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
18 items3 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment9
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Materials8
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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. Prepare sterile saline for injections in a separate group of control mice.
Day of injectionNot specified
Note: The highest recommended dose of MIA is 1 mg in 10 µl. Monoiodoacetate is very toxic - wear gloves and mask when handling powder and preparing solution. The 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
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 while under anesthesia.
Until anesthesia is confirmedNot specified
Note: Wear surgical gown, gloves, and mask while performing injection procedure. 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 and 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 well for disinfection. The patellar tendon (white line below the patella) will become visible.
Not specifiedNot specified
Note: Joint preference is not required.
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.”
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.
During injectionNot specified
Note: This stabilization is critical for accurate injection placement.
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 precise 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: The patellar tendon (white line below the patella) serves as a landmark.
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. After injection, massage the knee to ensure even distribution of the solution.
Not specifiedNot specified
Note: Discard the needle immediately in the sharps bin. 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
“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. After injection, massage the knee to ensure even distribution of the solution.”
8
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 is achievedHeated mat provided
Note: Continuous monitoring is required during recovery.
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.”
9
Pre-test habituation to behavioral apparatus
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 with 2 hr habituation per dayNot specified
Note: This training is essential to reduce stress and ensure consistent behavioral responses.
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.”
10
Baseline mechanical threshold assessment
On test days, habituate animals to the cubicles for up to 60 min prior to testing. Assess mechanical thresholds of both hind paws before MIA injection as baseline values using the von Frey hair testing procedure.
Up to 60 min habituation plus testing timeNot specified
Note: Wear gowns, gloves, and masks during all behavioral experiments. Use the same experimenter throughout to reduce stress.
View evidence from paper
“On test days, habituate animals to the cubicles for up to 60 min prior to testing. Following the procedure described above (2.2.1-2.2.4), assess mechanical thresholds of both hind paws before MIA injection as baseline values.”
11
Von Frey hair application - initial stimulus
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.
3 sec per hair or until withdrawalNot specified
Note: Use 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g fibers during testing. Start with 0.07 g filament.
View evidence from paper
“Apply calibrated von Frey hairs (flexible nylon fibers of increasing diameter that exert defined levels of force as calibrated by the manufacturing company and expressed as grams (g)) 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”
12
Von Frey hair application - ascending sequence
Apply in ascending order of force, up to 1 g (cut-off force), until a response is detected.
Not specifiedNot specified
Note: This is the first phase of the up-down method.
View evidence from paper
“Apply in ascending order of force, up to 1 g (cut-off force), until a response is detected.”
13
Von Frey hair application - descending sequence
Re-test the paw by repeating the procedure, starting 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.
Not specifiedNot specified
Note: This is the second phase of the up-down method.
View evidence from paper
“Re-test the paw by repeating step 2.2.1, starting 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.”
14
Von Frey hair application - final ascending sequence
Re-apply filaments in ascending order until a response is observed. Continue until a sequence of six responses is obtained (e.g., OXOXOX), in order to obtain the 'k' value by referring to tabular values.
Not specifiedNot specified
Note: The sequence of six responses (alternating X and O) is required to calculate the 50% withdrawal threshold.
View evidence from paper
“Re-apply filaments in ascending order until a response is observed. Continue until a sequence of six responses is obtained (e.g., OXOXOX), in order to obtain the 'k' value by referring to tabular values”
15
Calculate 50% paw withdrawal threshold
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 provides the mechanical withdrawal threshold value.
View evidence from paper
“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.”
16
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 thresholds 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.”
17
Weight bearing apparatus training
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. Allow the animals to move freely until they adopt a sitting posture. This training takes at least two days”
18
Calibrate weight bearing apparatus
Calibrate the weight incapacitance tester instrument 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 measurements.
View evidence from paper
“Calibrate the instrument before use with a 100 g check weight (or according to equipment instruction).”
19
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
“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.”
20
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.
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.”
Subjects / Specimens
- Species
- mouse
- Strain
- Not specified
- Age
- 8-10 week-old
- Sex
- unknown
- Count
- 8
- Weight
- Not specified
Housed in groups of 5 under 12 hr light/dark cycle with food and water ad libitum. Acclimatized for 1 week prior to experiment. Randomized and caged by body weight.