Simple and Effective Procedure for Hemostasis in Mouse Arteries

Summary

This study aims to develop an effective method for achieving hemostasis in mouse arteries.

Abstract

During arterial puncture or cannulation in mice, permanent ligation of the artery is often required due to difficulty achieving hemostasis after needle withdrawal. This leads to permanent ischemic injury in the supplied tissue region. Such localized ischemia frequently induces secondary complications, compromising experimental outcomes in animal models. The present method aims to seal the puncture site post-procedure while maintaining normal blood flow, thereby preventing postoperative ischemia. This method involves harvesting an adipose tissue fragment from the experimental animal itself, performing arterial puncture using standard techniques, and then wrapping the puncture site with the fat pad to achieve hemostasis while maintaining blood flow. In this study, bilateral common carotid artery puncture was performed on mice, followed by hemostasis using a fat-wrapping technique. Laser speckle contrast imaging(LSCI) revealed no differences in cerebral blood flow changes between the sham and operated groups of mice, and no significant changes were observed in preoperative versus postoperative cerebral blood flow measurements in the operated group. Histological assessment performed at 7, 14, and 28 days after surgery revealed that: (1) preserved brain architecture (H&E staining), (2) intact neuronal morphology without degenerative changes (Nissl staining), and (3) absence of pathological alterations in brain sections. This arterial hemostasis technique capitalizes on the pliable nature of adipose tissue and physiological coagulation mechanisms. The procedure involves: (1) wrapping autologous fat around the puncture site, (2) securing it with sutures to ensure optimal contact with the arterial adventitia, and (3) allowing escaping blood to permeate the fat-artery interface where it undergoes coagulation. This triple-action mechanism achieves reliable hemostasis while preserving arterial patency and preventing ischemic complications post-operatively. The method demonstrates notable procedural simplicity, clinical efficacy, and excellent translational potential.

Introduction

In animal models, creating arterial injury is an extensively utilized experimental approach for modeling various pathological conditions, including cerebral ischemia-reperfusion models, carotid (CCA) or aortic (AO) atherosclerosis models, and spinal cord ischemia models.

Taking the mice cerebral ischemia-reperfusion (MCAO) model as an example, the current standard modeling approach^1,2,3 involves: isolating the external carotid artery (ECA), ligating its distal end, clamping the proximal ECA with an arterial clip while placing a stay suture, creating a puncture hole between these points, inserting a filament retrogradely through the CCA, securing the vessel and filament with the stay suture, transecting the ECA between the puncture site and distal ligation, releasing the arterial clip, redirecting the filament into the internal carotid artery (ICA) to occlude the middle cerebral artery, and upon filament withdrawal, ligating the proximal ECA.

While this procedure achieves reperfusion, it only restores blood flow between the common and ICA, leaving the ECA permanently disconnected. This ECA transection causes ischemic damage to the ipsilateral facial regions, an unintended injury that not only complicates the surgical procedure but also introduces confounding trauma to experimental animals, potentially compromising study outcomes. More concerningly, in some experimental protocols^4,5, researchers perform a direct CCA puncture, a suture is inserted to establish the MCAO, and then immediately removed, followed by permanent ligation at both ends of the puncture site for hemostasis. However, this method directly increases vascular pressure, thereby elevating cardiac afterload and potentially leading to heart failure⁶.

Using the mouse CCA or AO endothelial injury model with a high-fat diet to establish atherosclerosis as an example, the conventional methodology^7,8,9,10 involves performing arterial puncture in the CCA, abdominal aorta (AA), or femoral artery (CFA), inserting a balloon catheter to induce endothelial denudation, and achieving hemostasis by permanently ligating both ends of the puncture site. This standard approach presents several critical limitations: the obligatory ligation creates localized ischemia distal to the occlusion site. More detrimentally, lipid accumulation occurs at the ligation sites. The validity of the compromise model is altered by affecting local hemodynamics, which interferes with drug delivery to target lesions and skews pharmacological evaluation outcomes.

In the murine spinal cord ischemia model, the procedure involves arterial puncture (via CCA, AA, or CFA access) followed by the insertion of a balloon catheter to induce ischemia at targeted locations¹¹. Following balloon withdrawal, conventional hemostasis is achieved by permanently ligating the puncture site bilaterally.

