Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis methods
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Methods
Adult male Wistar rats (230 ± 30 g, Charles River Laboratories, Barcelona, Spain) were housed in groups of three, with water and food ad libitum, at a constant temperature of 22°C and controlled lighting (12 h light/12 h dark cycle). A total of 99 animals were used in this study: 72 for behavioural testing and 27 for the pharmacological evaluation, 5 per drug (15 in total) and 4 per control (12 in total).
Osteoarthritis induction
Under brief isoflurane anaesthesia (5% isoflurane for anaesthesia induction, 2% for maintenance), an intra-articular injection was performed with the use of a Hamilton syringe, with a 26 G needle inserted through the patellar ligament into the joint space of the left knee. Animals received two injections, one on day 0 and another on day 3, with 25 µl of either sterile saline (control group), 250 U or 500 U of type II collagenase from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) dissolved in saline and filtered through a 0.22 µm membrane [ ]. Animals were randomly assigned to each group before the first injection. Doses of collagenase were chosen based on a previous study that assessed nociceptive alterations in rats [ ] injected with 500 U of collagenase. We tested the same dose and a smaller one, to evaluate if similar changes could be obtained...
Tissue processing
At one, two, four and six weeks after the first injection of saline, 250 U or 500 U of collagenase, animals were perfused with 4% paraformaldehyde (n = 6/group/treatment). Their ipsilateral DRG from lumbar segments L3, L4 and L5 were dissected, post-fixed in the same fixative (four hours) and kept in 30% sucrose with 0.01% sodium azide. DRG were serially sliced in 12 µm sections using a cryostat, with every 10 th section collected in the same slide. DRG were always cut longitudinally yielding 8 to 10 sections from each, on average. The injected knees were also dissected, post-fixed for 72 hours and then decalcified for 8 hours in a buffer containing 7% AlCl 3, 5% HCOOH and 8.5% HCl. The joints were then washed in 0.1 M phosphate buffer pH 7.2, and kept in 30% sucrose with 0.01% sodium azide until they were cut. Joints were cut into two approximately eq...
Immunofluorescence for TRPV1
Slides containing every 10 th section of ipsilateral L3, L4 or L5 DRG of animals sacrificed one or six weeks after collagenase or saline injection were used for immunofluorescence reactions against TRPV1. DRG sections were rinsed in 0.1 M phosphate-buffered saline (PBS; pH 7.4), followed by PBS + 0.3% triton X (PBST), and incubated in PBST with 10% normal serum (1 h 30). Sections were then incubated with guinea-pig anti-TRPV1 diluted in PBST + 2% normal serum (1:250, Chemicon, Temecula, CA, USA), overnight at room temperature. After thorough PBST washing, sections were incubated with Alexa Fluor 568 donkey anti-guinea-pig secondary antibody diluted in PBST + 2% normal serum (1:2,000, one hour, room temperature, Molecular Probes, Eugene, OR, USA). Slides were then rinsed in PBST followed by PBS, mounted with Prolong Gold Antifade mount...
Pharmacological experiments
A pharmacological evaluation was performed in different groups of animals at one, two, four and six weeks after 500 U collagenase injection using the opiate morphine, the local anaesthetic lidocaine and the NSAID diclofenac. Baseline values of nociception were determined before drug administration (t = 0 minutes). To confirm that the behavioural responses of OA animals in the CatWalk and Knee-Bend tests were induced by nociception, we evaluated if they could be reversed by a single administration of a non-sedative dose of morphine [ ]. Morphine (6 mg/mL in saline, Labesfal, Lisboa, Portugal) was subcutaneously (s.c.) injected in the upper dorsum in a dose of 6 mg/Kg [, ] (n = 5), and drug effects on both tests were assessed 30, 60, 90, 120 and 180 minutes after administration. Control animals were also injected with 500 U of collagenase and received...
Nociceptive behaviour
The Knee-Bend (Figure A-C) and CatWalk (Figure D-E) tests were used to evaluate loading- and movement-induced nociception following the i.a. injection of different doses of collagenase. In both tests and at all time-points, saline-injected control animals showed behavioural responses similar to those observed prior to the injection (day 0; 3.3 ± 0.3 for the ipsilateral Knee-Bend score; 51.0 ± 0.7% for the CatWalk). In collagenase-injected animals, behavioural changes were time-dependent, with the highest increase in nociception being observed one week after the first collagenase injection, and remaining higher than on day 0 or in control animals throughout the whole period of analysis for both collagenase doses (Figure ). For the 250 U, ipsilateral Knee-Bend scores (Figure B) increased from 3.3 ± 0.4 on day 0 t...
