Method Article

Reproducible Setup for In vivo Two-photon Imaging of the Contractility of Cervical Lymphatic Vessels and Lymph Flow in Mice

DOI:

10.3791/69482

⸱

April 10th, 2026

 ,  ,  ,  ,  ,  ,  , 
1Department of Biology, Saratov State University, 2Institute of Physics, Department of Optics and Biophotonics, Saratov State University, 3Department of Computer Science and Information Technology, Saratov State University, 4, Res Inst Intelligent Complex Syst, Fudan University, 5Potsdam Institute for Climate Impact Research

In This Article

Summary

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The manuscript demonstrates an innovative method for in vivo study of the contractility of cervical lymphatic vessels and lymph flow in mice for testing various physiological functions of cervical lymphatic vessels (cLVs) and the therapeutic strategies for modulation of the lymphatic regulatory mechanisms of brain drainage and clearance.

Abstract

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The contractility of cLVs is the driving force for lymph flow and a key mechanism underlying brain toxin cleansing. Here, we present an innovative method for in vivo two-photon imaging of the contractility of cLVs and the long-term observation of the removal of red blood cells with lymph flow in cLVs in mice. The components of the setup include a mini-heating pad adapted to the mouse's neck that allows for increasing the window for imaging up to 5 h while preserving the cLV contractility and the lymph flow; humidity control; a protective casing; and a positioning system that allows for maintaining stable optical visualization of cLVs. The method ensures reproducibility and compatibility with other types of two-photon microscopes and a wide range of objectives, maintaining high-quality images with depth up to 500 µm. The protocol includes detailed stages of surgical preparation of cLVs for maintaining the physiological environment, providing the normal cLV contractility.

Introduction

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The contractility of the cervical lymphatic vessels (cLVs) plays a key role in brain drainage and the lymphatic clearance of its tissues from metabolites and toxins1,2,3. Reduction of this main function of cLVs leads to suppression of brain toxin cleansing that is accompanied by many brain diseases, including Alzheimer's disease, traumatic brain injury, and brain cancer4,5,6,7,8,9. Therefore, studying the cLV contractility is an important approach in the development of new therapeutic strategies for modulation of the lymphatic regulatory mechanisms of brain drainage and clearance10,11,12.

This study presents an innovative method for real-time two-photon imaging of the cLV contractility and lymph flow in mice of different ages. The important components of the setup include a micro heating pad, which, when attached to the animal's neck, allows for an extended imaging time of up to 5 h while maintaining the contractile activity of the collector lymphatic vessels and the stability of lymph flow.

It should be noted that unlike the blood circulatory system, filled with blood that moves at a certain speed (from 10 mm/s in arterioles to 13 cm/s in the sagittal sinus in humans) through various types of blood vessels, the filling of lymphatic vessels with lymph depends on the increasing pressure gradient between empty lymphatic vessels and the volume of interstitial fluid generated during metabolism13,14,15,16,17,18,19. At the same time, lymph flow in the lymphatic vessels is intermittent, ranging from 0 to 1 mm/s20,21,22. Recent MRI studies in humans have shown that lymph flow in the meningeal lymphatic vessels (MLVs) is 1-3 mm23. However, it should be noted that even in a single person, lymph flow in MLVs varies at different times of the day and can drop to 0. Thus, lymph flow in the lymphatic vessels is inconsistent as it depends on the rate of metabolic processes, which in turn depends on temperature24. Indeed, the velocity of a biochemical reaction is deeply and directly affected by temperature, which therefore could also exert a significant effect on lymphatic vessel spontaneous contractions and thus alter lymph drainage and transport24,25,26.

Therefore, maintaining body temperature is an important condition for preserving the physiological environment of lymphatic vessels to support normal contractility and lymph flow. However, the use of commercially available heating plates does not ensure the maintenance of the necessary temperature in the deep and optically accessible areas of the neck where cLVs are located. This is especially important for two-photon imaging of the cLV contractility and lymph flow, as this is carried out in cold experimental rooms with an ambient temperature of 19 °C, which is required for this optical technique27.

