Source Paper
The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse
Thomas Pitcher, João Sousa-Valente, Marzia Malcangio
Journal of Visualized Experiments • 2016
Materials & Equipment Checklist
21 items3 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment9
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Materials11
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Software1
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Protocol Steps
1
Animal housing and acclimatization
House 8-10 week-old mice in groups of 5 under a 12 hr light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Allow mice to acclimatize for 1 week prior to starting the experiment.
1 weekNot specified
Note: Randomize and cage mice in groups of 5. Use animal numbers as codes to blind the experimenter to treatments. Use body weights as parameters for randomization.
View evidence from paper
“House 8-10 week-old mice in groups of 5 under a 12 hr light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Let the mice acclimatize for 1 week prior to starting the experiment.”
2
Prepare MIA solution
On the day of injection, freshly prepare the solution of monoiodoacetate in sterile saline (0.9% NaCl) at the desired concentrations. The solution should be sterile filtered with a 0.22 µm filter. Prepare sterile saline for control group injections.
Not specifiedNot specified
Note: Highest recommended dose is 1 mg in 10 µl. Monoiodoacetate is very toxic - wear gloves and mask when handling powder and preparing solution.
View evidence from paper
“On the day of injection, freshly prepare the solution of monoiodoacetate in sterile saline (0.9% NaCl) at the desired concentrations. The highest recommend dose of MIA is 1 mg in 10 µl. The solution should be sterile filtered with a 0.22 µm filter.”
3
Anesthetize mice
Anesthetize mice using an anesthetic trolley by first placing them in a chamber delivering 2% isoflurane in O2 mixture (flow rate 1.5 L/min) and then transfer mice to the nose cone section, which also delivers the 2% isoflurane-O2 mixture to maintain anesthesia during injection. Place vet ointment on the eyes to avoid drying out.
Not specifiedNot specified
Note: Confirm anesthesia by checking the animal's lack of response to a pinch stimulus on the hind paws.
View evidence from paper
“Anesthetize mice using an anesthetic trolley by first placing them in a chamber delivering 2% isoflurane in O2 mixture (flow rate 1.5 L/min) and then transfer mice to the nose cone section, which also delivers the 2% isoflurane-O2 mixture, and as such, maintains anesthesia during injection.”
4
Prepare injection site
Once the animal is under anesthesia, place it on its back. Trim and wipe the area surrounding the knee joint with alcohol. Povidone iodine or chlorhexidine can be used as alternative disinfectants. The patellar tendon (white line below the patella) will become visible.
Not specifiedNot specified
Note: Wear surgical gown, gloves, and mask while performing injection procedure.
View evidence from paper
“Once the animal is under anesthesia, place it on its back. Trim and wipe the area surrounding the knee joint with alcohol. Povidone iodine or chlorhexidine can be used as well for disinfection. The patellar tendon (white line bellow the patella) will become visible.”
5
Stabilize knee joint
In order to stabilize the injection site, keep the knee still in a bent position by placing the index finger beneath the knee joint and the thumb above the anterior surface of the ankle joint. Joint preference is not required.
Not specifiedNot specified
Note: Proper stabilization is critical for accurate injection.
View evidence from paper
“In order to stabilize the injection site, keep the knee still, in a bent position, by placing the index finger beneath the knee joint and the thumb above the anterior surface of the ankle joint.”
6
Locate injection site
To find the precise site of injection, run a 26 G needle attached to a syringe horizontally along the knee (so as not to pierce the skin with the tip) until it finds the gap beneath the patella. Apply gentle pressure to mark the area and then lift the needle and syringe vertically for the injection.
Not specifiedNot specified
Note: This step ensures accurate intra-articular placement.
View evidence from paper
“To find the precise site of injection, run a 26 G needle attached to a syringe horizontally along the knee (so as not to pierce the skin with the tip) until it finds the gap beneath the patella. Apply gentle pressure to mark the area and then lift the needle and syringe vertically for the injection.”
7
Perform intra-articular injection
Insert the needle in the marked area, through the patellar tendon, perpendicular to the tibia. No resistance should be felt. Use thumb as a guide and inject superficial to the site of entry. Inject 10 µl of MIA solution (or saline for controls) into the knee joint.
Not specifiedNot specified
Note: Proper technique is critical to ensure MIA enters the joint capsule and does not leak outside.