Nociceptive behaviour
Nociceptive behaviour. Nociception induced by movement and loading in control and collagenase-injected rats evaluated by the Knee-Bend and CatWalk tests. Knee-Bend score (A-C) is presented for both ipsilateral and contralateral knees of saline-injected control rats (A), and rats injected with 250 U (B) or 500 U (C) of collagenase. CatWalk data (D, E) are expressed as the percentage of total ipsilateral paw print intensity (%TIPPI). Collagenase-injected animals present increased movement- and loading-induced nociception. The number of animals per group at each time-point is indicated below the graphs. An initial number of 24 animals was used in each group. Animals were then sacrificed at different time-points for histopathology (one, two, four and six weeks; n = 6 per dose and per time-point). Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test...
Pharmacological evaluation
The effect of an acute administration of morphine, lidocaine and diclofenac on loading- and movement-induced nociception was evaluated at one, two, four and six weeks after injection of 500 U of collagenase (Figure ), since this was the dose that induced nociceptive changes that better correlated with relevant histopathological alterations. All animals showed increased nociceptive behaviour before drug or vehicle administration (t = 0 minutes, baseline). The effect of a s.c. injection of saline, a p.o. administration of bidistilled water or an intra-articular administration of saline was evaluated in animals injected with 500 U collagenase to monitor the vehicle's effects. We obtained similar results to those already published by our group for the monoiodoacetate (MIA) model of OA [ ], with no changes being observed (data not shown).
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Gait recovery and locomotor function
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Evidence quotes (8)
Adult male Wistar rats (230 ± 30 g, Charles River Laboratories, Barcelona, Spain) were housed in groups of three, with water and food ad libitum, at a constant temperature of 22°C and controlled lighting (12 h light/12 h dark cycle). A total of 99 animals were used in this study: 72 for behavioural testing and 27 for the pharmacological evaluation, 5 per drug (15 in total) and 4 per control (12 in total).
Under brief isoflurane anaesthesia (5% isoflurane for anaesthesia induction, 2% for maintenance), an intra-articular injection was performed with the use of a Hamilton syringe, with a 26 G needle inserted through the patellar ligament into the joint space of the left knee. Animals received two injections, one on day 0 and another on day 3, with 25 µl of either sterile saline (control group), 250 U or 500 U of type II collagenase from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) dissolved in saline and filtered through a 0.22 µm membrane [ ]. Animals were randomly assigned to each group before the first injection. Doses of collagenase were chosen based on a previous study that assessed nociceptive alterations in rats [ ] injected with 500 U of collagenase. We tested the same dose and a smaller one, to evaluate if similar changes could be obtained with a smaller amount of exogenous collagenase.
At one, two, four and six weeks after the first injection of saline, 250 U or 500 U of collagenase, animals were perfused with 4% paraformaldehyde (n = 6/group/treatment). Their ipsilateral DRG from lumbar segments L3, L4 and L5 were dissected, post-fixed in the same fixative (four hours) and kept in 30% sucrose with 0.01% sodium azide. DRG were serially sliced in 12 µm sections using a cryostat, with every 10 th section collected in the same slide. DRG were always cut longitudinally yielding 8 to 10 sections from each, on average. The injected knees were also dissected, post-fixed for 72 hours and then decalcified for 8 hours in a buffer containing 7% AlCl 3, 5% HCOOH and 8.5% HCl. The joints were then washed in 0.1 M phosphate buffer pH 7.2, and kept in 30% sucrose with 0.01% sodium azide until they were cut. Joints were cut into two approximately equal halves along the medial collateral ligament in the frontal plane. Three 10 µm frozen sections were cut from each half at 200 µm steps using a cryostat.