In all three aforementioned models, post-puncture hemostasis is uniformly achieved through bilateral ligation of the puncture site. While effective for bleeding control, this approach inevitably induces localized ischemia, leading to secondary complications that ultimately compromise model validity. The maintenance of vascular patency during hemostatic procedures is therefore physiologically and experimentally critical. Therefore, this article introduces an innovative, simple, and effective arterial hemostasis method for mouse experiments. The procedure involves first clamping both ends of the puncture site with arterial vascular clips, then wrapping the artery with a 3 mm × 3 mm × 1 mm fat pad to cover the puncture. The fat pad is secured in place with a ligature suture around the artery. The arterial clips are gradually released, and the tightness of the ligature is adjusted based on bleeding until complete hemostasis is achieved. This approach restores blood flow, maintains normal hemodynamics, and prevents ischemic damage to surrounding tissues caused by permanent vessel ligation. It is particularly advantageous for long-term post-modeling observation of mouse conditions and for assessing pharmacological interventions, thereby significantly enhancing the experimental rigor of preclinical studies.

Protocol

All experimental procedures were conducted in strict compliance with the Guidelines for the Care and Use of Laboratory Animals (Protocol Approval No. SNLL25040501; Animal Use License No. SYXK(Liao)2021 - 0010) established by the Institutional Animal Care and Use Committee (IACUC) of Shenyang Agricultural University. The reagents and the equipment used are listed in the Table of Materials.

1. Experimental preparation

1. Animal details and documentation: Document all animal research data following ARRIVE guidelines (Animal Research: reporting of In Vivo Experiments). Use male Balb/C mice aged 6-8 weeks, weighing 22 g ± 4 g. Maintain housing conditions at 24 ± 2 °C with 55% ± 5% relative humidity under a 12 h light/dark cycle.
2. Animal preparation
   1. Anesthetize the mice using an isoflurane vaporizer (following institutionally approved protocols). Induce anesthesia with 4% isoflurane, then maintain with 2% isoflurane. Use ophthalmic ointment on eyes to prevent dryness while under anesthesia. After induction of anesthesia, administer carprofen at a dose of 5 mg/kg subcutaneously to the mice for intraoperative and postoperative analgesia.
   2. After anesthesia, depilate the cervical and cranial regions and disinfect the exposed skin with povidone-iodine, followed by 75% ethanol to remove the residue.
   3. Place the mice on a 37 °C heating pad for all subsequent experimental procedures.
3. Measuring baseline cerebral blood flow in mice
   1. Position the mice in a prone posture. Confirm a surgical plane of anesthesia prior to starting surgery with appropriate monitoring (e.g., toe pinch). Make a midline scalp incision to expose the parietal bones. Carefully dissect and remove the overlying fascial layer from the skull surface.
   2. Adjust the LSCI system to scan the exposed mouse parietal bone at 10x magnification, and record the baseline cerebral blood flow data.
4. Harvest the fat pad and performing arterial puncture
   1. Reposition the mice in supine position. Make a midline cervical incision. Harvest a 3 mm × 3 mm × 1 mm subcutaneous adipose tissue graft for later use.
   2. Perform blunt dissection of the submandibular gland, sternohyoid muscle, and sternocleidomastoid muscle. Expose both CCA.
   3. Free the CCA to expose the ICA and ECA.
   4. Apply vascular clamp 1 to the distal end of the CCA, and place vascular clamp 2 on the proximal end.
   5. Puncture the CCA between the two clamps using a 23 G needle.
5. Applying the fat pad
   1. After the needle removal, wrap the CCA at the puncture site with the cut surface (non-serosa side) of the fat pad.
   2. Place a 4-0 suture beneath the fat-wrapped area; then tie a single throw gently over the fat surface to apply pressure for secure contact between the fat pad and the vessel wall; finally, adjust the ligature gradually to achieve pressure balance for hemostasis.
   3. Elevate the distal CCA with a moist cotton swab. Release vascular clamp 1 and slowly withdraw the swab to allow gradual blood flow toward the puncture site.
   4. Check for blood flow; if active bleeding was observed, reclamp the distal CCA with vascular clamp 1.
   5. Wait for 1 min, then slowly release the vascular clamp 1; if bleeding occurs, reposition the fat pad or slightly tighten the single throw of the ligature to increase pressure; continue these adjustments until complete hemostasis is achieved.
   6. Elevate the proximal CCA with a moistened cotton swab. Release vascular clamp 2 and gradually withdraw the swab to permit controlled blood flow toward the puncture site.
   7. If bleeding occurs, reclamp the distal CCA with a vascular clamp 2.
   8. Slightly tighten the single throw of the suture to increase pressure; wait for 1 min, then elevate the proximal CCA and release vascular clamp 2; slowly rolling the moistened cotton swab toward the puncture site.
   9. If bleeding persists, repeat the two preceding steps until achieving complete hemostasis.
   10. Place an additional throw with the suture to form a double knot over the initial one, thereby better securing the fat pad to the vessel wall and achieving hemostasis through balanced intravascular and external pressures.
   11. Confirm that the common carotid arteries on both sides of the fat pad were filled and exhibit consistent vessel diameter; consider this as successful blood flow restoration and hemostasis.
   12. Repeat the identical procedure to perform puncture hemostasis on the contralateral common carotid artery.
   13. Expose the bilateral common carotid arteries in sham mice without performing arterial puncture or fat pad wrapping, while maintaining all other procedures identical to the operated group.
   14. Suture the cervical surgical incision and apply an antiseptic.
6. Performing postoperative cerebral blood flow assessment
   1. Following the completion of bilateral CCA puncture hemostasis in the mice, adjust the LSCI system to scan the exposed parietal bone and acquire 10x magnified images; then record the cerebral blood flow changes.
   2. Suture the scalp incision and apply antiseptic, allow the mice to recover on a 37 °C heating pad before returning them to the housing cage for continued care.
7. Performing postoperative monitoring and tissue collection
   1. Monitor and record daily the survival status, motor activity, and any signs of bleeding in the cervicomandibular region of the mice during the postoperative period.
   2. On postoperative days 7, 14, and 28, euthanize the mice with carbon dioxide (following the institutionally approved protocols) and fix the brain tissues in 4% paraformaldehyde.

