Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs

Summary

This article presents a detailed protocol for dissecting uterosacral ligaments and other pelvic floor tissues, including the cervix, rectum, and bladder in mice, to expand the study of female reproductive tissues.

Abstract

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic floor are supported by different anatomical structures, including muscles, ligaments, and pelvic fascia. The uterosacral ligament (USL) is a critical load-bearing structure, and injury to the USL results in a higher risk of developing POP. The present protocol describes the dissection of murine USLs and the pelvic floor organs alongside the acquisition of unique data on the USL biochemical composition and function using Raman spectroscopy and the evaluation of mechanical behavior. Mice are an invaluable model for preclinical research, but dissecting the murine USL is a difficult and intricate process. This procedure presents an approach to guide the dissection of murine pelvic floor tissues, including the USL, to enable multiple assessments and characterization. This work aims to aid the dissection of pelvic floor tissues by basic scientists and engineers, thus expanding the accessibility of research on the USL and pelvic floor conditions and the preclinical study of women's health using mouse models.

Introduction

Approximately 50% of women are affected by pelvic organ prolapse (POP)^1,2. About 11% of these women fit the criteria for undergoing surgical repair, which has a poor success rate (~30%)^3,4. POP is characterized by the descent of any or all of the pelvic organs (i.e., bladder, uterus, cervix, and rectum) from their natural position due to the failure of the USL and the pelvic floor muscles to provide adequate support⁵. This condition involves anatomical dysfunction and disruption of the connective tissue, as well as neuromuscular....

Protocol

All animal experiments and procedures were performed according to protocol #2705, approved by the Animal Care and Use Committee of the University of Colorado Boulder. Six week old female C57BL/6J mice were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Animal preparation

1. Euthanize the animal following the institutionally approved method.
   NOTE: The present study used CO₂ .......

Discussion

The effect of structural damage on female reproductive tissues is understudied, and an easily accessible animal model for POP research is needed. The mouse is a cost-effective model that can mimic human reproductive studies¹⁶. Due to the growing interest in the study of the female reproductive system, there is a need for methods that aid the study of these tissues. To address this need, in this work, a method is established to dissect and prepare murine pelvic floor tissues for structural and func.......

Materials

Name	Company	Catalog Number	Comments
11 Blade	Fisher	3120030	Removable blade
1x PBS	Fisher	BP399-1	Diluted from 10x concentration
Chondroitinase ABC	Sigma	C3667-10UN	Enzyme
Collagenase Type I	Worthington Biochemical	LS004194	Enzyme
Confocal Microscope	Leica	STELLARIS 5	Upright configuration
Dissection Microscope	Leica	S9E	With camera
Dumont #5 Forceps	Fisher	NC9626652	Thin tip
Female C57BL/6J mice	Jackson Laboratory	strain #: 000664
FemtoTools Micromanipulator	FemtoTools	FT-RS1002	100 mN load cell
FST Curved Forceps	Fisher	NC9639443	Curved tip
FST Sharp 9 mm Scissors	Fisher	NC9639443	Dissection scissors
Ghost Dye 780	Tonbo	13-0865-T500	Free amine stain
Kimwipes	Fisher	06-666	Box of 50 wipes
OCT	Tissue Tek	4583	Used for tissue preservation
PDMS	Thermo Fisher	044764.AK	Follow manufacturer's instructions
Petri Dishes 35 mm	Fisher	FB0875711A	Used for dissected tissue
Polyglactin 5-0 Suture	Veter.Sut	VS385VL	With needle
Renishaw InVia Raman Microscope	Renishaw	PN192(EN)-02-A	With confocal objectives
Rocking Platform	VWR	10127-876	2 tier platform
Surgical Gloves	Fisher	52818	For dissection
Sytox	Thermo Fisher	S11381	Nuclear stain
T-pins	Fisher	S99385	For dissection
Transfer Pipets	Fisher	13-711-7M	For dissection
Underpads	Fisher	22037950	To cover dissection pad