In this regard, in the overwhelming majority of in vivo studies, the contractility of lymphatic collecting vessels using commercial heating plates is limited to 3-5 min (the maximum effect is usually observed within a few seconds3,28,29,30,31,32,33,34,35,36,37,38, after which the lymphatic filling and contractility decrease. This approach significantly limits the observation time of lymphatic vessel physiology and makes it impossible to assess long-term effects on their contractility and lymph flow, which motivated us to develop a mini-heating pad adapted to the mouse's neck with the aim of increasing the time window for imaging up to 5 h with preserving the cLV contractility and stable lymph flow.

Another component of the setup is the protective casing with humidity control and positioning system that allows for maintaining stable visualization of cLVs without water immersion, and with preservation of high-resolution images when long working distance air immersion lenses corrected for 0.17 mm cover glass (e.g., CFI Plan Apochromat Lambda D 10x (MRD70170) and CFI Plan Apochromat Lambda D 20x (MRD70270) is used.

The proposed method ensures reproducibility and compatibility with other types of two-photon microscopes and various objective range compatible with the casing taking into account the environmental features, including temperature/moisture control and the working distance from the lens, while maintaining high-quality images with depth up to 500 µm and time window (up to 5 h) which allows for long-term analysis of the contractility of cLVs and lymph flow with high quality in mice.

It is important to take into account the anatomical location of cLVs near the bifurcation of the carotid artery, where baroreceptors are located. The mechanical impact on baroreceptors leads to destabilization of blood pressure and heart rate. This can cause significant changes in hydrostatic pressure and, as a result, disruption of lymph flow39. Therefore, the proposed method includes a detailed description of the surgical protocol for the gentle isolation of cLVs in the deep neck area to avoid mechanical impact on the bifurcation of the nearby carotid artery and changes in blood pressure, which can lead to impaired lymph flow in the brain and, consequently, impaired lymph flow in cLVs and their contractility.

Results demonstrate the age-related decrease in the cLV contractility and its sensitivity to therapeutic photo-stimulating effects in mice. Furthermore, for the first time in long-term observation (over 5 h), we clearly demonstrate that cLVs act as tunnels for the removal of red blood cells from the right lateral ventricle, which is an important lymphatic pathway for brain toxin cleansing1,2,3. Overall, the method for in vivo two-photon imaging of the cLV contractility and lymph flow is intended for preclinical studies of various physiological functions of cLVs, including regulation of brain drainage and clearance, removal of metabolites and toxins from the brain, as well as for testing of the new therapeutic technologies for modulation of the cLV physiology.

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Protocol

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All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes, and the guidelines from the Ministry of Science and High Education of the Russian Federation (No. 742 from 13.11.1984), which have been approved by the Bioethics Commission of the Saratov State University (Protocol No. 7, 22.09.2022).

NOTE: Refer to the Table of Materials for details regarding all materials used in this protocol.

1. Technical device for fixing the mouse

  1. Use a steel optical rail (SOR, length 270 mm, width 43 mm; Figure 1A) as the base of the installation.
  2. Install two post holders (3PH-25) on top of the SOR at a distance of 175 mm from each other (Figure 1B). Place the mounting posts (3MP, diameter 12 mm) inside the 3PH-25 and then fix them firmly with the M6 screw (Figure 1C).
  3. Attach the post collar (3PC) to both mounting posts (3MP; Figure 1D). A post-collar is necessary for subsequent fixation of the catheter with a constant supply of saline solution during optical monitoring of the cervical lymphatic vessel.
    NOTE: This is necessary in order to flush the study area periodically to remove accumulating blood after surgical procedures, which can complicate optical monitoring.
  4. Attach the right-angle fixed post clamp (3RPC-12) to one of the 3MP mounting posts and fix it firmly with the M6 screw (Figure 1H).
    1. Insert the mounting post (diameter 12 mm) into the side hole of the 3RPC-12 (Figure 2B).
      NOTE: The mounting post is necessary for the subsequent fixation of the protective cover for a non-water-immersion two-photon lens.
    2. Attach the holder printed on a 3D printer to the side mounting post and fix it firmly using two pairs of M3 screws (Figure 2C).
    3. Using UV-curable glue, attach a cover glass with a diameter of 12 mm to the underside of the protective casing printed on a 3D printer (Figure 2D,F). After that, use the M4 screw-nut pair to attach the protective cover to the holder (Figure 2).
  5. Install the precision pantograph table for vertical movement 02TV004 (Figure 1E) on the SOR base using two table clips (3TC5) and sets of screws, nuts, and washers M6 (Figure 1F).
  6. Place a 3D-printed case (155 mm long, 110 mm wide) on the tabletop, which is used as a reservoir for the accumulation of saline solution (Figure 1G).

