To examine neuronal activation in the spinal cord due to secondary hyperalgesia resulting from intrajoint capsaicin injection, and the effect of physiotherapy manipulation, using functional magnetic resonance imaging (fMRI), in α-chloralose anesthetized rats.
Materials and Methods:
FMRI of the rat lumbar spinal cord was performed at 9.4 Tesla. Stimuli included injection of 25 μL of capsaicin (128 μg/mL in 7.5% dimethyl sulfoxide [DMSO]) into the right forepaw or 75 μL into the right ankle joint followed by a light touch stimulus, with and without physiotherapy manipulation.
Activation of pain areas of the spinal cord (dorsal horn) was found in all animals after injection of capsaicin into the plantar surface of the rat hindpaw and ankle joint. Overlay maps depicting activations and deactivations showed significant reproducibility between experiments. Greater overlay of activations were observed for intrajoint compared to intradermal capsaicin injection. The distribution of activations after stimulation of the hindpaw using a light touch stimulus was somewhat more varied; activation of the dorsal horn was evident, with greater overlap resulting when joint mobilization was not performed.
JOINT MOBILIZATION AND MANIPULATION has been used by physiotherapists and other practitioners for many years to manage painful musculoskeletal disorders. It appears that these treatments can exert a pain-relieving effect and contribute to improved function in patients with neck and back pain (1–4).
The mechanism of pain relief remains the subject of ongoing investigations. It has been suggested that activation of segmental pain inhibitory mechanisms and descending pain inhibitory systems projecting from brain to spinal cord might be important for mediating manipulation-induced analgesia in the period immediately after treatment application (5).
The results of multiple studies clearly demonstrate that mobilization of the cervical spine induces an immediate-onset hypoalgesic or antihyperalgesic effect in patients with lateral epicondylalgia; patients with insidious cervical joint pain; and in pain-free, normal volunteers (6–10). A consistent observation has been that application of joint mobilization treatments produces a significant elevation of pressure pain thresholds and other measures of mechanical hyperalgesia, but that these treatments have absolutely no influence on thermal pain measures. As well as influencing pain related measures, joint mobilization techniques also produce changes in measures related to motor function and autonomic nervous system function (6, 8). A strong correlation between changes in pain-related measures and changes in autonomic nervous system-related measures have been demonstrated.
Sluka and Wright (11) provided evidence of an antihyperalgesic effect of peripheral joint mobilization in the rat, using an intra-articular injection of capsaicin (0.2%, 50μL) as the pain model. The hyperalgesic effect of capsaicin and antihyperalgesic effect of treatment were evaluated by testing mechanical pain thresholds on the plantar surface of the foot using von Frey filaments. In this pain model, secondary mechanical hyperalgesia develops over the plantar surface of the foot within two hours after capsaicin injection. Results showed that treatment applied for a total of nine minutes (three × three minutes) or 15 minutes (three × five minutes) produced complete reversal of hyperalgesia induced by capsaicin injection. This effect was apparent within five minutes and lasted for up to 45 minutes. Given that there is evidence of an antihyperalgesic effect of knee joint mobilization in a model of articular pain in the rat, further research is required to investigate the neurophysiologic basis of that effect.
The occurrence of blood oxygen-level dependent (BOLD) contrast changes in the brain is well established (12–14). MRI studies of brain activation patterns in response to multiple capsaicin injections performed in rats clearly show activations in regions of the brain known to be associated with pain (15). Recently, the extension of functional magnetic resonance imaging (MRI) from the brain to the spinal cord has been demonstrated (16–20). This has opened the door to a new area of research in spinal cord function and functional recovery after spinal cord injury. In particular, functional imaging of the spinal cord (spinal fMRI) in animals allows for the development of new models to determine the extent of injuries, examination of physiology and function of damaged and healthy spinal cord models, and a new means of monitoring treatment of injury.
