Osteoarthritis (OA) is the most common rheumatological disease (Lawrence et al., 1998) that affects more than 80% of the population aged 55 years and older (Kryger, 2000). Pain in OA has been attributed to the deformation of periarticular tissues (Akeson et al., 1989) or subchondral bone (Gronblad et al., 1984), to raised intraosseous pressure (Arnoldi et al., 1980) and synovial inflammation (Smith et al., 1997). There are reports stating that many patients with spurs, lipping and joint space narrowing become pain free when their trigger points are treated, indicating a role of musculoskeletal tissues in the pain associated with osteoarthritis (Travell and Simons, 1983).
Several studies in acute experimental animal models with altered joint biomechanics and physically and chemically precipitated osteoarthritis are available. Neuronal responses from acute animal arthritis models are seen primarily in laminae V and VII of the dorsal horn cells (Lamour et al., 1983). In a similar study in a post kaolin and carrageenan induced arthritis of cats, new receptive fields responding to punctate stimuli and joint movement are attributed to awakening of the ‘silent nociceptors’ (Schaible and Schmidt, 1985). It is also shown that the spinal cord dorsal and ventral horn neurons responsible for the joint input respond increasingly to mechanical stimulation of areas outside their receptive fields, such as leg and thigh muscles in knee joint arthritic animals (Schaible et al., 1987; Neugebauer and Schaible, 1988, 1990). Extensive investigations in joint inflammation in animals have shown the altered neuro-chemical state of excitatory amino acids (EAAs) at the spinal level involving the N-methyl-D-aspartate (NMDA) receptor mechanisms (Dickenson et al., 1997). An excess release of EAAs and production of nitric oxide in the knee joint during inflammation suggest that both peripheral and central mechanisms subserving increases in nociception are associated with articular inflammation and pain (Lawand et al., 1999).
Several groups have studied muscle hyperalgesia (increased pain and enlarged referred areas) with the use of intramuscular (i.m.) hypertonic saline in chronic musculoskeletal pain syndromes such as whiplash (Johansen et al., 1999) and fibromyalgia (Sorensen et al., 1998; Graven-Nielsen et al., 2000), but no studies are yet available on lower extremity osteoarthritis. Use of i.m. hypertonic saline is a reliable experimental method for stimulation of deep tissue nociceptors (Kellgren 1937; Zhang et al., 1993; Stohler and Lund, 1995; Graven-Nielsen et al., 1997a). It is hypothesized that a persistent nociceptive input from the osteoarthritic joint leads to central sensitization, that might result in an increased responsiveness of dorsal horn neurons processing input from the joint and possibly other tissues such as muscles. Acceptance of this hypothesis implies increased muscle hyperalgesia and enlarged referred pain areas due to infusion of hypertonic saline in osteoarthritis patients as compared with healthy controls. The aim of the present study was to test this hypothesis.
2. Material and methods
Fourteen chronic OA patients, eight females and six males, aged 41.8±3.5 years; height 159.6±1.6 cm; weight 65.4±3.2 kg and having pain in one or more joints of the lower extremities for a duration of 3±0.93 years (mean±SEM) participated in the study. The OA patients had not taken medication within 72 h prior to the experiment. Fourteen unmedicated healthy subjects, eight females and six males, of comparable age (38.8±2.9 years), height (159.0±2.1 cm), and weight (63.2±2.5 kg), and not on any medication, were selected as controls. All subjects completed the medical history questionnaire and marked the pain areas on the pain charts. Two females in the OA group and one in the control group were post-menopausal and were not on hormonal replacement therapy. The other female subjects were in the reproductive age group, but the menstrual cycle phase at the time of the experiment was not determined. However, all had regular menstrual cycles and were not taking oral contraception. None of the OA patients or control subjects complained of depression or sleep problems, or of inability to carry out their daily work routines. The diagnosis of the OA patients was based on the findings from the previous chronological medical record fulfilling the criteria of the American College of Rheumatologists for osteoarthritis (Altman 1995). Ten OA patients (10/14) had pain in the knee joint (Fig. 1). Eight OA patients (8/14) also had pain in the thigh, leg or foot. The OA patients were further grouped into those having pain in the joints of both legs (5/14), pain only in the right leg joints (5/14), and pain only in the left leg joints (4/14). The mean total pain areas drawn by the patients were 1.78±0.75 AU (arbitrary units) (Table 1). None of the control subjects marked any pain areas at the time of enrolment. Each subject was given a written and verbal synopsis as to the nature of the study and received financial compensation for participation. All the subjects signed an informed consent form and could withdraw from the experiment at any time. The study was approved by the local ethics committee and was carried out according to the Helsinki declaration.
