Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials
- Native TSP1 protein, human (Novus Biologicals, Centennial, CO, USA; Cat No.: NBP1-99789; Thermo Scientific, Denver, IL, USA; Cat No.: RP-43151; Athens Research and Technology, Athens, GA, USA; Cat No.: 16-20-201319)
- CD47 antibody, clone mIAP301 (Novus Biologicals, Centennial, CO, USA; Cat No.: NBP1-43407)
- SIRPα antibody, clone C20 (Santa Cruz, Santa Cruz, TX, USA; Cat No.: sc-6922)
- Dextrose (Fisher Scientific, Tempe, AZ, USA; Cat No.: D-16-500)
- Calcium chloride dehydrate (Fisher Scientific, Tempe, AZ, USA; Cat No.: C614-500)
- Sodium bicarbonate (Fisher Scientific, Tempe, AZ, USA; Cat No.: S233-500)
- Potassium phosphate (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: P5379)
- Magnesium phosphate (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: M7506-500G)
- Potassium chloride KCl (EM Science, MA, USA; Cat No.: 7300)
- Sodium chloride (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: S5886)
- EDTA calcium Disodium salt (Santa Cruz, Santa Cruz, TX, USA; Cat. No.: SC-239970)
- Phenylephrine hydrochloride (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: P6126)
- Acetylcholine chloride (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: A6625)
- Forskolin (Sigma-Aldrich, Saint Louis, MO, USA; Cat No.: F6886)
- DMSO (Fisher Scientific, Tempe, AZ, USA; Cat No.: BP231-100)
- Meclofenamate sodium (Santa Cruz, Santa Cruz, TX, USA; Cat No.: SC-200532A)
- 0.9% saline (Fisher Scientific, Tempe, AZ, USA; Cat No.: Z1376)
- Isoflurane (Henry Schein, Melville, NY, USA; Cat No.: NDC11695-6776-1)
- 95% O2/5% CO2 (Airgas, Phoenix, AZ, USA; Cat No.: SGMOX5CO22)
2.2. Equipment
- Automated Multi Myograph System—620M (Danish Myo Technologies, DMT, Ann Arbor, MI, USA)
- PowerLab 8/35 (ADIntruments, Colorado Springs, CO, USA; Cat No.: PL3508)
- Force Calibration Kit (Danish Myo Technologies, Ann Arbor, MI, USA; Cat No.: 300041)
- Dissecting scope (Carl Zeiss Microscopy, White Plains, NY, USA; Cat No.: SteREO Discovery V8 with CL 1500 ECO)
- Dissecting dish (Living Systems Instrumentation, San Antonio, TX, USA; Cat No.: DD-90-S-BLK)
- Dumont #5 forceps (Fine Science Tools, Foster City, CA, USA; Cat No.: 11252-20)
- Dumont #7 forceps (Fine Science Tools, Foster City, CA, USA; Cat No.: 11271-30)
- Tissue forceps (Fine Science Tools, Foster City, CA, USA; Cat No.: 11021-12)
- Moria Miniature Vannas—curved (Fine Science Tools, Foster City, CA, USA; Cat No.: 15396-01)
- Vannas scissors—straight (Fine Science Tools, Foster City, CA, USA; Cat No.: 15009-08)
- Surgical scissors straight sharp/blunt (Fine Science Tools, Foster City, CA, USA; Cat No.: 14001-16)
- Surgical scissors straight sharp (Fine Science Tools, Foster City, CA, USA; Cat No.: 14002-13)
- Surgical tape (Various suppliers)
- 30G × 1 in needle (BD, NJ, USA; Cat No.: 305128)
- 3 mL syringe (BD, NJ, USA; Cat No.: 309657)
- Water bath with immersion circulator (Cole-Parmer, Vernon Hills, IL, USA; Cat No.: EW-12120-13)
- Alternate option to water bath: automatic buffer filler system—625FS (Danish Myo Technologies, Ann Arbor, MI, USA)
- Microfuge tube (Fisher Scientific, Phoenix, AZ, USA; Cat No.: 02-682-550)
2.3. Software
- LabChart 8 (ADInstruments Version 8)
- GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA)
3. Procedure
3.1. Preparation of the Instrument
- Optional: Myography Calibration
- On the day of experiment, power on PowerLab and myograph with HEAT OFF, and leave them on standby. Open the most recent calibration file in LabChart.
3.2. Preparation of Buffers
3.2.1. Preparation of Stock Solution
- KCl Stock
- Foskolin Stock
- Krebs Stock Salts
- Krebs Stock Phosphate
- Krebs Stock EDTA
3.2.2. Preparation of Fresh Solution
- Fresh Meclofenamate working stock
- Fresh phenylephrine hydrochloride (PE) and acetylcholine chloride (Ach) working stocks
- Dissolve 20.37 mg of PE or 18.17 mg of Ach into 1 mL of ddH2O to make 1 × 10−2 M PE or 1 × 10−2 M Ach stock solutions, respectively.
