Name: blocking lymph flow by suturing afferent lymphatic vessels in mice
Uploaded: 2020-05-14T14:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice at JoVE.com

The authors thank Ava Zardynezhad for proofreading of the manuscript. This work is supported by the Canadian Institute of Health Research (CIHR, PJT-156035), and the Canada Foundation for Innovation for SL (32930), and by the National Natural Science Foundation of China for Yujia Lin (81901576).

All animal work needs to be approved by institutional and governmental ethics and animal handling committee.&#160; This is a non-survival surgery.&#160;
1. Preparation of materials
<ol>
	<li>Prepare 100 mL of 70% ethanol by mixing 70 mL of 100% ethanol with 30 mL of sterile water. Autoclave all surgical tools before surgery and keep the tools in 70% ethanol before and during the surgery to maintain sterilization.</li>
	<li>Prepare an injection apparatus.
	<ol>
		<li>Cut ~30 cm of polyethylene tubing (0.28 mm in diameter). Connect the tip of a 30 G x &#189; needle (needle A) to one end of polyethylene tubing. Carefully dislodge another 30 G x &#189; needle (needle B) and connect the broken side to the other end of polyethylene tubing.</li>
		<li>Attach needle A to a 1 mL tuberculin syringe. 
		NOTE: For this polyethylene tubing, 1.6 cm of fluid in the tubing corresponds to 1 &#956;L20.</li>
	</ol>
	</li>
	<li>Prepare a 10:1 ketamine/xylazine mixture (10 mg/mL ketamine and 1 mg/mL xylazine) in saline (bacteriostatic 0.9% [w/v] sodium chloride). Prepare the solution freshly before use.</li>
</ol>
2. Preparation of the animal for surgery
NOTE: Use mice aged 6&#8722;10 weeks. Both female and male mice can be used. In this study, 6&#8722;10-week-old, C57BL/6 female mice were used. This method can be adapted for other strains of mice.
<ol>
	<li>Anesthetize the mouse by injecting 250 &#956;L of the ketamine/xylazine mixture intraperitoneally. Wait until the mouse is completely asleep. Ensure mouse does not react to a toe pinch to detect full anesthetization.</li>
	<li>Shave fur around the legs with hair clippers.</li>
	<li>Apply the depilatory cream around the leg and wait for 5 min. Wipe off the residual fur and the depilatory cream using a moist tissue and clean the leg with sterile water. Spray 70% ethanol around the leg to sterilize the operating area. The ethanol is restricted to the incision site.&#160;</li>
</ol>
3. Surgical suturing of afferent lymphatic vessels
NOTE: The right leg is sutured, and the left leg is used as the sham control. The lymphatic suture protocol (steps 3.1&#8722;3.8) takes 20&#8722;30 min.
<ol>
	<li>Keep mouse at a prone position and fix it with surgical tape to expose the operation area on the right leg.</li>
	<li>Intradermally inject 5 &#181;L of 1% Evans blue dye or 9 cm of the fluid of the injection apparatus tubing into the footpad. Gently massage the footpad to help Evans blue enter the lymphatic vessels. 
	NOTE: The insulin syringe is not easy to control for small volume injection. The volume can be controlled more accurately using the injection apparatus. Lymphatic vessels are visualized by blue dye under the skin. With extensive training, both afferent lymphatic vessels can be seen with the naked eye as transparent vessels in the adipose tissue, parallel to the Saphenous artery. With extensive training, it is possible to suture the vessels without injecting Evans Blue dye in cases where there are concerns of potential disturbances from the dye.</li>
	<li>Under a dissecting microscope, choose an incision site 5 mm from the bottom edge of the popliteal fossa. Make a small incision (~5 mm) in the skin with scissors. Using fine operation forceps, stretch the incision, and expose the collecting lymphatic vessels (Figure 1A). 
	NOTE: If necessary, a small skin fragment can be removed to expose the lymphatic vessels.</li>
	<li>Identify both afferent lymphatic vessels leading to the pLNs under the dissecting microscope (Figure 1B). 
	NOTE: There are two afferent lymphatic vessels from the footpad to the pLN. Both need to be sutured to block lymph flow completely.</li>
	<li>Using a needle holder, cautiously insert the suture needle (0.7 metric or smaller) between the afferent lymphatic vessel and the Saphenous artery and pull the needle gently out around the afferent lymphatic vessel. Gently pull the suture string and leave about 2 cm of the suture string behind. Use the needle holder to help tie the string tightly to suture one lymphatic vessel with a surgeon&#8217;s knot (Figure 1C). 
	NOTE: The tissue underneath the incision may dry out with prolonged exposure to air. Making the incision as small as possible and performing the suture quickly (i.e., within 5 min) will prevent the tissue from drying out. Maintain the tissue moisture by applying a small volume of saline with a cotton swab.</li>
	<li>Gently massage the footpad to ensure no Evans Blue dye passes the suture site and then cut the excess string with scissors.</li>
