Hind Limb Weight Bearing Research Articles

Tapping into the endocannabinoid system to ameliorate acute inflammatory flares and associated pain in mouse knee joints.

IntroductionDuring the progression of rheumatoid arthritis (RA), there are frequent but intermittent flares in which the joint becomes acutely inflamed and painful. Although a number of drug therapies are currently used to treat RA, their effectiveness is variable and side effects are common. Endocannabinoids have the potential to ameliorate joint pain and inflammation, but these beneficial effects are limited by their rapid degradation. One enzyme responsible for endocannabinoid breakdown is fatty acid amide hydrolase (FAAH). The present study examined whether URB597, a potent and selective FAAH inhibitor, could alter inflammation and pain in a mouse model of acute synovitis.MethodsAcute joint inflammation was induced in male C57BL/6 mice by intra-articular injection of 2% kaolin/2% carrageenan. After 24 hr, articular leukocyte kinetics and blood flow were used as measures of inflammation, while hindlimb weight bearing and von Frey hair algesiometry were used as measures of joint pain. The effects of local URB597 administration were then determined in the presence or absence of either the cannabinoid (CB)1 receptor antagonist AM251, or the CB2 receptor antagonist AM630.ResultsURB597 decreased leukocyte rolling and adhesion, as well as inflammation-induced hyperaemia. However, these effects were only apparent at low doses and the effects of URB597 were absent at higher doses. In addition to the anti-inflammatory effects of URB597, fatty acid amide hydrolase (FAAH) inhibition improved both hindlimb weight bearing and von Frey hair withdrawal thresholds. The anti-inflammatory effects of URB597 on leukocyte rolling and vascular perfusion were blocked by both CB1 and CB2 antagonism, while the effect on leukocyte adherence was independent of cannabinoid receptor activation. The analgesic effects of URB597 were CB1 mediated.ConclusionsThese results suggest that the endocannabinoid system of the joint can be harnessed to decrease acute inflammatory reactions and the concomitant pain associated with these episodes.

α-Conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement

Neuropathic pain affects millions of people worldwide, causing substantial disability and greatly impairing quality of life. Commonly used analgesics or antihyperalgesic compounds are generally characterized by limited therapeutic outcomes. Thus, there is a compelling need for novel therapeutic strategies able to prevent nervous tissue alterations responsible for chronic pain. The α9α10 nicotinic acetylcholine receptor antagonist α-conotoxin RgIA (RgIA), a peptide isolated from the venom of a carnivorous cone snail, induces relief in both acute and chronic pain models. To evaluate potential disease-modifying effects of RgIA, the compound was given to rats following chronic constriction injury (CCI) of the sciatic nerve. Two or 10nmol RgIA injected intramuscularly once a day for 14days reduced the painful response to suprathreshold stimulation, increased pain threshold to nonnoxious stimuli, and normalized alterations in hind limb weight bearing. Histological analysis of the sciatic nerve revealed that RgIA prevented CCI-induced decreases of axonal compactness and diameter, loss of myelin sheath, and decreases in the fiber number. Moreover, RgIA significantly reduced edema and inflammatory infiltrate, including a decrease of CD86+ macrophages. In L4-L5 dorsal root ganglia, RgIA prevented morphometric changes and reduced the inflammatory infiltrate consistent with a disease-modifying effect. In the dorsal horn of the spinal cord, RgIA prevented CCI-induced activation of microglia and astrocytes. These data suggest that RgIA-like compounds may represent a novel class of therapeutics for neuropathic pain that protects peripheral nervous tissues as well as prevents central maladaptive plasticity by inhibiting glial cell activation.