- Make 10× serial dilution of PE and Ach working stocks by mixing 20 µL of PE or Ach and 180 µL of Krebs buffer. For example, 20 µL of 1 × 10−1 M PE is mixed with 180 µL of Krebs buffer to make 200 µL of 1 × 10−2 M PE.
3.2.3. Fresh Krebs–Henseleit Solution
- To prepare 1 L of fresh Krebs buffer, fill a calibrated Erlenmeyer flask with 750 mL of ddH2O, dissolve 1.25 g of NaHCO3 and 0.99 g of dextrose, followed by 12 mL of KH2PO4 stock and 1 mL of EDTA stock, while stirring.
- Gas the solution with 95% O2/5% CO2, gradually add 75 mL of stock salts.CRITICAL STEP Slow addition of stock salts while stirring and bubbling is required to prevent precipitation.
- Add the remaining volume of 162 mL of ddH2O to make a final volume of 1 L, and add 310 µL of meclofenamate stock. Continue bubbling for around 5 min, seal the flask with parafilm, and store at 4 °C until use.
3.2.4. TSP1 Stock (Novus or Athens) Preparation
- Spin down.
- Add 270.6 µL of ddH2O to the vial containing 25 µg of TSP1, vortex to completely dissolve, and spin down. An aliquot of 50 µL should be kept and used on ice, with the rest stored at −20 °C.
3.2.5. Drug Candidate Preparation
3.3. Tissue Preparation and Mounting of the Artery
3.3.1. Animal Dissection
- Euthanasia with isoflurane inhalation.
- Lay the mouse on its back onto a blue under pad. Tape down paws for a complete exposure of the ventral side.
- Wipe the chest and abdomen with Kim wipes wet with water or alcohol.
- Grasp the skin with tissue forceps, cut open the abdomen with straight sharp/blunt surgical scissors, and exsanguinate by cutting off the abdominal aorta and inferior vena cava with straight sharp surgical scissors.
- Cut open the rib cage with straight sharp/blunt surgical scissors to reveal the chest cavity. Slowly inject 3 mL of cold saline into the left ventricle through the heart apex. The lung turns from pink to whitish with blood flush-out.
3.3.2. Tissue Harvest
- Add freshly prepared cold Krebs buffer into a dissecting dish. Generally, 3–5 mL is enough for immersion of a harvested vessel. Adjust the volume according to the size of your dissecting dish. Set aside, preferably on ice.
- Add 5 mL of freshly prepared cold Krebs buffer into myograph chamber and gas with 95% O2/5% CO2. Keep the myograph remaining in HEAT OFF.
- Remove the lungs and heart to expose the descending thoracic aorta.
- From the right or the left side of the body, slide the curvy part of a Dumont #7 forceps underneath the aorta and make sure to lay the forceps against the spine.
- Repeat closing and releasing the forceps to loosen the connective tissue/fat between the aorta and spine. Repeat step 4 and 5 along the thoracic aorta.
- Make a cut at the aorta hiatus at the diagram with straight Vannas scissors.
- Grab the fat tissue on top of the loose end of the aorta with a Dumont #5 forceps in the left hand, and slightly raise the loosened tissue. Start cutting with a curved Moria Miniature Vannas in the right hand from the loose end up to the neck.
3.3.3. Tissue Preparation and Mounting
- Immerse the isolated aorta into cold Krebs solution in the dissecting dish. Secure the aorta by pinning down fat/connective tissues at two ends of the isolated vessel. Fasten an approximately 2 cm long plastic ruler along the isolated vessel.CRITICAL STEP Do not stretch the isolated vessel to avoid injury or damage to the intimal layer of the vessel, especially when the vasoreactivity of the endothelium is of interest.
- Clear off fat/connective tissues by gently pulling the fat/connective tissues away from the vessel using Dumont #5 forceps and cutting it off with a curved Moria Miniature Vannas whose tip is kept away from the vessel. The vessel branching remains uncut for the purpose of seizing in step 5. The fat/connective tissue at two ends generally is not used owing to possible injury during the process of harvest.
- Cut the aorta into segments 2.5 to 3 mm in length with straight Vannas scissors, starting at least 1 mm from each end. Keep aortic segments in cold Krebs buffer until use.
- Remove a chamber off the myograph interface. Adjust the micropositioner of the myograph chamber to bring the two pins as close together as possible, but not touching each other, under the dissecting microscope.
- Hold a vessel branching of one aortic segment with Dumont #5 forceps to transfer it from the dissecting dish to the myograph chamber and mount it onto pins.
