Analgesic properties of intrathecal glucocorticoids in three well established preclinical pain models
-
Mienke Rijsdijk
,
Camilla I Svensson
Abstract
Background and aim
Glucocorticoids, a group of anti-inflammatory agents, are frequently administered in pain medicine. Of interest is the reported activity after intrathecal delivery in patients with neuropathic pain syndromes such as postherpetic neuralgia, though its efficacy is controversial. After the publication of two randomized clinical trials in postherpetic neuralgia patients treated with similar intrathecal methylprednisolone acetate (MPA) dosing regimes with conflicting results; one showing significant pain reduction (Kotani N, Kushikata T, Hashimoto H, Kimura F, Muraoka M, Yodono M, Asai M, Matsuki A: Intrathecal methylprednisolone for intractable postherpetic neuralgia. N Engl J Med 2000;23: 1514–9), the other increased pain sensations (Rijsdijk M, van Wijck AJ, Meulenhoff PC, Kavelaars A, van der Tweel I, Kalkman CJ: No beneficial effect of intrathecal methylprednisolone acetate in postherpetic neuralgia patients. Eur J Pain 2013;38:175–200), we decided additional research was warranted. Present study sought to determine effects of intrathecally delivered methylprednisolone on pain-like behaviour and pain-associated markers in three well established rodent pain models: (1) intraplantar carrageenan, (2) intraplantar formalin, and (3) ligation of L5/L6 spinal nerves (SNL model).
Methods
Male rats with intrathecal catheters were examined for (1) tactile allodynia after unilateral hindpaw intraplantar carrageenan injection (2%), (2) flinching and subsequent long term tactile allodynia after unilateral hindpaw intraplantar formalin injection (2.5%) or (3) tactile allodynia after unilateral ligation of the L5 and L6 spinal nerves. Rats were treated with the maximum tolerable intrathecal dose of the soluble methylprednisolone sodium succinate (MP) or the particulate methylprednisolone acetate (MPA). Dorsal root ganglia and spinal cords were harvested for immunohistochemistry to assess markers of neuronal damage (ATF3) and glial activation (GFAP, Iba1).
Results
During dose finding, severe generalized allodynia was observed with high intrathecal doses of both MPA and MP in naive rats. MPA had no effect upon tactile allodynia after carrageenan. MP and MPA did not reverse tactile allodynia in the SNL model, and did not reduce flinching in the formalin model. MP and MPA prevented the delayed (7–day) tactile allodynia otherwise observed in the formalin-injected paw. Systemic MP or perineural MP or MPA did not reduce pain-like behaviour in the SNL model. No reduction of neuronal injury (ATF3) in the dorsal root ganglion or astrocyte activation (GFAP) in the spinal dorsal horn with intrathecal MP or MPA was observed. There was a decrease in microglial activation (Iba1) in the spinal dorsal horn with MPA after SNL.
Conclusion
Severe generalized allodynia was observed after high intrathecal doses of MP and MPA in naive rats. No acute analgesic effects with intrathecal glucocorticoids were observed in three well established pain models. Only a late antiallodynic effect was present in the formalin model, 7 days after formalin injection and drug treatment.
Implications
Our results do not support use of intrathecal methylprednisolone in the treatment of pain.
1 Introduction
Glucocorticoids, a group of anti-inflammatory agents, are frequently administered in pain medicine. Of interest is the reported activity after neuraxial (epidural and intrathecal) delivery in patients with low back pain and in patients with neuropathic pain syndromes such as postherpetic neuralgia and complex regional pain syndrome [1,2,3,4], though their efficacy is controversial [5,6,7,8]. This controversy is surprising, given that glucocorticoids act upon a variety of crucial biological links in the neuraxial pathways after inflammation and nerve injury leading to hyper-pathic states [9]. Glucocorticoids can act through transrepression of pro-inflammatory genes (interacting with activator protein-1 and nuclear factor kappa B (NFκB)), transactivation of anti-inflammatory genes (lipocortin I, p11/calpactin-binding protein) and nongenomic effects (interacting with G-protein coupled receptors, mitogen-activated protein (MAP) kinases, phosholipases and protein kinases (SRC)), to down regulate inflammatory processes [10], leading to a reduced production and secretion of inflammatory products such as cyclooxygenase-2, interleukin (IL)-1β, IL-2, IL-6, IL-8, tumour necrosis factor (TNF), interferon-gamma, and inducible nitric oxide synthase [11,12]. These effector products are considered to be important components in neuropathic pain signalling. If these neuroinflammatory products are indeed reduced, it is not clear why the analgesic effects of glucocorticoids are varying and often disappointing.
Our research team encountered disappointing analgesic effects with glucocorticoids in a randomized controlled clinical trial conducted in patients suffering from postherpetic neuralgia [4]. Four intrathecal injections with methylprednisolone acetate (MPA) with 7 day intervals were administered in patients with intractable neuropathic pain. Patients treated with intrathecal MPA reported increased pain and with statistical evidence of futility, the trial was ended early. Our results were in sharp contrast with results of an earlier trial with a similar drug and dosing regime, showing pain reduction in 92% of patients in the intrathecal MPA treated group [13]. Since we did not understand the differences in results between the two trials, we decided to conduct a preclinical study using a similar MPA formulation. Additional to (i) the MPA formulation, a suspension with depot characteristics and reduced preservative concentrations, we also studied (ii) methylprednisolone sodium succinate (MP), a solution without preservatives, both frequently used in pain medicine.
We argued that since the aetiology of postherpetic neuralgia is not clear and we were interested to see if intrathecally delivered MPA had any effect on pain like behaviour and surrogate markers in a severe pain state, we should study the efficacy of glucocorticoids in multiple models inducing pain like behaviour with; (i) an inflammatory, (ii) a neurotoxic, and (iii) a direct nerve injury stimulus.
Three well established preclinical pain models in rats were selected: (i) intraplantar carrageenan leading to a robust inflammation and tactile allodynia of the injected paw [14]; (ii) intraplantar formalin leading to a biphasic flinching behaviour acutely after injection and an evolving tactile allodynia that develops over the ensuing 7 days [15,16]; (iii) unilateral ligation of the L5/6 spinal nerves (SNL model) yielding a unilateral mononeuropathy characterized by a robust tactile allodynia [17]. We further examined the effects of glucocorticoids on the expression of well characterized markers including activation transcription factor 3 (ATF3) in the dorsal root ganglion (DRG) and induced astrocyte (glial fibrillary acidic protein (GFAP)) and microglia activation (ionized calcium-binding adapter molecule 1 (Iba1)) in the spinal dorsal horn. ATF3 is upregulated in injured DRG neurons after peripheral nerve injury and has a survival function driving neurite outgrowth [18]. Glial cells are activated by cytokines such as IL-1β, IL-6, TNFα and monocyte chemoattractant protein (MCP)-1 produced through MAP kinases (p38 and SRC-family kinases) phosphorylation and NFkB activation. We hypothesized that intrathecal glucocorticoids would reverse the hyperpathic states and the indices of DRG and dorsal horn neuroinflammation.
2 Methods
The protocol of the present study has been approved by the AAALAC accredited (International, Association for Assessment and Accreditation of Laboratory Animal Care) Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, USA.
2.1 Animals
Male Harlan Sprague-Dawley rats (200–225 g for the carrageenan and 80–100 g for the SNL model) and male Holtzman rats (200–225 g for the formalin model) (Indianapolis, IN, USA) were maintained 2 per cage in standard cages at room temperature on a 12:12 h light/dark cycle with free access to food and water. After arrival at the housing facility, they were allowed at least 2–3 days of acclimation before use.
2.2 Drug administration
In all three pain models rats received intrathecal drug treatment. Only in the SNL model, additional groups of rats received intraperitoneal or perineural drug treatment;
For intrathecal drug injections, rats were surgically implanted with intrathecal catheters as described previously under general anaesthesia (inhalation of isoflurane 2.4% in a room air/oxygen mixture) [19]. Intrathecal catheters were externalized for injection. Rats were given post-operative subcutaneous fluids including analgesics (lactated Ringers + 5 mg/kg Carpro-fen) and then housed individually for post-operative recovery. Following implantation, catheters were flushed with saline and rats were monitored daily for viability, allowing at least 5 days of recovery before testing. Animals showing any evidence of motor dysfunction or distress after catheter placement were immediately euthanized in a carbon dioxide chamber.