Testosterone Dose Dependently Prevents Bone and Muscle Loss in Rodents after Spinal Cord Injury

Androgen administration protects against musculoskeletal deficits in models of sex-steroid deficiency and injury/disuse. It remains unknown, however, whether testosterone prevents bone loss accompanying spinal cord injury (SCI), a condition that results in a near universal occurrence of osteoporosis. Our primary purpose was to determine whether testosterone-enanthate (TE) attenuates hindlimb bone loss in a rodent moderate/severe contusion SCI model. Forty (n=10/group), 14 week old male Sprague-Dawley rats were randomized to receive: (1) Sham surgery (T9 laminectomy), (2) moderate/severe (250 kdyne) SCI, (3) SCI+Low-dose TE (2.0 mg/week), or (4) SCI+High-dose TE (7.0 mg/week). Twenty-one days post-injury, SCI animals exhibited a 77-85% reduction in hindlimb cancellous bone volume at the distal femur (measured via μCT) and proximal tibia (measured via histomorphometry), characterized by a >70% reduction in trabecular number, 13-27% reduction in trabecular thickness, and increased trabecular separation. A 57% reduction in cancellous volumetric bone mineral density (vBMD) at the distal femur and a 20% reduction in vBMD at the femoral neck were also observed. TE dose dependently prevented hindlimb bone loss after SCI, with high-dose TE fully preserving cancellous bone structural characteristics and vBMD at all skeletal sites examined. Animals receiving SCI also exhibited a 35% reduction in hindlimb weight bearing (triceps surae) muscle mass and a 22% reduction in sublesional non-weight bearing (levator ani/bulbocavernosus [LABC]) muscle mass, and reduced prostate mass. Both TE doses fully preserved LABC mass, while only high-dose TE ameliorated hindlimb muscle losses. TE also dose dependently increased prostate mass. Our findings provide the first evidence indicating that high-dose TE fully prevents hindlimb cancellous bone loss and concomitantly ameliorates muscle loss after SCI, while low-dose TE produces much less profound musculoskeletal benefit. Testosterone-induced prostate enlargement, however, represents a potential barrier to the clinical implementation of high-dose TE as a means of preserving musculoskeletal tissue after SCI.

NF-κB but not FoxO sites in the MuRF1 promoter are required for transcriptional activation in disuse muscle atrophy

The muscle-specific ring finger protein 1 (MuRF1) gene is required for most types of skeletal muscle atrophy yet we have little understanding of its transcriptional regulation. The purpose of this study is to identify whether NF-κB and/or FoxO response elements in the MuRF1 promoter are required for MuRF1 gene activation during skeletal muscle atrophy due to the removal of hindlimb weight bearing ("unloading"). Both NF-κB -dependent and FoxO-dependent luciferase reporter activities were significantly increased at 5 days of unloading. Using a 4.4-kb MuRF1 promoter reporter construct, a fourfold increase in reporter (i.e., luciferase) activity was found in rat soleus muscles after 5 days of hindlimb unloading. This activation was abolished by mutagenesis of either of the two distal putative NF-κB sites or all three putative NF-κB sites but not by mutagenesis of all four putative FoxO sites. This work provides the first direct evidence that NF-κB sites, but not FoxO sites, are required for MuRF1 promoter activation in muscle disuse atrophy in vivo.

Pre-emptive, early, and delayed alendronate treatment in a rat model of knee osteoarthritis: effect on subchondral trabecular bone microarchitecture and cartilage degradation of the tibia, bone/cartilage turnover, and joint discomfort

Bisphosphonates are considered potential disease modifying osteoarthritis (OA) agents. The present study investigated the efficacy of pre-emptive, early, and delayed alendronate (ALN) treatment initiation on subchondral trabecular bone and cartilage in low-dose monosodium iodoacetate (MIA)-induced knee OAin rats. Male rats received pre-emptive (n=12, day 0-end of week 2), early (n=12, end of week 2-end of week 6), or delayed (n=12, end of week 6-end of week 10) ALN treatment (30μg/kg/week). Pre-emptive ALN-treated rats were scanned using invivo micro-computed tomography (micro-CT) after 2 weeks and then sacrificed, early ALN-treated rats were scanned after 2 and 6 weeks and sacrificed, and the delayed ALN-treated rats were scanned after 2, 6, and 10 weeks of OA induction and sacrificed. After sacrifice, bone histomorphometry and histology of the tibia and biomarker analyses were undertaken. Changes in hind limb weight-bearing were assessed from day-1 until day 14. MIA-induced pathological features similar to progressive human OA in the cartilage and subchondral bone. Pre-emptive ALN treatment preserved subchondral trabecular bone microarchitecture, prevented bone loss, decreased bone turnover and joint discomfort. Pre-emptive ALN treatment had moderate effects on cartilage degradation. Early and delayed ALN treatments prevented loss of trabeculae and decreased bone turnover, but had no significant effect on cartilage degradation. ALN prevented increased bone turnover and preserved the structural integrity of subchondral bone in experimental OA. The time point of treatment initiation is crucial for treating OA. Treating both the subchondral bone and cartilage in OA would be clinically more beneficial.