	<li>Perform the same suture steps (i.e., steps 3.5 and 3.6) on the other afferent lymphatic vessel (Figure 1D). Close the skin incision with the same suture that was used to suture the vessels in step 3.5 (Figure 1E).</li>
	<li>For the sham control, intradermally inject 5 &#181;L of 1% Evans blue dye at the left footpad and massage the footpad to visualize the lymphatic vessels. Open the skin with an excision and then close the wound without suturing the vessel (Figure 1F).</li>
	<li>Optionally, monitor the operated mice for 2&#8722;4 h. The suture side of the leg should show edema with Evans Blue spread to the thigh, while the control leg will show restricted Evans blue dye in the footpad.&#160;If mice&#160;awaken, an additional ketamine/xylazine&#160;mix will be injected to keep anesthetized until euthanasia.</li>
</ol>
4. Tracking of the lymph flow
<ol>
	<li>Immediately after the surgery, intradermally inject 10 &#181;L of 2% fluorescein isothiocyanate (FITC) in the footpad of both the control and the lymphatic sutured leg.</li>
	<li>Euthanize the mice with 400 &#956;L of ketamine/xylazine mixture and perform cervical dislocation when the mice are fully anesthetized.</li>
	<li>Collect pLNs from the popliteal fossa and carefully remove the perinodal adipose tissue around the pLNs under the dissection microscope at 2, 6, and 12 h after FITC injection.</li>
	<li>Embed the pLNs with the medullary sinus area facing to the side of the cryomold in optimal cutting temperature (OCT) compound (Figure 1G,H).</li>
	<li>Prepare 20 &#181;m frozen sections using a cryotome.</li>
	<li>Image the cryosections under a confocal microscope to determine FITC distribution.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Blocking lymph flow will have broad applications in manipulating antigen delivery to the LN in healthy and diseased conditions. It is possible to use this method to control the timing of antigen delivery in order to study how continuous lymph flow regulates immune response in draining LNs. This method of lymph flow interruption can also be used to study how lymph impacts cell compartmentalization, cell activation, cell migration, and cell-cell interactions in the LN.
Mice specifically expressing human diphtheria toxin receptor (DTR) in their lymphatic endothelial cells (Flt4-cre-dtr) have been developed; these can be used to specifically deplete lymphatic vessels to study lymphatic function22. Administration of DT kills lymphatic endothelial cells along the lymphatic vessels and in the LN. The depletion of lymphatic endothelial cells can completely abrogate lymph flow regionally or systemically to study lymphatic function. This method causes significant fluid accumulation in tissue and serves as a great model to study lymphedema and lymphatic function.
Compared to the lymphatic endothelial cell-specific DTR expression model, the advantage of the lymphatic suture method is that it interrupts lymph flow with minimal damage to lymphatic endothelial cells or any other lymphatic vessel around the area. The intervention does not directly impact cells in the draining LN, so the resulting impact on the LN microenvironment or immune cell communication is a consequence of the lymph flow blockade rather than potential cell death induced by DT. Another benefit of this method is that lymph flow is instantly blocked after the surgery, so the timing of the lymph flow blockade can be better controlled.
The limitation of this method is that it can only be used to study regional intervention of lymph flow in afferent lymphatic vessels from the footpad to the pLN. This method needs identification of the exact location of the collecting lymphatic vessels. Collecting lymphatic vessels are difficult to identify in some anatomical locations, and thus this technique requires extensive anatomical and surgical training before successful identification of afferent lymphatic vessels to block lymph flow. Another limitation is that this method cannot directly block lymph entering the initial lymphatic vessels. After the suture, the blocked lymph flow may increase the interstitial fluid pressure and change the lymph flow direction in the initial lymphatic vessels. Thus, the intact lymphatic vessels around the area may compensate for the function of the interrupted lymphatic vessels and change the direction of lymph flow.
Moreover, the injection of Evans Blue dye increases the interstitial fluid pressure, which may interfere with the subsequent tracer or antigen injection. The autofluorescence of Evans Blue dye may interfere with other fluorescent tracers or other fluorophores used for immunofluorescent staining. To avoid any interaction between Evans Blue dye and potential antigens, other tracers, or potential molecular mechanisms of lymphatic function regulation, it is possible to identify the collecting lymphatic vessels without Evans Blue dye with the naked eye. This can be achieved with extensive training to identify the vessels. Other dyes, such as isosulfan blue dye can also be used to replace the Evans Blue dye.