2. Heating element

  1. Assembly of peripheral devices.
    1. Cover the connection point of the wires to the heating element 1 with a thin layer of silicone (Figure 3). Fix the temperature sensor 1 to the mat and seal it with silicone (Figure 3B).
    2. Solder the wires of the heating element and temperature sensors to a multi-wire cable, fill the connection point with silicone, and close with shrink wrap (Figure 3).
    3. Place the heating element 2 and the temperature sensor 2 in the foam roller (Figure 4).
  2. Assembly of the control unit
    NOTE: Link to download files for printing the case of the control https://disk.yandex.ru/d/06BURfMlQpl0Jg.
    1. Print the case of the control unit on a 3D printer (Figure 5B).
    2. Fix the connectors for connecting the power cable and the power button on the walls of the case, as shown in Figure 5C,D).
  3. Install a 24 V DC power supply, a DC step-down converter, and 2 MOSFET modules inside the enclosure (Figure 5A,G,H,I).
  4. To assemble the circuit, follow these steps (Figure 6).
    1. Connect the step-down converter to the source and set the voltage to 9 V (Figure 6).
    2. Connect the VIN pin on the Arduino UNO to the OUT+ connector on the converter and GND to the OUT connector, respectively (Figure 6).
    3. Connect the V+ pin on the power supply to the VIN+ pins on the MOSFET modules, and the V pin to the VIN pins, respectively (Figure 6).
    4. Connect the V pin+ on heating element 1 to VOUT+ on module MOSFET 1 and V- to VOUT, respectively (Figure 6).
    5. Repeat the steps for heating element 2 and MOSFET 2 (Figure 6).
    6. Connect the GND pin on the MOSFET 1 and 2 module to the GND on the Arduino UNO, and the PWM MOSFET 1 to the D11 connector on the Arduino UNO, and the PWM MOSFET 2 to the D10 connector, respectively (Figure 6).
    7. Connect the VIN pin of the temperature sensor 1 to the GND connector on the Arduino UNO, VIN+ to the 5 V connector on the Arduino UNO, and SIG to D3 (Figure 6).
    8. Connect the SIG contact of the temperature sensor 2 to A1 on the Arduino UNO, connect the GND sensor to the GND on the Arduino UNO, and also connect the 100 kΩ pull-up resistor (Figure 6).
  5. Fix the Arduino UNO and the keyboard screen in the lid housing (Figure 7I).

3. Temperature control software guide

  1. Download the Arduino sketch (.ino file) and open it in the Arduino development environment (IDE version: 2.3.5-nightly-20241212).
  2. Select the correct COM port and download the firmware. The interface contains two buttons. To move between them, use the left and right buttons (Figure 7B,D). The selection indicator is located to the left of the column (Figure 7F).
  3. The temperature selection field is located in the left column of the screen (Figure 7H). Use the Up and Down buttons to adjust (Figure 7C).
  4. The START/shutdown field is located in the right column of the screen (Figure 7G). To select this column, press the Right button on the keyboard, and then press Select (Figure 7A). When the process is running, the activity indicator will blink, and the label on the RUN button will change to OFF.

4. Fixing the animal in the technical device

NOTE: The experiments were conducted on male BALB/c mice (25-28 g, 2, 18, and 24 months old); in each group, there were 7 animals.