Intraperitoneal drug injections were performed in awake rats. The injection was given in the lower left abdominal quadrant. Injection of the drug was preceded by careful aspiration to determine correct placement of the needle tip.
Perineural drug administration was performed under general anaesthesia with inhalation of isoflurane 2.4% in a room air/oxygen mixture during the nerve ligation procedure (described below). Directly after nerve ligation, a 32 gauge blunt needle was positioned in parallel to the spinal nerve, proximal to the ligation, aiming towards the foramen and cautiously blindly advanced until a resistance was felt. There the drug was administered. This was performed directly after the L5 and L6 spinal nerve ligation.
2.3 Drug preparation and dosing
The drugs used in the present study, are methylprednisolone acetate, MPA (Depo-medrol® from Pfizer) a slow release formulation, and the solution methylprednisolone sodium succinate (MP). The following preparations were employed:
MPA contains the preservatives polyethylene glycol and myristyl-gamma-picoliniumchloride, which are potentially neurotoxic. To remove these preservatives 1 ml of the general formulation of MPA 40 mg/ml (Pfizer) was centrifuged in a mini centrifuge (Fisher Scientific, Cat no 05-090-128,14.000 rpm) for 10 min. The supernatant was aspirated with a 20 G needle and syringe and discarded. The residual pellet of 40 mg MPA was resuspended in 1 ml saline to give a stock solution of 20 mg/ml.
MP, a powder, was dissolved in saline to a concentration of 40 mg/ml. Since this solution has a limited stability, this was done less than 10 min before administration.
Based on preliminary studies, the highest tolerable dose of either drug was administered in the present study. The highest tolerable intrathecal dose of MPA was 400 μg. With higher doses (800 μg) severe generalized allodynia developed, causing the animals to vocalize and adapt aggressive guarding behaviour when stroking their fur. The 400 μg MPA dose easily passed through the intrathecal catheters. Initially a comparable dose of MP was chosen, 400 μg. The animals did not tolerate this dose and showed a similar generalized allodynia as was seen with the 800 μg MPA dose. The highest tolerable dose of MP was found to be 40 μg. Since the highest tolerable intrathecal dose of MP was 40 μg, we decided to give a similar perineural dose per treated spinal nerve root, so a total of 80 μg. The perineural MPA dose was adjusted to a total dose of 80 μg.
2.4 Drug pharmacokinetics
To be able to time the clearance of MPA during the initial phase after the pain stimulus, we started our experiments with a pharmacokinetics study measuring methylprednisolone plasma levels. Rats were given a 400 μg intrathecal MPA dose and 1, 3, 6, 24, 48, 72 and 144h after administration, a cardiac blood sample was obtained under general inhalational anaesthesia with isoflurane 2.4% in a room air/oxygen mixture. Also four blood samples from rats with intrathecal catheters were included in which no drug treatment was administered. All rats, still under general anaesthesia, were immediately euthanized in a carbon dioxide chamber after the blood sample was drawn. Blood samples were centrifuged at low g-force for 5 min and plasma was aspirated from the sample and stored in a —80°C freezer. The methylprednisolone concentrations in blood plasma were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Neogen Corporation, Lexington, KY, USA).
2.5 Animal models
Rats were randomly assigned to one of three pain models: (1) carrageenan model to study inflammatory induced pain, (2) formalin model to study chemically induced pain and (3) spinal nerve ligation model (SNL model) to study neuropathic pain after direct nerve injury.
2.5.1 Carrageenan model
After 1 h drug pretreatment with either intrathecal saline or intrathecal MPA, the paw thickness of both hind paws were measured using callipers after which rats received a plantar injection with a 30 G needle in the left hind paw with 0.1 ml of 2% carrageenan under general inhalational anaesthesia with isoflurane 2.4% in a room air/oxygen mixture. Directly after the injection, rats were allowed to recover and tested for the development of thermal hyperalgesia and mechanical allodynia at 1, 2, 4 and 8 h. Eight hours after the carrageenan injection, paws thickness was measured again under anaesthesia and rats were sacrificed in a carbon dioxide chamber.
2.5.2 Formalin model
After 1 h pretreatment of intrathecal saline, intrathecal MP or MPA, measurement of the baseline paw thickness and acclimation to the model in individual Plexiglas chambers for at least 30 min, rats received an injection with a 30 G needle in the dorsal side of the left hind paw of 0.05 ml of 2.5% formalin. Formalin induced flinching behaviour was recorded during the hour after injection using an automated detection system [20]. Flinches were counted in 1-min intervals for 60 min. The data are expressed as total number of flinches observed during phase 1 (0–9 min) and phase 2 (10–60 min) [21,22]. Rats were allowed to recover for seven days after which their mechanical thresholds were measured and rats were sacrificed (procedure described below) for tissue collection for immunohistochemistry. Previous work has shown the development of a late phase tactile allodynia [15].
2.5.3 SNL model
Spinal nerve injury was induced by the procedure described by Kim and Chung [17]. Briefly, the left L5/L6 lumbar spinal nerves were exposed in isoflurane/oxygen-anesthetized rats and tightly ligated with 6.0 silk suture at a point distal to their DRGs and proximal to their conjunction to form the sciatic nerve. An intrathecal catheter was implanted 13 days after SNL. Intrathecal injections (Saline/MP/MPA), starting 18 days after SNL, were given twice with a 3-day interval. Intraperitoneal injections (Saline/MP) were given on the first postoperative day after SNL for four consecutive days, and perineural injections (Saline/MP/MPA) were given during SNL (Fig. 1 ). Spinal cord and DRG tissue were collected for immunohistochemistry seven days after drug treatment in all rats.
Different dosing regiments in the SNL model. Intrathecal dosing: Thirteen days after SNL, an intrathecal (IT) catheter was implanted. Intrathecal drug treatment started on post-operative day 18 and was repeated on day 21. Tissue (spinal dorsal horn and DRG) was collected 7 days after the first drug dose. Perineural dosing: perineural injections were given during the SNL and tissue was collected 11 days later. Intraperitoneal dosing: Drugs were given daily for four consecutive days starting on the first postoperative day. Tissue was collected 11 days after SNL.
2.6 Behavioural measurements
All behavioural measurements were made by observer (MR) blinded to the treatment groups and were conducted at fixed times (9:00 a.m.–5:00 p.m.). To measure mechanical allodynia, thresholds were measured with a series of calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA), ranging from 3.16 to 5.18 (0.41–15.0 g). The animals were acclimated for 45 min in the test chamber on mesh flooring suspended above the observer, and von Frey filaments were applied perpendicularly against the plantar surface of the paw. The “up-down” method of Dixon as described by Chaplan et al. [23] was used to determine the value at which paw withdrawal occurred 50% of the time, interpreted as the mechanical threshold. Severe generalized allodynia was defined as vocalization within the first hour after intrathecal drug administration in combination with aggressive guarding behaviour when stroking the animal’s fur.
