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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

Abstract

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories. Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 ± 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

Introduction

There is an unmet need for preclinical animal models for research in vascular diseases such as peripheral artery disease (PAD). Despite the advanced developments in diagnosis and treatment, there were more than 200 million patients with PAD in 20181, and their number is constantly increasing. Although several novel therapeutic approaches2,3,4,5,6,7 have been described, successful translation of these therapeutic modalities into clinical application remains a daunting task. Therefore, reliable and relevant in vivo experimental models simulating the human disease condition are required to investigate the potential mechanism and efficiency of these new therapeutic approaches to treat PAD6,7.

Hyperlipidemia and atherosclerosis (AS) are the main risk factors for the development of PAD. ApoE-/- mice (on a high-fat diet) display abnormal fat metabolism and hyperlipidemia and subsequently develop atherosclerotic plaques rendering ApoE-/- mice as the best choice to simulate the clinically relevant PAD. Preclinical HLI animal models are generated through double ligation of the femoral artery (DLFA), which is the most widely used approach in laboratories all over the world8,9,10,11,12,13,14,15 to simulate acute-on-chronic ischemia. However, this approach usually requires a relatively large and invasive incision. Furthermore, it inevitably leads to the animals (especially mice) suffering from increased pain injury and inflammation, which also influences the subsequent experimental results5,6,16,17. This paper describes an acute-on-chronic HLI model in APOE-/- mice by using a very small incision.

Protocol

NOTE: All experimental procedures were performed according to the EC guideline EC 2010/63/EU and have been approved by the local German legislation (35-9185.81/G[1]239/18). Ten male ApoE-/- mice with the C57BL/6J background, weighing 29.6-38.0 g, were housed on a 12 h light/dark cycle and fed a western diet (1.25% cholesterol and 21% fat) and water ad libitum for 12 weeks from the age of 8 weeks. HLI was performed on 20-week-old mice as described below.

1. Induction of HLI in ApoE-/- mice

  1. Prepare the required equipment and tools for surgery (see the Table of Materials and Figure 1). Sterilize the surgical instruments via autoclaving before use and use a glass Bead Sterilizer during the operation.
  2. Anesthetize the mouse with a subcutaneous injection (S.C.) of a mixture of midazolam (5 mg/kg), medetomidine (0.05 mg/ml/kg), and fentanyl (0.5 mg/kg) before all surgical procedures.
    1. After onset of anesthesia, use the vet ointment on the eyes to prevent dryness, and confirm the absence of the pedal withdrawal reflex in the forelimb and hind limb.
  3. Afterwards place the mouse on a heating pad to keep the core body temperature at approximately 37 °C. Using cotton swabs and hair removal cream, carefully remove hair from the hind limb skin on the right side.
    NOTE: The hair removal cream should be used in sufficient quantity to cover the hindlimb, especially the inguinal region where the incision will be made. The hair removal cream should be used for a duration of less than 3 minutes and should be subsequently removed with moistened cotton swabs 2 to 3 times.
  4. Lay the mouse in the supine position on the heating pad under a dissecting microscope. Use a skin antiseptic (see the Table of Materials) to disinfect the skin of the mouse. Afterwards, use pointed forceps and surgical scissors to make an approximately 3-4 mm incision in the middle of the inguinal region. See Figure 2 for a schematic of the procedure.
  5. Remove the subcutaneous fat tissue carefully with help of fine pointed forceps to expose the proximal femoral neurovascular bundle. Carefully use the fine pointed forceps to pierce the membrane of the femoral sheath. Use a cotton swab moistened with saline to move the femoral artery (FA) carefully away from the femoral nerve (FN) and femoral vein (FV).
  6. Pass two 7-0 absorbable sutures through the proximal FA, and make double knots using spring scissors to transect the FA between the two ties.
  7. To expose the distal FA, pass a 7-0 absorbable suture through the lower edge of the incision and gently drag the incision to the region of the right side of the knee of the hind limb.
  8. Move the subcutaneous tissue aside carefully to expose the neurovascular bundle. Use fine pointed forceps to pierce the membrane of the femoral sheath, and dissect the FA from the FV and FN.
  9. Pass two 7-0 absorbable sutures through the distal FA, and make double knots. Use spring scissors to transect the FA between the two ties.
    NOTE: No ligation was performed on the left limb, which served as a control in each mouse.
  10. Afterwards, use 6-0 absorbable sutures to stitch the incision. Place the mouse on a heating pad in a clean cage and continue to monitor its vital parameters until recovery. Provide postoperative analgesics: Buprenorphine s.c. (0.1 mg/kg body weight every 8 hours for 48 h). 48 h after the operation, administer metamizole in drinking water (24 mg/5mL of water corresponds to a dose of 200 mg/kg 4 times daily).