- Adjust the micropositioner to slightly stretch the mounted vascular segment in order to prevent it from sliding off. Install the chamber back onto the myograph interface, resume gas, and continue keeping HEAT OFF. A vessel segment mounted onto myograph and stretched to the optimal resting tension is exhibited in step 2 of Section 3.4.1 Establishment of optimal resting tension.
- Repeat steps 4 to 6 for the remaining vessels.
3.4. Experiment
3.4.1. Establishment of Optimal Resting Tension
- After all vessel segments are mounted, click “start” in LabChart to start recording; zero all channels following the direction shown on myograph display screen. Turn on myograph heat, and proceed to experiment setup by beginning timing. Turn on the water bath to warm up Krebs buffer with continuous gassing with 95% O2/5% CO2, if DMT Automatic Buffer Filler System—625FS is not available. The vessels segments are incubated in warming up Krebs buffer for 20 min, counted as the first 20 min equilibration period.
- Change buffer. Start timing for the second 20 min equilibration period. Zero all channels. Gradually stretch vessel rings by adjust micropositioner to add resting tension to the vessels. Resting tension starts from 0 to 500 mg, with a 100 mg increase and an interval of 30 s between each stretch, while closely monitoring reading changes in the LabChart interface. Continue adjusting every few minutes until the tension stays constant at 500 mg (Figure 2).
- 3.
- Change buffer to start the third 20 min equilibration period. Adjust tension to 500 mg if necessary. Until now, the aortic segments are incubated for a total of 60 min.
3.4.2. Standard Start (Also Called Tissue Priming)
- Add exactly 5 mL of Krebs buffer to each myograph chamber and adjust the micropositioner to maintain the resting tension at 500 mg. Zero all channels.
- Add 100 µL of 3 M KCl stock to make a final concentration of 60 mM in each chamber, and mark in LabChart. Wait for 5–10 min and wash out three times with approximately 5 mL of Krebs buffer. If the tension falls below 0, bring it back up to 0 mg, and allow the vessel rings to relax for 10–15 min.
- Repeat the second KCl stimulation for 20 min, followed by washout three times and relaxation for 20 min. Adjust tension to 0 mg, if necessary.
3.4.3. Endothelium Intactness Examination
- Add exactly 5 mL of Krebs buffer to each chamber and adjust the resting tension to 0 mg, if necessary. Add 5 µL of 1 × 10−3 M PE to each chamber and mark in LabChart. Wait for the PE-induced contraction to reach a plateau, and write down the force of tension.
- Without changing the buffer, add 5 µL of 1 × 10−3 M Ach to each chamber and mark in LabChart. Wait for Ach-induced relaxation to reach a plateau, and write down the force of tension. Calculate the percentage of relaxation induced by Ach with the following equation:
- 3.
- Proceed to washout three times. Let the vessel rings relax for 30 min with replacement of Krebs buffer every 10 min. Adjust the resting tension to zero if necessary.
- 4.
- Proceed to the next experiment setup, described under either Section 3.4.4.1 Pharmacological screening of potential drug candidates/pre-treatment experiment, or Section 3.4.4.2 Therapeutic treatment experiment/post-treatment experiment, as needed.
3.4.4. Experiment
3.4.4.1. Pharmacological Screening of Potential Drug Candidates/Pre-Treatment Experiment
- Add exactly 5 mL of buffer to each chamber.Note: The volume of Krebs buffer may need to be adjusted according to the volume of pharmacological agent to be added, in order to ensure the concentration accuracy of the pharmacological agent. Our laboratory applies a volume rule of 1% or less. For instance, the volume of Krebs buffer is reduced to 4950 µL when 50 µL of TSP1 is added to a myograph chamber.
- Add 10 µL of CD47 antibody, 10 µL of SIRPα antibody, or 5 µL of selected drug candidate to make a final concentration of 1 µg/mL, 2 µg/mL, or 6.6 × 10−9 M, noted as dose 1, respectively. Mark the treatment at the corresponding channel in LabChart. Start the timer.Note: This step was skipped in the tests of reactivity of TSP1 from different vendors.
- Precisely 15 min later, add 50 µL of TSP1 into three chambers pre-treated with CD47 antibody, SIRPα antibody, or the drug candidate, or Krebs buffer only, respectively. TSP1 incubation time is set at 60 min. Generally, a fourth vessel segment is left untreated as a negative control.Note: Volume of TSP1 needs to be adjusted based on the concentration of original package or stock solution.
- Mark treatment at corresponding channels in LabChart.
- Add 5 µL of 1 × 10−3 M PE into each chamber without changing the buffer, mark in LabChart, and wait for PE-induced contraction to reach a plateau.
- Start Ach doses, starting from a final concentration of 1 × 10−9 M up to 1 × 10−5 M, cumulatively in half-log increments, following the Table 1. Mark each dose of Ach in LabChart.OPTIONAL STEP At the end of the experiment, add 4.1 µL of 12.18 mM forskolin to the vessel ring received TSP1 treatment only to make a final concentration of 1 × 10−5 M. Forskolin is included occasionally to prove the viability of the vessel, which does not respond to Ach stimulation.
