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

Summary

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

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

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 toxins^1,2,3. 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 cancer^4,5,6,7,8,9

Protocol

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 optica....

Results

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 .......

Discussion

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 flow^3,28,29,30,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D printer	Bambu Lab, China	P1S
5pin connector	Connfly electronic (Zhenqin), China	DS1110-01
A set of heat shrinkable tubes	Jupiter, China	JP7230-01
Arduino IDE	Arduino LLC	2.3.5-nightly-20241212
BALB/c mice	"Andreevka" Branch FSBIS SCBT FMBA, Russia	3-15-16	Male, 6-month-old
Chlorhixedin	BIOPHARMACEUTICAL PLANT, Russia	N/A
Dumont forceps	Stoelting, USA	52100-07
Evans Blue dye	Sigma-Aldrich, St. Louis, MO, USA	N/A
Fine Forceps	Stoelting, USA	52102-02P
Hamilton	Hamilton Bonaduz AG, Switzerland	29 G needle
Heating element	China	LFH-9415sg
Insulin needle	INSUPEN, Italy	31G, 0.25mm*6mm
Ketamine	MOSCOW ENDOCRINE PLANT, FSUE, Russia		General anesthesia
Keypad Shield	diymore, China	1602 LCD Keypad Shield
Lidocaine	ORGANICA, Russia	N/A	Analgesic drug
Lockable switch button	China	N/A
Metal-film resistors	TZT, China	N/A
Micro Dissecting Vannas Spring Scissors	Stoelting, USA	52130-00P
MOSFET Module	China	MOSFET Module D4184
Mounting post	Standa, Lithuania	3MP, diameter 12 mm
Mounting wire	AB retail, Russia	HB-1
Non-sharp tweezer	Stoelting, USA	52108-83P
Plastic for 3D printer	Bambu Lab, China	A00-D0-1.75-1000-SPL.
Post collar	Standa, Lithuania	3PC
Post holder	Standa, Lithuania	3PH-25
Power connector	RUICHI, China	AC-015
Power supply	SANPU, China	PS200-H1V24
Precision pantograph table for vertical movement	"Laser components", Russia	02TV004
Right-angle fixed post clamp	Standa, Lithuania	3RPC-12
Screw M3	Standa, Lithuania	N/A
Screw M4	Standa, Lithuania	N/A
Screw M6	Standa, Lithuania	N/A
Shaving machine	Braun,	Series 3310s
Sodium chloride	Kraspharma, Russia	N/A
Solder	Production of metal powders, Russia	tin-lead solder 61
Soldering flux	Solins, Russia	LTI-120 22 ml with brush
Soldering station	AOYUE, China	N/A
Soldering station	AOYUE, China	N/A
Steel optical rails (SOR)	Standa, Lithuania	N/A
Step-down voltage converter	RUICHI, China	EM-825
Straight dissecting scissors	Stoelting, USA	52132-10P
Temperature sensor	Liludin, China	DS1820
Tetracycline	JSC Tatkhimfarmpreparaty, Russia		Eye ointment
The Controller	Arduino, China	UNO R3
Tweezer	Stoelting, USA	52100-03
Two-photon  microscop	Nikon, Japan	A1R MP
Wire	Partner-Electro, Russia	P020G-0305-C050
Xylazine	"Alfasan International B.V.", Netherlands		Muscle relaxant
Catheter	Scientific Commodities Inc., USA	PE-10, 0,28 mm ID × 0,61 mm OD