2. Magnetic resonance imaging

NOTE: One day after DLFA, the mice must undergo MRI scans to assess FA blockage.

  1. Place the mouse in a transparent induction chamber, and anesthetize the mouse with 1.5-2% isoflurane in ambient air until loss of righting reflex.
  2. Place the mouse on a heated animal bed equipped with a bite holder and positioned toward the magnet with a laser-controlled system. Maintain the body temperature at 37±1 °C.
  3. During image acquisition, maintain anesthesia with 1.5-2% isoflurane in ambient air, and monitor the respiration using a pressure probe.
  4. Acquire images in the transverse slice orientation using a three-dimensional (3D) time of flight (TOF) angiography sequence with parameter echo time (TE)/repetition time (TR)/flip angle (FA) = 2 ms/12 ms/13°, four averages, an acquisition matrix of 178 x 144 reconstructed to 256 x 192 and 121 slices, resulting in an isotropic resolution of 0.15 mm3. To suppress the signal from the veins, place a saturation slice distally to the hind limbs.

3. Clinical evaluation and follow-up

  1. Estimate the functional recovery in the 1st, 3rd, 5th, and 7th days after the surgery by using the functional scoring Tarlov scale18,19 (Table 1).

4. Histologic evaluation

  1. Seven days after the surgery, apply pentobarbital injection (115 mg/kg) to euthanize the mice.
  2. Perfuse phosphate-buffered saline (PBS) containing 1% paraformaldehyde (PFA) through the left cardiac ventricle (100 mL per mouse). Fix the bilateral gastrocnemius (Gm) of the mice in 4% PFA overnight at 4 °C.
  3. Embed the sample in paraffin according to the previously described protocol20.
    1. Cut 4-5 µm thick sections of the paraffin-embedded tissue block on a microtome. With the help of a round paint brush, place the cut tissue sections in the water bath maintained at 42°C.
    2. Insert the microscope slide into the water at a 45° angle, and carefully position it underneath the group of sections to be collected.
    3. Carefully lift the slide from the water, and allow the sections to attach to the slide and dry overnight in the benchtop incubator at 37 °C.
  4. Perform hematoxylin/eosin (HE) staining of the paraffin sections.
    1. Place the slides containing the sections in slide holders. Prepare 3 containers of fresh xylene, and place the slides in each container for 5 min to deparaffinize the sections.
    2. Rehydrate the sections by dipping the slices successively in 96%, 80%, 70%, 50%, 30% ethanol, and deionized water for 5 min each.
    3. Stain in hematoxylin solution for 10 min.
    4. Transfer the slices to a container of deionized water and rinse by placing under running tap water for 5 min.
    5. Using a microscope, check the intensity of hematoxylin staining. If the staining enables identification of the cell nuclei clearly, continue to the next step. If the staining intensity does not facilitate identification of cell nuclei or if the intensity of staining is faint, place the slide in the hematoxylin solution for 1 min, repeat the washing with water (step 4.4.4), and then check again.
    6. Counterstain in eosin-Y solution for 5 min.
    7. Dehydrate the sections by dipping the slices successively into containers containing deionized water and 30%, 50%, 70%, 80%, and 96% ethanol successively for a duration 10 s each. Next, place the sections sequentially in three containers of fresh xylene for 10 s each.
    8. Place the slides horizontally on microscopic slide storage maps with the sections facing upwards. Add enough mounting medium on the slide, and mount the slides with coverslips.
