Free
Education  |   November 2003
Cyclooxygenase-1 in the Spinal Cord Is Altered after Peripheral Nerve Injury
Author Affiliations & Notes
  • Xiaoying Zhu, M.D.
    *
  • James C. Eisenach, M.D.
  • *Graduate Student, Program in Neuroscience, †F.M. James III Professor of Anesthesiology, Wake Forest University School of Medicine.
  • Received from the Program of Neuroscience, Department of Anesthesiology, and Center for the Study of Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
Article Information
Education
Education   |   November 2003
Cyclooxygenase-1 in the Spinal Cord Is Altered after Peripheral Nerve Injury
Anesthesiology 11 2003, Vol.99, 1175-1179. doi:
Anesthesiology 11 2003, Vol.99, 1175-1179. doi:
NEUROPATHIC pain is a physically and emotionally debilitating condition for which treatment is often inadequate. The mechanisms that underlie neuropathic pain are poorly understood, but several animal models have been developed to probe these mechanisms. Partial sciatic nerve transsection (PSNT) and L5–L6 spinal nerve ligation (SNL) in rats, two widely used models, result in hyperalgesia (i.e.  , increased pain intensity in response to noxious stimuli) and allodynia (i.e.  , pain in response to normally innocuous stimuli). 1,2 
Prostaglandins are synthesized in the spinal cord during acute nociceptive stimulation, 3 peripheral inflammation, 4–7 intrathecal injection of substance P 8 or kainic acid, 9 and mechanical injury to the spinal cord. 10,11 Cyclooxygenase (COX), the rate-limiting enzyme in prostaglandin synthesis, is constitutively expressed as isoenzymes COX-1 and COX-2 in the spinal cord. 4,12,13 Accumulating evidence indicates that COX-2 plays an important role in hypersensitivity induced by peripheral inflammation. 5,12,14–17 Thus, COX-2 but not COX-1 mRNA and protein are increased in spinal cord homogenates after peripheral inflammation, and hypersensitivity is prevented or treated by intrathecal injection of selective COX-2 but not COX-1 inhibitors.
The role of COX-1 and COX-2 in neuropathic pain remains unclear and has received little attention. Intrathecal injection of the nonselective COX inhibitor, indomethacin, near the time of peripheral nerve injury delays for many days the onset of hypersensitivity, 18 and intrathecal injection of a selective COX-1 inhibitor at the time of nerve injury permanently inhibits the development of hypersensitivity. In addition, intrathecal injection of a COX-1-preferring inhibitor, ketorolac, is effective in attenuating thermal hyperalgesia and cold allodynia induced by sciatic nerve injury in rats 19 and reverses tactile allodynia induced by partial sciatic nerve ligation. 20 Based on these observations, we tested whether COX-1 expression is altered in the spinal cord in animal models of neuropathic pain. We previously observed no change in spinal COX-1 expression after SNL, 18 but that result was in tissue homogenates. The purpose of the current study was to examine whether localized changes in COX-1 expression occur after PSNT or SNL injury, using immunohistochemistry.
Materials and Methods
Male Sprague-Dawley rats (220–260 g) were used in this study with the approval of the Animal Care and Use Committee of Wake Forest University.
Rats were placed in clear plastic cages on an elevated mesh floor and allowed to accommodate for 30 min. Paw withdrawal threshold in response to probing with von Frey filaments was measured using the up-and-down method as previously described. 21 One group of rats was then anesthetized with 2–3% halothane and the left sciatic nerve partially transected using a slightly modified procedure of that previously described. 1,2 Briefly, the sciatic nerve was exposed at the midthigh level, and the medial half of the nerve was transected with finely tipped scissors at a point just proximal to the branch running to the musculus biceps femoris. The skin was then sutured and the animals recovered in their cages. Another group of rats underwent L5–L6 SNL as described by Kim and Chung. 22 The left L5 and L6 spinal nerves were isolated and ligated tightly with 5-0 silk suture. Sham control animals received the same anesthesia and incision in the skin and muscles, without any manipulation of the nerves. Three or four rats were used in each group, and a group of three normal rats was also studied.
