabling antegrade EVH from the lower leg. Following minimally invasive surgical access through a bridging vein harvest technique, an endoscopic optical dissector is inserted antegrade into the wound. The main vessel and side branches are dissected under continuous optical control of vein quality status and the working channel. After, an endoscopic optical retractor is inserted with an internal bipolar electrocoagulation device for precise, safe, and tissue-protective interruption of side branches. After release of the vein, the vessel is cut off at the proximal and distal ends under optical control, retrieved from the wound, then cannulated and flushed with heparinized saline. Finally, all side branches of the vein graft are double-clipped. Vascular histology is analyzed in a randomized selection of vein samples. After applying this standardized EVH protocol, the learning curve was shown to be steep, and graft quality was sufficient for coronary artery bypass grafting in every case. There was no conversion to surgical harvesting and low risks for tissue damage and bleeding. Leg positioning and synergizing EVH with bridging vein harvesting improved procedural success and vein graft quality. In our hands, antegrade EVH from the lower leg was feasible, demonstrating straightforward graft dissection as well as adequate macroscopic and microscopic graft quality with preserved endothelial integrity. In conclusion, the introduced technique is safe, shows excellent vein autograft quality, and illustrates feasibility for elective and urgent isolated CABG and combined CABG scenarios.

Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

A Murine Tail Lymphedema Model

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.

Lymphedema is extremity swelling caused by lymphatic dysfunction. We describe a chronic murine tail model of lymphedema and the novel use of tissue nanotransfection technology (TNT) for genetic cargo delivery to the tail.

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. ...