  5. Perform the immunohistochemical (IHC) staining of the paraffin sections.
    1. Repeat deparaffinization and rehydration steps 4.4.1-4.4.2. Then, immerse the sections in a container with 10 mM sodium citrate buffer, pH 6, and bring the sample to a boil in a microwave.
      NOTE: As over- or under-heating of samples can cause inconsistent staining, maintain the temperature at just below the boiling point for 10 min.
    2. Next, cool the sections on a benchtop for 30 min. Thereafter, wash the sections in PBS three times for 5 min. Carefully dry the area around the sample, and draw a large circle around the sample using a hydrophobic pen. .
      NOTE: Never touch the sample. Marking with a hydrophobic pen creates a hydrophobic boundary, which facilitates the use of a smaller volume of antibody solution.
    3. Quench endogenous peroxidase activity by placing the section in 0.3% H2O2 in PBS for 10 min. Block sections with 400 µL of block buffer (PBS contain 3% bovine serum albumin and 0.3% of non-ionic detergent) for 1 h at room temperature in a humidified chamber.
    4. Wash the sections in PBS for 5 min. Next, add 100-400 µL of diluted anti-CD31 antibody (1:250) enough to cover the section. Following this, incubate sections overnight at 4 °C in a humidified chamber.
      NOTE: Ensure that the section is completely covered with the antibody solution.
    5. Remove the primary antibody, and wash the sections thrice in PBS for a duration of 5 min each.
    6. Prepare the 3, 3'-diaminobenzidine (DAB) mixture by adding 1 drop of the DAB concentrate to 1 mL of the DAB diluent, and mix well. Afterwards, add 100-400 µL of DAB mixture to the sections, and monitor closely by eye for 2 min until an acceptable staining intensity is observed.
      NOTE: Ensure that the section is completely covered with the DAB mixture.
    7. Afterwards, rinse under running tap water for 5 min. Perform hematoxylin staining as described in steps 4.4.3-4.4.5.
    8. Perform eosin-Y staining described in step 4.4.6. Perform dehydration steps described in step 4.4.7.
    9. Mounted the sections with coverslips by using mounting medium. Use ImageJ to estimate the percent of CD31 positive area (%) in 5 randomly selected fields (40x) that can be regarded as microvascular density as previously described21.

5. Statistical analysis

  1. Use statistical analysis software to express the results as mean ± standard deviation and to perform unpaired t-test on the comparisons. Consider P < 0.05 to be statistically significant.

Results

Characteristics of ApoE-/- mice
DLFA surgeries were successfully performed on 10 mice to establish the HLI model, and none of the mice died after the procedure. To follow changes in body weight, mice were weighed before the DLFA procedure (Pre-DLFA) and 7 days after the DLFA surgery (Post-DLFA). Pre-DLFA weights ranged from 29.6 to 38.0 g (mean 34.74 ± 2.47 g), and post-DLFA weights ranged from 26.5 to 34.1 g (mean 30.77 ± 2.15 g), which were s...

Discussion

This study reports a modified, simplified, and surgically efficient approach to establish an HLI model in ApoE-/- mice using double ligation in the proximal and distal regions of the FA through a 3-4 mm incision without any required laboratory upgrades. The main characteristic of this method is the smaller size of the incision compared to previously reported studies describing mouse HLI models8,9,10,

Disclosures

The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgements

Authors thank Viktoria Skude, Alexander Schlund, and Felix Hörner for the excellent technical support.