- Click “Stop” in LabChart. Save the file for further analysis. Generally, the file name includes experiment date, treatment, and dose. If the name is too long, make a note in the lab notebook with details.
- Proceed to Section 3.4.5 Clean up.
3.4.4.2. Therapeutic Treatment Experiment/Post-Treatment Experiment
- Add exactly 5 mL of buffer to each chamber.Note: The rule of thumb of 1% or less in volume consistently applies. The volume of Krebs buffer may need to be adjusted according to the volume of pharmacological agent to be added, in order to ensure the concentration accuracy of the pharmacological agent.
- Add 50 µL of TSP1 into three chambers to be treated with CD47 antibody, or the drug candidate, or Krebs buffer only, respectively. Mark the treatment at the corresponding channel in LabChart. Start the timer. TSP1 incubation time is set for 60 min. Generally, a fourth vessel segment is left untreated as a negative control.Note: Volume of TSP1 needs to be adjusted based on the concentration of original package or stock solution.
- Precisely 15 min later, add 10 µL of CD47 antibody, 10 µL of SIRPα antibody, or 10 µL of selected drug candidate to make a final concentration of 1 µg/mL, 2 µg/mL, or 1.32 × 10−8 M, noted as dose 2, respectively.Note: This step was skipped in the tests of reactivity of TSP1 from different vendors.
- Mark each dose treatment at the corresponding channels in LabChart. The co-incubation period for CD47 antibody, SIRPα antibody, or drug candidate with TSP1 is 45 min.
- Add 5 µL of 1 × 10−3 M PE into each chamber without changing the buffer, mark in LabChart, and wait for PE-induced contraction to reach a plateau.
- Start Ach doses, starting from a final concentration of 1 × 10−9 M up to 1 × 10−5 M, cumulatively in half-log increments, following Table 1. Mark each dose of Ach in LabChart.OPTIONAL STEP At the end of the experiment, add 4.1 µL of 12.18 mM forskolin to the vessel ring receiving TSP1 treatment only to make a final concentration of 1 × 10−5 M. Forskolin is included occasionally to prove the viability of the vessel, which does not respond to Ach stimulation.
- Click “Stop” in LabChart. Save the file for further analysis. Generally, the file name includes experiment date, treatment, and dose. If the name is too long, make a note in the lab notebook with details.
- Proceed to Section 3.4.5 Clean up.
3.4.5. Clean Up
3.5. Data Analysis
- Open data file in LabChart and quantify vessel contractile/relaxing responses using the Dose Response Module. A step-by-step video instruction on how to use the Dose Response Module in LabChart is available [21], which reports a variety of pharmacological parameters such as half maximal effective concentration (EC50) and hill slope.
- Create a new GraphPad file and copy the dose and matching force to the GraphPad file for statistical analysis. Concentration–response curves were plotted using non-linear regression fitting. Comparisons of concentration–response curves between the groups were performed using two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. The logarithms of EC50 values were compared using a two-tailed Student’s t-test, and a p-value smaller than 0.05 was taken as significant.
4. Representative Results
4.1. Examination of Bioactivity of TSP1
4.2. Pharmacological Screening of TSP1-Targeting Drug Candidates
4.3. CD47-Based Candidate Drug as a Potential Therapy
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Acetylcholine, Working Stock Concentration (M) | Volume of Working Stock to Be Added (µL) | Acetylcholine, Final Concentration (M) |
---|---|---|
1 × 10−6 | 5 | 1 × 10−9 |
1 × 10−5 | 1.0 | 3 × 10−9 |
1 × 10−5 | 3.5 | 1 × 10−8 |
1 × 10−4 | 1.0 | 3 × 10−8 |
1 × 10−4 | 3.5 | 1 × 10−7 |
1 × 10−3 | 1.0 | 3 × 10−7 |
1 × 10−3 | 3.5 | 1 × 10−6 |
1 × 10−2 | 1.0 | 3 × 10−6 |
1 × 10−2 | 3.5 | 1 × 10−5 |
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Yao, M.; Ganguly, S.; Shin, J.H.S.; Elbayoumi, T. Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System. Methods Protoc. 2021, 4, 74. https://doi.org/10.3390/mps4040074
Yao M, Ganguly S, Shin JHS, Elbayoumi T. Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System. Methods and Protocols. 2021; 4(4):74. https://doi.org/10.3390/mps4040074
Chicago/Turabian StyleYao, Molly, Samayita Ganguly, Jane Hae Soo Shin, and Tamer Elbayoumi. 2021. "Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System" Methods and Protocols 4, no. 4: 74. https://doi.org/10.3390/mps4040074