Unrestricted Weight Bearing as a Method for Assessment of Nociceptive Behavior in a Model of Tibiofemoral Osteoarthritis in Rats

Background: Novel preclinical models for prediction of osteoarthritis-like pain are necessary for the elucidation of osteoarthritis (OA) pathology and for assessment of novel analgesics. A widely used behavioral test in rat models of tibiofemoral OA is hind limb weight bearing (WB). However, this method evaluates WB in an unnaturally restricted manner. The aim of this study was therefore to characterize the Tekscan Pressure Measurement System as a means to assess OA-like tibiofemoral pain in rats by determination of plantar pressure distribution in a more natural and unrestricted position, defined as unrestricted WB. Methods: Intra-articular injections of 1 mg monosodium iodoacetate (MIA) or saline were administrated in the left hind knee of 84 male Sprague Dawley rats. Changes in unrestricted WB between ipsilateral and contralateral hindlimbs were determined. Morphine (5 mg/kg administered subcutaneously) and naproxen (60 mg/kg per-oral) were examined for their ability to reverse WB changes. Results: Changes in hind limb unrestricted WB were observed 14 (P P P Conclusion: This study indicated that unrestricted WB assessed by the Tekscan system can be utilized as a clinically relevant method to assess aberrations in WB induced by intra-articular MIA injections in rodents. Therefore, this system may be useful for understanding the mechanisms of OA pain in humans and may also assist in the discovery of novel pharmacological agents.

Functional Changes in Muscle Afferent Neurones in an Osteoarthritis Model: Implications for Impaired Proprioceptive Performance

BackgroundImpaired proprioceptive performance is a significant clinical issue for many who suffer osteoarthritis (OA) and is a risk factor for falls and other liabilities. This study was designed to evaluate weight-bearing distribution in a rat model of OA and to determine whether changes also occur in muscle afferent neurones.Methodology/Principal FindingsIntracellular recordings were made in functionally identified dorsal root ganglion neurones in acute electrophysiological experiments on the anaesthetized animal following measurements of hind limb weight bearing in the incapacitance test. OA rats but not naïve control rats stood with less weight on the ipsilateral hind leg (P = 0.02). In the acute electrophysiological experiments that followed weight bearing measurements, action potentials (AP) elicited by electrical stimulation of the dorsal roots differed in OA rats, including longer AP duration (P = 0.006), slower rise time (P = 0.001) and slower maximum rising rate (P = 0.03). Depolarizing intracellular current injection elicited more APs in models than in naïve muscle afferent neurones (P = 0.01) indicating greater excitability. Axonal conduction velocity in model animals was slower (P = 0.04).Conclusions/SignificanceThe present study demonstrates changes in hind limb stance accompanied by changes in the functional properties of muscle afferent neurones in this derangement model of OA. This may provide a possible avenue to explore mechanisms underlying the impaired proprioceptive performance and perhaps other sensory disorders in people with OA.

Spontaneous firing in C-fibers and increased mechanical sensitivity in A-fibers of knee joint-associated mechanoreceptive primary afferent neurones during MIA-induced osteoarthritis in the rat