2.7 Immunohistochemistry
Animals were anesthetized with isoflurane 4.0% in a room air/oxygen mixture and transcardially perfused with saline 1 ml/gram bodyweight followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) 1 ml/gram bodyweight. Spinal cord and DRGs of L5 and L6 roots were removed, post-fixed overnight in the same fixative and moved to 30% sucrose for at least 72 h. Free floating transverse sections (30 μm) were taken from the spinal cord using a microtome. DRGs were embedded in Tissue-Tek® (O.C.T. Compound, Sakura® Finetek, PA, USA) frozen and cut (10μm) on a Leica CM1800 Cryostat (IMEB, CA, USA) and directly mounted on glass slides. Both free floating sections as mounted DRG tissue were permeabilized with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA), blocked with 5% goat serum in PBS and incubated with the following antibodies; a marker for neuronal damage, ATF3 (rabbit, 1:500, cat. no. sc188, Santa Cruz Biotechnology Inc., CA, USA), markers for astrocytes GFAP (mouse, 1:4000, cat. no. #MAB360, Chemicon, USA), microglia Iba1 (rabbit, 1:2000, cat. no. #019-19741, WAKO, VA, USA), and neurons NeuN (mouse, 1:1000, cat. no. MAB377, EMD Millipore Corporation, MA, USA) overnight at room temperature. Binding sites were visualized with secondary antibodies conjugated with fluoro-Alexa-594 (1:1000, cat. no. A11032, Invitrogen, NY, USA), fluoro-Alexa-488 (1:1000, cat. no. A11058, Invitrogen, NY, USA) or streptavidin conjugated fluoro-Alexa-488 (1:1000, cat no. S-32354, Life Technologies, CA, USA). A streptavidin/biotin blocking kit (Vector Labs, CA, USA) was utilized as appropriate before biotinylated ATF3. To confirm that the marker for neuronal damage, ATF3, was only observed in neurons, sections were double labelled with the NeuN antibody. Images were captured using a fluorescence microscope (Nikon TE300 fluorescence microscope (Nikon Corp, Tokyo, Japan)) and overlay performed with Adobe Photoshop Creative suite (CS6; Adobe Systems Incorporated). The investigators (MR and EdG) were blinded for the experimental conditions during quantification of ATF3, Iba1 and GFAP staining. Quantification of ATF3 in DRGs (by MR and EdG) was assessed by counting the number of cells with ATF3 positive nuclei over the total number of NeuN positive cells. L5 and L6 DRGs of the ipsi- and contralateral sides (4-6 sections per side) were group mounted for individual animals onto slides. All sections on a slide were separated by at least 60 μm. Three sections per DRG per side (ipsi-and contralateral) per animal were counted (total of 12 sections per animal). The quantification of Iba1 and GFAP in the spinalcord (MR) was performed by measuring the total integrated signal intensity of pixels in lamina I-II of the dorsal horn after subtraction of the background signal intensity in this area using ImageJ 1.47 software. The total signal intensity was measured in at least 6 sections of the L4–6 areas of the ipsi- and contralateral dorsal horn (total of 12 sections per animal). Per section, 3 background signal intensity measurements were performed in the lamina I-II area and pooled to a mean for subtraction. An increase in the signal intensity for Iba1 and GFAP staining was interpreted to signify microglia and astrocyte reactivity, respectively.
2.8 Data analysis
Data are presented as means with 95% confidence intervals (CI). Significance was ascribed for p < 0.05. Behavioural time-course data in the carrageenan and SNL model (tactile thresholds) was analyzed using two-way ANOVA with repeated measures across time. If statistical main effects were observed, the analysis was followed by Bonferroni post hoc tests (e.g., unpaired t-tests with Bonferroni corrections) at each time point unless otherwise stated. Differences in paw thickness between groups in the carrageenan model were analyzed with an unpaired two tailed t-test. For behavioural time course data in the formalin model (flinching behaviour) we calculated the area under the curve (AUC). Differences between group AUCs were calculated with an unpaired two tailed t-tests. Differences in tactile thresholds previous to formalin compared to 7 days after formalin within one group were analyzed with a paired two tailed t-test. Differences in signal intensity (immunohistochemistry; GFAP, Iba1) and ATF3 count between treatment groups were calculated using two-way ANOVA with repeated measures per side (ipsi versus contralateral). If statistical main effects were observed, the analysis was followed by Bonferroni post hoc tests for each side. All analyses were carried out using Graphpad Prism version 5.
3 Results
3.1 Pharmacokinetics of intrathecal MPA
The pharmacokinetics of intrathecal administered MPA, show that there is a rapid diffusion out of the intrathecal space into the blood plasma (Fig. 2). Peak plasma values are reached 1-3 h after intrathecal injection. Methylprednisolone plasma levels go below detection limits by 72 h after injection.
Pharmacokinetics after intrathecal MPA injection. Time in hours (h) on the x-axis. Methylprednisolone (MP) plasma levels in ng/ml on the j/-axis. The n indicates the number of rats that were sampled from at the corresponding time point.
3.2 High doses of intrathecal MP and MPA cause severe generalized allodynia in rats
While searching for the highest tolerable intrathecal dose of both MP (40 μg) and MPA (400 μg), we encountered severe generalized allodynia (MPA dose of 800 μg, MP of 400 μg, 200 μg) in rats, which lasted for 30 min up to an hour. Since both the MP solution and the MPA suspension had this allodynic effect, it is unlikely that particles, found in the MPA suspension, caused this phenomenon. The presence of preservatives was minimized in the MPA formulation [24] and absent in the MP formulation, and therefore not expected to play a role in the development of severe allodynia. Saline, MP and MPA were analyzed for their pH and osmolarity (Table 1 ). None of these variables are known to cause allodynia. To rule out an effect of the higher osmolarity values observed in the higher MP doses, we injected mannitol with a similar osmolarity (measured with a 5002 Osmette) in the intrathecal space. No adverse reaction was observed with equivalent mOsm mannitol doses, so we conclude that the severe generalized allodynia is caused by the glucocorticoid itself (Table 2).
MP and MPA drug characteristics; pH and mOsm.
| Drug | pH | mOsm |
|---|---|---|
| Saline | 6.0 | 272 |
| MP 4 μg (0.4 mg/ml) | 6.4 | 274 |
| MP 40 μg (4 mg/ml) | 7.2 | 287 |
| MP 400 μg (40 mg/ml) | 7.6 | 399 |
| MPA 400 μg (40 mg/ml) | 6.2 | 226 |
-
MP = methylprednisolone sodium succinate, MPA = methylprednisolone acetate
Mannitol doses with equivalent mOsm values to MP doses.
| Drug | N | mOsm | Behaviour |
|---|---|---|---|
| Saline | >50 | 272 | No adverse behaviour |
| Mannitol 5% | 2 | 276 | No adverse behaviour |
| Mannitol 7.2% | 2 | 397 | No adverse behaviour |
| MP 4 μg (0.4 mg/ml) | >50 | 274 | No adverse behaviour |
| MP 40 μg (4 mg/ml) | 5 | 287 | 2 out of 5 rats had severe generalized allodynia |
| MP 400 μg (40 mg/ml) | 2 | 399 | Both rats had severe generalized allodynia |
-
MP = methylprednisolone sodium succinate, N = number of rats.
3.3 There is no acute analgesic effect of intrathecal MPA in any of the three pain models and only a late moderate antiallodynic effect in the formalin model
Intraplantar carrageenan: In the inflammatory carrageenan model, rats displayed a significant inflammation of the injected paw (as measured by paw thickness) and tactile allodynia which developed over 2–3 h. Pretreatment with intrathecal MPA had no significant effect on tactile thresholds compared to saline controls during the 8-h follow up (two-way rm-ANOVA; time p≤ 0.0001, drug p = 0.50, interaction p = 0.21) (Fig. 3A). However the increase in paw thickness was significantly smaller in the intrathecal MPA pretreated animals Δ = 3.0mm versus saline controls Δ = 4.7mm (difference 1.7 mm; 95% CI 1.2–2.1; p < 0.0001) (Fig. 3B).
Effect of intrathecal MPA treatment on pain-like behaviour in the carrageenan, formalin and SNL model. (A) Carrageenan model: Tactile thresholds in grams (y-axis) over time in minutes. Tx indicates drug treatment 1 h before the injection of carrageenan (CAR) in the hind paw. Baseline thresholds have been assessed twice; once before drug treatment and once 45 min after drug treatment. (B) Increase in paw thickness in mm 8 h after carrageenan injection. (C) Formalin model: Mean number of flinches per minute (y-axis) averaged over 5 min (x-axis) after formalin injection (FOR). (D)The area under the curve (AUC) of phase 2 flinching (11-60 min after formalin injection). (E) Formalin model: Tactile thresholds in grams (y-axis) per drug treatment; saline (white), MPA (black). Saline/MPA pre represent the baseline thresholds before formalin injection after intrathecal saline/MPA injection. Saline/MPA 7-dy represent the thresholds 7 days after formalin injection and drug treatment. (F) SNL model: Tactile thresholds in grams (y-axis) overtime in hours (x-axis). Tx indicates drug treatment on day 0 (=18 days after nerve ligation)and day 3 (=21 days after nerve ligation). All graphs: White square = saline controls, Black diamond = MPA treatment, n = number of animals, data are plotted as mean, error bars are the standard deviation of the mean.