Because tactile allodynia develops in rats within 1 week after L5–L6 SNL 22 and 2 weeks after PSNT 1 surgery, and remains stable for 4 weeks, rats with PSNT or sham operation were perfused 4 weeks after surgery. Rats with SNL or sham operation were perfused 4 h, 4 days, and 2 weeks after surgery to determine the time course of changes. Before perfusion, paw withdrawal threshold to von Frey filaments was measured. Rats were then deeply anesthetized with pentobarbital and perfused transcardially with 0.01 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in ice-cold 0.1 M phosphate buffer (PB) (pH 7.4). The caudal lumbar spinal cords were removed and postfixed in the same fixative for 3 h. After cryoprotection in 30% sucrose in 0.1 M PB at 4°C for 24 h, these tissues were cut on a cryostat at a 40 μm thickness.
A series of one of every 10 sections were collected in PBS. After pretreatment with 0.3% H2O2, sections were blocked with 5% normal goat serum, incubated for 24 h at 4°C in a mouse monoclonal antiovine COX-1 antibody (1:2000; Cayman, Ann Arbor, MI) diluted in PBS containing 0.3% Triton-X 100 (PBS+T), and 5% normal goat serum. Subsequently, the sections were sequentially incubated with secondary goat anti-mouse antibody and ABC reagent (Vectastain ABC; Vector, Burlingame, CA) according to the instructions of the manufacturer. Between incubations, sections were washed twice in PBS+T for 10 min. Finally, the immunoprecipitates were developed by 3,-3′ diaminobenzidine and enhanced by nickel. After immunostaining, sections were dehydrated in ethanol and cleared xylene, cover-slipped, and examined by light microscopy.
For quantification of COX-1-immunoreactive cells (COX-1-IR) in the spinal cord of normal and SNL rats, four sections at the L5 level were chosen from each rat and digitally imaged. The L5 level was chosen because changes in immunostaining were greatest at this level, tapering to no change two or three dermatomes cephalad and caudad. An area with the same size was specified in the superficial laminae (I, II, and III) and deep laminae (IV, V, and VI) in spinal dorsal horns. By using Sigma Scan (Jandel Scientific, Carpinteria, CA), the COX-1-IR cells were automatically counted using a fixed threshold for all sections from the normal, 4-h, and 2-week groups. The threshold for the 4-day group was reduced to include the lightly stained cells, because at this time point, all the positively stained cells were lightly stained. Each data point is compared to that in the baseline (normal) rats. Data are presented as mean ± SEM and were analyzed using one-way ANOVA followed by a Tukey test. P  < 0.05 was considered to be significant.
Results
Paw withdrawal threshold decreased from 37.33 ± 0 g before surgery to less than 4 g 4 weeks after PSNT and 2 weeks after SNL. Four days after surgery, rats with SNL showed slight allodynia with a paw withdrawal threshold of 17.11 ± 6.58 g.
COX-1 was constitutively expressed in the spinal cord in normal rats (fig. 1A). COX-1-IR was located in cells with glial morphology throughout the spinal cord gray and white matter and in cells with motor neuron morphology in the ventral horn. High magnification of the glial-like profiles showed that COX-1-IR extended from the cell body cytoplasm to the processes (fig. 1B and C).
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D  ) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar  :A, D  , 200 μm; B, C, E  , and F  , 20 μm.
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D 
	) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E 
	) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F 
	) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar 
	:A, D 
	, 200 μm; B, C, E 
	, and F 
	, 20 μm.
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D  ) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar  :A, D  , 200 μm; B, C, E  , and F  , 20 μm.