Osteoarthritis (OA) pain mechanisms are poorly understood. We used the monosodium iodoacetate (MIA) model of knee OA to characterize changes in excitability during the course of OA in different classes of mechanosensitive afferents projecting to joint-associated tissues, and examine whether these afferent responses and pain behavior are correlated. Rats were injected intra-articularly with MIA (1mg in 50 μl). Hind-limb weight bearing was studied 3 (MIA3) and 14 (MIA14) days after MIA, followed by deep anesthesia and teased-nerve-fiber recordings. Spontaneous activity (SA) and mechanically evoked responses of A- and C-mechanosensitive fibers (AM and CM respectively, probably nociceptive) innervating tissues associated with the ipsilateral knee joint were examined. MIA3 and MIA14 rats exhibited reduced ipsilateral weight bearing. SA (>0.02 impulses/s) occurred in ∼50% of CMs from MIA rats vs 0% in normals. SA firing rates in CMs were significantly higher than normal; decreased weight bearing was correlated with increased CM SA rates. Neither percentages of AMs with SA (20%) nor their firing rates (0-0.01 impulses/s) significantly increased after MIA. In contrast, in MIA rats AMs, but not CMs, exhibited decreased mechanical thresholds and increased firing rates in response to suprathreshold mechanical stimulation. These findings of increased SA firing rate in CMs but not AMs and increased mechanical sensitivity of AMs, but not CMs, have not previously been reported. These are two distinct important physiological mechanisms that may underpin spontaneous pain (CMs) and stimulus-evoked pain (AMs) in OA. Our data contribute to a mechanism-based understanding of OA pain.

Monosodium iodoacetate-induced osteoarthritis produces pain-depressed wheel running in rats: Implications for preclinical behavioral assessment of chronic pain

Pain stimulates some behaviors (e.g., withdrawal responses) and depresses other behaviors (e.g., feeding and locomotion). We are developing methods for testing candidate analgesics using measurements of pain-depressed behaviors. Such assays may model important aspects of clinical pain and complement traditional procedures that measure pain-stimulated behaviors. The present study characterized the effects of a chronic pain manipulation (monosodium iodoacetate (MIA)-induced osteoarthritis) on wheel running in rats. Rats had 24h voluntary access to running wheels. Duration of running wheel acquisition was manipulated such that rats had either 21 or 7days of running wheel access prior to MIA administration. Wheel running was monitored for an additional 21days following MIA administration. MIA produced concentration- and acquisition length-dependent decreases in wheel running. Parallel experiments demonstrated that MIA produced concentration-dependent tactile allodynia and shifts in hind limb weight bearing. MIA was differentially potent across assays with a potency rank: weight-bearing≥von Frey>running wheel. MIA produced greater depression of wheel running in rats with relatively high baseline running rates compared to rats with relatively low baseline running rates. The differential potency of MIA across assays and apparent rate-dependent effects in running wheels may impact our traditional interpretations of preclinical nociceptive and antinociceptive testing.

Pre-treatment with capsaicin in a rat osteoarthritis model reduces the symptoms of pain and bone damage induced by monosodium iodoacetate

A rat model of osteoarthritis was used to investigate the effect of pre-treatment with capsaicin on the symptoms of osteoarthritis induced by the injection of monosodium iodoacetate. This model mimics both histopathology and symptoms associated of human osteoarthritis. Injection of monosodium iodoacetate, an inhibitor of glycolysis, into the femorotibial joints of rodents promotes loss of articular trabecular bone and invokes pain symptoms similar to those noted in human osteoarthritis. Twenty rats were divided in two groups either receiving placebo or monosodium iodoacetate. Each group was subdivided in two groups either receiving pre-treatment with capsaicin two weeks before monosodium iodoacetate injection or not, resulting in four groups of five rats each. The impact of a single intra-articular administration of capsaicin (0.5%) on the generation of evoked mechanical pain (hind limb weight bearing, automated von Frey monofilament and RotaRod tests) and bone lesions (micro-CT scan radiographic analyses of bone structure) following monosodium iodoacetate-induced osteoarthritis in rats was determined. Evoked mechanical pain as monitored over a period of 4 weeks after monosodium iodoacetate injection was abolished in capsaicin pre-treated animals and pain values are comparable to those of capsaicin controls. Chronic joint pathological changes such as bone erosion and trabecular damage were significantly reduced by pre-treatment with a single administration of capsaicin. Decrease of bone volume was considerably ameliorated and trabecular connectivity was substantially better in capsaicin pre-treated animals. Capsaicin, an agonist activator of the vanilloid nociceptors (TRPV1), appears to be effective in protecting bone from arthritic damage. The present results support the hypothesis that capsaicin-sensitive sensory neurons contribute to bone lesions in the monosodium iodoacetate-induced osteoarthritis rat model.

