Injury to peripheral nerves arising from disease or physical trauma leads to abnormal ‘neuropathic’ pain states. In animal models, this long-lasting syndrome is principally characterized by the emergence of tactile allodynia and thermal hyperalgesia, conditions reminiscent of human neuropathic pain states. Allodynia indicates a nociceptive-like response to stimuli which are normally not painful, such that light touch might be interpreted as noxious, and hyperalgesia is defined as increased nociceptive response to a normally noxious stimulus (Merskey and Bogduk, 1994; Payne, 1986). Importantly, convergent evidence has accumulated to indicate that these pathological manifestations of neuropathic pain may be mediated by different neuronal pathways, which may help explain the apparent resistance of tactile allodynia to treatment by opioids. One indication is that morphine given spinally is inactive against tactile allodynia, even at supramaximal anti-nociceptive doses in normal conditions, yet is able to modulate tactile allodynia when given either systemically or intracerebroventricularly (Bian et al., 1995, 1999; Lee et al., 1995). In contrast, spinal morphine completely blocks thermal nociception in nerve-injured rats, and produces antinociception, although with decreased potency (Bian et al., 1995, 1999; Mao et al., 1995). Transection of the spinal cord at T8 has been shown to block tactile allodynia in rats with SNL of the L5 and L6 spinal nerves or of the sacral spinal nerves without abolishing thermal hyperalgesia (Bian et al., 1998; Malan et al., 2000; Sung et al., 1998). These animals retained spinal reflexes to nociceptive pinch or thermal stimuli, indicating that loss of allodynia was probably not due to immobility of the hindlimb. Spinal transection attenuated the mechanical hyperexcitability of wide dynamic range neurons as well as tactile allodynia in response to the application of mustard oil to the hindpaw, suggesting that a supraspinal system is involved in mechanical hyperalgesia (Mansikka and Pertovaara, 1997; Pertovaara, 1998).
There is also considerable evidence that tactile allodynia is mediated chiefly through large diameter, Aβ afferent fibers whereas thermal hyperalgesia is likely to be mediated through small diameter, unmyelinated C-fibers. The long-term selective desensitization of C-fibers with resiniferatoxin (RTX) produced thermal hypoalgesia but did not alter nociceptive mechanical thresholds in normal rats (Xu et al., 1997). Wind-up induced by conditioning stimuli of C-fibers was also attenuated by RTX (Xu et al., 1997), and reflected RTX-induced desensitization of capsaicin-sensitive C-fibers. The systemic injection of RTX also resulted in long-term loss of sensitivity to thermal stimuli in rats with L5/L6 SNL; however, tactile allodynia was not reduced by RTX (Ossipov et al., 1999). In a similar type of experiment, rats that received capsaicin neonatally, which significantly reduces C-fibers, failed to develop thermal hyperalgesia, but did show heightened responses to mechanical nociceptive stimuli after chronic constriction injury (CCI of the sciatic nerve) (Shir and Seltzer, 1990).
The large myelinated fibers are normally involved in mediation of non-noxious sensory inputs through the dorsal columns projecting ipsilaterally to second order cells in the brainstem, including cells in the nucleus gracilis and nucleus cuneatis. Transection of the sciatic nerve has led to increased expression of preprotachykinin in the nucleus gracilis, probably as a result of de novo synthesis in large diameter, myelinated afferent fibers projecting directly into this region (Noguchi et al., 1995). The observation of elevated CGRP in the terminal regions of the afferent fibers (i.e. the dorsal column nuclei) demonstrate a supraspinal role in neuronal plasticity following peripheral nerve injury (Miki et al., 1998). Supraspinal increases in preprotachykinin mRNA in projection neurons caused by inflammation were interpreted as evidence that tachykinins may act as neurotransmitters at supraspinal sites through projection pathways (Noguchi and Ruda, 1992). Furthermore, there exists evidence that ascending pathways through the dorsal column nuclei activate descending nociceptive modulatory pathways by way of the rostroventral medial medulla (RVM) (Rees and Roberts, 1993). In light of these observations, it seemed reasonable to propose that peripheral nerve injury may indeed cause neuroplastic changes at supraspinal loci involved in sensory pathways. The present experiments were therefore designed to further explore the role of the spinal dorsal columns and one site of neuronal termination, the nucleus gracilis, in the manifestation of tactile allodynia following SNL.