The spatial organization of the lymphatic system provides structural and functional support to efficiently remove extracellular fluid and transport antigens and antigen-presenting cells (APCs) to the draining LNs. The initial lymphatic vessels (also named lymphatic capillaries) are highly permeable due to their discontinuous intercellular junctions, which facilitate the effective collection of fluids, cells, and other materials from surrounding extracellular spaces1. The initial lymphatic vessels merge into collecting lymphatic vessels, which have tight intercellular junctions, a continuous basement membrane, and lymphatic muscle coverage. Collecting lymphatic vessels are responsible for transporting collected lymph to the draining LNs and eventually returning lymph to the circulation2,3. The collecting lymphatic vessels that propel lymph into the draining LN are the afferent lymphatic vessels4,5,6,7. Obstruction of afferent lymphatic vessels can block lymph flow into the LNs, which is a useful technique when studying the function of lymph flow.
Previous studies have shown that lymph flow plays a significant role in transporting antigens and APCs, as well as maintaining LN homeostasis. It is well understood that tissue-derived APCs, typically activated migrating dendritic cells (DCs), travel through the afferent lymphatic vessels to the LN to activate T cells8. The idea that free-form antigens, such as microbes or soluble antigens, passively flow with lymph to the LN to activate LN-resident APCs has been gaining acceptance in the past decade9,10,11,12. Free-form antigens traveling with lymph take minutes after the infection to travel to the LN, and the LN-resident cell activation may occur within 20 min after the stimulation. This is much faster than the activation of migrating DCs, which takes more than 8 h to enter the draining LN9. Besides transporting antigens to initiate immune protection, lymph also carries cytokines and DCs to the LN to maintain its microenvironment, and to support immune cell homeostasis13,14. Previously, blocking lymph flow by suturing the afferent lymphatic vessels demonstrated that lymph is required to maintain the HEV phenotype required for supporting homeostatic T cell and B cell homing to the LN15,16,17. CCL21 is a critical chemokine that directs DC and T cell positioning in the LN8,18. Blocking lymph flow interrupts CCL21 expression in the LN and potentially interrupts DC and T cell positioning and/or interaction in the LN19. Thus, blocking lymph flow can directly or indirectly abrogate antigen/DC access to the draining LN by disrupting the LN microenvironment that regulates immune responses in the LN. To better investigate the function of lymph flow, an experimental protocol is presented (Figure 1) to block lymph flow in mice by suturing the afferent lymphatic vessels from the footpad to the pLN. This method can be an important technique for future studies on lymphatic function in healthy and diseased conditions.

