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 injury, in addition to predisposing factors^3,6. POP is associated with multiple factors such as age, weight, parity, and delivery type (i.e., vaginal or caesarian births). These factors are thought to affect the mechanical integrity of all the pelvic floor tissues, with pregnancy and parity thought to be the main drivers of POP^5,7,8.

The uterosacral ligaments (USLs) are important supportive structures for the uterus, cervix, and vagina and tether the cervix to the sacrum⁴. Damage to the USLs puts women at increased risk of developing POP. It is believed that pregnancy and childbirth impose additional strain on the USL, which potentially induces injury and increases the chances of POP. The USL is a complex tissue composed of smooth muscle cells, blood vessels, and lymphatics distributed heterogeneously along the ligament, which can be divided into three distinct sections: cervical, intermediate, and sacral region⁹. The mechanical integrity of the USL is derived from extracellular matrix (ECM) components like collagens, elastin, and proteoglycans^5,9,10. Type I collagen fibers are known to be a major load-bearing tensile component of ligamentous tissues and are, therefore, likely involved in USL failure and POP¹¹.

There is a lack of knowledge regarding the causes, prevalence, and effects of POP in women. The development of an appropriate animal model of POP is necessary to advance our understanding of the female pelvic floor. Mice and humans have similar anatomical landmarks within the pelvis, such as the ureters, rectum, bladder, ovaries, and round ligaments⁹, as well as similar intersection points of the USL with the uterus, cervix, and sacrum. Further, mice offer ease of genetic manipulation and have the potential to be an easily accessible, cost-effective model for the study of POP⁹.

This study developed a method to access and isolate the USL and the different pelvic floor tissues from nulliparous (i.e., never pregnant) mice. The extracted USLs were subjected to enzymatic digestion (i.e., to remove collagens and glycosaminoglycans), tested to determine the mechanical response under tensile loading, and evaluated for biochemical composition in a proof-of-concept study. The ability to isolate intact tissues will facilitate further mechanical and biochemical characterizations of the pelvic floor components, which is a crucial first step toward improving our understanding of the injury risks related to childbirth, pregnancy, and POP.

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 inhalation in alignment with the American Veterinary Medical Association's guidelines (a displacement rate of 30% to 70% of the chamber volume with CO₂ per minute), followed by cervical dislocation, to ensure successful euthanization.
   1. Work under a hood, if possible, to minimize the spread of mice allergens. Once the mouse stops moving and breathing, allow 2 min or more to verify the lack of response.
      NOTE: If the mouse is pregnant or post-partum, the pups must be individually euthanized. Pups E15.5 and older must be decapitated during the dissection.
2. Prepare the dissection setup with a dissection pad, an 11-blade scalpel, curved thin sharp scissors, two pairs of forceps, curved forceps, 5-0 polyglactin suture, a dissecting microscope, and six pins (Figure 1, see Table of Materials).
3. Place the mouse on the pad, and pin the forelimbs down (Figure 2A). Make an incision of approximately 1-1.5 cm in the abdomen with scissors (Figure 2B). Gently use the scissors to separate the skin at the cranial, caudal, and lateral sides of the incision (Figure 2C, D).
4. Flip the mouse to its dorsal side, and gently peel back the skin toward the hindlimbs to remove the skin away from the dissection site (Figure 2E-H).
5. Pin the mouse at the limbs (Figure 2I), and make an incision of approximately 1 cm into the abdomen from the thorax to the pelvis (Figure 2J).
   NOTE: Ensure not to damage the underlying organs.
6. Gently push the organs toward the thorax to clear the field of view (Figure 2K).
   NOTE: Irrigate the tissues with 1x PBS to maintain hydration.
7. Clear all of the fat tissue from the pelvic floor (Figure 2L-N).
   ​NOTE: Use forceps to gently pull and clear fat off the organs and tissues of interest.

Figure 1: A clean workspace with all the tools needed to perform the dissections. Please click here to view a larger version of this figure.

