Name: a new best practice for validating tail vein injections in rat with near-infrared-labeled agents
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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><tbody><tr><td>100% Oxygen air tank</td><td>AirGas Heathcare</td><td>n/a</td><td>For ventilation of animal.</td></tr><tr><td>70% Ethanol</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Alcohol Pads</td><td>Henry Schein</td><td>112-6131</td><td></td></tr><tr><td>Artificial Tears</td><td>Henry Schein</td><td>100-2634</td><td>This protects the rats eyes while it is anesthetized.</td></tr><tr><td>Beaker</td><td>Multiple sources</td><td>n/a</td><td>This holds warm water to dilate the tail veins.</td></tr><tr><td>Distilled water</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Exhaust Fans</td><td>Hazard Technologies</td><td>n/a</td><td>For ventilation of lab, if it is not built in.</td></tr><tr><td>Face Mask</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Gas Chamber with tubing and face mask</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Gauze Pads</td><td>Henry Schein</td><td>100-2634</td><td></td></tr><tr><td>Hair Bonnet</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Heating Lamp</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Heating Pad</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Isoflurane</td><td>Southmedic Inc.</td><td>ND66794-013-25</td><td></td></tr><tr><td>Padded Bench Cloth</td><td>Box Board Products Inc.</td><td>026755100I</td><td></td></tr><tr><td>Pearl Small Animal Imager</td><td>Li-COR Biosciences</td><td></td><td></td></tr><tr><td>Pearl Trilogy Small Animal Imaging System</td><td>LI-COR Biosciences</td><td>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><td>Scrubs, lab coat, shoe covers</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Sharps container</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>special diet</td><td>Research Diets, Inc, New Brunswick, NJ</td><td></td><td></td></tr><tr><td>Sprague-Dawley rats</td><td>Hilltop Animals, Springdale, PA</td><td></td><td></td></tr><tr><td>Sterile injection cap</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Sterile needle, 27 G</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>SURFLO IV Catheter, 24 G, yellow</td><td>TERUMO</td><td>SR+OX2419C1</td><td>This is an alternative to using a sterile needle. It provides additional indication of correct venous insertion.</td></tr><tr><td>Surgical gloves</td><td>Multiple sources</td><td>n/a</td><td></td></tr><tr><td>Surgical Tape</td><td>Multiple sources</td><td>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/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/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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21.0K 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

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Cancer Research

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

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Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Biology

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

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Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

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Neuroscience

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

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Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

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There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly ...

AAV-Based Therapies via Intravenous Injection in Preclinical Mouse Models

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse&#39;s lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose.
Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Author Spotlight: Developing Gene Therapies for Muscular Dystrophies and Neuromuscular Disorders

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...

Compound Administration I

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
As many research protocols require that a substance be injected into an animal, the route of delivery and the amount of the substance must be accurately determined. There are several routes of administration available in the mouse and rat. Which route to use is determined by several factors of the substance to be injected: the pH of the solution, the volume required for the desired dosage, and the viscosity of the solution. Severe tissue damage can occur if a substance is administered incorrectly. This video looks at the various restraint methods and technical details for the most commonly used injection routes.

Compound Administration in Rodents- Injection Techniques

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
As many research protocols require that a substance ...