2.2. Intra-muscular hypertonic saline infusion
Marks with non-toxic ink were placed at the belly of the tibialis anterior (TA) muscle 14 cm distal to the inferior lateral edge of the patella on both legs. To prevent local skin pain, the infusion site was anaesthetized with an intradermal injection of 0.1 ml intradermal 2% xylocaine injection (Astra-IDL, Sweden) given 1 min before the intramuscular saline infusion. At the marked site in the TA a 24G - 40mm needle was inserted vertically until a piercing of the muscle fascia was felt at a depth of approximately 20 mm from the skin surface. Then the plunger of the syringe was withdrawn to ensure that the needle was deep in the muscle and not in a blood vessel. The needle was then connected through a polyethylene extension tube (Vygon, France, No. 1155.70) to a 10 ml syringe fitted in a computer-controlled auto-infusion syringe pump (Terumo Terufusion syringe pump, model STC-S27, Type CG). A total volume of 0.5 ml sterile 6% hypertonic saline (58.5 mg/ml, Sygehus Apotekerne, Denmark) was infused at a rate of 90 ml/h over 20 s into the TA muscle (Zhang et al., 1993; Stohler and Lund, 1995; Graven-Nielsen et al., 1998; Svensson et al., 1998a).
2.3. Pain intensity of experimental muscle pain
The pain intensity response was scored on a 0–10 cm visual analogue scale (VAS) after the infusion. On the VAS ‘0 cm’ indicated ‘no pain’ and ‘10 cm’ indicated ‘worst imaginable pain’. The time between the onset of infusion and maximum VAS score was defined as the time to peak pain, and the time to the disappearance of pain was defined as the pain offset time.
2.4. Spatial distribution of experimental muscle pain
The subjects were asked to mark the painful region(s) on pain maps after infusion (Fig. 2). Local pain was defined as the pain area drawn at the infusion site, referred pain was defined as the pain areas drawn away from the infusion site and radiating pain was defined as the pain areas drawn radiating from the local site into the other regions of the leg. The areas of marked pain were digitized (ACECAD D9000+digitizer, Taiwan) and the pain area calculated in cm2 (Sigma-Scan, Jandel Scientific, Canada). The pain areas were however expressed in arbitrary units (AU) as the size of the pain drawing did not match the actual limb size.
2.5. Study design
Circadian influences in OA patients are known (Bellamy et al., 1990), and therefore all the patients were tested between 09:00 and 12:00 h. The sequence of right and left leg was chosen in a randomized way to minimize order effects. The experiment was conducted in one leg at a time, by giving a single bolus infusion of hypertonic saline with a time interval of approximately 20 min between each leg. The experiment on the second leg was started only after the experimentally induced pain had completely subsided in the first limb. Pain intensity and spatial distribution of pain were recorded immediately after infusion, at 2, 5, 10 and 20 min after the infusion and then every 10 min until the experimentally induced pain disappeared.
The results were expressed as mean±SEM. A repeated measures analysis of variance (RMANOVA) or Friedman RMANOVA on ranks test was performed. Post-hoc analysis was performed with the Student–Newman–Keul's test (SNK). Sample group comparison was performed by a t test or Mann–Whitney Rank Sum Test. Correlations were performed using Spearman's test. Significance was accepted at P<0.05.
3.1. Intensity of experimental muscle pain
Pain intensity VAS increased significantly in the right leg of OA patients compared with the control subjects at 2 min (t(26)=−2.27; P=0.032) and at 5 min (Mann–Whitney's T=150, n=14, P=0.016) (Fig. 3). RMANOVA analysis of pain intensity VAS indicated an effect of hypertonic saline infusion in both the right and left leg of the OA patients and control subjects (P<0.001). Post-hoc analysis revealed that significant pain occurred immediately after and at 2 min in both OA and control groups as compared with 5, 10 and 20 min after infusion (SNK: P<0.05). The OA patients still had significant pain in the left leg at 5 min as compared with 10 and 20 min after infusion (SNK: P<0.05). The total VAS intensities of the experimental pain for the right and left leg at 2 min after infusion correlated with the clinical habitual osteoarthritic pain duration in OA group (Spearman's R=−0.53, P=0.047).