Male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 200–300 g at the time of testing, were maintained in a climate-controlled room on a 12 h light/dark cycle (lights on at 06:00 h) and food and water were available ad libitum. All of the testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain (IASP) and the National Institutes of Health (NIH) guidelines for the handling and use of laboratory animals and received approval from the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona.
2.1. Spinal nerve ligation
Spinal nerve ligation (SNL) injury was induced using the procedure of Kim and Chung (1992). Anesthesia was induced with 2% halothane in O2 at 2 l/min and maintained with 0.5% halothane in O2. After surgical preparation of the rats and exposure of the dorsal vertebral column from L4 to S2, the L5 and L6 spinal nerves were tightly ligated distal to the dorsal root ganglion using 4-0 silk suture. The incision was closed and the animals were allowed to recover for 5 days. Rats that exhibited motor deficiency (such as paw dragging) or failure to exhibit subsequent tactile allodynia were excluded from further testing (less than 5% of the animals were not used). Sham control rats underwent the same operation and handling as the experimental animals, but without SNL.
2.2. Surgical preparation
All rats were prepared for drug microinjection into the nucleus gracilis by placing anesthetized (ketamine/xylazine 100 mg/kg, i.p.) animals in a stereotaxic headholder. For intracranial drug administrations, the skull was exposed and a 26-gauge guide cannula (Plastics One Inc., Roanoke, VA) was directed toward the nucleus gracilis (AP −5.5 mm from interaural line, ML ±0.5 mm from midline, 2 mm above the interaural line); these coordinates were obtained from the atlas of Paxinos and Watson (1986). The guide cannula was cemented in place and secured to the skull by small stainless steel machine screws. The animals were allowed to recover for 5 days post-surgery before any pharmacological manipulations were made. Lidocaine (4% w/v) was microinjected in a volume of 0.5 μl through a 33-gauge injection cannula inserted through the guide cannula and protruding an additional 1 mm into fresh brain tissue to prevent backflow of drug into the guide cannula. At the termination of all experiments, the animals were sacrificed, Toluidine Blue dye was microinjected into the region and cannula placement was verified histologically. Only those animals with correct cannula placement were included in behavioral analysis.
2.3. Tactile allodynia
The assessment of tactile allodynia (i.e. decreased threshold to paw withdrawal following probing with non-noxious mechanical stimuli) consisted of measuring the withdrawal threshold of the paw ipsilateral to the site of nerve injury in response to probing with a series of calibrated von Frey filaments. Each filament was applied perpendicularly to the plantar surface of the ligated paw of rats kept in suspended wire-mesh cages. Measurements were taken both before and after administration of drug or vehicle. The withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength (‘up–down’ method), analyzed using a Dixon non-parametric test (Chaplan et al., 1994) and expressed as the mean withdrawal threshold.
2.4. Thermal hyperalgesia
The method of Hargreaves et al. (1988) was employed to assess paw withdrawal latency to a thermal nociceptive stimulus. Rats were allowed to acclimate within a plexiglass enclosure on a clear glass plate maintained at 30°C. A radiant heat source (i.e. high intensity projector lamp) was activated with a timer and focused onto the plantar surface of the hindpaw of a rat. The paw withdrawal latency was determined by a motion detector that halted both lamp and timer when the paw was withdrawn. The latency to withdrawal of the paw from the radiant heat source was determined both before and after drug or vehicle administration. A maximal cut-off of 40 s was employed to prevent tissue damage.