Intraplantar formalin: Intraplantar formalin injection resulted in a robust biphasic flinching of the injected hind paw. Pretreatment with intrathecal MPA had no effect upon phase 1 (0–10 min after injection; mean AUC saline = 205 vs AUC MPA =188; Δ = 17, 95% CI —132 to 166) or phase 2 flinching (11–60 min after injection; mean AUC saline = 854 vs AUC MPA = 599; Δ = 255; 95% CI –222 to 733) compared to saline controls (Fig. 3C and D). When measuring tactile thresholds 7 days after the formalin injection in saline controls, a significant decline in tactile thresholds was observed indicating development of tactile allodynia (previous to formalin injection; 14.5 g vs 7-day follow up 6.1 g; Δ = 8.4,95% CI 4.7–12.2, p = 0.001). In contrast, intrathecal MPA pretreated rats (pre; 12.6 gvs post; 9.4 g; Δ = 3.2, 95% CI –2.9 to 9.2, p = 0.24) did not develop such allodynia (Fig. 3E).
SNL mononeuropathy: Following nerve ligation, all rats developed a robust tactile allodynia that was maximum by postoperative day 7. Intrathecal MPA delivered on postoperative day 18 and 21 (corresponding with, respectively, 0 h and 72h in the graph), had no effect upon the tactile thresholds observed in the first hours or the following days after treatment compared to saline controls (Fig. 3F).
3.4 Intrathecal injection of the soluble MP did not decrease pain-like behaviour in the formalin or SNL model
Concurrently with the MPA experiments, we ran experiments with the soluble MP to determine if the drug characteristics had any influence on pain-like behaviour in the formalin and SNL model. With the maximum tolerable dose of soluble MP (40 μg), no difference in flinching behaviour during either phase 1 (0-10 min after injection; mean AUC saline = 205 vs AUC MP = 296; Δ = 91, 95% CI –15 to 197) or phase 2 (11-60 min after injection; mean AUC saline = 854 vs AUC MP = 881; Δ = 27; 95% CI –328 to 383) was observed as compared to saline controls (Fig. 4A and B). The development of mechanical allodynia 7-days after formalin injection was, like in the MPA pretreated rats, also attenuated in MP pretreated rats (pre; 12.8g vs post; 10.3g; Δ =2.5, 95% CI ,p = 0.39) (Fig. 4C). No decrease in pain-like behaviour was observed in the SNL model (Fig. 4D).
Effect of intrathecal MP treatment on pain-like behaviour in the formalin and SNL model. (A) Formalin model: Mean number of flinches per minute (y-axis) averaged over 5 min (x-axis) after formalin injection (FOR). (B) The area under the curve (AUC) of phase 2 flinching (11-60 min after formalin injection). (C) Formalin model: Tactile thresholds in grams (y-axis) per drug treatment; saline (white), MP (grey). Saline/MP pre represent the baseline thresholds before formalin injection after intrathecal saline/MPA injection. Saline/MP 7-dy represent the thresholds 7 days after formalin injection and drug treatment. (D) SNL model: Tactile thresholds in grams (y-axis) over time in hours (x-axis). Tx indicates drug treatment on day 0 (=18 days after nerve ligation) and day 3 (=21 days after nerve ligation). All graphs: White square = saline controls, Black triangles = MP treatment, n = number of animals, data are plotted as mean, error bars are the standard deviation of the mean.
3.5 Systemic administration ofMP or perineural administration ofMP or MPA did not reduce pain-like behaviour in the SNL model
We conducted additional experiments to examine the effects of systemic and perineural glucocorticoids. There was no analgesic effect of systemic MP (two-way rm-ANOVA; time p = 0.77, drug p = 0.73, interaction p = 0.08) or perineural MP or MPA (two-way rm-ANOVA; time p< 0.0001, drug p = 0.74, interaction p = 0.69) compared to saline controls in the SNL model (Fig. 5A and B).
Effect of (A) intraperitoneal (IP) MP treatment and (B) perineural (PN) MP and MPA treatment on pain-like behaviour in the SNL model. Tactile thresholds in grams (y-axis) overtime in days (x-axis). “SNL” indicates the moment where SNL was performed. Tx indicates drug treatment. White square = saline controls, black triangles = MP, black diamonds = MPA, black star=rats with SNL without drug treatment. Data are plotted as mean, error bars are the standard deviation of the mean.
3.6 There was no effect on DRGATF3 expression after intrathecal MP or MPA treatment in the formalin or SNL model
Intraplantar formalin: After intraplantar formalin injection, a few ATF3 containing neurons were observed in the ipsilateral DRG in all three groups (percentage of ATF3 stained neurons: intrathecal saline group 2.1%, MP 1.7%, and MPA 1.5%). No ATF3 containing neurons were observed in the contralateral DRG in all three groups. The incidence of ATF3 containing neurons was too small to make a statistically meaningful comparison between groups (Fig. 6).
Effect of intrathecal MP and MPA on neuronal injury (ATF3) in DRG in the formalin model. (A) Contralateral DRG of saline control rat. (B) Ipsilateral DRG of saline control rat. (C) Ipsilateral DRG of intrathecal MP treated rat. (D) Ipsilateral DRG of intrathecal MPA treated rat. (E) Percentage of neuronal nuclei containing ATF3 (y-axis) for saline (white), MP (grey) and MPA (black) treated animals in the formalin model. Data are plotted as mean, error bars are the standard deviation of the mean, **0.001 <p< 0.0001.
SNL mononeuropathy: After SNL the percentage of ATF3 (+) neuronal nucleï was significantly higher in the ipsilateral DRGs compared to the contralateral DRGs in all groups (saline ipsi mean 61% vs contra 0.04%, MP ipsi 58% vs contra 0.1%, MPA ipsi 62% vs contra 0%; two-way rm-ANOVA; side p = 0.0002, drug p = 0.97, interaction p = 0.96). In the contralateral DRG, ATF3 (+) neuronal nucleï were observed in saline and MP treated animals, but not in intrathecal MPA treated animals. There were no differences in ATF3(+) containing neuronal nucleï in the ipsilateral DRGs between groups (p = 0.97) (Fig. 7).
Effect of intrathecal MP and MPAon neuronal injury (ATF3) in DRG in the SNL model. (A) Contralateral DRG of saline control rat. (B) Ipsilateral DRG of saline control rat. (C) Ipsilateral DRG of intrathecal MP treated rat. (D) Ipsilateral DRG of intrathecal MPA treated rat. (E) Percentage of neuronal nuclei containing ATF3 (y-axis) for saline (white), MP (grey) and MPA (black) treated animals. Data are plotted as mean, error bars are the standard deviation of the mean, **0.001 <p< 0.0001.
3.7 Intrathecal MPA treatment reduced microglial activation in the SNL model, but there was no effect of intrathecal MP or MPA treatment on astrocyte activation
Intraplantar formalin: Seven days after intraplantar formalin injection, GFAP and Iba1 immuno reactivity in the spinal dorsal horn did not increase on the ipsilateral side as compared to the contralateral side in either saline treated animals or in glucocorti-coid treated animals.
SNL mononeuropathy: After SNL, GFAP protein levels in the spinal dorsal horn were significantly elevated on the ipsilateral side compared to the contralateral side as measured at 25 days after injury (saline mean ipsi 11.4 vs contra 9.0, MP ipsi 11.7 vs contra 10.2, MPA ipsi 12.1 vs contra 8.6; two-way rm-ANOVA; side p = 0.009, drug p = 0.88, interaction p = 0.47), but there were no differences between groups (Fig. 8). Iba1 protein levels were also significantly increased on the ipsilateral side (saline mean ipsi 3.8 vs contra 1.6, MP ipsi 1.8 vs contra 0.7, MPA ipsi 1.3 vs contra 0.6; two-way rm-ANOVA; side p = 0.02, drug p = 0.13, interaction p = 0.28) and Iba1 protein levels were significantly lower on the ipsilateral side after intrathecal MPA treatment compared to saline controls (Bonferroni p< 0.05) (Fig. 8).
