The overall goal of this procedure is to demonstrate how to non-invasively image lymphatics in small animals using a near infrared fluorescence imaging system. This is accomplished by first reconstituting the fluorescent dye to the appropriate concentration immediately before imaging. Next, the sedated animal is injected intradermally with the dye in the location.
That will enable visualization of the lymphatic vessels of interest in the final step. Images of the vessels are acquired and then analyzed. Ultimately, near infrared.
Fluorescent lymphatic imaging can be used to demonstrate and reveal the function and architecture of the lymphatic vasculature. Though this method can provide insight into the role the lymphatic system plays in healthy animals. It can also be applied in a variety of disease conditions such as cancer metastases, acute and chronic immune response wound healing, and transgenic.
Models of lymphatic associated disorders On the day of imaging begin by reconstituting incy and green with sterile water, then dilute with normal saline to a concentration of 645 micromolar. Then after adjusting the nose cone on the sedated animal, turn out the lights if necessary. A small desk Hagen light can be used to see for the injections.
The single most difficult aspect of this procedure is injecting the mice into dermally instead of subcutaneously in the correct location for uptake by the lymphatic vessels that are to be visualized. To ensure success, acquire two to three baseline images to confirm the location of the lymphatic vessels in each animal before beginning the experiment. Now use an insulin syringe with a 31 gauge needle to inject 10 microliters of the dye into the left side of the base of the tail to visualize the lymphatics draining from the inguinal region to the axillary region in general.
To visualize the left side of the animal, inject in location 5, 6, 9, or 10. To visualize the right side, inject the dye into location 7, 8, 11, or 12 locations, one through four, maybe two inferior on the tail for optimum uptake to visualize lymphatic drainage from the inguinal region to the axillary region. If the animal is not under the imaging system during the injection, immediately place it thereafter.
Next, cover the injection site with black paper to help observe the lymphatic vessels using v plus plus software and a small animal near infrared fluorescence imaging system. Acquire lymphatic images for up to one hour. Finally allow the animals to recover on a snuggle heat pad and then return them to their cage while small animal near infrared fluorescent imagers are commercially available.
In this video, a customized small animal Nerf imaging system consisting of a 785 nanometer laser diode outfitted with an aspheric lens diffuser and filter to create a uniform excitation field that illuminates the animal at an incident fluence rate of less than 1.4 milliwatts per square centimeter was used and electron multiplying charge couple device camera system with 2 830 nanometer filters and a 28 millimeter nyco lens is used to capture lymphatic images with integration times of 200 milliseconds for dynamic imaging and up to 800 milliseconds for static imaging of fluorescent lymph. When incy in green or cyclic albumin binding domain peptide IR dye 800 is injected intradermally at the base of the tail of a normal mouse. The lymphatic vasculature between the injection site at the base of the tail and the inguinal lymph node should be immediately visualized within a few seconds to minutes after the injection.
The lymphatic vessel between the inguinal lymph node and the axillary lymph node should be visualized as seen here since the lymphatics in mice vary from animal to animal as they do in humans. Variations in architecture between animals may be seen as demonstrated in this figure. These panels show images obtained on the day of injection one, and these panels show images obtained two days later on the day of injection two, using the same mice and imaging protocol.
While the lymphatic architecture varies between mouse 1 24 and 1 28, the images obtained using the near infrared fluorescence imager are consistent for each mouse. On days one and three, when the imaging agent is injected intradermally on the dorsal aspect of the hind paw of a normal mouse, two lymphatic vessels can be visualized draining to the popliteal lymph node as shown here. In some cases, it is difficult to distinguish both vessels because of their proximity as illustrated in this magnification of the image of the animal's hind limb.
For the representative mouse shown here, 10 microliters of ICG was injected in the dorsal aspect of the left hind paw as well as into the left side of the tail base. At times, visualization of the lymphatics is delayed most commonly due to the injection being administered subcutaneously instead of intradermally. When subcutaneous injections are given, lymphatic transport may not be immediately visualized as illustrated in this image because of the additional time required for the dye to reach and be taken up by the lymphatic capillaries in the skin.
Thus, it is important to take care the dye be injected intradermally not subcutaneously. On occasion, abnormal lymphatic vessels are observed. For example, in this image on the non-injured side of the animal, the inguinal lymph node can be visualized as well as the relatively straight EENT lymph vessel draining up toward the axillary lymph nodes On the mouse's injured side.
However, the normal lymph vasculature was interrupted due to wounding and appears abberant due to tissue repair acquired images of the visualized lymphatic vessels can be loaded into Image J or MATLAB for data analysis. As shown in these images, constant area circular regions of interest are selected or drawn along the entire length of the fluorescent lymphatic vessel. In human and animal lymphatic imaging, a region of interest is selected such that its diameter is approximately the diameter of the image of the fluorescent vessel.
The mean fluorescence intensity within each region of interest is then plotted as a function of imaging time to assess the propulsive velocity and frequency of the packets of dia laden lymph propelled along the lymphatic vessels. The perturbations in fluorescence intensity across the ROIs represent a lymphatic pulse propagating through the regions of interest and are parallel to the arrows to assess the lymphatic propagation velocity and frequency of lymphatic propulsion. Two regions of interest with clearly defined maxima or fluorescent intensity variations representing the propagation of packets of lymph are selected and their fluorescent intensity profiles are plotted as can be observed in these two graphs.
The propagation velocity is computed by taking the ratio of the distance between the two regions of interest and the transit time for a packet of lymph to propagate between them. By assessing the number of fluorescent pulses or packets arriving at a single region of interest per time, the contractile frequency is computed After its development. This technique paved the way for researchers to non-invasively and longitudinally elucidate the role of the lymphatic system in a variety of diseases in health conditions.