Slides containing every 10 th section of ipsilateral L3, L4 or L5 DRG of animals sacrificed one or six weeks after collagenase or saline injection were used for immunofluorescence reactions against TRPV1. DRG sections were rinsed in 0.1 M phosphate-buffered saline (PBS; pH 7.4), followed by PBS + 0.3% triton X (PBST), and incubated in PBST with 10% normal serum (1 h 30). Sections were then incubated with guinea-pig anti-TRPV1 diluted in PBST + 2% normal serum (1:250, Chemicon, Temecula, CA, USA), overnight at room temperature. After thorough PBST washing, sections were incubated with Alexa Fluor 568 donkey anti-guinea-pig secondary antibody diluted in PBST + 2% normal serum (1:2,000, one hour, room temperature, Molecular Probes, Eugene, OR, USA). Slides were then rinsed in PBST followed by PBS, mounted with Prolong Gold Antifade mounting medium (Molecular Probes) and cover-slipped. Negative controls were performed by following the same procedure without the primary antibody. DRG sections were viewed using a Zeiss Imager.Z1 fluorescence microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and images were acquired using an Axi...
A pharmacological evaluation was performed in different groups of animals at one, two, four and six weeks after 500 U collagenase injection using the opiate morphine, the local anaesthetic lidocaine and the NSAID diclofenac. Baseline values of nociception were determined before drug administration (t = 0 minutes). To confirm that the behavioural responses of OA animals in the CatWalk and Knee-Bend tests were induced by nociception, we evaluated if they could be reversed by a single administration of a non-sedative dose of morphine [ ]. Morphine (6 mg/mL in saline, Labesfal, Lisboa, Portugal) was subcutaneously (s.c.) injected in the upper dorsum in a dose of 6 mg/Kg [, ] (n = 5), and drug effects on both tests were assessed 30, 60, 90, 120 and 180 minutes after administration. Control animals were also injected with 500 U of collagenase and received a s.c. injection of saline (n = 4). Behavioural assessment was performed after 30, 60 and 120 minutes. To determine if nociception in this model originates from knee articular tissues, we intra-articularly injected the local anaesthetic lidocaine in the knee of rats injected with 500 U o...
The Knee-Bend (Figure A-C) and CatWalk (Figure D-E) tests were used to evaluate loading- and movement-induced nociception following the i.a. injection of different doses of collagenase. In both tests and at all time-points, saline-injected control animals showed behavioural responses similar to those observed prior to the injection (day 0; 3.3 ± 0.3 for the ipsilateral Knee-Bend score; 51.0 ± 0.7% for the CatWalk). In collagenase-injected animals, behavioural changes were time-dependent, with the highest increase in nociception being observed one week after the first collagenase injection, and remaining higher than on day 0 or in control animals throughout the whole period of analysis for both collagenase doses (Figure ). For the 250 U, ipsilateral Knee-Bend scores (Figure B) increased from 3.3 ± 0.4 on day 0 to 13.1 ± 0.9 on week 1 ( P <0.001), slightly decreasing throughout time, but remaining significantly different from the contralateral knee and control animals until the sixth week (10.2 ± 1.7, P <0.001). For the 500 U dose, ipsilateral Knee-Bend scores (Figure...
Nociceptive behaviour. Nociception induced by movement and loading in control and collagenase-injected rats evaluated by the Knee-Bend and CatWalk tests. Knee-Bend score (A-C) is presented for both ipsilateral and contralateral knees of saline-injected control rats (A), and rats injected with 250 U (B) or 500 U (C) of collagenase. CatWalk data (D, E) are expressed as the percentage of total ipsilateral paw print intensity (%TIPPI). Collagenase-injected animals present increased movement- and loading-induced nociception. The number of animals per group at each time-point is indicated below the graphs. An initial number of 24 animals was used in each group. Animals were then sacrificed at different time-points for histopathology (one, two, four and six weeks; n = 6 per dose and per time-point). Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test for comparisons between groups at each time-point. # P <0.05, ### P <0.001, for comparisons between control and the 250 U collagenase group; * P <0.05, *** P <0.001, for comparisons between control and the 500 U collagenase group.
The effect of an acute administration of morphine, lidocaine and diclofenac on loading- and movement-induced nociception was evaluated at one, two, four and six weeks after injection of 500 U of collagenase (Figure ), since this was the dose that induced nociceptive changes that better correlated with relevant histopathological alterations. All animals showed increased nociceptive behaviour before drug or vehicle administration (t = 0 minutes, baseline). The effect of a s.c. injection of saline, a p.o. administration of bidistilled water or an intra-articular administration of saline was evaluated in animals injected with 500 U collagenase to monitor the vehicle's effects. We obtained similar results to those already published by our group for the monoiodoacetate (MIA) model of OA [ ], with no changes being observed (data not shown).
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