FMRI of the rat cervical spinal cord has been recently examined. Activations were observed in the left dorsal horn after capsaicin injection into the left forepaw (17). Spinal neurons have been shown to increase in firing rate and response to both noxious and innocuous stimuli after intradermal capsaicin injection (21–23). Moreover, the mechanism of action of intradermal capsaicin injection and the secondary hyperalgesia response is well documented (24–27). The greatest amount of neuronal activation known to be involved in noxious mechanical stimulation of the rat hindlimb appears typically in ipsilateral spinal cord segments L4 and L5 and laminae I–II and V–VI of the dorsal horn (28–31).
We hypothesized that fMRI of the spinal cord after painful stimulation will result in activation of the regions associated with pain, such as the dorsal horn. We also hypothesized that secondary hyperalgesia resulting from capsaicin injection will lead to mechanical allodynia (i.e., increased pain from innocuous stimuli) and the intensity and/or distribution of the activation in the spinal fMRI as a response to secondary hyperalgesia would be reduced after physiotherapy joint mobilization.
The present study investigated spinal cord activation detectable with fMRI in response to subcutaneous and intrajoint capsaicin injection in rats, and the effect of joint mobilization on spinal cord neuronal activation resulting from secondary hyperalgesia and allodynia. Functional imaging of the lumbar spinal cord can provide a means of assessing spinal cord injury, and is a useful tool in animal models of pain, injury, and therapy.
MATERIALS AND METHODS
Specific pathogen-free Sprague-Dawley rats were obtained from Charles River (Canada) and housed in the Institute for Biodiagnostics (IBD) Animal Facility for a minimum of seven days in order to acclimatize before use. Animals were treated according to the Canadian Council for Animal Care guidelines, and the protocol was reviewed and approved by the IBD Animal Care Committee. Each animal was anesthetized with isoflurane (3%–4% induction, 1.5%–2% maintenance) in oxygen (0.4 liters/minute) and nitrogen (0.6 liters/minute). Rectal temperature was monitored and maintained at 37 ± 0.5°C using a circulating water blanket and heating lamp. Catheters (PE 50) containing heparinized saline were placed in the femoral artery and vein. Arterial blood samples, mean arterial blood pressure, and heart rate were obtained from the arterial catheter. Fluids and drugs were administered via the venous catheter. Bupivacaine (0.25%) was administered into the wound site before closure. The animal was intubated and ventilated using a small animal ventilator (Columbus Instruments, OH). The ventilation volume (3–4 mL) was adjusted to maintain the animal at normal arterial blood gases (PO2 of 100–120 mmHg; PCO2 of 35–45 mmHg) while keeping the ventilation rate (60 breaths per minute) constant. In order to minimize movement, the animals were anesthetized with α-chloralose, as has been reported previously in fMRI activation studies in rats (32). The anesthetic α-chloralose (30 mg/mL, 80 mg/kg) was administered intravenously over approximately five minutes, and the isoflurane was discontinued. Anesthesia was maintained with additional doses of α-chloralose (40 mg/mL every 90 minutes). Physiologic parameters were recorded every 10 minutes. Upon completion of the MR experiment, the animal was euthanized with pentobarbital (120 mg/kg intravenously).
Capsaicin stimulation experiments involved placement of PE20 tubing fitted with a small needle (26-gauge) into either the plantar surface of the right hindpaw (subcutaneously) or the lateral aspect of the right ankle joint, and secured with surgical tape. The tubing was filled with capsaicin (128 μg/mL in 7.5% dimethyl sulfoxide [DMSO]) with a slight air bubble at the tip, in order to prevent any premature stimulation.
Experiments were performed using a 9.4 T/21 cm horizontal bore magnet (Magnex, UK) with Avance (Bruker, Germany) console and a surface coil tuned to 400.5 MHz centered over the lumbar spine. The animal was placed supine over the surface coil and held in place by securing a water blanket over the animal. Tape was also placed across the animal's chest, and secured to the holder, in order to restrict motion near the imaging coil. Acquisition of MR image data was also gated to the respiration.