3.2. Pain duration and time to peak
The mean pain-offset time was significantly longer in OA patients (11.3±7.9 min) as compared with the controls (6.04±2.12 min) (t(26)=−2.37; P=0.025). The time to peak pain in OA patients (1.4±0.1 min) was significantly longer (Mann–Whitney's T=156.5, n=14, P=0.03) than in the controls (1.1±0.1 min). Time to peak correlated with the maximum pain intensity (Spearman's R=−0.65, P=0.011) in OA patients.
3.3. Pain areas of experimental muscle pain
A RMANOVA analysis indicated significantly increased referred pain areas immediately after and 2 min post saline infusion as compared with 5, 10, and 20 min in the OA and control groups (Friedman's P=0.001, SNK P<0.05) (Fig. 4). Significantly increased referred pain was seen in the right leg of OA patients and control subjects immediately after post-infusion compared with the local pain (SNK P=0.013). Two minutes after infusion, referred and radiating pain areas significantly increased as compared with local pain areas in both the legs of OA patients, but no changes were seen in the control subjects (SNK P<0.05). Similarly, significant referred pain areas were still present in OA patients 5 min after infusion in the right leg and after 5 and 10 min in the left leg compared with the remaining post-infusion follow-up times (Friedman's P<0.001, SNK P<0.05).
3.4. Frequency of referred pain responses
The frequency of the occurrence of pain referral sites was analyzed in the right (n=14) and left (n=14) legs of both OA and control groups. Most commonly referred pain occurred at the ankle in both the control (16/28) and the OA (14/28) group immediately after the infusion. Referred pain at the hips and toes was seen in 3/28 and 7/28, respectively, of OA patients as compared with the 0/28 in the healthy control subjects.
This study shows significantly higher local pain duration and intensity, larger pain areas and significantly increased referred and radiating pain intensity after infusion of hypertonic saline in the legs of the OA patients as compared with the controls.
4.1. Intra-muscular hypertonic saline
The present study showed significant enhancement of the pain responses to i.m. hypertonic saline in OA patients, suggesting the presence of muscle hyperalgesia. Muscle hyperalgesia with i.m. hypertonic saline has been demonstrated earlier in other chronic painful conditions such as fibromyalgia (Sorensen et al., 1998) and whiplash syndromes (Johansen et al., 1999). Hypertonic saline effectively excites small diameter (group III/IV) muscle nociceptors and other fine afferents (Paintal, 1960; Iggo, 1961; Mense, 1993a). It has been shown that that hypertonic saline injection into the masseter and TA muscle causes a transient increase in both surface and intramuscular electromyographic activity, but that this activity is not correlated to perceived pain intensity as measured on the VAS (Graven-Nielsen et al., 1997a; Svensson et al., 1998b). Rossi et al. (1999) suggested that the chemically activated muscle nociceptive discharge actually depresses the activity of group Ib interneurons in humans (Rossi et al., 1999). Overall, it appears that the muscle electrical activity caused by injection of hypertonic saline may be insufficient to cause muscle spasm that may constrict the blood vessels and obstruct the washout of metabolic wastes resulting in the sensitization of nociceptors. It has been shown that it requires repeated and protracted work against increasing load to reach a state of ischaemia resulting in pain due to isometric muscle contraction (Vecchiet et al., 1983). Sodium magnetic resonance imaging has shown that patients with osteoarthritis have increased sodium concentration in the bursa and tissues around the knee joint as compared with healthy controls (Connstantinides et al., 2000). This may also be another factor contributing to the differences in responses in the OA patients and control subjects in the present study. An influence of the long term impaired motor control patterns in OA patients as compared with non-symptomatic subjects may also result in differences in muscular responses (Robon et al., 2000).