2. Pathological examination

1. After 24 h fixation, dehydrate the brain tissues through an ethanol gradient, clear with xylene, and embed in paraffin for sectioning.
2. Perform H&E staining on hippocampal sections and examine the brain tissue morphology under a microscope.
3. Perform Nissl staining on hippocampal sections and examine neuronal architecture under a microscope.

Results

Analysis of the pre- and post-operative laser speckle contrast imaging data and relative perfusion measurements in both the sham and operated groups revealed no significant changes, demonstrating that this arterial hemostasis technique successfully sealed the common carotid artery puncture site without compromising cerebral blood flow (Figure 1).

Mice in the operated group were continuously monitored for 28 days post-operatively, with no mortality observed. Their ...

Discussion

In MCAO models, preserving the ECA better recapitulates clinical pathology by preventing facial ischemic damage associated with ECA ligation and transection. While the conventional approach of filament insertion via CCA puncture offers technical simplicity, it necessitates bilateral ligation of the puncture site, resulting in cerebral perfusion that depends on Circle of Willis collateral circulation, a suboptimal representation of true stroke pathophysiology. The fat pad hemostasis technique presented in this st...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-0 with thread sewing needle	Shanghai Pudong Jinhuan Medical Supplies Co., Ltd
Balb/C mouse	Beijing Huafukang Biotechnology Co., Ltd		6-8w,male
DHG Series Heating and Drying Oven	Shanghai Yiheng Technology Co., Ltd	DHG-9123A
Hematoxylin and Eosin (H&E) High Definition Staining Kit	Servicebio	G1076-500ML
Isoflurane	Shenzhen Ruiwode Life Technology Co., Ltd	R510-22
Laser speckle analyzer	Shenzhen Ruiwode Life Technology Co., Ltd	RFLSI ZW
Nissl dye solution	Servicebio	G1036-100ML
Organizational embedding machine	Leica	HistoCore Arcadia H
Slide scanning imaging system	Shenzhen Shengqiang Technology Co., Ltd	SQS-40R
Small animal anesthesia machine	Shenzhen Ruiwode Life Technology Co., Ltd	R500
Tissue dehydrator	Leica	HistoCore pearl
Ultrapure Water System	Thermo	50132370
Ultra-Thin Semiautomatic Microtome	Leica	HistoCore MULTICUT