View evidence from paper
“Insert the needle in the marked area, through the patellar tendon, perpendicular to the tibia. No resistance should be felt. Use thumb as a guide and inject superficial to the site of entry.”
8
Post-injection massage and needle disposal
After injection, massage the knee to ensure even distribution of the solution. Discard the needle immediately in the sharps bin.
Not specifiedNot specified
Note: Massage helps distribute MIA throughout the joint space.
View evidence from paper
“After injection, massage the knee to ensure even distribution of the solution. Discard the needle immediately in the sharps bin.”
9
Post-injection recovery
Place mice back into a clean home cage on a heated mat and allow them to recover. Keep constant vigilance on the animals until they regain suitable consciousness, which is measured by them regaining sternal recumbency. Once animals are recovered, return to their cage.
Until sternal recumbency achievedHeated mat
Note: Continuous monitoring is essential during recovery from anesthesia.
View evidence from paper
“Place mice back into a clean home cage on a heated mat and allow them to recover. Keep constant vigilance on the animals until they regain suitable consciousness, which is measured by them regaining sternal recumbency.”
10
Training for weight bearing assessment
Train each mouse to walk into a Plexiglass chamber on the weight incapacitance tester apparatus and sit in the holding box. Place the mouse in front of the holding box, lift the entrance up 45°, and allow the mouse to walk in and close the box. Allow the animals to move freely until they adopt a sitting posture.
At least two daysNot specified
Note: This training guarantees that the animal is still and not leaning on either side of the chamber during measurement.
View evidence from paper
“Train each mouse to walk into a Plexiglass chamber on the apparatus and sit in the holding box. Place the mouse in front of the holding box, lift the entrance up 45°, and allow the mouse to walk in and close the box. This training takes at least two days and guarantees that the animal is still and not leaning on either side of the chamber.”
11
Calibrate weight incapacitance tester
Calibrate the instrument before use with a 100 g check weight or according to equipment instruction. Make sure that each hind paw is placed on the appropriate recording pad.
Not specifiedNot specified
Note: Proper calibration is essential for accurate measurements.
View evidence from paper
“Calibrate the instrument before use with a 100 g check weight (or according to equipment instruction). Make sure that each hind paw is placed on the appropriate recording pad.”
12
Baseline weight bearing measurement
Assess weight bearing changes before MIA injection as baseline values. Collect three measurements of the weight borne on each hind paw from the recording pad for each recording session and use the mean value to calculate the difference in weight borne by ipsilateral and contralateral paws.
1 sec per measurementNot specified
Note: The duration of each measurement takes 1 sec, as per the manufacturer's instructions. Express values as the difference between contralateral and ipsilateral paws in grams.
View evidence from paper
“Assess weight bearing changes before MIA injection as baseline values. The duration of each measurement takes 1 sec, as per the manufacturer's instructions. Collect three measurements of the weight borne on each hind paw from the recording pad for each recording session and use the mean value to calculate the difference in weight borne by ipsilateral and contralateral paws.”
13
Post-injection weight bearing assessment
Repeat weight bearing assessments at regular intervals over several weeks to ascertain the development of gait changes. For example, measurements can be taken on days 0, 3, 5, 7, 10, 14, 21, and 28 after MIA injection.
Up to 28 days post-injectionNot specified
Note: A normal weight bearing value of 50% represents equal weight distribution across ipsilateral and contralateral hindlimb. Animals considered hypersensitive display a weight bearing change of approximately 45%.
View evidence from paper
“Then, repeat assessments at regular intervals over several weeks to ascertain the development of gate changes. For example, we report thresholds measured on 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection.”
14
Optional post-mortem confirmation
For best practice and training purposes, use a dye and perform immediate post-mortem dissection to confirm correct localization of injection.
Not specifiedNot specified
Note: This step is optional but recommended for validation of injection technique.
View evidence from paper
“It is suggested for best practice and training purposes that a dye is used and immediate post-mortem dissection performed to confirm correct localization of injection.”
Subjects / Specimens
- Species
- mouse
- Strain
- Not specified
- Age
- 8-10 weeks
- Sex
- unknown
- Count
- 8
- Weight
- Not specified
Housed in groups of 5 under 12 hr light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Acclimatized for 1 week prior to experiment. Randomized and caged by body weight.