Sequential T2*-weighted scout images were acquired using a multi-slice gradient-echo fast imaging sequence (TE = 25 msec, TR = 100 msec, 30° flip angle, 128 × 128 matrix) to select transverse slices through the lumbar spinal cord from the 13th thoracic vertebrae to the 2nd lumbar vertebrae (T13–L2). Slices were centered on the lumbar vertebrae and on the intervertebral discs. Sequential images were acquired using a multi-slice fast spin-echo imaging sequence with rapid acquisition with relaxation enhancement (RARE) phase encoding (TEeff = 85 msec, TR = 1800 msec, 128 × 64 matrix). Six 2-mm thick slices were acquired simultaneously with a field of view of 2 cm and an acquisition time of approximately eight seconds.
A series of fast spin-echo images (NR = 40) were collected in which either 25 μL of capsaicin (128 μg/mL in 7.5% DMSO) was injected into the right plantar surface of the rat hindpaw or 75 μL into the lateral aspect of the right ankle joint, after a number of images acquired initially at baseline. Ten images were collected at baseline, followed by capsaicin injection and acquisition of the remaining 30 images. Functional imaging experiments involving capsaicin injection into the hindpaw were repeated three times in each animal, allowing for 30 minutes between the start of the experiments. In functional imaging experiments involving injection into the ankle joint, a light stimulus was applied to the plantar surface of the right hindpaw after injection. In the first group involving ankle injection, fMRI experiments using the light touch stimulus were repeated for 4 hours after injection (control). In the second group, the light touch stimulus was repeated for 2 hours after ankle injection, then physiotherapy mobilization of the knee joint was performed, followed by light touch stimulus fMRI repeated until four hours after capsaicin-injection. Table 1 illustrates the different groups of experiments.
Table 1. Experimental Groups
1 (N = 7)
Capsaicin injection into right plantar surface of hindpaw
2 (N = 9)
Capsaicin injection into right ankle
Light stimulus of right plantar hindpaw (4 hours)
3 (N = 4)
Capsaicin injection into right ankle
Light stimulus of right plantar hindpaw (2 hours)
Light stimulus of right plantar hindpaw (2 hours)
The light touch stimulation device was a sharp screw tip, which could be turned in the holder from outside of the magnet bore. Trials on the bench preceded every experiment in order to determine the amount of rotation required in order for the tip of the screw to just rest on the plantar surface of the hindpaw. The hindpaw was secured so that movement was restricted, ensuring that the degree of rotation would be sufficient to just touch to surface of the paw without causing any painful sensation. FMRI experiments were also performed before injection of capsaicin to show that no activations in the dorsal horn were observed when the light touch stimulus was applied.
Physiotherapy Joint Mobilization
For joint mobilization treatment groups, the animals were removed from the magnet, and mobilization of the knee joint performed three times, three minutes per mobilization, interleaved with one-minute rest periods. The joint mobilization involved extension of the knee joint with an anterior-posterior glide of the tibia on the femur ipsilateral to the side of injection (11). The femur was stabilized with one hand and the mobilization performed by moving the tibia on the femur. The knee joint was rhythmically flexed and extended to the end of the range of extension while the tibia was simultaneously moved in an anterior to posterior direction. This procedure has previously been shown to produce an antihyperalgesic effect (11). Care was taken to avoid contact with either the foot or the ankle.
The fast spin-echo fMRI time course data were analyzed by means of direct correlation on a pixel-by-pixel basis to a paradigm (33) (using custom-made software developed in IDL; Interactive Data Language, Research Systems Inc., Boulder, CO). For the capsaicin injection experiments, the stimulus paradigm was defined with the initial 10 time points acquired during the baseline condition, and the next five time points were considered the transition period. These were not included in the correlation calculation so that no model intensity time course had to be assumed. The next 20 time points were considered to be the active period, and the remaining time points were again excluded from the correlation because these may or may not have started to return to baseline. A correlation coefficient (R) of 0.312 was used for the correlation, resulting in a P ≤ 0.05. For the light stimulus experiments, three rest periods were alternated with two activation periods, during which time the tip of the screw rested against the plantar surface of the right hindpaw.