×
Partial sciatic nerve transsection dramatically increased the number of COX-1-IR glial-like profiles in the superficial laminae of the ipsilateral spinal dorsal horn of L4–L6 spinal cord (fig. 1D). High magnification showed that these COX-1-IR profiles showed the same morphology as those in normal controls (fig. 1E). Interestingly, PSNT concomitantly decreased COX-1-IR in the deep dorsal horn, ventral horn, and white matter. Indeed, after PSNT, there were no COX-1-IR profiles in the deep dorsal horn (fig. 1D and F). PSNT did not affect COX-1-IR staining in the spinal cord contralateral to injury (fig. 1D).
Spinal nerve ligation resulted, 2 weeks after injury, in a pattern of change in COX-1-IR similar to that observed after PSNT. Four hours after SNL, COX-1-IR was no different than normal and sham-operated rats (fig. 2A and Bcompared with fig. 1A). Four days after SNL, the number of COX-1-IR cells increased diffusely throughout the entire spinal cord ipsilateral to injury. These cells were lightly stained, and no intensely stained cells were observed (fig. 2C and D). Two weeks after SNL, the pattern of change in COX-1-IR in the ipsilateral spinal cord was the same as that seen in the rats with PSNT. COX-1-IR profiles in the superficial dorsal horn ipsilateral to nerve injury were increased in number compared with those in sham-operated rats, and at this time, the intensity of staining was greater than that 4 days after injury. In the deep dorsal horn, COX-1-IR disappeared, and in the ventral horn and white matter, COX-1-IR was dramatically decreased (figs. 1D and 2D–F). Quantification of areas of immunostaining for COX-1 in the spinal cord showed statistical significance for these changes (fig. 3). High magnification showed the COX-1-IR profiles in sham-operated and SNL rats at all the time points tested had the same morphology as that in normal rats (figs. 1B and C, and 4). The contralateral spinal cord showed no difference from normal and sham-operated rats (figs. 1D, 2B and D, and F).
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B  ) Sham-operated and SNL rats 4 h after surgery. (C, D  ) Sham-operated and SNL rats 4 days after surgery. (E, F  ) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole  in the ventral white matter  . The area of quantification in the superficial and deep laminae is indicated by the upper  and lower rectangles  in F  . Scale bar  , 200 μm.
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B 
	) Sham-operated and SNL rats 4 h after surgery. (C, D 
	) Sham-operated and SNL rats 4 days after surgery. (E, F 
	) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole 
	in the ventral white matter 
	. The area of quantification in the superficial and deep laminae is indicated by the upper 
	and lower rectangles 
	in F 
	. Scale bar 
	, 200 μm.
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B  ) Sham-operated and SNL rats 4 h after surgery. (C, D  ) Sham-operated and SNL rats 4 days after surgery. (E, F  ) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole  in the ventral white matter  . The area of quantification in the superficial and deep laminae is indicated by the upper  and lower rectangles  in F  . Scale bar  , 200 μm.
×
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar  ) and deep laminae (IV, V, and VI) (open bar  ) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar  represents the mean, and error bars  indicate the SEM. Significant differences between the each time point and baseline are indicated by **P  < 0.01, ***P  < 0.001 (one-way ANOVA) (n = 3 in each group).
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar 
	) and deep laminae (IV, V, and VI) (open bar 
	) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar 
	represents the mean, and error bars 
	indicate the SEM. Significant differences between the each time point and baseline are indicated by **P 
	< 0.01, ***P 
	< 0.001 (one-way ANOVA) (n = 3 in each group).
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar  ) and deep laminae (IV, V, and VI) (open bar  ) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar  represents the mean, and error bars  indicate the SEM. Significant differences between the each time point and baseline are indicated by **P  < 0.01, ***P  < 0.001 (one-way ANOVA) (n = 3 in each group).
×
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar  , 20 μm.
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar 
	, 20 μm.
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar  , 20 μm.
×
Discussion
Consistent with previous investigations, 4,12,13,23,24 the current study shows that COX-1 is constitutively expressed in the spinal cord. Aside from the large motor neuron COX-1-IR profiles, the remaining small COX-1-IR cells are evenly distributed in the gray and white matter. This distribution and cell morphology suggest that these cells are glia, in agreement with previous observations of COX-1-IR in mouse spinal cord 23 and colocalization of COX-1-IR in rat spinal cord with markers for microglia (unpublished data).