<table><tbody><tr><td>&lt;strong&gt;0.9% Sodium Chloride Saline&lt;/strong&gt;</td><td>Baxter</td><td>JB1323</td><td></td></tr><tr><td>&lt;strong&gt;100% ethanol&lt;/strong&gt;</td><td>Greenfield Global</td><td></td><td>University of Calgary distribution services UN1170.</td></tr><tr><td>&lt;strong&gt;Depilatory cream&lt;/strong&gt;</td><td>Nair</td><td></td><td>Nair Sensitive Formula Hair Removal Cr&amp;egrave;me with Sweet Almond Oil and Baby Oil, 200-ml. Or similar product.</td></tr><tr><td>&lt;strong&gt;Evans Blue dye&lt;/strong&gt;</td><td>Sigma Life Science</td><td>E2129-10G</td><td>For 1 ml of Evans blue dye, add 0.1g Evans blue to 10 ml PBS. The Evens Blue solution will be filtered through 0.22 mm filters and kept sterile in 1ml aliquots.</td></tr><tr><td>&lt;strong&gt;Fluorescein isothiocyanate isomer I (FITC)&lt;/strong&gt;</td><td>Sigma Life Science</td><td>F7250-1G</td><td></td></tr><tr><td>&lt;strong&gt;Forceps Dumont #3&lt;/strong&gt;</td><td>WPI</td><td>500337</td><td></td></tr><tr><td>&lt;strong&gt;Forceps Dumont #5&lt;/strong&gt;</td><td>WPI</td><td>500233</td><td></td></tr><tr><td>&lt;strong&gt;Injection apparatus&lt;/strong&gt;</td><td></td><td></td><td>Connect one end of polyethylene tubing to 30G &amp;times; &amp;frac12; needle. Attach a 1ml TB syringe to the needle. Dislodge needle shaft from another 30G &amp;times; &amp;frac12; needle. Insert the blunt end of the 30G &amp;times; &amp;frac12; needle shaft into the other end of the tubing. The inside diameter of this tubing is 0.28mm. Thus, 1.6 cm of fluid in the tubing is 1 &amp;mu;l.</td></tr><tr><td>&lt;strong&gt;Insulin syringe&lt;/strong&gt;</td><td>Becton Dickinson and Company (BD)</td><td>329461</td><td></td></tr><tr><td>&lt;strong&gt;IRIS Forcep straight&lt;/strong&gt;</td><td>WPI</td><td>15914</td><td></td></tr><tr><td>&lt;strong&gt;IRIS scissors&lt;/strong&gt;</td><td>WPI</td><td>14218-G</td><td></td></tr><tr><td>&lt;strong&gt;Ketamine&lt;/strong&gt;</td><td>Narketan</td><td>DIN 02374994</td><td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td></tr><tr><td>&lt;strong&gt;Needles (26Gx3/8)&lt;/strong&gt;</td><td>Becton Dickinson and Company (BD)</td><td>305110</td><td></td></tr><tr><td>&lt;strong&gt;Needles (30Gx1/2)&lt;/strong&gt;</td><td>Becton Dickinson and Company (BD)</td><td>305106</td><td></td></tr><tr><td>&lt;strong&gt;Paton Needle Holder&lt;/strong&gt;</td><td>ROBOZ</td><td>RS6403</td><td>Straight, Without Lock; Serrated</td></tr><tr><td>&lt;strong&gt;Phosphate-Buffered Saline (PBS)&lt;/strong&gt;</td><td>Sigma Life Science</td><td>P4417-100TAB</td><td></td></tr><tr><td>&lt;strong&gt;Polyethylene tubing&lt;/strong&gt;</td><td>Becton Dickinson and Company (BD)</td><td>427401</td><td></td></tr><tr><td>&lt;strong&gt;Surgical tape (1.25cmx9.1m )&lt;/strong&gt;</td><td>Transpore</td><td>1527-0</td><td></td></tr><tr><td>&lt;strong&gt;Surgical tape (2.5cmx9.1m )&lt;/strong&gt;</td><td>Transpore</td><td>1527-1</td><td></td></tr><tr><td>&lt;strong&gt;Suture&lt;/strong&gt;</td><td>Davis and Geck CYANAMID Canada</td><td>11/04</td><td>0.7 metric monofilament polypropylene</td></tr><tr><td>&lt;strong&gt;Syringe (1ml)&lt;/strong&gt;</td><td>Becton Dickinson and Company (BD)</td><td>309659</td><td></td></tr><tr><td>&lt;strong&gt;VANNAS scissors&lt;/strong&gt;</td><td>World Precision Instruments (WPI)</td><td>14122-G</td><td></td></tr><tr><td>&lt;strong&gt;Xylazine&lt;/strong&gt;</td><td>Rompun</td><td>DIN02169606</td><td>The suppliers of Ketamine and Xylazine are usually under institutional and governmental regulation.</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissecting microscope&lt;/strong&gt;</td><td>Olympus</td><td></td><td>Olympus S261 (522-STS OH141791) with light source: Olympus Highlight 3100</td></tr><tr><td>&lt;strong&gt;Confocal microscope&lt;/strong&gt;</td><td>Leica</td><td></td><td>SP8</td></tr></tbody></table>

blocking lymph flow by suturing afferent lymphatic vessels in mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