2.5. Spinal lesions
Spinal hemisections at the T8 level were performed in halothane-anesthetized nerve-ligated or sham-operated rats. A laminectomy was made at the T8 level to expose the spinal cord and a 1 mm section of the spinal cord was removed. Hemostasis was confirmed and the wound over the exposed spinal cord was packed with gelfoam and closed. Rats with spinal hemisections were monitored for signs of shock after recovery and the bladder was periodically expressed manually. These rats were tested 24 h following spinal transection. Dorsal column lesions were performed with the aid of a dissecting microscope and by careful incision with the point of a #11 scalpel blade. Sham spinal surgery was performed by exposing the vertebrae and performing the laminectomy, but without cutting any neuronal tissue. All lesions were verified histologically at the termination of the experiment by fixing the spinal sections obtained from the lesion site in paraffin. Sections (40 μm thick) were mounted and stained with Luxor Fast Blue myelin stain to visualize intact and disrupted white matter. Behavioral results were included in analysis only in animals that had an appropriately placed hemisection or dorsal column lesion.
Rats with L5/L6 SNL demonstrated tactile allodynia with paw withdrawal thresholds to probing with von Frey filaments in the range of 0.87±0.15 g (Fig. 1), a value significantly lower than their pre-SNL baseline (15±0 g). Hemisection of the spinal cord ipsilateral to SNL resulted in a complete blockade of tactile allodynia. Paw withdrawal thresholds were significantly (P≤0.05) increased to 15±0 g (Fig. 1). The absence of response to von Frey filaments was not likely to be due to hindpaw paralysis since noxious pinch to the toes still produced a vigorous flexor reflex response in these animals. Spinal hemisection performed contralateral to SNL did not alter tactile allodynia; withdrawal thresholds were 2.06±0.21 g after hemisection (Fig. 1). Likewise, sham section did not alter response thresholds to probing with von Frey filaments (Fig. 1).
Bilateral lesions of the dorsal columns produced a complete blockade of tactile allodynia in SNL rats (Fig. 2). Paw withdrawal thresholds were significantly (P≤0.05) increased from a mean of 1.12±0.25 g before dorsal column lesion to 14.2±0.82 g after dorsal column lesion. The rats appeared normal in behavior and gait, with no apparent motor deficits. Lesions restricted to the dorsal columns ipsilateral to SNL also resulted in a reversal of tactile allodynia (Fig. 3). Paw withdrawal thresholds were significantly (P≤0.05) increased to 12.3±1.7 g from a pre-lesion value threshold of 1.06±0.13 g. Restriction of the dorsal column lesion to the side contralateral to SNL did not affect tactile allodynia (Fig. 3). The paw withdrawal thresholds were 1.75±0.23 and 1.37±0.19 g before and after contralateral dorsal column lesion, respectively, changes which were not significant.
The implantation of guide cannulae directed toward the nucleus gracilis did not produce any overt changes in behavior or mobility of the rats. The microinjection of 0.5 μl of lidocaine (4% w/v) into the nucleus gracilis on the side ipsilateral to SNL blocked tactile allodynia (Fig. 4a). Paw withdrawal thresholds were significantly (P≤0.05) increased from 1.85±3.2 to 14±1.0 g within 10 min of the microinjection. The anti-allodynic action of lidocaine was reversible, and paw withdrawal thresholds returned to baseline within 30 min of microinjection (Fig. 4a). The microinjection of normal saline (0.5 μl) into the nucleus gracilis did not produce any changes over the same time period. Neither lidocaine nor saline microinjected into the nucleus gracilis contralateral to SNL had any effect on paw withdrawal thresholds in SNL rats. Thresholds remained in the range of 1.2±0.15 and 1.6±0.26 g over the 60 min observation period (Fig. 4a).