An emerging series of observations suggest there are fundamental differences in spinal COX isoenzymes involved in different pain states. A dominant, perhaps exclusive, role for spinal COX-2 occurs with peripheral inflammation. 5,12,14–17 In contrast, COX-1-IR is increased after incisional surgery, and tactile hypersensitivity after this surgery is blocked by selective COX-1 inhibitors, but unaffected by intrathecal injection of selective COX-2 inhibitors. 25 Hypersensitivity from nerve injury is delayed 18 or prevented by intrathecal injection of nonisoenzyme-selective, or COX-1-selective inhibitors but not by COX-2-selective inhibitors. The current study shows a long-term change in COX-1 in the spinal cord after nerve injury, consistent with the hypothesis that COX-1 is important in the development of hypersensitivity induced by nerve injury. Should this model be predictive of the human experience, it may lead to new treatment strategies to prevent neuropathic pain when the time of injury is known, such as amputation or thoracic surgery.
The current study suggests COX-1 may also be important in the maintenance of hypersensitivity after nerve injury. In contrast, previous studies 5,12,14,24 have shown that COX-1 does not play a role in inflammatory pain. This may be the result of the different mechanisms involved in these two types of pain. PSNT and SNL induce stable neuropathic hypersensitivity in rats, which lasts 6 to 8 weeks. 1 Four weeks after PSNT or 2 weeks after SNL, when rats had stable hypersensitivity, the number of COX-1-IR cells dramatically increased in the ipsilateral superficial laminae compared with normal or sham control. Because superficial laminae are the sites for the termination of nociceptive Aδ and C fiber primary afferents, prostaglandins produced by COX in the superficial laminae may be important in maintaining hyperalgesia from altered neurotransmission in this region. This is supported by the behavioral studies that show that intrathecal ketorolac, a COX-1-preferring inhibitor, restores morphine efficacy in rats that underwent SNL. 26 Intrathecal ketorolac alone also effectively attenuates thermal hyperalgesia and cold allodynia induced by sciatic nerve injury in rats 19 and reverses tactile allodynia induced by partial sciatic nerve ligation. 20 We are currently completing safety trials of intrathecal ketorolac in patients with neuropathic pain, to be followed by controlled trials to examine its efficacy.
The pattern of COX-1-IR increase in the dorsal horn ipsilateral to nerve injury is different from that in the postoperative pain model of paw incision. The latter shows a diffuse increase in the entire ipsilateral spinal dorsal horn, with a more prominent increase in the medial region. 25 In the current study, the increase of COX-1-IR is located only in the ipsilateral superficial laminae. These COX-1-IR glial cells only increase in number, without changing in morphology, unlike the altered morphology observed after surgery. 25 Whether this reflects differences in timing (days in the postoperative model and weeks in the nerve injury models) or activation mechanisms specific to the injury is unknown.
The decrease in the current study in COX-1-IR in the deep laminae of the spinal cord ipsilateral to the nerve injury was striking. We suspect that previous studies that have failed to observe changes in total COX-1 protein in the spinal cord after nerve injury missed important regional anatomic changes lost when using tissue homogenates. Peripheral nerve injury has been shown to cause a considerable degree of anatomic plasticity in the spinal cord. Using transganglionic tracers, Shortland and Fitzgerald 27 showed that transsection of the sciatic nerve in neonate rats caused the A and C fibers of the intact saphenous nerve to extend their arborizations into the territory of the transected sciatic nerve, whereas the sciatic afferents that survived neonatal axotomy retained a normal terminal distribution. It is noteworthy that the Aβ fibers that had sprouted into the sciatic territory, and the surviving sciatic Aβ fibers, had sprouted dorsally into the superficial laminae. In contrast, the labeled C fibers had not sprouted into the deeper laminae occupied by Aβ fibers and indeed showed a reduced intensity of terminal labeling. This type of change could occur, probably to a lesser extent, after PSNT and SNL in adult rats. In this case, spontaneous firing in the Aβ and C fibers would concentrate on the neurons in the superficial laminae. Nonetheless, it not clear whether upregulation of COX-1 in the superficial laminae and decrease in COX-1 in the deep laminae cause Aβ and C fibers sprouting or vice versa  or whether these two are caused by a third factor.