Lymphatic vessel suture has been used in previous studies15,16,17,19, where it served as an important tool to study the function of lymph flow before the molecular biology of lymphatic vessels was better understood. Blocking lymph flow interrupts LN homeostasis, which leads to HEVs losing the critical gene expression needed for optimal lymphocyte homing to the LN15,16,17. Since then, it took another two decades to demonstrate that DCs traveling with lymph are crucial in maintaining the HEV gene expression profile and lymphocytes homing to the LN13. The shear stress provided by the lymph flow is critical to stimulate chemokine expression in the LN. Blocking lymph flow interrupts chemokine CCL21 expression in the LN19, which is critical in directing DC and T cell positioning in the LN. Therefore, interrupted flow may compromise DCs and T cells positioning in the LN8,18.
Immediately after the surgery, a small molecular weight fluorescent tracer, FITC, was used to track lymph flow. FITC (10 &#181;L of 2% FITC) was injected intradermally in the footpad of the sham control and the lymphatic sutured leg. The draining pLNs were collected 2, 6, and 12 h later. The draining pLNs were embedded in OCT, and 20 &#181;m frozen sections were prepared. Confocal images showed substantially reduced FITC accumulation in the pLNs after suture. The residual FITC in the pLNs was preferentially accumulated in the LN sinuses (Figure 2).
How the lymph flows through the adipose tissue surrounding the LN was investigated using lymphatic suture. The afferent lymphatic vessels leading to the pLNs were sutured to block lymph flow, and it was determined that the perinodal adipose tissue could support a small amount of lymph flow when lymphatic vessels were blocked21. The lymph flow through the adipose tissue to the capsule of the LN was mapped; it appeared to feed into the LN sinuses. Small amounts of lymph may have flowed into the LN sinuses over time (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61178/61178fig01.jpg" /> 
Figure 1: Steps of popliteal LN (pLN) afferent lymphatic vessel suture.&#160;Briefly, after mice were anesthetized with a ketamine and xylazine mixture, their legs were shaved, and the residual fur was removed by a depilatory cream. The right leg was used for suture and the left leg was the sham control. The right side of the footpad was intradermally injected with 5 &#181;L of 1% Evans Blue dye prepared in PBS. (A) By gently massaging the footpad, Evans Blue dye filled the afferent lymphatic vessels. (B) A small skin cut was performed 5 mm away from the pLN to expose the lymphatic vessels, which are indicated by the two white arrows. (C,D) Both afferent lymphatic vessels were sutured. (E) The skin excision was closed by sutures. (F) The control leg received Evans blue injection, skin excision, and suture closure without suturing the lymphatic vessels. (G) The success of the lymph flow blockage was indicated by Evans blue dye, which entered the pLN of the control leg but not the sutured leg. (H,I) The collected pLNs were embedded in OCT compound with subcapsular sinus (SCS) and medullary sinus (MS) facing the side of the cryomold before snap freezing in liquid nitrogen. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61178/61178fig01large.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/61178/61178fig02.jpg" /> 
Figure 2: FITC distribution in the draining pLNs of the sham or sutured leg.&#160;Confocal images of pLNs collected 2, 6, and 12 h after FITC injection showed substantially reduced FITC accumulation in the pLNs after suture. The residual FITC in the pLNs was preferentially found in the LN sinuses. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61178/61178fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61178/61178fig03.jpg" /> 
Figure 3: FITC distribution in the perinodal adipose tissue (PAT), around the draining pLNs of the sham or sutured leg.&#160;Confocal images of the PAT and the LN showed that FITC enters the PAT and the LN sinuses but was not effectively distributed throughout the LN when lymphatic vessels were blocked. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61178/61178fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

blocking-lymph-flow-by-suturing-afferent-lymphatic-vessels-in-mice

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

5.5K Views.  Harbin Medical University. A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Video: Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

Medicine

Lymphatic and Blood Network Analysis During Obesity

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still ...

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. ...

Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction

Lymphedema is a common condition often associated with cancer and its treatment, which leads to damage to the lymphatic system, and current treatments are mostly palliative rather than curative. Its high incidence among oncologic patients indicates the need to study both normal lymphatic function and pathologic dysfunction. To reproduce chronic lymphedema, it is necessary to choose a suitable experimental animal. Attempts to establish animal models are limited by the regenerative capacity of the lymphatic system. Among the potential candidates, the rabbit hindlimb is easy to handle and extrapolate to the human clinical scenario, making it advantageous. In addition, the size of this species allows for better selection of lymphatic vessels for vascularized lymph node resection.
In this study, we present a procedure of vascular lymph node resection in the rabbit hindlimb for inducing secondary lymphedema. Anesthetized animals were subjected to circumferential measurement, patent blue V infiltration, and indocyanine green lymphography (ICG-L) using real-time near-infrared fluorescence, a technique that allows the identification of single popliteal nodes and lymphatic channels. Access to the identified structures is achieved by excising the popliteal node and ligating the medial and lateral afferent lymphatics. Special care must be taken to ensure that any lymphatic vessel that joins the femoral lymphatic system within the thigh without entering the popliteal node can be identified and ligated. 
Postoperative evaluation was performed at 3, 6, and 12 months after induction using circumferential measurements of the hindlimb and ICG-L. As demonstrated during follow-up, the animals developed dermal backflow that was maintained until the 12th month, making this experimental animal useful for novel long-term evaluations in the management of lymphedema. In conclusion, the approach described here is feasible and reproducible. Additionally, during the time window presented, it can be representative of human lymphedema, thus providing a useful research tool.

