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Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS-lesioned and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.
A significant percentage of spinal cord injury (SCI) patients report pain, including pain with neuropathic components . Both spontaneous pain and cutaneous hypersensitivity have been reported above, at and below the level of the SCI and the mechanism of neuropathic SCI pain, particularly below-level, appears to have a few similarities with that of peripheral neuropathic pain. For example, the loss of descending tonic inhibition at the level of the spinal dorsal horn leads to hyper-responsiveness of dorsal horn neurons to peripheral stimulation, increased unevoked, spontaneous firing and elevated transcriptional activity [3, 4, 10, 25]. These neurons maybe found several segments distal to the SCI [5, 25]. Restoring bulbospinal inhibition is one method of attenuating neuropathic SCI pain [10, 11].
A second important source of spinal inhibition is GABAergic neurons found in lamina II. These neurons appear to be particularly sensitive to the excitotoxic events following peripheral nerve injury. The excitotoxic cascade that occurs within the spinal cord following nerve injury includes increased extracellular glutamate, dynorphin and other excitatory neurotransmitters, and a concurrent decrease in postsynaptic inhibition, leading to cutaneous hypersensitivity . Animals with a chronic sciatic nerve injury develop increased hind paw sensitivity to noxious and innocuous stimuli in parallel with a significant reduction in dorsal horn GABAergic immunoreactivity .
Since a similar excitatory cascade has been observed following SCI, it is possible that a part of neuropathic SCI pain may be due to a profound dysfunction or loss of inhibitory dorsal horn neurons. To evaluate this possibility, a SCI was induced by unilateral dorsal horn injections of the non-selective glutamate receptor agonist quisqualic acid (QUIS), which leads to persistent neuropathic pain-related behaviors . To induce a prolonged nociceptive stimulus, the hind paw ipsilateral to the lesion was injected with formalin. Formalin injection also evokes the expression of the immediate early gene product Fos, believed to be a marker of neural transcriptional activity . Fos has been implicated in the production of other gene products, such as dynorphin, which maintain the injury cascade and, thus, the pain state . To determine if there is a difference in spinal neural activity between rats that were either QUIS-lesioned or sham-lesioned, spinal Fos immunostaining between these rats was compared. Possible loss of dorsal horn neurons, particularly GABAergic neurons, was also evaluated.
All procedures were reviewed by the University of Miami Animal Care and Use Committee and followed guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats were obtained from Charles River (MA) and were 200–225 g upon arrival. Rats prior to and after surgery were allowed free access to food and water and were housed 2 per cage.
Rats were anesthetized in isoflurane/O2 and the lumbar area of the back shaved. Using aseptic technique, the dorsal thoraco-lumbar spinal cord was exposed via laminectomy. The dura was cut and 0.4 μl of QUIS (125 mM in phosphate-buffered saline; Sigma-Aldrich Co., MO) was injected three times into the right dorsal horn . The injections were spaced 0.5 mm apart and were made 0.5 mm lateral to the midline at a depth of 1 mm (n = 8 QUIS-lesioned rats). In sham-lesioned rats (n = 8), phosphate-buffered saline was injected into the dorsal horn.
Behavioral testing was performed two weeks after surgery. Rats were briefly restrained in a towel as 50 μl of 5% formalin was injected into the plantar hind paw ipsilateral to the dorsal horn surgery. The number of flinches within one minute were counted at five minute intervals and rats were observed for one hour following formalin injection .
Ninety minutes following formalin injection, rats were deeply anesthetized with pentobarbital and perfused with saline followed by phosphate-buffered 4% paraformaldehyde at 4°C . Spinal cords were removed and post-fixed for 24 hours at 4°C. The lumbar segment (L4–L5) was isolated and cut with a cryostat in 10 μm coronal sections. Sections were incubated in rabbit anti-fos antibody (1:5,000; Oncogene Science, MA) overnight at 4°C, rinsed with phosphate-buffered saline and incubated with biotinylated goat anti-rabbit antibody (1:200; Vector Labs, CA). Sections were then incubated in a solution of diaminobenzedine and hydrogen peroxide to visualize the secondary antibody. Profile quantification of 12 randomly selected sections was performed by an observer who was unaware of the source of the sections.