In summary, two types of peripheral nerve injury associated with hyperalgesia and allodynia show similar changes in COX-1 expression weeks after injury. COX-1-IR increases in the superficial dorsal horn ipsilateral to injury and decreases in the deeper dorsal horn, ventral horn, and white matter. These data support a change in COX-1 in the spinal cord after nerve injury, unlike findings with peripheral inflammation, and suggest targeting spinal COX-1 inhibition could relieve neuropathic pain.
References
Lindenlaub T, Sommer C: Partial sciatic nerve transection as a model of neuropathic pain: A qualitative and quantitative neuropathological study. Pain 2000; 89: 97–106Lindenlaub, T Sommer, C
Seltzer Z, Dubner R, Shir Y: A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990; 43: 205–18Seltzer, Z Dubner, R Shir, Y
Coderre TJ, Gonzales R, Goldyne ME, West J, Levine JD: Noxious stimulus-induced increase in spinal prostaglandin E2 is noradrenergic terminal-dependent. Neurosci Lett 1990; 115: 253–8Coderre, TJ Gonzales, R Goldyne, ME West, J Levine, JD
Ebersberger A, Grubb BD, Willingale HL, Gardiner NJ, Nebe J, Schaible HG: The intraspinal release of prostaglandin E2 in a model of acute arthritis is accompanied by an up-regulation of cyclo-oxygenase-2 in the spinal cord. Neuroscience 1999; 93: 775–81Ebersberger, A Grubb, BD Willingale, HL Gardiner, NJ Nebe, J Schaible, HG
Hay CH, Trevethick MA, Wheeldon A, Bowers JS, de Belleroche JS: The potential role of spinal cord cyclooxygenase-2 in the development of Freund's complete adjuvant-induced changes in hyperalgesia and allodynia. Neuroscience 1997; 78: 843–50Hay, CH Trevethick, MA Wheeldon, A Bowers, JS de Belleroche, JS
Yang LC, Marsala M, Yaksh TL: Characterization of time course of spinal amino acids, citrulline and PGE2 release after carrageenan/kaolin-induced knee joint inflammation: A chronic microdialysis study. Pain 1996; 67: 345–54Yang, LC Marsala, M Yaksh, TL
Hay CH, de Belleroche JS: Dexamethasone prevents the induction of COX-2 mRNA and prostaglandins in the lumbar spinal cord following intraplantar FCA in parallel with inhibition of oedema. Neuropharmacology 1998; 37: 739–44Hay, CH de Belleroche, JS
Hua XY, Chen P, Marsala M, Yaksh TL: Intrathecal substance P-induced thermal hyperalgesia and spinal release of prostaglandin E2 and amino acids. Neuroscience 1999; 89: 525–34Hua, XY Chen, P Marsala, M Yaksh, TL
Yang LC, Marsala M, Yaksh TL: Effect of spinal kainic acid receptor activation on spinal amino acid and prostaglandin E2 release in rat. Neuroscience 1996; 75: 453–61Yang, LC Marsala, M Yaksh, TL
Nishisho T, Tonai T, Tamura Y, Ikata T: Experimental and clinical studies of eicosanoids in cerebrospinal fluid after spinal cord injury. Neurosurgery 1996; 39: 950–6Nishisho, T Tonai, T Tamura, Y Ikata, T
Tonai T, Taketani Y, Ueda N, Nishisho T, Ohmoto Y, Sakata Y, Muraguchi M, Wada K, Yamamoto S: Possible involvement of interleukin-1 in cyclooxygenase-2 induction after spinal cord injury in rats. J Neurochem 1999; 72: 302–9Tonai, T Taketani, Y Ueda, N Nishisho, T Ohmoto, Y Sakata, Y Muraguchi, M Wada, K Yamamoto, S