The consistency of functional experiments between animals was established by overlaying the activations or deactivations of each of the experiments. The overlay of activations was accomplished by manually lining up the slices from each of the functional maps. The areas of activation and deactivation from each animal were summed and overlaid onto the reference image. Only pixels above a chosen threshold were included. The threshold of 1.5 in a 3 × 3 × 3 volume around a pixel was used to show overlay of activations after capsaicin injection. In this volume, the voxel in question will have 26 nearest neighbors (a total volume of 27 pixels). If there is a threshold of 1.5, then there must be 41 or more active pixels combined from all of the animals, within that specific volume of 27 pixels, in order for there to show any region of activation on the overlay map. Three-by-three boxcar smoothing was performed to obtain the final overlay map. The resulting overlay map is color-coded with the pixels that have a high degree of consistency between experiments in red, followed by blue, and then green.
The mean arterial blood gas values were maintained within physiologic limits throughout the experiments (Table 2). As previously illustrated, injection of capsaicin results in an immediate increase in mean arterial pressure, which returned to pre-capsaicin injection level within 3–4 minutes after the injection (15). Stimulation using light touch did not result in any visible changes in mean arterial pressure.
Table 2. Blood Gas Values and Mean Arterial Blood Pressure (MAP) Changes as a Result of Noxious and Innocuous Stimulation
Capsaicin injection right ankle
Capsaicin injection right hindpaw
Light stimulus right hindpaw
40.00 ± 19.47
46.11 ± 16.05
7.44 ± 0.03
7.43 ± 0.04
108.6 ± 9.4
105.2 ± 9.4
43.9 ± 6.7
44.2 ± 7.9
Consistent regions of activity in the spinal cord were observed for all animals after capsaicin injection. Figure 1 shows the transverse sections of the spinal cord segments that were imaged. Stimulation of the right hindpaw and ankle joint with capsaicin-injection produced significant functional activation, mainly in the right side of the spinal cord at the level of the 13th thoracic to 2nd lumbar vertebrae, corresponding to the 3rd to 6th lumbar and 1st sacral spinal cord segments (Fig. 2). Regions of the spinal cord known to be involved in the transmission of painful information, namely the dorsal horn, clearly showed activation in several animals. Greater overlap was observed in the dorsal horn region after injection of capsaicin into the ankle joint compared to injection into the plantar surface of the hindpaw. Some activations were also noted centrally, as well as on the contralateral side, of the spinal cord. Figure 2 shows the greatest amount of overlap between individual activations marked in red. Arrows point to the greatest areas of overlap of interest. As evident in the figures, consistent regions of activation were observed throughout the imaging slices from the L2 to T13 vertebrae, corresponding to the spinal cord segments denoted above. The majority of activations occurred from vertebral level T13 to L1.
Functional imaging experiments involving capsaicin injection into the hindpaw were repeated three times for each animal (N = 7), allowing 30 minutes between capsaicin injections for recovery of the vanilloid receptors. Of these, only experiments showing clear activation upon injection of capsaicin (that is correlation with the paradigm) were included in the combined activation maps (N = 15). All animals showed clear correlation with the paradigm in at least one capsaicin injection experiment.
The dorsal horn region of the spinal cord, involved in sensory information, also showed activation for several hours after capsaicin injection with the application of a non-painful stimulus (light touch stimulus). Following joint mobilization, compared to control animals with no joint mobilization, there does appear to be greater overlap and increased regions showing activation in response to hyperalgesia without physiotherapy mobilization (Fig. 3).
Figure 3 shows activations after mobilization, again mainly in the right side of the spinal cord, with some contralateral activation. The greatest amount of overlap between individual activations appeared in the slices corresponding to T13, between T13 and L1, and L1 vertebrae. Again, the combined activation map shows the pixels that have a high degree of consistency (red) between experiments, with arrows pointing to the main regions of consistent activations. In this case, a cut-off value of 0.33 was used; the 3 × 3 × 3 volume surrounding a pixel would have to contain a total of nine or more active pixels in all of the activation maps being combined to be visible in the overlay images (green). Of the nine experiments involving injection into the ankle joint in the no-joint-mobilization group, four did not show activations in the cord upon capsaicin injection. One of the remaining five animals did not show activation with the light touch stimulus three hours after capsaicin injection. Therefore, a total of four animals were included in the combined activation maps from this group. All four animals showed activation upon capsaicin injection into the ankle in the physiotherapy joint mobilization group, and are included in the overlay maps.