Effects of wheel running duration on osteoarthritis‐induced shifts in hind limb weight bearing in rats

We are developing methods to characterize the potency/efficacy of chronic pain states to suppress feeding, locomotor activity and wheel running. The present study describes a serendipitous finding that wheel running, when manipulated as an independent variable, attenuates osteoarthritis‐induced shifts in hind limb weight bearing. Rats were given intra‐articular saline or monosodium iodoacetate (MIA, 3.2mg) into the left hind knee. Rats were divided into three groups. Group 1 had 24 hr voluntary access to running wheels for 42 consecutive days (21 days acquisition followed by 21 days post‐MIA). Group 2 had 24 hr voluntary access to running wheels for 21 consecutive days (0 days acquisition followed by 21 days post‐MIA). Group 3 had no access to running wheels for 21 consecutive days (21 days post‐MIA). Weight bearing was measured on post‐MIA days 3, 7, 14 and 21. MIA produced robust and reliable shifts in weight bearing in non‐runners. In contrast, runners demonstrated a duration‐dependent attenuation of MIA‐induced shifts in weight bearing. These results suggest that voluntary wheel running may be protective against osteoarthritis pain. Parallel experiments are being conducted to determine the effects of wheel running duration on MIA‐induced damage to cartilage, subchondral bone and muscle. Funded by NIAMS grant R15 AR054975‐01 (GWS).

Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception

Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH 2 into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1β concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH 2 evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH 2, injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1β production are independent of this channel.

The quantitative assessment of functional impairment and its correlation to infarct volume in rats with transient middle cerebral artery occlusion

The purpose of this study was to quantitatively assess motor activity and sensory functions and to determine their relationships to infarct severity by measuring infarct volume in rats with transient ischemic stroke. Male Sprague–Dawley rats (11 weeks old, n = 42) were randomly divided into 4 separate groups; a sham operation group, and 1-h, 2-h and 3-h middle cerebral artery occlusion (MCAO) groups. Percent weights borne on paretic hind limbs were measured consecutively for 7 days starting from the day before the induction of ischemia. Fifty percent withdrawal threshold values of forepaws and hindpaws were measured using von-Frey hairs. Infarct volumes in the three ischemic groups, which were significantly different ( p < 0.01) from each other, were found to increase in size with ischemic time. The percent weight borne on paretic hind limb in the three MCAO groups were significantly lower than that in the sham group, and this functional deficit remained significant throughout the observational period ( p < 0.01). A significant correlation was found between infarct volumes and percent weight borne on paretic hind limbs after ischemia (− 0.7 < rho < − 0.4, p < 0.05). In contrast the 50% withdrawal threshold values of paretic forepaws and hindpaws in the three ischemic groups were not significantly different from those of the sham controls. Motor test findings, which were used to assess reductions in paretic hind limb weightbearing, were shown to be correlated with infarct volume. The present study shows that this test could be used as a tool to objectively and quantitatively assess functional outcome in MCAO rats.

A Fracture Pain Model in the Rat

Because of the relative lack of understanding of the mechanisms that drive skeletal pain, the purpose of this study was to adapt a previously validated closed femur fracture model to quantitatively evaluate skeletal pain in female and male rats. Three-month-old female and male Sprague-Dawley rats were anesthetized, and a stainless steel pin was inserted into the intramedullary space of the left femur. Three weeks later, the rats were reanesthetized, and left femoral diaphyses were fractured using a standardized impactor device. At 1-21 days after fracture, skeletal pain was measured by quantitatively assessing spontaneous guarding, spontaneous flinching, and weight bearing of the fractured hind limb. Females and males showed highly robust pain behaviors that were maximal at day 1 after fracture and returned gradually to normal nonfractured levels at days 14-21 after fracture. The magnitude of fracture pain was not significantly different at most time points between female and male rats. In both females and males, the pain-related behaviors were attenuated by subcutaneous morphine in a dose-dependent manner. This model may help in developing a mechanism-based understanding of the factors that generate and maintain fracture pain in both females and males and in translating these findings into new therapies for treating fracture pain.