In a subset of randomly selected spinal cord sections from QUIS-lesioned formalin-injected rats, GABA immunocytochemistry was done using a guinea pig polyclonal antibody to anti-GABA (1:100; Protos Immunoresearch, CA) and a monoclonal antibody to neuron-specific nuclear protein (Neu-N; 1:200; Chemicon, CA).
Immunocyctochemistry for these antigens were done to qualitatively illustrate the extent of neuronal changes following QUIS injection.
To determine statistical significance, a one-way ANOVA was performed on data from the treatment groups and Dunnett’s test was used for post-hoc comparisons. Statistical significance was taken at P < 0.05.
Following formalin injection, rats with either a QUIS or sham lesion displayed bi-phasic hind paw flinching (Fig. 1). Sham-lesioned rats displayed decreased flinching towards the end of the observation period, whereas QUIS-lesioned rats displayed sustained flinching (P < 0.05 vs. sham).
Fos immunoreactivity was visualized as a dark coloration limited to the nucleus of the neuron [4, 20]. Ipsilateral to formalin injection, there were numerous Fos immunoreactive profiles in superficial (laminae I–II) and deep (laminae III–V) dorsal horn and around central canal (lamia X) (Fig. 2). In the contralateral spinal cord, there were a few Fos-immunoreactive profiles found in superficial and deep dorsal horn. No significant difference in Fos-immunoreactive profiles was observed between QUIS and sham-lesioned rats (Fig 2; P > 0.05).
Injection of 50 μl of saline into the ipsilateral hind paw in QUIS-lesioned rats did not lead to significant hind paw flinching (n = 6; data not shown). Also, hind paw saline injection ipsilateral did not induce Fos immunoreactivity in either QUIS-lesioned or sham-lesioned rats (data not shown). Thus, the induction of Fos was dependent on a robust formalin-mediated nociception .
A reduction of Neu-N immunoreactivity confirms previous observations that QUIS injection into the dorsal horn lead to a marked loss of ipsilateral dorsal grey matter, in particular, deep dorsal horn laminae IV–V (Fig. 3; [23, 24]). Compared to the contralateral dorsal horn, the reduction of Neu-N immunoreactivity following QUIS injection indicated large-scale destruction of neurons. (Injection of an equal volume of vehicle did not affect either the morphology of deep dorsal horn (data not shown).) With the general loss of neurons, a paucity of GABAergic profiles was also apparent (Fig. 3), compared to the abundance of GABAergic profiles in the contralateral dorsal horn. GABAergic axons were present in both the ipsilateral and contralateral dorsal horn, possibly from bulbospinal neurons, indicating the selectivity of QUIS for soma over axons .
Formalin-evoked spinal dorsal horn neuron activation, as indicated by Fos-immunoreactivity, was similar between QUIS- and vehicle-lesioned rats. Thus, despite a lesion that led to a marked decrease in dorsal horn neurons, a considerable number of neurons were still activated by peripheral noxious stimulation. Towards the end of the observation period, significant hind paw flinching was observed in rats with a QUIS lesion, whereas rats with a sham lesion showed decreased flinching. Since the neural activity, suggested by Fos immunoreactivity, was similar between QUIS- and sham-lesioned rats, the sustained duration of flinches could be due to a reduction of spinal inhibition.
The loss of extrinsic and intrinsic inhibition at the level of the dorsal horn following SCI leads to exaggerated responses of dorsal horn neurons and the development of neuropathic SCI pain. A loss of GABAergic tone was clearly evident following a spinal cord contusion injury and a significant decrease in GABAergic neurons is observed after a transient spinal cord ischemia, both of which were accompanied by prominent below-level cutaneous hypersensitivity [6, 26]. In the case of transient ischemia, as the severity of cutaneous hypersensitivity decreases over time, the number of GABAergic neurons in the dorsal horn increased to eventually return to control-uninjured levels. A significant drop in spinal GABA inhibition was observed in other chronic neuropathic pain models, which appears to be reversed with either treatment or over time [14, 19].