Regions of negative functional activation, or deactivation, were also apparent in these animals (Figs. 2 and 3). Regions corresponding to negative functional activation were frequently observed in pixels adjacent to those showing activation.
Table 3 shows the average percentage signal changes upon capsaicin injection and physiotherapy manipulation, from the functional imaging time courses. The percentage signal change upon injection of capsaicin into the anklejoint and hindpaw were similar. Lower percentage signal changes were observed with the light touch stimulus compared to capsaicin injection, but no differences were observed between the manipulation and no manipulation groups with respect to signal change.
Table 3. Mean Percentage Signal Changes, ΔS/S (%), and Standard Deviation (SD) From fMRI Timecourses Following Either Capsaicin Injection or Light Touch Stimulation
Time following injection (minutes)
ΔS/S (%) ± S.D.
18.6 ± 10.2
15.9 ± 7.5
13.5 ± 10.0
19.0 ± 1.8
7.2 ± 1.0
10.7 ± 4.6
11.1 ± 8.4
13.0 ± 10.0
Clear areas of functional activation of the lumbar spinal cord are observed with capsaicin-induced nociceptive stimulation. The regions of activation in the spinal cord are consistent with spinal cord physiology, with the majority of activations appearing in the dorsal horn ipsilateral to the site of the stimulation. The cell bodies of the unmyelinated C-fibers, or poorly myelinated Aδ fibers involved in nociception are located in the dorsal root ganglion. Pain related impulses are transmitted through the dorsal root to the dorsal horn, mainly to Rexed's laminae I and II at the tip of the dorsal horn. The neuronal activation determined by the functional imaging experiments involving noxious stimulation of the rat hindlimb are in good agreement with physiologic studies of neuronal activation, with ipsilateral activations in spinal cord segments L3 to S2, with the majority of activations appearing from L3 to L6, and clear activations in the dorsal horn (17, 28–31).
The large number of interneurons in the spinal cord also play a crucial role in response to external stimuli. Many pathways involving pain also involve reflex arcs (34). This can account, in part, for activation appearing both in ventral regions and the center of the spinal cord upon stimulation. Furthermore, the percentage signal changes in the spinal functional imaging experiments (approximately 20%–30% for capsaicin injection) are consistent with the signal changes noted in functional imaging experiments of the brain of anesthetized animals at 9.4 T (15, 35).
As previously determined, subsequent activations of pain regions in the brain and spinal cord can be seen with repeated capsaicin injection, provided that sufficient time is allowed between capsaicin injections (15). Therefore, repeated injections were performed in animals in which capsaicin was injected intradermally into the plantar surface of the hindpaw. This allows for a greater amount of information to be acquired, while a minimum number of animal experiments are performed. The reproducibility is evident in the combined activation maps (Fig. 2), illustrating a high degree of overlap in functional activation in the spinal cord between experiments.
We were also interested in the response due to secondary hyperalgesia in rats that underwent intrajoint injection; therefore, only one injection of capsaicin was performed in these animals. Greater overlap was observed in animals after intrajoint injection of capsaicin compared to intradermal injection. This is consistent with a trend towards a greater area of activation in brain regions associated with the processing of pain observed in animals after injection into the ankle compared to hindpaw injection; these regions include the anterior cingulate (bilateral), frontal cortex (bilateral), and hindlimb (ipsilateral) (36).