Figure 2: Removal of the skin and opening of the pelvic and thoracic cavities of the mouse. (A) Pinning down all the limbs. (B) Initial incision. (C) Separating the skin from underlying fascia using scissors. (D) Cutting of the skin and preparing for removal. (E-G) Pulling the skin off by going around the mouse. (H) Completely removing the skin from the dorsal side. (I) Complete removal of the skin from the torso, and re-pinning of the mouse limbs. (J) Opening of the abdomen. (K) View of the open abdomen. (L) Moving the organs out of the field of view. (M) Removing the fat. (N) View of the cleared pelvic floor. Please click here to view a larger version of this figure.

2. USL harvesting

1. Cut the uterine horns from the ovaries (Figure 3B), pull away from the field of view, and cut off at the cervical connection (Figure 3C).
   NOTE: The uterine horns can be identified following the schematic in Figure 3A. Irrigate the tissues with 1x PBS to maintain hydration.
2. Cut the ureters away from the bladder connection (Figure 3D).
   NOTE: This is to avoid confusion with the USL.
3. Cut the colon as close to the cervix as possible (Figure 3E,F).
   NOTE: Irrigate the tissues with 1x PBS to maintain hydration.
4. Place the mouse along with the dissection pad under the dissecting scope to visualize the USLs (Figure 3G).
5. Gently use the forceps to clean the surrounding fat from the USLs.
   NOTE: Use a second pair of forceps to hold the cervix up at a small angle to enhance the visualization of where the USL intersects with the cervix. Irrigate the tissues with 1x PBS to maintain hydration.
6. Tie a 5-0 polyglactin suture around the cervical end of both USLs (Figure 4B, C).
   NOTE: The USLs can be identified using the schematic and magnification images (Figure 4I-K)
7. In this study, one USL is used for morphological or biochemical analyses (i.e., Raman microscopy, immunohistochemistry, histology). Cut the cervical end of the USL, leaving a piece of the cervix attached, and cut a piece of muscle from the bottom of the USL (Figure 4D). Place the dissected tissue in a bath with 1x PBS to keep the tissue hydrated (Figure 4G, H).
8. Use the remaining USL for mechanical testing and imaging. Cut the cervical end of the USL, leaving a piece of cervix attached, to facilitate the mechanical setup (Figure 4D).
   NOTE: The cervical tissue will act as an anchor to secure the USL during the mechanical test.
9. Once all the tissues of interest are harvested (steps 3-5), dislocate the femurs from the pelvis (Figure 4E).
   NOTE: One should hear a faint clicking sound when the femoral head is disarticulated from the acetabular cup.
10. Cut the pelvic bone from the distal and proximal ends of the pelvic bone, leaving about 10 mm of total tissue (Figure 4F). Place the dissected tissue in 1x PBS.

Figure 3: Cleared pelvic floor for USL dissection. (A) Schematic of the anatomy. (B) Cutting the uterine horns at the ovarian connection. (C) Cutting off uterine horns. (D) Cutting of the ureters. (E) Cutting of the colon. (F) A clear view of the rectum and USLs. (G) Placing the mouse and dissection pad under the dissecting scope. Please click here to view a larger version of this figure.

Figure 4: View of the USL and surrounding tissues and dissection of the USLs. (A) Schematic of anatomical landmarks surrounding the USL. (B) Tying a suture around the cervical ends. (C) Slicing off the cervical ends of the USL. (D) Slicing off the USL to be used for the biochemical analyses at the sacral connection. (E) Cutting of the femurs from the pelvic bone. (F) Cutting off the proximal end of the pelvis. (G) Dissecting the USL in a 35 mm Petri dish. (H) The USL with the attached pelvis in a 35 mm petri dish. (I) The USL and rectum at 0.75x magnification. (J) Removing fat from the USL. (K) Cleaning of the USLs at 1.0x magnification. Scale bar = 2 mm. Please click here to view a larger version of this figure.