Here, the surgical induction of stable acquired lymphedema in the rabbit hindlimb is described. This experimental animal can be used to further investigate the effect of lymphedema treatment by microsurgical techniques.

Lymphedema is a common condition often associated with cancer and its treatment, which leads to damage to the lymphatic system, and current treatments are ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Biology

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

Murine Tail Lymphedema Model: An In Vivo Technique to Generate Sustained Lymphedema in Murine Tail

Source: Hassanein, A. H. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/61848">A Murine Tail Lymphedema Model.</a> J. Vis. Exp. (2021).
This video describes an in vivo technique to generate murine tail lymphedema, an abnormal fluid accumulation in the interstitial tissue space leading to swelling in the tail. The model is applied to study mechanisms and therapy for lymphedema.

Model obrzęku limfatycznego mysiego ogona: technika in vivo generowania trwałego obrzęku limfatycznego w mysim ogonie

Source: Hassanein, A. H. et al., A Murine Tail Lymphedema Model. J. Vis. Exp. (2021).
This video describes an in vivo technique to generate murine tail ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

On the day before the surgery, cut the cannulas to the appropriate lengths, and use a sterile surgical blade to make a bevel at the tip of each piece of tubing. Next, to prepare the intraduodenal cannula, loop the cannula back onto itself, and then, heat the cannula to create an anchor point in the tubing.
On the day of the surgery, shave the fur from the right side of the abdomen and neck and left clavicle regions of an anesthetized adult rat, and then, clean the surgical regions aseptically with three successive povidone-iodine solution and 70% ethanol scrubs.
To cannulate the mesenteric lymph duct, place the animal with its right side facing up and use a sterile scalpel blade to open the top layer of the abdominal muscle wall with a straight four-centimeter incision extending from the xiphoid process to the right flank, approximately two centimeters below the costal margin.
Retract the small intestine under the left abdominal wall, and use two to three pieces of sterile gauze saturated with normal saline to keep it in place. Then, bridge the rat over a 10-milliliter plastic syringe placed horizontally under the animal&#39;s back at the level of the right kidney to assist in the visualization of the mesenteric lymph duct.
Locate the superior mesenteric lymph duct, which is approximately 0.5 to 1 millimeter in diameter, perpendicular to the right kidney, and immediately rostral and parallel to the dark red pulsating mesenteric artery. Note that in non-fasted rats, as shown here, the superior mesenteric lymph duct is white, opaque, and easier to visualize than in fasted rats.
Next, pass a pair of straight forceps through the peri-renal fat bed at the lower margin of the right kidney parallel to the superior mesenteric lymph duct, and through the connective tissue layers, immediately below the vena cava. Then, using the tip of the forceps, pull the lymph cannula through the peri-renal fat bed with one end immediately adjacent to the mesenteric lymph duct, and the other exteriorized from the animal at the level of the right kidney.
Then, using a surgical microscope and jeweler&#39;s forceps, isolate the mesenteric lymph duct through the overlying layers of connective and fat tissue by blunt dissection, starting at the lower end and taking care not to damage the lymph duct. When the duct has been isolated, use the tip of the fine-tip forceps to make a small hole in the lymph duct. After ensuring that the lymph cannula is completely filled with anticoagulant solution and no air gaps, use a small pair of forceps to insert the lymph cannula approximately two to four millimeters inside the mesenteric lymph duct via the puncture.
If the cannulation is successful, a gradual flow of intestinal lymph will be observed from the free-collecting end of the cannula. When this occurs, place a small drop of cyanoacrylate glue over the entrance hole into the lymph duct to secure the cannula, taking care not to occlude the vessel with excess glue.

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

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Rozdziały w tym wideo

Murine Superficial Lymph Node Surgery

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Lymphatic and Blood Network Analysis During Obesity

A Murine Tail Lymphedema Model

Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Model obrzęku limfatycznego mysiego ogona: technika in vivo generowania trwałego obrzęku limfatycznego w mysim ogonie

Kaniulacja krezki limfatycznej w modelu szczura w celu pobrania płynu limfatycznego