Thermal sensitivity was also tested after microinjection of lidocaine into the nucleus gracilis ipsilateral to SNL. Rats with SNL demonstrated significantly (P≤0.05) decreased paw withdrawal latencies to radiant heat, which is indicative of thermal hyperalgesia. The mean baseline paw withdrawal latency of the SNL rats was 10.7±0.40 s, whereas that of the sham-operated group was 18.4±0.79 s. The microinjection of lidocaine into the nucleus gracilis did not produce any changes in paw withdrawal latencies over the 60 min observation period in either group of rats (Fig. 4b).
The results of the present study extend our previous observations that supraspinal modulation is required for the manifestation of light tactile input as allodynia after peripheral nerve injury. The requirement for supraspinal input is consistent with observations involving transection of the spinal cord which indicated a block of tactile allodynia, though without affecting thermal hyperalgesia, in SNL rats (Bian et al., 1998; Mansikka and Pertovaara, 1997; Pertovaara, 1998; Sung et al., 1998). It seems possible that both supraspinal and spinal mechanisms may mediate thermal hyperalgesia, while tactile allodynia is mediated predominately as a result of ascending inputs through supraspinal loci. The observations reported here are also consistent with the evidence indicating that tactile allodynia is chiefly mediated through the large diameter, myelinated Aβ afferent fibers, whereas thermal hyperalgesia is mediated through the unmyelinated C-fiber nociceptors (Ossipov et al., 1999; Shir and Seltzer, 1990). The C-fibers are generally regarded as primary afferent nociceptors, and are a logical target for the treatment of abnormal pain. Indeed, thermal hyperalgesia is readily managed by opioids, and opioid receptors are present on the peripheral and central terminals of these afferent fibers (Besse et al., 1990; Mansour et al., 1994). In contrast, tactile allodynia is much more difficult to manage, is generally non-responsive to opioids, and this observation is consistent with the apparent lack of opioid receptors on large fibers (Zhang et al., 1998).
An important observation made here is that tactile allodynia was blocked by a spinal hemisection made ipsilateral, but not contralateral, to SNL. If allodynia was transmitted to supraspinal sites through the spinothalamic pathways that are normally associated with ascending nociceptive input, then one would expect that contralateral, rather than ipsilateral, hemisection would block tactile allodynia. Such would be the case if, for instance, the afferent large diameter Aβ fibers were to form abnormal collateral sprouts and innervate the superficial laminae of the spinal cord. In this case, these abnormal physiopathic synapses would form with second-order neurons which would be expected to decussate across the spinal cord and transmit touch-evoked nociception through the spinothalamic tract. An alternate route to supraspinal sites involves the spinal dorsal columns. The large diameter sensory afferents are known to enter through the dorsal horn of the spinal cord and project ipsilaterally through the dorsal columns, either as direct projections or through post-synaptic neurons that also project through this pathway (Willis and Westlund, 1997). Although generally involved in the transmission of light touch and vibration, these pathways have also recently been shown to transmit visceral nociceptive input as well (Willis and Westlund, 1997). More importantly, however, is the observation that the dorsal columns may transmit abnormal pain. Recently, it was demonstrated that lesions of the dorsal column blocked tactile allodynia in a model of bone pain, but without altering behaviors suggestive of spontaneous pain (Houghton et al., 1999). It was therefore interpreted that supraspinal input through the dorsal columns was essential for the development of tactile-evoked pain (Houghton et al., 1999). The observations presented here further extend the role for ascending input along the dorsal columns in the mediation of allodynia. In the present study, we also found that ablation of only the ipsilateral dorsal column blocked tactile allodynia after SNL, consistent with the projections of the dorsal columns. It is unclear whether such ascending input is the result of direct projections to dorsal column nuclei, or whether a synapse occurs within the dorsal horn, or both. Spinal morphine clearly blocks thermal hyperalgesia following SNL, and it does not alter tactile allodynia providing some basis for a direct projection of large fibers. However, conflicting evidence suggests the possibility of blockade of tactile allodynia by spinal NMDA (Chaplan et al., 1997) or AMPA (Nichols, Ossipov and Porreca, unpublished data) antagonists, or even a high-efficacy μ-opioid agonist (DAMGO) (Nichols et al., 1995), suggesting the need for further experimentation prior to drawing firm conclusions on this issue.