3. Bladder harvesting

1. After the fat is cleared, hold the bladder with the forceps, and gently lift it at an angle of approximately 40° (Figure 5A).
2. With the scissors, slice off the bladder from the distal side, right above the cervix (Figure 5B).
3. Place the tissue in a bath with 1x PBS to keep the tissue hydrated (Figure 5H).

4. Rectum harvesting

1. Once the USLs are disconnected from the cervix and the bladder is dissected, lift the cervix at an angle of approximately 40° with the forceps. There is the rectovaginal fascia that connects the rectum and the cervix. With the scalpel, gently cut this connection (Figure 5C, D).
2. Cut the pubic bone at the pubic symphysis using scissors. Gently widen the workspace to increase visual access to the tissue insertions.
3. With the forceps, gently pull the rectum toward the thorax, and use the scissors to follow the rectum from its posterior side to the anus. Cut the rectum at the anus (Figure 5E).
4. Place the tissue in 1x PBS to keep the tissue hydrated (Figure 5I).

5. Cervix-vagina complex harvesting

1. After the USLs are removed from the cervix, use the forceps to hold the cervix. Cut the cervix as close to the vulva as possible using scissors (Figure 5F, G).
   NOTE: Ensure to cut the pubic symphysis to see the distal end of the vagina visually.
2. Place the tissue in 1x PBS to keep the tissue hydrated (Figure 5J).

Figure 5: Bladder, rectum, and cervix/vagina dissections. (A) Holding the bladder at an angle. (B) Cutting off the bladder. (C) Cutting the tendon connecting the cervix and rectum. (D) The tendon at 1.0x magnification. (E) Cutting the rectum. (F) Holding onto the cervix with forceps. (G) Cutting at the distal end of the vagina. (H) The bladder in a 35 mm Petri dish. (I) The rectum in a 35 mm Petri dish. (J) The cervix-vagina tissue complex in a 35 mm Petri dish. Please click here to view a larger version of this figure.

6. Sample preparation for tissue characterization

1. Mechanical and visual analyses of the USL
   1. Place the USL with the pelvic attachment over a T-shaped wall within a custom staining well to ensure full immersion in the staining solution (CAD drawings of the well can be found in Supplementary Coding File 1 and Supplementary Coding File 2).
      NOTE: Use suture and forceps to help with the placement.
   2. Dilute a commercially available dye that stains free amine groups (5 µL, see Table of Materials) in 2.5 mL of 1x PBS, add the solution to the custom staining well, and stain the tissue for 2 h on a rocker at 4 °C.
      NOTE: Vortex the solution before adding it to the staining well.
   3. During the last 15 min of staining, add 2.5 µL of a commercially available dead cell nuclei stain (see Table of Materials) to the solution.
      NOTE: Vortex the solution prior to adding to the staining well.
2. Raman analysis of the USLs
   1. Pin the USL in a straight line on a polydimethylsiloxane (PDMS) block that is contained in a custom well.
      NOTE: The PDMS is used as a soft substrate to enable the pinning of the sample in the desired configuration. Blocks of varying dimensions can be made by mixing the two components following the manufacturer's instructions (see Table of Materials), casting in a Petri dish, and, after polymerization, cutting the PDMS into the required geometry with a scalpel blade.
   2. Pin with insect pins at the suture loop and at the pelvic muscle. Hydrate the tissue with 1x PBS.
3. Remaining tissues
   1. Snap-freeze the remaining tissues with liquid nitrogen or in an appropriate embedding compound, depending on the desired analyses.
   2. Save the tissues at −80 °C until subsequent analyses (e.g., immunohistochemical or biochemical assays).

Results

Each step of the dissection of a wild-type mouse is detailed in the associated video and figures related to the protocol. For this study, 6 week old female C57BL/6J mice were used (Supplementary Table 1). Three sample groups with USLs treated with different enzymes were analyzed: control (no treatment), collagenase-treated, and chondroitinase-treated groups. The smooth muscle, nerves, and lymphatics in the USL are surrounded by an ECM rich in fibrillar collagens and glycosaminoglycans (GAGs)

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