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

We present the need for a new imaging of cervical lymphatic vessel contractility and lymphatic removal of red blood cells in mice. Studying the functions of cervical lymphatic vessels is challenging, particularly when long-term observation of the contractility of lymph flow and the tracer circulations is required. To begin, place a steel optical rail measuring 270 millimeters in length and 43 millimeters in width as the base of the device.

Install two post holders on top of the steel optical rail, ensuring a distance of 175 millimeters between them. Insert two mounting posts with a diameter of 12 millimeters into the post holders and secure each one using an M6 screw. Then attach a post collar to each of the two mounting posts.

Fix a right-angle, fixed-post clamp to one of the mounting posts and fasten it firmly using an M6 screw. Insert a 12-millimeter mounting post into the side hole of the right-angle post clamp. Attach a 3D-printed holder to the inserted side-mounting post and fasten it securely using two pairs of M3 screws.

Using UV-curable glue, affix a 12-millimeter-diameter cover glass to the underside of a protective 3D-printed casing. Then secure the protective casing to the 3D-printed holder using an M4 screw/nut pair. Install a precision pantograph table for vertical movement on the steel optical rail base using two table clips and sets of M6 screws, nuts, and washers.

Then place a 3D-printed case measuring 155 millimeters in length and 110 millimeters in width onto the tabletop. This case serves as a reservoir for accumulating saline solution. Cover the connection point of the wires to heating element one with a thin layer of silicone.

Fix temperature sensor one to the mat and seal it with silicone. Solder the wires of the heating element and temperature sensors to a multi-wire cable. Fill the connection point with silicone and close it using shrink wrap.

Place heating element two and temperature sensor two into the foam roller. Fix the connectors for the power cable and the power button to the walls of the control-unit case. Install a 24-volt direct-current power supply, a direct-current step-down converter, and two MOSFET modules inside the enclosure.

After assembling the circuit and soldering the required components, fix the Arduino Uno and the keyboard screen into the lid housing. Locate the temperature-selection field. Adjust the temperature and press run.

After anesthetizing the mouse, apply eye ointment to both eyelids to prevent drying of the eyeballs during surgery. Once the hind-leg withdrawal reflex and tail-tucking reflex cease, place the mouse on its back on the heating element to provide access to the abdominal region. Plug the control unit into an electrical outlet with an input voltage of 175 to 240 volts alternating current.

Using medical tape, fix the animal's body in a position parallel to the table surface, and place the head at an angle to the sagittal axis so that one side of the neck rests on the roller. Wrap a ligature around the upper incisors and attach it to the back of the device and use medical tape to help maintain the animal's body temperature. Next, shave the fur from the neck region.

Then remove any remaining loose hairs and cover the animal to preserve the sterile field. For primary disinfection, disinfect the neck skin using an alcohol swab. Then apply a 0.5%chlorhexidine solution or a 2%iodine solution for antiseptic treatment and repeat the disinfection process two more times.

After confirming the anesthetic depth by toe-pinch reflex, perform a dermatotomy by making a longitudinal incision along the neck skin from the lower jaw to the collarbone, approximately two centimeters in length. Then perform a fasciotomy using microsurgical curved scissors. Using curved tweezers, gently push aside the salivary glands and superficial neck muscles to expose the sternocleidomastoid muscle.

Ensure that visible blood vessels are not damaged, and continuously moisten the surgical field with saline to prevent tissue damage. Use microsurgical scissors to perform a myotomy of the sternocleidomastoid muscle, providing access to the region between the trachea and carotid artery. Continue spreading the muscles and fascia, performing both myotomy and fasciotomy.

Next, locate the deep cervical lymph node along with the afferent and efferent lymphatic vessels at a depth of five to seven millimeters. Place the device holding the fixed animal onto the movable microscope table. And secure the device in place by firmly fastening it with an M6 screw to ensure stability during imaging.

Attach the catheter, pre-filled with saline solution, to the mounting post using medical tape. Place a roller with a diameter of one centimeter under the heating element on the side of the animal's head to maintain a 45-degree tilt relative to the microscope table. Then position the fixed catheter into the surgically opened neck area so that it lies near the collarbone region of the animal and turn on the saline supply to fill the open neck cavity with saline solution.

Lower the protective casing over the neck area filled with saline so that the lymphatic vessel lies at the center of the viewing field. Then place the protective casing on the trachea without exerting pressure. Finally, lower the non-water-immersion two-photon lens into the protective casing to the correct depth based on the working distance of the lens and perform two-photon visualization of the cervical lymphatic vessel.

After the procedure, successful identification of the deep cervical lymph nodes was confirmed by Evans Blue accumulation in the tissue. Two-photon monitoring of the cervical lymphatic vessel contractility was performed before and after photobiomodulation. Photobiomodulation significantly increased the cervical lymphatic vessel contractility in 2-and 18-month-old mice with similar intensity.

Quantitative analysis showed that cervical lymphatic vessel contractility was significantly lower in 24-month-old mice compared to 2-and 18-month-old mice. However, the values were not significantly different between 2-and 18-month-old mice. Photobiomodulation significantly increased cervical lymphatic vessel contractility in 2-and 18-month-old mice, but had no effect in 24-month-old mice.

Red blood cells injected into the right lateral ventricle were observed in cervical lymphatic vessels four hours after injection and continued moving through the lumen at five hours, confirming directional flow from the brain to the periphery. Our protocol facilitates in vivo monitoring of cervical lymphatic vessel function while maintaining the contractility for up to five hours of observation. Our method offer good reproducibility and is compatible with various two-photon microscopes and objective lenses while maintaining high-quality image with depths up to 500 micrometers.

This technique can potentially become a universal tool for studying the physiology of the brain drainage system and the functions of cervical lymphatic vessels.