Effect of intrathecal MP and MPA onglial cell activation (GFAP, Iba1) in the SNL and formalin model. (A) Represents the immuno reactivity of GFAP (red; astrocyte activation) in the spinal dorsal horn (SDH) of saline controls (left) and intrathecal MPA (right) treated animals in the SNL model. contra = contralateral side, ipsi = ipsilateral side. There is no difference between the contra and ipsilateral side and between the two treatment groups shown inC. (C)The quantitative signal intensity of GFAP immuno reactivity (y-axis) for saline (white), MP (grey) and MPA (black) treated animals. (B) Represents the immuno reactivity of Iba1 (green; microglial activation) in the SDH of saline controls (left) and intrathecal MPA (right) treated animals in the SNL model. There is an increase in signal intensity on the ipsilateral side and a decrease in signal intensity comparing MPA treated animals with saline controls. (D)The quantitative signal intensity of Iba1 immuno reactivity (y-axis) for saline (white), MP (grey) and MPA (black) treated animals. (Eand F) Signal intensity of GFAP (E) and Iba1 (F) in the formalin model for saline (white), MP(grey) and MPA (black) treated animals. No difference between the contra and ipsilateral side and between the two treatment groups are observed. Data are plotted as mean, error bars are the standard deviation of the mean.
4 Discussion
4.1 Overview of results
We observed severe generalized allodynia when high intrathecal doses of either MP or MPA were injected. At behaviorally tolerated doses, we did not observe analgesic or antihyperalgesic effects in any of the three models; carrageenan, formalin or SNL There was a reduction in allodynia 7-days after formalin injection in intrathecal MP and MPA treated rats. No reduction was noted in indices of neuronal injury in DRG or astrocyte activation in the spinal dorsal horn with intrathecal MP or MPA. There was a decrease in microglial activation in spinal dorsal horn in intrathecal MPA treated rats in the SNL model. The importance of these observations will be considered below.
4.2 Generalized allodynia
The severe generalized allodynia that was observed in the present study after intrathecal injections of glucocorticoids has been described only once before, after intrathecal bolus injections of doses of 400 μg MP in rats [25]. In addition, increased pain sensitivity in the paws has been reported after continuous intrathecal infusion of 0.02 mg/kg/day dexamethasone for 7 days in rats [26]. In the present study, rats tolerated a substantially higher dose of MPA (400 μg) as compared to MP (40 μg). MPA is hydrolyzed by cholinesterases into active methylprednisolone to become soluble and clinically active, giving the formulation its depot characteristics [27]. MP, methylprednisolone sodium succinate, is soluble and thereby biologically active when injected. The free fraction of methylprednisolone in the MP formulation is higher as compared to the MPA formulation. This explains why a lower dose of MP is tolerated intrathecally, as compared to the MPA dose. Why severe allodynia is observed at higher doses is unknown. As considered above, this hyperpathia is unlikely to be caused by pH, osmolar-ity, preservatives or particles in the suspension. It might be an effect of the glucocorticoid itself. Safety studies in dogs showed that intrathecal glucocorticoids cause neuro-inflammation when given at high doses [28,29,30,31] even with minimal concentrations of preservatives present [24].
4.3 Inflammatory model and glucocorticoid action
In the carrageenan model there was no difference in the development of mechanical allodynia between intrathecal MPA treated rats and saline controls in the first 8 h after carrageenan injection. No studies are available examining the efficacy of intrathecal glu-cocorticoids in this model. The effect of systemic glucocorticoid administration has been described, showing a complete prevention of the development of hyperalgesia 3 h after carrageenan injection [32]. Since the plasma levels of methylprednisolone were low after intrathecal MPA administration (18ng/ml), this might have prevented a strong peripheral effect. However, while methylprednisolone plasma levels were low, MPA treated rats did have less development of paw oedema as compared to saline controls confirming a drug action. Similar results were observed in a sciatic transection model where chronic high dose MP did not affect noci-ceptive thresholds but did prevent the development of neuropathic oedema [33]. Reduced peripheral oedema after a very low intrathecal dose of MPA in the carrageenan model could be explained by the fact that only low doses are necessary for oedema reduction or that the reduction of peripheral oedema can be mediated neuraxi-ally. There are examples of neuraxial administered drugs reducing peripheral oedema such as morphine and MAP kinase inhibitors [34,35].
4.4 Formalin model and glucocorticoid action
In agreement with previous work, there was no effect in flinching behaviour during the first phase after formalin between intrathecal MPA and MP versus saline controls [25,36,37]. Also, phase 2 flinching behaviour in intrathecal MPA and MP treated animals was not reduced as compared to saline controls. In other studies, intrathecal glucocorticoids; dexamethasone [36] and repeated triamcinolone 250 μg [25], but also systemic glucocorticoids; single dose dexamethasone 2.5-6.25 mg/kg [37] and 30 mg/kg [38] have been reported to reduce phase 2 flinching.There is also data showing modest or no reduction in flinching behaviour after a single intrathecal dose of 400 μg MP [25] or twice intraperi-toneal dosing of 250 μg/kg dexamethasone [39].The single doses of intrathecal MP and MPA as used in our study, were the maximum tolerable intrathecal doses, but low as compared to the intraperi-toneal or repeated intrathecal doses in studies showing analgesic effects. The late reduction in mechanical allodynia we observed in intrathecal MPA/MP treated rats compared to saline, has not been hitherto reported before. A possible explanation for the later analgesic onset of the treatment with MP is that a majority of the therapeutic effects of glucocorticoids are mediated through transactivation and repression of genes which takes 24–48 h to occur [10].
4.5 Nerve injury model and glucocorticoid action
Of the six reports on intrathecal administration of glucocorticoids in neuropathic pain models (SNL, chronic constriction injury (CCI) or spared nerve injury (SNI)) in rats, three reports mention a reduction, two an increase of thermal hyperalgesia and/or mechanical allodynia, and one no effect [26,40,41,42,43,44]. Analgesic effects were observed with different glucocorticoid formulations (prednisolone, methylprednisolone, or dexamethasone) either bolus or continuous infusion, all started on the day of nerve injury or within three days after nerve injury [40,41,42]. After local (perineural) [43,45,46], epidural [47], or systemic [48,49,50] administration of glucocorticoids in neuropathic pain models (SNL, CCI, or SNI) in rats, most report a reduction of nociceptive behaviour. However in three out of seven studies the analgesic effect was either very short lived (only at one time point) and/or less than 25% reduction of painlike behaviour [43,47,50]. Further, others have reported no effect [33,51] or that glucocorticoids have pain enhancing capabilities [26,44,52]. In conclusion, regarding the large body of data on glucocorticoid treatment for neuronal injury collected over more than 60 years and the contradicting results published, one may say that if there is any analgesic effect of glucocorticoids after nerve injury it is small and short lived. In our study with well documented pre-clinical models, no effect on pain behaviour was observed after any route of administration.
4.6 Effect of glucocorticoids on surrogate markers
The origin of afferent traffic associated with neuropathic pain after SNL reflects ectopic activity arising from the injury site and from the DRG of the injured axon. In the face of spinal nerve injury, local inflammatory products arising from the nerve injury site, activate DRG cell populations and induce activity of ATF3. ATF3, rapidly upregulated in all injured DRG neurons after peripheral nerve injury confirmed in our study, has a survival function driving neurite outgrowth [18]. Glucocorticoid treatment did not affect the ATF3 measured in DRG neurons as compared to saline control. This may be explained as the dose of MP reaching the DRG was too low. The peak plasma level of MP (18ng/ml) and an unknown amount of local diffusion after intrathecal MPA administration are probably insufficient to prevent neuronal damage occurring in the DRG.