Vasoactive intestinal peptide (VIP) is a modulator of joint pain in a rat model of osteoarthritis

Osteoarthritis (OA) is a debilitating disease in which primarily weight-bearing joints undergo progressive degeneration. Despite the widespread prevalence of OA in the adult population, very little is known about the factors responsible for the generation and maintenance of OA pain. Vasoactive intestinal peptide (VIP) was identified in the synovial fluid of arthritis patients nearly 20 years ago and the aim of this study was to examine whether VIP could be involved in the generation of OA pain. Hindlimb weight bearing was used as a measure of joint pain, while von Frey hair algesiometry applied to the plantar surface of the ipsilateral hindpaw tested for secondary mechanical hyperalgesia. Intra-articular injection of VIP into normal rat knee joints caused a significant shift in weight bearing in favour of the contralateral non-injected hindlimb as well as causing a reduction in ipsilateral paw withdrawal threshold. These pain responses were blocked by co-administration of the VPAC receptor antagonist VIP 6–28. Induction of OA by intra-articular sodium monoiodoacetate injection resulted in a reduction in weight bearing on the affected leg, but no evidence of secondary hyperalgesia in the paw. Treatment of OA knees with a single injection of VIP 6–28 diminished hindlimb incapacitance while increasing paw withdrawal threshold. This study showed for the first time that peripheral application of VIP causes increased knee joint allodynia and secondary hyperalgesia. Furthermore, antagonists that inhibit VIP activity may prove beneficial in the alleviation of OA pain.

Nociceptin/orphanin FQ evokes knee joint pain in rats via a mast cell independent mechanism

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like neuropeptide that has been shown to cause peripheral sensitization of knee joint afferents; however, the effect of the peptide on joint pain behaviour is unknown. In addition to having a direct effect on peripheral nerves, N/OFQ has also been shown to activate connective tissue mast cells causing the local release of potentially pain causing mediators. The present study tested the effect of peripherally administered N/OFQ on joint pain and examined whether synovial mast cells contribute to these responses. Hindlimb weight bearing and von Frey hair algesiometry were measured before and following a single injection of N/OFQ in the vicinity of the right knee of male Wistar rats. Compared to saline-treated controls, N/OFQ caused a conspicuous shift in hindlimb weight bearing in favour of the contralateral non-injected leg. Similarly, paw withdrawal threshold and latency were significantly reduced following N/OFQ administration indicative of secondary hyperalgesia. To test the involvement of synovial mast cells in these pain reactions, a separate group of rats were treated with the mast cell stabilizer cromolyn (20 mg/kg s.c.) 5 min prior to N/OFQ injection. Cromolyn treatment had no significant effect on N/OFQ-induced weight bearing deficit nor secondary hyperalgesic responses. In conclusion, these data support the premise that N/OFQ has a pain causing effect in the periphery which occurs independently of mast cell activation.

Hindlimb unweighting for 2 weeks alters physiological properties of rat hindlimb motoneurones.

We sought to determine whether decreased neuromuscular use in the form of hindlimb unweighting (HU) would affect the properties of innervating motoneurones. Hindlimb weight-bearing was removed in rats for a period of 2 weeks via hindlimb suspension by the tail. Following this the electrophysiological properties of tibial motoneurones were recorded under anaesthesia in situ. After HU, motoneurones had significantly (P < 0.05) elevated rheobase currents, lower antidromic spike amplitudes, lower afterhyperpolarization (AHP) amplitudes, faster membrane time constants, lower cell capacitances, and depolarized spike thresholds. Frequency-current (f-I) relationships were shifted significantly to the right (i.e. more current required to obtain a given firing frequency), although there was no change in f-I slopes. 'Slow' motoneurones (AHP half-decay times, > 20 ms) were unchanged in proportions in HU compared to weight-bearing rats. Slow motoneurones had significantly lower minimum firing frequencies and minimum currents necessary for rhythmic firing than 'fast' motoneurones in weight-bearing rats; these differences were lost in HU rats, where slow motoneurones resembled fast motoneurones in these properties. In a five-compartment motoneurone model with ion conductances incorporated to resemble firing behaviour in vivo, most of the changes in passive and rhythmic firing properties could be reproduced by reducing sodium conductance by 25% and 15% in the initial segment and soma, respectively, or by increasing potassium conductance by 55% and 42%, respectively. This supports previous conclusions that changes in chronic neuromuscular activity, either an increase or decrease, may result in physiological adaptations in motoneurones due to chronic changes in ion conductances.