  1. Prepare a mixture of Ketamine and Xylazine (100 mg/kg; 10-15 mg/kg, respectively) and inject it by intraperitoneal injection according to the weight of the mouse.
  2. Apply eye ointment to the eyelids to prevent the eyeballs from drying out during surgery. Repeat this procedure if necessary.
    NOTE: After anesthesia, the depth of anesthesia was assessed. The surgical procedure requires the absence of a flexion reflex and a reaction to pinching the tail, as well as a decrease/absence of muscle tone in the extremities, slow, even heartbeat and breathing.
  3. Place the mouse on the heating element on the back so as to provide access to the abdominal part of the body.
    NOTE: The mouse's body temperature is constantly monitored using a heating element (36-37 °C), as well as using a thermal imager. The procedure lasts for 5 h from the moment the mouse is placed under the microscope lens. 1 h before the start of visualization, the animal is connected to the supply of 1% isoflurane through a face mask, after which the animal is under anesthesia until the end of the study. During the entire time, the depth of anesthesia is assessed every 30 min.
  4. Plug the control unit into an outlet (input voltage range 175-240 V AC, frequency range 50-60 Hz, AC current 1.8A/230 V AC). Set the operating temperature on the device to 37-38 °C.
  5. Using medical tape, fix the animal so that the body is parallel to the surface of the table, and position the head at an angle relative to the sagittal axis of the body so that the right or left side of the neck lies on the roller (Figure 8A).
    NOTE: This fixation of the mouse's head is necessary so that the right or left side of the neck (depending on the experimental conditions) is raised to a height of about 5 mm towards the microscope lens. Thus, the area of dcLN and cLV will be almost on the same level as the trachea.
  6. Wrap the ligature around the upper incisors and attach it to the back of the device to maintain the animal's body temperature using medical tape.
  7. Using a shaving machine, shave the area of the hairline in the neck area. Remove any remaining hair after shaving, then cover the animal to protect the sterile field. For surgical procedures and subsequent optical control, inject 0.5 - 1 mL of lidocaine/bupivacaine (7-8 mg/kg and 0.25 mg/mL, respectively) subcutaneously into the incision site.
    1. For primary disinfection, degrease and partially disinfect the skin with an alcohol swab, then, for antiseptic treatment, apply a solution of chlorhexidine 0.5% or iodine 2% to the skin. Repeat all the sterilization steps two more times.
  8. Using straight dissecting scissors, perform a dermatotomy of the neck skin with a longitudinal incision from the lower jaw to the collarbone area about 2 cm long (Figure 8B).
  9. Performing fasciotomy with microsurgical curved scissors, using curved tweezers, push apart the salivary glands and superficial muscles of the neck, exposing the sternocleidomastoid muscle. Avoid damaging visible blood vessels to prevent bleeding. Constantly moisten the surgical area with saline solution to prevent excessive tissue injury.
  10. Using microsurgical scissors, perform a myotomy of the sternocleidomastoid muscle to provide access to the area between the trachea and the carotid artery of the animal.
  11. Continuing to spread the muscles and fascia and performing myo- and fasciotomy, locate the deep cervical lymph node and afferent and efferent lymph vessels at a depth of 5-7 mm (Figure 8C).

5. Fixing the mouse under a microscope

  1. Place the device with the fixed animal on the movable table of the microscope and firmly connect them using the M6 screw. This is necessary to ensure a permanent installation position on the movable table.
  2. Plug the control unit into an outlet (input voltage range 175-240 V AC, frequency range 50-60 Hz, AC current 1.8A/230 V AC). Set the operating temperature on the device to 36-37 °C.
  3. Attach the catheter filled with saline solution to the mounting post using medical tape.
  4. Place a 1 cm diameter roller under the heating element on the side of the animal's head to ensure a 45° tilt angle relative to the movable microscope table. This is necessary in order to place the cervical lymph vessel in a horizontal position and reduce the subsequent pressure on the trachea.
  5. Place the fixed catheter in the neck area open after surgical procedures so that it is located on the side of the collarbones of the animal.
    1. Turn on the supply of saline solution, wait until the open neck area is completely filled with saline solution.
    2. Lower the protective casing over the neck area filled with saline solution so that the area of the lymph vessel is located in its center, and the animal's trachea is not compressed (Figure 9A).
    3. To install the protective casing, it is recommended to use a binocular connected to a two-photon microscope. Place the protective casing on the trachea so that it does not press on it. Make sure that no air bubbles accumulate under the protective casing as it is lowered. Make sure that the protective plate is located on the upper muscles of the neck and does not press cLVs, allowing the heated saline solution to flow into the vessel and preventing mechanical deformation of cLVs.
  6. Lower the non-water-immersion two-photon lens into the protective casing to a depth corresponding to the working distance of this lens and then perform a two-photon visualization of the cervical lymphatic vessel (Figure 9B).
    NOTE: When conducting the first scan, it is necessary to make sure that the lymphatic vessel has a complete structure along its entire length and is mobile when the mouse is breathing. This will mean that it is not deformed or pinched by a protective casing.