The inflammatory cascade activated after nerve injury may activate DRG cell populations such as satellite cells and pericel-lular glia, resulting in a further increase of local expression of pro-inflammatory substances such as cytokines, chemokines, and growth factors. Although injury is limited to the primary afferent and/or DRG, these interventions can lead to persistent changes in the spinal dorsal horn, including glial activation. Activation of spinal glia after peripheral nerve injury occurs in the first week after injury. Microglia are activated by day 3 with a maximum on day 7 and remain activated for at least three weeks [26]. Astrocytes follow a similar timeframe. It has been reported that glucocorticoids attenuate glial activation. The activation of astrocytes was reduced in the SNL and SNI model [42,43,46]. Microglia activation was attenuated after perineural treatment with 200 μg triamcinolone acetonide in the SNL model [46] but not after daily intraperitoneal injections of 2.5 mg/kg dexamethasone in the SNI model [26]. In the present study only microglial activation was attenuated after intrathecal MPA treatment in the SNL model.
4.7 Study strength and weaknesses
Combining all data, the number of rats without an analgesic effect after intrathecal glucocorticoid injection is substantial. We chose to pretreat rats 1 h before carrageenan and formalin injection. This was based on the pharmacokinetic data of intrathecal MPA showing peak plasma levels from 1 to 3 h after administration. The peak plasma levels would be present during the initial part of the pain stimulus/model. However, we are aware of the fact that glucocorticoids also have later onset therapeutic effects through transactivation and repression of genes, which can take over 24 h [12]. This might explain why we did not observe any analgesic affect after glucocorticoid administration but only a later effect on mechanical allodynia 7 days after formalin injection. In the SNL model, we sacrificed rats receiving intraperitoneal and perineural glucocorticoids 11 days after nerve ligation. It is known that at this time point glial cell activation is around its peak. However, since the glucocorticoid treatment was earlier we might have missed a treatment effect on glial cell activation.
5 Conclusion
In this study severe generalized allodynia was observed after high doses of MP and MPA in naive rats. No acute analgesic effects were observed with intrathecal MP and MPA in any of the three established rodent pain models; carrageenan, formalin or SNL model. Only late antiallodynic effects were observed in the formalin model 7 days after formalin injection. Our results do not support clinical use of intrathecal glucocorticoids in the treatment of pain.
Highlights
Efficacy of intrathecal glucocorticoids in neuropathic pain patients is debated.
Intrathecal methylprednisolone on pain-like behaviour is studied in 3 pain models.
Generalized allodynia is observed after high intrathecal methylprednisolone doses.
Intrathecal methylprednisolone has no effect upon inflammatory or nerve pain.
Our results do not support use of intrathecal methylprednisolone in pain treatment.
DOI of refers to article: http://dx.doi.org/10.1016/j.sjpain.2015.10.007.
-
Authors’ contributions
Author: M. Rijsdijk, MD
Contribution: Study design, data collection, data analysis, preparation of the first draft of the manuscript, preparation of revisions of the manuscript after review by the co-authors.
Attestation: M. Rijsdijk approved the final manuscript. M. Rijsdijk attests to the integrity of this manuscript. M. Rijsdijk is the designated archival author who is responsible for maintaining the study records.
Conflicts of Interest: None.
Author: C.I. Svensson, PhD
Contribution: Has supervised data collection and analysis, preparation of the first draft of the manuscript and critically reviewed the manuscript.
Attestation: C.I. Svensson approved the final manuscript.
Conflicts of Interest: None.
Author: AJ.M. van Wijck, MD, PhD
Contribution: Critical revision of the manuscript.
Attestation: AJ.M. van Wijck approved the final manuscript.
Conflicts of Interest: None.
Author: CJ. Kalkman, MD, PhD
Contribution: Critical revision of the manuscript.
Attestation: CJ. Kalkman approved the final manuscript.
Conflicts of Interest: None.
Contribution: Has supervised the study design, data collection, data analysis, preparation of the first draft of the manuscript, and critically reviewed the manuscript.
Attestation: T.L Yaksh approved the final manuscript.
Conflicts of Interest: None.
M. Rijsdijk performed the research, M.Rijsdijk, T.L. Yaksh and C.I. Svensson designed the research study, C.I. Svensson, CJ. Kalkman and T.L. Yaksh contributed essential reagents and tools, M. Rijsdijk, C.I. Svensson, A.J.M. van Wijck, CJ. Kalkman and T.L. Yaksh analyzed the data, M. Rijsdijk wrote the paper and C.I. Svensson, A.J.M. van Wijck, CJ. Kalkman and T.L. Yaksh revised the manuscript. All authors approved the final manuscript.
Acknowledgements
This study was funded by a collaborative research grant from the European Society of Regional Anaesthesia and Pain Therapy, the Stiftelsen Lars Hiertas minne grant (FO2013-0235) from the Karolinska Institutet and the Department of Anesthesiology, University Medical Centre, Utrecht, The Netherlands. Funders did not play a role designing the study, analyzing the data or preparing the manuscript.
We thank Jorrit Huisman for his technical support with the illustrations and Esther de Groot for her hard work during the quantification of ATF3 staining.
References
[1] Cohen SP, Bicket MC, Jamison D, Wilkinson I, Rathmell JP. Epidural steroids: a comprehensive, evidence-based review. Reg Anesth Pain Med 2013;38:175–200.Suche in Google Scholar
[2] Dworkin RH, O’Connor AB, Kent J, Mackey SC, Raja SN, Stacey BR, Levy RM, Backonja M, Baron R, Harke H, Loeser JD, Treede RD, Turk DC, Wells CD. Inter-ventional management of neuropathic pain: NeuPSIG recommendations. Pain 2013;154:2249–61.Suche in Google Scholar
[3] Munts AG, van der Plas AA, Ferrari MD, Teepe-Twiss IM, Marinus J, van Hilten JJ. Efficacy and safety of a single intrathecal methylprednisolone bolus in chronic complex regional pain syndrome. Eur J Pain 2010;14:523–8.Suche in Google Scholar
[4] Rijsdijk M, van Wijck AJ, Meulenhoff PC, Kavelaars A, vanderTweel I, Kalkman CJ. No beneficial effect of intrathecal methylprednisolone acetate in posther-petic neuralgia patients. EurJ Pain 2013;38:175–200.Suche in Google Scholar
[5] Baron R, Wasner G. Prevention and treatment of postherpetic neuralgia. Lancet 2006;367:186–8.Suche in Google Scholar
[6] Dworkin RH, Johnson RW, Breuer J, Gnann JW, Levin MJ, Backonja M, Betts RF, Gershon AA, Haanpaa ML, McKendrick MW, Nurmikko TJ, Oaklander AL, Oxman MN, Pavan-Langston D, Petersen KL, Rowbotham MC, Schmader KE, Stacey BR, Tyring SK, van Wijck AJ, Wallace MS, Wassilew SW, Whitley RJ. Recommendations for the management of herpes zoster. Clin Infect Dis 2007;44(Suppl 1):S1–26.Suche in Google Scholar
[7] Lampe JB, Hindinger C, Reichmann H. Intrathecal methylprednisolone for postherpetic neuralgia. N Engl J Med 2001;344:1019–20.Suche in Google Scholar
[8] Nelson DA, Landau WM. Intrathecal methylprednisolone for postherpetic neuralgia. N EnglJ Med 2001;344:1019–22.Suche in Google Scholar
[9] Mensah-Nyagan AG, Meyer L, Schaeffer V, Kibaly C, Patte-Mensah C. Evidence for a key role of steroids in the modulation of pain. Psychoneuroendocrinology 2009;34(Suppl. 1):S169–77.Suche in Google Scholar
[10] Rijsdijk M, van Wijck AJ, Kalkman CJ, Yaksh TL. The effects of glucocorticoids on neuropathic pain: a review with emphasis on intrathecal methylprednisolone acetate delivery. Anesth Analg 2014;118:1097–112.Suche in Google Scholar
[11] De Bosscher K, van den Berghe W, Haegeman G. The interplay between the glucocorticoid receptor and nuclear factor-kappaB or activator protein-1: molecular mechanisms for gene repression. Endocr Rev 2003;24:488–522.Suche in Google Scholar