Materials

NameCompanyCatalog NumberComments
10x Phosphate buffer salineRoth9143.1Used for haematoxylin and eosin stain and immunohistochemistry stain
30% H2O2Roth9681.2Used for immunohistochemistry stain
6-0 absorbable suturesPROLENE8776HUsed for stitching the skin
6-0 absroable suturePROLENEEP8706Used in Surgery
7-0 absorbable suturesPROLENEEH8021EUsed for ligating the artery
7-0 absroable suturePROLENEEP8755Used in Surgery
Acetic acidRoth6755.1Used for haematoxylin and eosin stain
Albumin Fraktion VRoth8076.2Used for immunohistochemistry stain
AutoclaveSystec GmbHSystec VX-150Used for the sterilisation of the surgical instruments
Axio vert A1 microscopeCarl ZeissZEISS Axio Vert.A1Used for viewing and taking the pictures from haematoxylin and eosin stain and immunohistochemistry stain
Bruker BioSpec 94/20 AVIIIBruker Biospin MRI GmbHN/AScan the femoral artery blockage
Buprenovet Sine 0,3mg/mlBayer AG2542 (WDT)Used in post operative pain-management. Dose - 0.1 mg/kg body weight every 8 hours for 48 h after operation
CD31 antibodyAbcamab28364Used for immunohistochemistry stain
Eosin Y solution 0.5 % in waterRothX883.1Used for haematoxylin and eosin stain
Epitope Retrieval Solution pH 6Leica Biosystems6046945Used for immunohistochemistry stain
Ethanol ≥ 99,5 %Roth5054.1Used for haematoxylin and eosin stain and immunohistochemistry stain
FentanylCayman Chemical437-38-7Used for anesthesia
Fine point forcepsMedixplus93-4505SUsed for separating the artery from nerve and vein
Glass bead sterilisatorSimon KellerType 250Used for sterilisation of the surgical instruments
Graefe iris forceps curvedVUBUVUBU-02-72207Used for blunt separation of skin and subcutaneous tissue
Hair Remover cream, Veet (with aloe vera)Reckitt Benckiser108972Remove hair from mice hind limbs
Heating plateSTÖRK-TRONIC7042092Keep the satble temperature of mice
HematoxylinRothT865.2Used for haematoxylin and eosin stain and immunohistochemistry stain
Leica surgical microscopeLeicaM651Enlarge the field of view to facilitate the operation
Liquid DAB+Substrate Chromogen SystemDakoK3468Used for immunohistochemistry stain
Male ApoE-/- miceCharles River LaboratoriesN/AUsed for establish the Peripheral artery disease mice model
MedetomidineCayman Chemical128366-50-7Used for anesthesia
Micro Needle HolderBlack & Black SurgicalB3B-18-8Holding the needle
Micro suture tying forcepsLife Saver Surgical IndustriesPS-MSF-145Used to assist in knotting during surgery
MicrotomeBiobaseBk-Mt268mUsed for tissue sectioning
MidazolamRatiopharm44856.01.00Used for anesthesia
MR-compatible Small Animal Monitoring and Gating System Model 1025SA InstrumentsN/amonitoring vital signs of animal during MRI scan
Octeniderm farblosSchülke & Mayr GmbH180212used for disinfection of the skin
Ointment for the eyes and noseBayer AG1578675Keep the eyes wet under the anesthesia
ParaformaldehydeRoth0335.1Used for fixation of the tissue
PentobarbitalNembutal76-74-4Used for anesthesia
SalineDeltaSelect1299.99.99Used for anesthesia
Spring handle scissors with fine, sharp tipsBlack & Black SurgicalB66167Used for cutting the artery
SuperCut ScissorsBlack & Black SurgicalB55992Used for cutting the skin
Triton X-100Roth9002-93-1Used for immunohistochemistry stain
Western diet, 1.25% Cholesterolssniff Spezialdiäten GmbHE15723-34Diet for the mice
XyleneRoth4436.3Used for haematoxylin and eosin stain and immunohistochemistry stain

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