6. Two-photon in vivo monitoring of the cLV contractility

NOTE: The 2-photon images were obtained using a multiphoton microscope (pulsed laser, excitation wavelength 970 nm). The image was obtained with a resolution of 20x (f/0.75). Each image consisted of 1 field of view zoom x 3.0 (212.13 × 212.13 µm), consisting of 512 x 512 dots. The images of cLVs were obtained by shooting a video at 30 frames per second, lasting 1 min. The 2-photon images were obtained in one channel with an excitation of fluorescent LYVE-1 in cLVs at a wavelength of 488 nm and its emission at a wavelength of 525 nm.

  1. cLV diameter analysis and automated contractility assessment
    1. Measure the diameter of cLVs in the region of interest (ROI) using 30 min movies at 30 frames/s and 3x magnification. Using a custom Python code, measure the time profile of cLV diameter for the same segments used for lymph flow calculation.
    2. Select manually two points corresponding to the opposite walls of the сLVs. Determine their position in subsequent frames using the Lucas-Kanade optical flow algorithm (calcOpticalFlowPyrLK function in OpenCV), which provides sub-pixel accuracy for the measurements. Calculate the diameter as the distance between the tracked points, converted from pixels to micrometers. Use a sliding window median filter to eliminate motion artifacts. There should be at least 20 diameter measurements in one ROI.
    3. To determine the internal frequency of pulsations, use the smoothed time series of diameter. Detect the signal's maxima and minima automatically using the find_peaks function from the SciPy library, which identifies local extremes of the time signal based on amplitude thresholds and the minimum distance between peaks.
    4. Exclude from the analysis the cLV contractions with an amplitude of less than 3% of the average diameter and an interval of less than 2 s. Calculate the average frequency over 30 min intervals as the frequency of contractions per minute.

7. Two-photon analysis of lymph flow in cLVs

NOTE: Lymph flow analysis was assessed by the movement of red blood cells in cLVs during 5 h after the introduction of autologous blood into the right lateral ventricle via the chronic catheter.

  1. Anaesthetize the mouse with 1% isoflurane (see protocol published in40). Implant the catheter into the right lateral ventricle according to the protocol described in40.
  2. Prepare the mouse for two-photon in vivo imaging of cLVs in accordance with steps 7.1, 7.3-7.5.
  3. Collect blood from the tail vein by needle puncture in an amount of 10 µL in a sterile micro-centrifuge tube pre-flushed with heparin to avoid coagulation during blood sampling and injection.
  4. Inject blood into the right lateral ventricle (AP = −0.5 mm; ML= −1.06 mm; DV = 2.5 mm) using a microinjection pump at a rate of 0.1 µL/min.
  5. Conduct a two-photon analysis of red blood cell movement in cLVs. Calculate lymph flow rate using protocols published in41,22.

8. Photobiomodulation of the cLV contractility

  1. For the PBM of cLVs, use the previously published technology and protocols in40.

9. Euthanasia

  1. After conducting the study, euthanize the mouse with a CO2 euthanasia chamber.

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Results

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The use of protective casing provides the most important advantages for long-term monitoring of cervical lymphatic vessels in vivo, such as isolation of the surgical area from the external environment, allowing for the maintenance of immersion of the lymph vessel and surrounding tissues in saline solution with a constant supply of saline solution through a catheter attached to the body. In addition, due to its attachment to the microscope slide and close mechanical contact with the tissues surrounding the lymph ...