Mechanical hyperalgesia of the paw is known to occur after intra-articular injection in rats (11), and intradermal injection of capsaicin also produces secondary hyperalgesia (21). Similar areas of activation, as with capsaicin injection, were observed in the spinal cord with the light touch stimulus after hyperalgesia derived from capsaicin injection. Several second messenger pathways in the spinal cord are thought to be involved in secondary mechanical allodynia and hyperalgesia that contributes to the activation mainly in the region of the dorsal horn (21). Studies have shown that protein kinase C inhibitor reverses allodynia but has little or no effect on secondary mechanical hyperalgesia. Intraspinal injections of phorbol esters or intradermal capsaicin injections increase responses of primate spinothalamic tract cells to weak, but not strong, mechanical stimuli (21). As with capsaicin injection, the light touch stimulus produced neuronal activation in the spinal cord with a high degree of overlap observed in the functional activation between rats.
A previous study by Sluka and Wright (11) showed that after capsaicin injection into the rat ankle joint, secondary hyperalgesia develops within two hours, as reflected by sensitivity on the plantar surface of the paw. Subsequent physiotherapy joint mobilization reduced this hyperalgesic effect immediately after manipulation. The secondary hyperalgesia, however, returned to pre-manipulation levels within two hours of the mobilization (11). Therefore, some decrease in either degree or amount of functional MR activation in regions of the spinal cord associated with pain was expected after physiotherapy manipulation with a light touch to the plantar surface of the hindpaw as the stimulus. Figure 3 shows that there does appear to be greater activation in the dorsal horn in animals that did not undergo physiotherapy manipulation, as represented by greater overlap in activation in the region in individual experiments, compared to those in which physiotherapy joint manipulation was performed. It must be noted, however, that this only refers to the region of activation and not the extent of pain or analgesic relief that may be provided by the physiotherapy manipulation.
Interestingly, injection of capsaicin produced an increase in blood pressure, as previously observed (15); however, no increase in blood pressure response was observed with the light touch stimulus, even when clear activations were observed (as a result of mechanical allodynia and hyperalgesia) in regions of the spinal cord associated with the presentation of the stimulus. Similar increases in blood pressure were observed for both injection into the ankle joint and intradermal injection. The percentage signal change upon injection of capsaicin into the anklejoint and hindpaw were similar. Lower signal changes were observed in general with the light touch stimulus compared to capsaicin injection, as expected. Despite greater amounts of overlap of activations in regions of pain in animals that did not undergo physiotherapy manipulation, there were no significant differences in the percentage signal change observed between manipulation and no manipulation during light touch stimulation. Perhaps the amount of activation is similar for the manipulation and no manipulation cases, but different mechanisms are triggered. For instance, it is possible that more afferent fibers are activated in one case and greater, non-painful sensory fiber activation occurs in the other; therefore, percentage signal change is not as important as the actual location of the neuronal activation within the spinal cord.
Negative functional MR changes were consistently observed after capsaicin injection, as well as the light touch stimulus. There are an increasing number of studies utilizing fMRI in the brain that have identified regions of “deactivations” (37–39). While the nature of the BOLD activations reflect neuronal excitation, the origin of this paradigmatic decrease in BOLD signal remains speculative. It is interesting to note, however, that paradigmatic decreases are also observed in functional imaging of the spinal cord, and appear immediately adjacent to the regions of activation (17). It seems likely, therefore, that the mechanism of negative activations results from blood being shunted to regions requiring additional oxygenation as a result of stimulation. The physiologic mechanism involved in negative activation in the spinal cord requires much more thorough examination.
In conclusion, neuronal activation in the spinal cord can be observed after capsaicin injection into the plantar surface of the hindpaw or into the ankle joint. Greater overlap between experiments is observed after injection into the ankle compared to injection into the hindpaw, particularly in the ipsilateral dorsal horn region; however, this does not necessarily reflect greater pain sensation. Functional activation was also noted after hyperalgesia-induced painful stimulation by the light touch stimulus, both with and without joint mobilization. Regions of negative signal intensity changes appear surrounding the regions of functional activation, perhaps representative of blood being shunted from one region of the spinal cord to another during activation. The amount of overlap in functional activation of regions of the spinal cord associated with pain between animals was reduced after physiotherapy manipulation compared to those animals that did not undergo joint mobilization. Functional imaging of the lumbar spinal cord can provide a means of assessing spinal cord injury and is a useful tool in animal models of pain, injury, and therapy.