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
19 items4 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment8
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Materials10
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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 monoiodoacetate solution
On the day of injection, freshly prepare the solution of monoiodoacetate in sterile saline (0.9% NaCl) at the desired concentrations. Prepare sterile saline for injections in a separate group of control mice.
Day of injectionNot 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. Solution should be sterile filtered with a 0.22 µm filter.
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
Place mice in a chamber delivering 2% isoflurane in O2 mixture (flow rate 1.5 L/min). Transfer anesthetized 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.
Until anesthesia is confirmedNot 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
Position mouse for injection
Once the animal is under anesthesia, place it on its back. Wear surgical gown, gloves, and mask while performing injection procedure.
ImmediateNot specified
Note: Confirm anesthesia by checking the animal's lack of response to a pinch stimulus on the hind paws.
View evidence from paper
“Once the animal is under anesthesia, place it on its back. Wear surgical gown, gloves, and mask while performing injection procedure.”
5
Disinfect knee joint area
Trim and wipe the area surrounding the knee joint with alcohol. Povidone iodine or chlorhexidine can be used as alternative disinfectants.
Not specifiedNot specified
Note: The patellar tendon (white line below the patella) will become visible after disinfection.
View evidence from paper
“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.”
6
Stabilize knee joint
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.
During injectionNot specified
Note: This positioning 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.”
7
Locate precise injection site
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 identifies the correct anatomical location for intra-articular injection.
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.”
8
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 the MIA solution (10 µl at desired concentration).
Not specifiedNot specified
Note: Absence of resistance indicates correct intra-articular placement. Discard the needle immediately in the sharps bin after injection.
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.”
9
Massage knee joint
After injection, massage the knee to ensure even distribution of the solution.
Not specifiedNot specified
Note: This step ensures uniform distribution of MIA throughout the joint space.
View evidence from paper
“After injection, massage the knee to ensure even distribution of the solution.”
10
Recovery from anesthesia
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: 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.
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.”
11
Habituation to behavioral testing environment
Bring mice to the behavioral room and let unrestrained animals acclimatize in acrylic cubicles (8 cm x 5 cm x 10 cm) atop a wire mesh grid. Train mice by handling and 2 hr habituation to the cubicles for two days prior to von Frey hair application in order to limit stress and ambulation during application of von Frey hairs.
2 days prior to testing, 2 hr per dayNot specified
Note: On test days, habituate animals to the cubicles for up to 60 min prior to testing. Wear gowns, gloves, and masks during all behavioral experiments.
View evidence from paper
“Train mice by handling and 2 hr habituation to the cubicles for two days prior to von Frey hair application in order to limit stress and ambulation during application of von Frey hairs. On test days, habituate animals to the cubicles for up to 60 min prior to testing.”
12
Baseline mechanical threshold assessment
Assess mechanical thresholds of both hind paws before MIA injection as baseline values using the up-down method with von Frey hairs.
Not specifiedNot specified
Note: This establishes baseline withdrawal thresholds for comparison with post-injection measurements.
View evidence from paper
“Following the procedure described above (2.2.1-2.2.4), assess mechanical thresholds of both hind paws before MIA injection as baseline values.”
13
Apply von Frey hairs - up-down method
Starting with a stimulus strength of 0.07 g, apply von Frey hairs according to the up-down method: mark as X a withdrawal response and O an absence of response. Apply in ascending order of force, up to 1 g (cut-off force), until a response is detected. Re-test the paw by repeating with the filament that exerts a force below the one that produced a withdrawal. Then, apply the remaining filaments sequentially, by descending force, until no withdrawal occurs. Re-apply filaments in ascending order until a response is observed. Continue until a sequence of six responses is obtained (e.g., OXOXOX).
Not specifiedNot specified
Note: Hold each hair in place for 3 sec or until the paw is withdrawn, the latter defining a positive response. Use 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g fibers during testing.
View evidence from paper
“Apply calibrated von Frey hairs to the plantar surface of the hind paw until the fiber bends. Hold each hair in place for 3 sec or until the paw is withdrawn, the latter defining a positive response. Starting with a stimulus strength of 0.07 g, apply hairs according to the up-down method: mark as X a withdrawal response and O an absence of response.”
14
Calculate 50% paw withdrawal threshold
Obtain the 'k' value by referring to tabular values. Express paw withdrawal values as 50% paw withdrawal thresholds in grams using the formula (10 [Xr + K δ])/10,000 where Xr = value of last von Frey filament used in the sequence (in log units), k = tabular value, and δ = mean difference in forces between fibers. Where no response is detected, use the maximal response of 1 g.
Not specifiedNot specified
Note: This calculation converts the up-down method response pattern into a quantitative threshold value.
View evidence from paper
“obtain the 'k' value by referring to tabular values. Express paw withdrawal values as 50% paw withdrawal thresholds in grams. Use the formula (10 [Xr + K δ])/10,000 where Xr = value of last von Frey filament used in the sequence (in log units), k = tabular value, and δ = mean difference in forces between fibers.”
15
Post-injection mechanical threshold assessment
After injection, assess thresholds of the ipsilateral and contralateral paws at regular day intervals for several weeks after MIA to ascertain the development of mechanical allodynia. For example, assess at 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection.
Multiple time points over 28 daysNot specified
Note: Animals are considered allodynic when they display a response to 0.1 g or less. Normal responses fall within 0.6-1 g range.
View evidence from paper
“After injection, assess thresholds of the ipsilateral and contralateral paws at regular day intervals for several weeks after MIA to ascertain the development of mechanical allodynia. For example, we report thresholds measured 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection.”
16
Train mice 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.
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.”
17
Calibrate weight incapacitance tester
Calibrate the weight incapacitance tester before use with a 100 g check weight or according to equipment instruction.
Before each testing sessionNot specified
Note: Proper calibration is essential for accurate weight bearing measurements.
View evidence from paper
“Calibrate the instrument before use with a 100 g check weight (or according to equipment instruction).”
18
Baseline weight bearing assessment
Assess weight bearing changes before MIA injection as baseline values. Make sure that each hind paw is placed on the appropriate recording pad. 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.
1 sec per measurement, 3 measurements per sessionNot specified
Note: Express values as the difference between contralateral and ipsilateral paws in grams.
View evidence from paper
“Make sure that each hind paw is placed on the appropriate recording pad. 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.”
19
Post-injection weight bearing assessment
Repeat weight bearing assessments at regular intervals over several weeks to ascertain the development of gait changes. For example, assess at 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection. Express values as the difference between contralateral and ipsilateral paws in grams.
Multiple time points over 28 daysNot specified
Note: A normal weight bearing value of 50% represents an 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.”
20
Combined behavioral measurements
Measurements of mechanical thresholds and weight bearing deficits can be performed in the same mice, as neither end point affects the other.
Not specifiedNot specified
Note: This allows for comprehensive assessment of pain-like behavior in individual animals.
View evidence from paper
“Measurements of mechanical thresholds and weight bearing deficits can be performed in the same mice, as neither end point affects the other.”
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.