The supraspinal terminals of the dorsal columns are the nucleus cuneatus and nucleus gracilis, with the latter encountering the input arising from the caudal (i.e. lumbar) sites (Miki et al., 2000). Although inputs to the nucleus gracilis are generally considered to be of the non-noxious, light tactile type (Willis and Westlund, 1997), it has been found that the nucleus gracilis may act as a relay for mechanical nociceptive input from the spinal cord through to the thalamic nuclei after peripheral nerve injury (Miki et al., 2000). The nucleus gracilis may also act as a site where nociceptive visceral and somatic inputs are integrated (Al-Chaer et al., 1997); therefore, the possibility that the nucleus gracilis may also act in ascending nociceptive inputs seems reasonable (Willis et al., 1999). Neuroplastic changes in the nucleus gracilis have also been noted after peripheral nerve injury (Noguchi et al., 1994). The results of the present studies extend these observations and more closely link the dorsal column and associated nucleus gracilis to tactile allodynia. The finding here that lidocaine microinjected into the ipsilateral nucleus gracilis blocked tactile allodynia supports this hypothesis. The fact that microinjection of lidocaine into the contralateral nucleus gracilis did not alter allodynia strongly indicates that the effects observed were not because of a generalized local anesthetic effect within the CNS, but localized within the specific nucleus that was injected. Furthermore, the lack of effect of lidocaine on thermal nociception or hyperalgesia is consistent with the expectation that these inputs are transmitted through the spinothalamic tract and not via the dorsal column nuclei.
Since nociceptive processes, whether arising from ‘normal’, acute defensive nociceptive stimuli or due to neuropathic origins, involve several levels within the CNS, then the possibility exists that supraspinal descending modulation, including activation of facilitatory influences from the RVM, may ultimately act to provoke the decreased withdrawal thresholds to tactile stimuli seen after SNL. In this regard, it should be noted that projections of the spinothalamic tract and the dorsal column/medial lemniscus converge on the same neurons of the ventrobasal thalamus (Ma et al., 1987). This convergence provides a basis for an interaction between somatosensory and nociceptive inputs at supraspinal centers. In fact, it has also been proposed that an ascending loop may occur through the anterior pretectal nucleus, which gives rise to descending modulatory influences (Rees and Roberts, 1993). Further, evidence exists in the nerve-injured rat to indicate that tactile allodynia is likely mediated through large diameter, myelinated afferent fibers (Ossipov et al., 1999; Shir and Seltzer, 1990), which would be expected to reach the thalamus via the dorsal column-lemniscal pathway. Thus, regardless of the site of origin of nociceptive input, the same descending cascade activating spinopetal facilitatory and inhibitory mechanisms may be activated ‘downstream’ from the ventrobasal thalamus. Therefore, it is conceivable that ascending tactile input may ultimately be perceived as nociceptive and activate descending influences that may involve the RVM. This supposition is supported by our previous observations showing a blockade of nerve injury-induced allodynia and hyperalgesia by administration of lidocaine in the RVM (Kovelowski et al., 2000). Such findings also lend support to the concept of descending modulation of both allodynia and hyperalgesia, in spite of apparently different ascending pathways.
In conclusion, it has become apparent that two principal manifestations of neuropathic pain, tactile allodynia and thermal hyperalgesia, are likely to be mediated through separate mechanisms and neuronal pathways. Although many spinal and peripheral changes have been considered in the studies underlying mechanisms of neuropathic pain, the supraspinal consequences of peripheral nerve injury remain largely unknown. It has become clear that the terminations of the dorsal columns are important to the manifestation of tactile allodynia. However, the multiple interconnections between the dorsal column nuclei and other supraspinal sites involved in somatosensory and nociceptive processing after peripheral and/or central nerve injuries remain to be elucidated.
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