It is possible that diminished numbers of specific dorsal horn neurons occurred following QUIS lesioning. Since QUIS receptors are found in lamina II and GABAergic neurons are also found in this lamnia, QUIS injection may lead to a significant attenuation of GABAergic neurons . In the current study, there was a striking diminution of GABAergic profiles in the ipsilateral dorsal horn. Combined with the decreased Neu-N immunostaining, the data suggest that GABAergic interneurons may be particularly sensitive to QUIS. Alternatively, diminished GABA synthesis, rather than neural death, may have occurred following QUIS injection. Zhang et al. pointed out that following spinal ischemia there was no evidence of “morphological damage” and that the number of GABAergic neurons in their study recovered to pre-injury levels . More extensive morphological and neurochemical studies may be able to determine the extent to which QUIS causes either outright GABAergic cell loss or dysfunction.
In parallel with the loss of inhibition, there is increased activation of dorsal horn neurons in neuropathic SCI pain . Elimination of bulbospinal descending inhibition by a complete thoracic level transaction significantly increased formalin-evoked Fos, bilaterally, compared to Fos levels in sham-transected rats . In rats following a severe contusion injury of the thoracic spinal cord, which eliminates most descending tracts, formalin-evoked dorsal horn Fos was greater than in uninjured rats [1, 2]. Following either QUIS injection or contusion, abnormal evoked activity and increased unevoked background activity have been noted in dorsal horn neurons, which mediate, in part, below-lesion cutaneous hypersensitivity [6, 25]. The Fos-immunoreactive profiles in the current study could be markers of dysfunctional interneurons or supraspinally projecting neurons .
Dorsal horn Fos levels were similar between QUIS- and sham-lesioned rats following formalin injection which contrasts to observations from other types of SCI. There are at least two possibilities that could explain the lack of difference in the current study. First, although the number of dorsal horn inhibitory neurons may have diminished following QUIS lesioning, descending tracts were sufficiently intact to allow for some inhibition at the level of the dorsal horn, thus suppressing a potential additional increase in Fos expression . However, QUIS-lesioned rats displayed a prolonged duration of hind paw flinching, which suggests that descending inhibition may have not been entirely effective in suppressing sustained neural activation due to injury-induced plasticity of the (e.g.) serotonergic system .
The second possibility is that Fos does not directly reflect the actual nociceptive state and that other molecular markers may more direct indicators. For example, in spinally transected rats, formalin-evoked Fos was greatly increased but flinching was significantly less compared to control rats . In contusion-injured rats, although Fos was increased following formalin injection, it is not known if behavioral response were greater or less than that of uninjured rats (rats were anesthetized) . The lack of Fos expression in QUIS-lesioned rats injected in the hind paw with saline suggests a lack of basal activity of dorsal horn neurons, yet robust spontaneous activity and prolonged after-discharges to innocuous cutaneous stimulation have been reported in these rats . Indeed, vigorous stimulation, such as visceral distention and electrical stimulation, in addition to formalin injection, is needed to induce dorsal horn Fos expression [2, 4, 16]. By contrast, extracellular recordings demonstrated that hind paw brushing evoked significant exaggerated neural responses, but the same stimulus did not evoke Fos expression [2, 6, 25]. Similar levels of Fos in spinal dorsal horn have been reported following innocuous mechanical stimulation and no stimulation above the contusion injury . Also, studies in non-SCI rats which directly suppressed spinal Fos expression demonstrated conflicting behavioral responses to formalin [12, 13]. Other molecular markers, such as extracellular signal-regulated kinase (ERK), may display greater distinction between the injured and uninjured state to a given stimulus and also reveal the involvement of non-neural cells and the associated injury cascade in neuropathic SCI pain [5, 27].
The current data demonstrated a prolonged hind paw response to noxious stimulation in rats with a dorsal horn QUIS lesion. Based on findings in other SCI models, the exaggerated responses to noxious stimuli in the current study and in clinical SCI suggest a significant loss of segmental inhibition in conjunction with increased excitation . Thus, to restore inhibitory tone as a long-term method of reducing neuropathic SCI pain, one could consider either enhancing recovery of intrinsic inhibitory neurons or transplanting GABA secreting cells [8, 14].
We thank Ms. C.D. Carrasco and Dr. S. Chen for expert technical assistance. Sponsored by The Miami Project to Cure Paralysis and by NIH grant NS51667.
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