[12] Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids - new mechanisms for old drugs. N EnglJ Med 2005;353:1711–23.Suche in Google Scholar
[13] Kotani N, Kushikata T, Hashimoto H, Kimura F, Muraoka M, Yodono M, Asai M, Matsuki A. Intrathecal methylprednisolone for intractable postherpetic neuralgia. N EnglJ Med 2000;23:1514–9.Suche in Google Scholar
[14] Ferreira SH, Zanin T, Lorenzetti BB, de Souza MZ, Medeiros MC, Leme JG. Increased vascular permeability, oedema and hyperalgesia caused by car-rageenin in the rat’s paw [proceedings]. Agents Actions 1978;8:159.Suche in Google Scholar
[15] Fu KY, Light AR, Maixner W. Relationship between nociceptor activity, peripheral edema, spinal microglial activation and long-term hyperalgesia induced by formalin. Neuroscience 2000;101:1127–35.Suche in Google Scholar
[16] Yamamoto T, Yaksh TL. Comparison of the antinociceptive effects of pre- and posttreatment with intrathecal morphine and MK801, an NMDA antagonist, on the formalin test in the rat. Anesthesiology 1992;77:757–63.Suche in Google Scholar
[17] Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355–63.Suche in Google Scholar
[18] Seijffers R, Allchorne AJ, Woolf CJ. The transcription factor ATF-3 promotes neurite outgrowth. Mol Cell Neurosci 2006;32:143–54.Suche in Google Scholar
[19] Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6.Suche in Google Scholar
[20] Yaksh TL, Ozaki G, McCumber D, Rathbun M, Svensson C, Malkmus S, Yaksh MC. An automated flinch detecting system for use in the formalin nociceptive bioassay. J Appl Physiol (1985) 2001;90:2386–402.Suche in Google Scholar
[21] Malmberg AB, Yaksh TL. Antinociceptive actions of spinal nonsteroidal anti-inflammatory agents on the formalin test in the rat. J Pharmacol Exp Ther 1992;263:136–46.Suche in Google Scholar
[22] Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: characteristic biphasic pain response. Pain 1989;38:347–52.Suche in Google Scholar
[23] Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55–63.Suche in Google Scholar
[24] Rijsdijk M, van Wijck AJ, Kalkman CJ, Meulenhoff PC, Grafe MR, Steinauer J, Yaksh TL. Safety assessment and pharmacokinetics of intrathecal methylprednisolone acetate in dogs. Anesthesiology 2012;116:170–81.Suche in Google Scholar
[25] Abram SE, Marsala M, Yaksh TL. Analgesic and neurotoxic effects of intrathecal corticosteroids in rats. Anesthesiology 1994;81:1198–205.Suche in Google Scholar
[26] Scholz J, Abele A, Marian C, Haussler A, Herbert TA, Woolf CJ, Tegeder I. Low-dose methotrexate reduces peripheral nerve injury-evoked spinal microglial activation and neuropathic pain behavior in rats. Pain 2008;138: 130–42.Suche in Google Scholar
[27] Meyers C, Lockridge O, La Du BN. Hydrolysis of methylprednisolone acetate by human serum cholinesterase. Drug Metab Dispos 1982;10:279–80.Suche in Google Scholar
[28] Barros GA, Marques ME, Ganem EM. The effects of intrathecal administration of betamethasone over the dogs’ spinal cord and meninges. Acta Cir Bras 2007;22:361–5.Suche in Google Scholar
[29] Schaefer RB, Penn RD. Chronic intrathecal administration of dexamethasone sodium phosphate: pharmacokinetics and neurotoxicity in an animal model. Neurosurgery 2000;46:178–82.Suche in Google Scholar
[30] Latham JM, Fraser RD, Moore RJ, Blumbergs PC, Bogduk N. The pathologic effects of intrathecal betamethasone. Spine 1997;22:1558–62.Suche in Google Scholar
[31] Lima RM, Navarro LH, Carness JM, Barros GA, Marques ME, Solanki D, Ganem EM. Clinical and histological effects of the intrathecal administration of methylprednisolone in dogs. Pain Phys 2010;13:493–501.Suche in Google Scholar
[32] Prado FC, Araldi D, Vieira AS, Oliveira-Fusaro MC, Tambeli CH, Parada CA. Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation. Neuropharmacology 2013;67:252–8.Suche in Google Scholar
[33] Kingery WS, Castellote JM, Maze M. Methylprednisolone prevents the development of autotomy and neuropathic edema in rats, but has no effect on nociceptive thresholds. Pain 1999;80:555–66.Suche in Google Scholar
[34] Boyle DL, Jones TL, Hammaker D, Svensson CI, Rosengren S, Albani S, Sorkin L, Firestein GS. Regulation of peripheral inflammation by spinal p38 MAP kinase in rats. PLoS Med 2006;3:e338.Suche in Google Scholar
[35] Brock SC, Tonussi CR. Intrathecally injected morphine inhibits inflammatory paw edema: the involvement of nitric oxide and cyclic-guanosine monophos-phate. Anesth Analg 2008;106:965–71.Suche in Google Scholar
[36] Coderre TJ, Yashpal K. Intracellular messengers contributing to persistent noci-ception and hyperalgesia induced by L-glutamate and substance P in the rat formalin pain model. Eur J Neurosci 1994;6:1328–34.Suche in Google Scholar
[37] Yashpal K, Coderre TJ. Influence of formalin concentration on the antinociceptive effects of anti-inflammatory drugs in the formalin test in rats: separate mechanisms underlying the nociceptive effects of low- and high-concentration formalin. EurJ Pain 1998;2:63–8.Suche in Google Scholar
[38] Karim F, Kanui TI, Mbugua S. Effects of codeine, naproxen and dexamethasone on formalin-induced pain in the naked mole-rat. Neuroreport 1993;4:25–8.Suche in Google Scholar
[39] Taylor BK, Akana SF, Peterson MA, Dallman MF, Basbaum AI. Pituitary-adrenocortical responses to persistent noxious stimuli in the awake rat: endogenous corticosterone does not reduce nociception in the formalin test. Endocrinology 1998;139:2407–13.Suche in Google Scholar
[40] Gu X, Peng L, Yang D, Ma Q, Zheng Y, Liu C, Zhu B, Song L, Sun X, Ma Z. The respective and interaction effects of spinal GRs and MRs on radicular pain induced by chronic compression of the dorsal root ganglion in the rat. Brain Res 2011;1396:88–95.Suche in Google Scholar
[41] Ma ZL, Zhang W, Gu XP, Yang WS, Zeng YM. Effects of intrathecal injection of prednisolone acetate on expression of NR2B subunit and nNOS in spinal cord of rats after chronic compression of dorsal root ganglia. Ann Clin Lab Sci 2007;37:349–55.Suche in Google Scholar
[42] Takeda K, Sawamura S, Sekiyama H, Tamai H, Hanaoka K. Effect of methylprednisolone on neuropathic pain and spinal glial activation in rats. Anesthesiology 2004;100:1249–57.Suche in Google Scholar
[43] Wang QS, Jiang YH, Wang TD, Xiao T, Wang JK. Effects of betamethasone on neuropathic pain in a rat spare nerve injury model. Clin Exp Pharmacol Physiol 2013;40:22–7.Suche in Google Scholar
[44] Wang S, Lim G, Zeng Q, Sung B, Ai Y, Guo G, Yang L, Mao J. Expression of central glucocorticoid receptors after peripheral nerve injury contributes to neuropathic pain behaviors in rats. J Neurosci 2004;24:8595–605.Suche in Google Scholar
[45] Johansson A, Bennett GJ. Effect of local methylprednisolone on pain in a nerve injury model. A pilot study. Reg Anesth 1997;22:59–65.Suche in Google Scholar
[46] Li JY, Xie W, Strong JA, Guo QL, Zhang JM. Mechanical hypersensitivity, sympathetic sprouting, and glial activation are attenuated by local injection of corticosteroid near the lumbar ganglion in a rat model of neuropathic pain. Reg Anesth Pain Med 2011;36:56–62.Suche in Google Scholar
[47] Xie W, Liu X, Xuan H, Luo S, Zhao X, Zhou Z, Xu J. Effect of betamethasone on neuropathic pain and cerebral expression of NF-kappaB and cytokines. Neurosci Lett 2006;393:255–9.Suche in Google Scholar
[48] Clatworthy AL, Illich PA, Castro GA, Walters ET. Role of peri-axonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain. Neurosci Lett 1995;184:5–8.Suche in Google Scholar
[49] Kingery WS, Agashe GS, Sawamura S, Davies MF, Clark JD, Maze M. Glucocorti-coid inhibition of neuropathic hyperalgesia and spinal Fos expression. Anesth Analg 2001;92:476–82.Suche in Google Scholar
[50] Li H, Xie W, Strong JA, Zhang JM. Systemic antiinflammatory corticosteroid reduces mechanical pain behavior, sympathetic sprouting, and elevation of proinflammatory cytokines ina rat model of neuropathic pain. Anesthesiology 2007;107:469–77.Suche in Google Scholar