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Discussion

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The study of the physiology of lymphatic vessels requires the use of non-invasive methods while maintaining special environmental conditions that allow for normal contractility and stable lymph flow. The vast majority of methods for studying the functions of lymphatic vessels are based on optical monitoring of their contractility and assessment of the speed of lymph flow3,28,29,30,

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This research was supported by a grant from the Russian Science Foundation (No. 23-75-30001).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
3D printerBambu Lab, ChinaP1S
5pin connectorConnfly electronic (Zhenqin), ChinaDS1110-01
A set of heat shrinkable tubesJupiter, ChinaJP7230-01
Arduino IDEArduino LLC2.3.5-nightly-20241212
BALB/c mice"Andreevka" Branch FSBIS SCBT FMBA, Russia3-15-16Male, 6-month-old
Chlorhixedin BIOPHARMACEUTICAL PLANT, RussiaN/A
Dumont forceps Stoelting, USA52100-07 
Evans Blue dye Sigma-Aldrich, St. Louis, MO, USAN/A
Fine ForcepsStoelting, USA52102-02P
HamiltonHamilton Bonaduz AG, Switzerland29 G needle
Heating elementChinaLFH-9415sg
Insulin needleINSUPEN, Italy31G, 0.25mm*6mm
Ketamine MOSCOW ENDOCRINE PLANT, FSUE, RussiaGeneral anesthesia 
Keypad Shielddiymore, China1602 LCD Keypad Shield
Lidocaine ORGANICA, RussiaN/A Analgesic drug
Lockable switch buttonChinaN/A
Metal-film resistorsTZT, ChinaN/A
Micro Dissecting Vannas Spring ScissorsStoelting, USA52130-00P
MOSFET ModuleChinaMOSFET Module D4184
Mounting postStanda, Lithuania3MP, diameter 12 mm
Mounting wireAB retail, RussiaHB-1
Non-sharp tweezerStoelting, USA52108-83P 
Plastic for 3D printerBambu Lab, ChinaA00-D0-1.75-1000-SPL.
Post collar Standa, Lithuania3PC
Post holderStanda, Lithuania3PH-25
Power connectorRUICHI, ChinaAC-015
Power supplySANPU, ChinaPS200-H1V24
Precision pantograph table for vertical movement"Laser components", Russia02TV004 
Right-angle fixed post clamp Standa, Lithuania3RPC-12
Screw M3Standa, LithuaniaN/A
Screw M4Standa, LithuaniaN/A
Screw M6Standa, LithuaniaN/A
Shaving machine Braun, figure-materials-1Series 3310s
Sodium chlorideKraspharma, RussiaN/A
Solder Production of metal powders, Russiatin-lead solder 61
Soldering fluxSolins, RussiaLTI-120 22 ml with brush
Soldering stationAOYUE, ChinaN/A
Soldering station AOYUE, ChinaN/A
Steel optical rails (SOR)Standa, LithuaniaN/A
Step-down voltage converterRUICHI, ChinaEM-825
Straight dissecting scissors Stoelting, USA52132-10P 
Temperature sensorLiludin, ChinaDS1820
TetracyclineJSC Tatkhimfarmpreparaty, RussiaEye ointment 
The ControllerArduino, ChinaUNO R3
TweezerStoelting, USA52100-03 
Two-photon  microscopNikon, JapanA1R MP
WirePartner-Electro, RussiaP020G-0305-C050
Xylazine "Alfasan International B.V.", NetherlandsMuscle relaxant
CatheterScientific Commodities Inc., USAPE-10, 0,28 mm ID × 0,61 mm OD

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Two Photon ImagingCervical Lymphatic VesselsLymph FlowIn Vivo ImagingLymphatic ContractilityMouse ModelBrain Toxin ClearanceSurgical PreparationOptical VisualizationImaging Reproducibility
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