4.2. Hyperalgesia and osteoarthritis
Recently the relationship between OA and hyperalgesia has been studied by Farrell et al. (2000a,b) using mechanical, thermal, and electrical stimuli over the carpo-metacarpal joint of patients affected with OA pain. However, they were not able to demonstrate hyperalgesia in the forearm (Farrell and Richards, 1986, 2000a). The difference in the results from the present study could be due to the fact that in the present study we used selective deep tissue stimulation by i.m. hypertonic saline, which was not applied in the study by Farrell et al. (2000a). In another study, Farrell et al. showed a substantial increase in spontaneous pain in response to resisted movement of the carpo-metacarpal joint in OA patients (Farrell et al., 2000b). They also selectively blocked Aβ fibers by tying a ribbon over the wrist and found that this resulted in an increase in the mechanical and thermal thresholds as compared with OA patients without the block. On the basis of this, it is suggested that Aβ fibers are involved in the alteration of stimulus response characteristics in OA patients (Farrell et al., 2000b). Further, Farrell et al. (2000b) proposed that reduced thermal pain thresholds over the painful joints may be dependent on central sensitization (Farrell et al., 2000b).
4.3. Referred and radiating pain areas
Pain maps have been used previously to study the pattern of pain referral (Fortin et al., 1994a,b; Demco 2000). The time course enlargement of referred and radiating pain areas in response to i.m. hypertonic saline in OA patients showed significant enhancement of pain areas at immediately after, 2, 5, and 10 min after hypertonic saline infusion, while in controls this enlargement took place only immediately after the injection. These differences in the effectiveness of pain measurement by VAS and pain pattern areas may be due to the increased variability in marking pain areas as compared with VAS markings. The VAS has a distinct advantage as it consists of 100 points of 1 mm each, and very small changes in pain intensity could theoretically be noted on this measure (Jensen et al., 1998). Other studies also demonstrate that the pain map areas (pain extensity) do not correlate with the pain intensity measured on the VAS (0–10 scale) (Weiner et al., 1998). The VAS is also known to be a reliable measure of subjective states including pain, nausea, well-being, and mood (Cella and Perry, 1986). It may be that the pain mapping and VAS ratings appear to tap different components of pain. It has been suggested that referred pain, which is intense, appears to be radiating and involves larger areas (McMahon, 1994). It is proposed that long term nociceptive input from the OA joints in the lower limb may result in central sensitization in the spinal cord (Schaible et al., 1987; Woolf et al., 1988; Neugebauer and Schaible, 1990; Coderre et al., 1993). In the present study, the pattern of pain spread in the control subjects was restricted to the lower leg and ankle, coinciding to the patterns obtained in previous studies (Kellgren, 1937, 1939; Graven-Nielsen et al., 1997a,b). Similarly, the proximal spread in some OA patients and larger spread distally to the foot correspond with patterns obtained in fibromyalgia (Sorensen et al., 1998) and whiplash syndromes (Johansen et al., 1999). High intensity stimulation of muscle nociceptors in rats resulted in expansion of muscle receptive fields (Hoheisel and Mense, 1989; Hoheisel et al., 1993). It has been suggested that the development of referred pain sites caused by i.m. noxious stimulation could be due to unmasking of anatomically existing, but functionally ineffective, fiber connections between the spinal neurons and the periphery (Mense, 1994). Other mechanisms, such as a decrease in the efficacy of the descending anti-nociceptive system or heterotopic facilitation caused by active nociceptive fibers outside the receptive fields may result in muscle hyperalgesia (Mense, 1993b; 1994). A recent study compared the effects of diffuse inhibitory control (DNIC) in acute and chronic monoarthritis in animals and demonstrated that input activated during chronic monoarthritis fails to recruit DNIC (Danziger et al., 1999).
One limitation of the present study is that the chronic OA patients and healthy subjects included subjects both with and without a past history of trauma. A study involving well defined groups of OA patients suffering from acute, subacute or subacute on chronic OA, OA only with movement, those with primary or secondary OA or those classified by pain descriptors or psychosocial state could have given a better insight into the underlying mechanisms of hyperalgesia in OA.
In conclusion, patients with osteoarthritis show manifestations of central sensitization to muscle nociceptor input demonstrated by the increased pain intensity, enlarged radiating, and referred pain areas to intramuscular infusion of hypertonic saline. Together with new pharmacological treatment regimes these findings may result in new and more effective treatment strategies for painful OA.
The authors acknowledge the Danish National Research Foundation and the Mermaid Institute, India for sponsoring the study. We would like to thank Professor Siegfried Mense, Dr. med., Institut für Anatomie und Zellbiologie, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany, for valuable comments on the manuscript.
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