[51] Lee JB, Choi SS, Ahn EH, Hahm KD, Suh JH, Leem JG, Shin JW. Effect of perioperative perineural injection of dexamethasone and bupivacaine on a rat spared nerve injury model. Korean J Pain 2010;23:166–71.Suche in Google Scholar
[52] Loram LC, Taylor FR, Strand KA, Frank MG, Sholar P, Harrison JA, Maier SF, Watkins LR. Prior exposure to glucocorticoids potentiates lipopolysaccharide induced mechanical allodynia and spinal neuroinflammation. Brain Behav Immun 2011;25:1408–15.Suche in Google Scholar
© 2015 Scandinavian Association for the Study of Pain
Artikel in diesem Heft
- Editorial comment
- Plasma pro-inflammatory markers in chronic neuropathic pain: Why elevated levels may be relevant for diagnosis and treatment of patients suffering chronic pain
- Original experimental
- Plasma pro-inflammatory markers in chronic neuropathic pain: A multivariate, comparative, cross-sectional pilot study
- Editorial comment
- Genetic variability of pain – A patient focused end-point
- Observational study
- COMT and OPRM1 genotype associations with daily knee pain variability and activity induced pain
- Editorial comment
- Complex Regional Pain Syndrome (CRPS) after viper-bite in a pregnant young woman: Pathophysiology and treatment options
- Clinical pain research
- Complex regional pain syndrome following viper-bite
- Editorial comment
- An investigation into enlarging and reducing the size of mirror reflections of the hand on experimentally induced cold-pressor pain in healthy volunteers
- Original experimental
- An investigation into enlarging and reducing the size of mirror reflections of the hand on experimentally-induced cold-pressor pain in healthy human participants
- Editorial comment
- Multimodal Rehabilitation Programs (MMRP) for patients with longstanding complex pain conditions – The need for quality control with follow-up studies of patient outcomes
- Observational study
- Patients with chronic pain: One-year follow-up of a multimodal rehabilitation programme at a pain clinic
- Editorial comment
- Advancing methods for characterizing structure and functions of small nerve fibres in neuropathic conditions
- Clinical pain research
- Structural and functional characterization of nerve fibres in polyneuropathy and healthy subjects
- Editorial comment
- Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy
- Original experimental
- Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy
- Editorial comment
- Targeting glial dysfunction to treat post-surgical neuropathic pain
- Topical review
- Glial dysfunction and persistent neuropathic postsurgical pain
- Editorial comment
- Mechanisms of cognitive impairment in chronic pain patients can now be studied preclinically by inducing cognitive deficits with an experimental animal model of chronic neuropathic pain
- Original experimental
- Impaired recognition memory and cognitive flexibility in the ratL5–L6 spinal nerve ligation model of neuropathic pain
- Editorial comment
- Pain treatment with intrathecal corticosteroids: Much ado about nothing? But epidural corticosteroids for radicular pain is still an option
- Original experimental
- Analgesic properties of intrathecal glucocorticoids in three well established preclinical pain models
- Editorial comment
- The obesity epidemic makes life difficult for patients with herniated lumbar discs – and for back-surgeons: Increased risk of complications
- Observational study
- Obesity has an impact on outcome in lumbar disc surgery
- Editorial comment
- Finnish version of the fear-avoidance-beliefs questionnaire (FABQ) and the importance of validated questionnaires on FAB in clinical praxis and in research on low-back pain
- Clinical pain research
- Translation and validation of the Finnish version of the Fear-Avoidance Beliefs Questionnaire (FABQ)
- Editorial comment
- Pain, sleep and catastrophizing: The conceptualization matters Comment on Wilt JA et al. “A multilevel path model analysis of the relations between sleep, pain, and pain catastrophizing in chronic pain rehabilitation patients”
- Clinical pain research
- A multilevel path model analysis of the relations between sleep, pain, and pain catastrophizing in chronic pain rehabilitation patients
Artikel in diesem Heft
- Editorial comment
- Plasma pro-inflammatory markers in chronic neuropathic pain: Why elevated levels may be relevant for diagnosis and treatment of patients suffering chronic pain
- Original experimental
- Plasma pro-inflammatory markers in chronic neuropathic pain: A multivariate, comparative, cross-sectional pilot study
- Editorial comment
- Genetic variability of pain – A patient focused end-point
- Observational study
- COMT and OPRM1 genotype associations with daily knee pain variability and activity induced pain
- Editorial comment
- Complex Regional Pain Syndrome (CRPS) after viper-bite in a pregnant young woman: Pathophysiology and treatment options
- Clinical pain research
- Complex regional pain syndrome following viper-bite
- Editorial comment
- An investigation into enlarging and reducing the size of mirror reflections of the hand on experimentally induced cold-pressor pain in healthy volunteers
- Original experimental
- An investigation into enlarging and reducing the size of mirror reflections of the hand on experimentally-induced cold-pressor pain in healthy human participants
- Editorial comment
- Multimodal Rehabilitation Programs (MMRP) for patients with longstanding complex pain conditions – The need for quality control with follow-up studies of patient outcomes
- Observational study
- Patients with chronic pain: One-year follow-up of a multimodal rehabilitation programme at a pain clinic
- Editorial comment
- Advancing methods for characterizing structure and functions of small nerve fibres in neuropathic conditions
- Clinical pain research
- Structural and functional characterization of nerve fibres in polyneuropathy and healthy subjects
- Editorial comment
- Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy
- Original experimental
- Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy
- Editorial comment
- Targeting glial dysfunction to treat post-surgical neuropathic pain
- Topical review
- Glial dysfunction and persistent neuropathic postsurgical pain
- Editorial comment
- Mechanisms of cognitive impairment in chronic pain patients can now be studied preclinically by inducing cognitive deficits with an experimental animal model of chronic neuropathic pain
- Original experimental
- Impaired recognition memory and cognitive flexibility in the ratL5–L6 spinal nerve ligation model of neuropathic pain
- Editorial comment
- Pain treatment with intrathecal corticosteroids: Much ado about nothing? But epidural corticosteroids for radicular pain is still an option
- Original experimental
- Analgesic properties of intrathecal glucocorticoids in three well established preclinical pain models
- Editorial comment
- The obesity epidemic makes life difficult for patients with herniated lumbar discs – and for back-surgeons: Increased risk of complications
- Observational study
- Obesity has an impact on outcome in lumbar disc surgery
- Editorial comment
- Finnish version of the fear-avoidance-beliefs questionnaire (FABQ) and the importance of validated questionnaires on FAB in clinical praxis and in research on low-back pain
- Clinical pain research
- Translation and validation of the Finnish version of the Fear-Avoidance Beliefs Questionnaire (FABQ)
- Editorial comment
- Pain, sleep and catastrophizing: The conceptualization matters Comment on Wilt JA et al. “A multilevel path model analysis of the relations between sleep, pain, and pain catastrophizing in chronic pain rehabilitation patients”
- Clinical pain research
- A multilevel path model analysis of the relations between sleep, pain, and pain catastrophizing in chronic pain rehabilitation patients