Development and pharmacological characterization of a rat model of osteoarthritis pain

Osteoarthritis (OA) is an age-related joint disease characterized by degeneration of articular cartilage and is associated with chronic pain. Although several experimental models of OA have been employed to investigate the underlying etiologies of the disease, there has been relatively little investigation into development of animal models of OA to study the pain associated with the condition. In the present study, we investigated OA induced by injection of either iodoacetate or papain into the knee joint of rats, and assessed the joint degeneration with radiographic analyses and measured pain behavior using hind limb weight bearing. We found that injection of iodoacetate, but not papain, resulted in a chronic joint degeneration as measured by decreased bone mineral content and bone mineral density, necrosis of articular cartilage and osteophyte formation. These pathological changes were associated with pain that manifested as time- and concentration-dependent alterations in hind limb weight bearing. These alterations in hind limb weight bearing were reversed with morphine, but were not significantly affected by acute administration of either indomethacin or celecoxib. However, administration of 30 mg/kg celecoxib twice daily for 10 days resulted in a significant restoration of hind limb weight bearing. We conclude that the iodoacetate model of OA is a relevant animal model to study pain associated with OA, and can be used to test potential therapeutic agents.

A rat model of bone cancer pain

This study describes the first known model of bone cancer pain in the rat. Sprague–Dawley rats receiving intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra-tibial injections of 3×10 3 or 3×10 4 syngeneic MRMT-1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10–14 after inoculation of 3×10 3 MRMT-1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate-resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat-killed MRMT-1 cells. Intra-tibial injections of 3×10 3 or 3×10 4 MRMT-1 cells, heat-killed cells or vehicle did not show changes in body weight and core temperature over 19–20 days. The general activity of animals after injection with live or heat-killed MRMT-1 cells was higher than that of the control group, however, the activity of the MRMT-1 treated group declined during the progress of the disease. Rats receiving intra-tibial injections of MRMT-1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12–14 or 10–12 following injection of 3×10 3 or 3×10 4 cells, respectively. These symptoms were not observed in rats receiving heat-killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti-nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1–3 mg/kg, subcutanously (s.c.)) produced a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra-tibial injections of 3×10 3 MRMT-1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10–30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3×10 3 MRMT-1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT-1 mammary tumour cell line provides a valid pre-clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX-2 inhibitor, celebrex, has no influence on the pain-related behavioural changes in this model.

Activity-induced fiber regeneration in rat soleus muscle.

In an attempt to understand why muscle recovery is limited following atrophy due to limb immobilization, satellite cell activity and muscle fiber regeneration were analyzed in rat soleus muscles. Adult rat hindlimbs were immobilized in plaster casts for a period of two to ten weeks. Soleus muscles were examined by electron microscopy for evidence of fiber degeneration or regeneration, and to quantify satellite cell nuclei. Immunocytochemical localization of embryonic myosin was used to identify regenerating myofibers. Soleus muscle wet weight to body weight ratios for the casted muscles significantly decreased over the 10-week immobilization period. The casted muscles displayed ultrastructural evidence of minor fiber damage, including myofibrillar atrophy, Z-disc disruption, and abnormal triadic junctions. No ultrastructural evidence of regeneration was seen in the casted animals. The number of satellite cells in the casted muscles significantly decreased from 6.4% to 3. 3% by eight to 10 weeks of immobilization. Approximately 1.0% of extrafusal fibers in the control soleus muscles appeared to be regenerating since they expressed embryonic myosin and were of a small diameter, while in casted muscles, only 0.1% of the fibers were embryonic myosin-positive. Following release from immobilization, a reappearance of embryonic myosin-positive fibers was noted within four days of renewed activity. In contrast to control muscles, embryonic myosin-positive fibers in the recovery muscles included both small and large diameter fibers. Subtle changes in functional activity influence muscle damage and subsequent myofiber regeneration. Reduced activity reduces muscle fiber regeneration, while increased activity, as seen by increased hindlimb weight bearing and return to normal activity following immobilization, increase regenerating fibers and also the expression of embryonic myosin in adult fibers.