Beiche F, Scheuerer S, Brune K, Geisslinger G, Goppelt-Struebe M: Up-regulation of cyclooxygenase-2 mRNA in the rat spinal cord following peripheral inflammation. FEBS Lett 1996; 390: 165–9Beiche, F Scheuerer, S Brune, K Geisslinger, G Goppelt-Struebe, M
Willingale HL, Gardiner NJ, McLymont N, Giblett S, Grubb BD: Prostanoids synthesized by cyclo-oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability. Br J Pharmacol 1997; 122: 1593–604Willingale, HL Gardiner, NJ McLymont, N Giblett, S Grubb, BD
Yaksh TL, Dirig DM, Conway CM, Svensson C, Luo ZD, Isakson PC: The acute antihyperalgesic action of nonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2 is mediated by the inhibition of constitutive spinal cyclooxygenase-2 (COX-2) but not COX-1. J Neurosci 2001; 21: 5847–53Yaksh, TL Dirig, DM Conway, CM Svensson, C Luo, ZD Isakson, PC
Yamamoto T, Nozaki-Taguchi N: Role of spinal cyclooxygenase (COX)-2 on thermal hyperalgesia evoked by carrageenan injection in the rat. Neuroreport 1997; 8: 2179–82Yamamoto, T Nozaki-Taguchi, N
Samad TA, Moore KA, Sapirstein A, Billet S, Allchorne A, Poole S, Bonventre JV, Woolf CJ: Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity. Nature 2001; 410: 471–5Samad, TA Moore, KA Sapirstein, A Billet, S Allchorne, A Poole, S Bonventre, JV Woolf, CJ
Dirig DM, Isakson PC, Yaksh TL: Effect of COX-1 and COX-2 inhibition on induction and maintenance of carrageenan-evoked thermal hyperalgesia in rats. J Pharmacol Exp Ther 1998; 285: 1031–8Dirig, DM Isakson, PC Yaksh, TL
Zhao Z, Chen SR, Eisenach JC, Busija DW, Pan HL: Spinal cyclooxygenase-2 is involved in development of allodynia after nerve injury in rats. Neuroscience 2000; 97: 743–8Zhao, Z Chen, SR Eisenach, JC Busija, DW Pan, HL
Parris WC, Janicki PK, Johnson B Jr, Horn JL: Intrathecal ketorolac tromethamine produces analgesia after chronic constriction injury of sciatic nerve in rat. Can J Anaesth 1996; 43: 867–70Parris, WC Janicki, PK Johnson, B Horn, JL
Ma W, Du W, Eisenach JC: Role for both spinal cord COX-1 and COX-2 in maintenance of mechanical hypersensitivity following peripheral nerve injury. Brain Res 2002; 937: 94–9Ma, W Du, W Eisenach, JC
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55–63Chaplan, SR Bach, FW Pogrel, JW Chung, JM Yaksh, TL
Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992; 50: 355–63Kim, SH Chung, JM
Maihofner C, Tegeder I, Euchenhofer C, deWitt D, Brune K, Bang R, Neuhuber W, Geisslinger G: Localization and regulation of cyclo-oxygenase-1 and −2 and neuronal nitric oxide synthase in mouse spinal cord. Neuroscience 2000; 101: 1093–108Maihofner, C Tegeder, I Euchenhofer, C deWitt, D Brune, K Bang, R Neuhuber, W Geisslinger, G
Beiche F, Brune K, Geisslinger G, Goppelt-Struebe M: Expression of cyclooxygenase isoforms in the rat spinal cord and their regulation during adjuvant-induced arthritis. Inflamm Res 1998; 47: 482–7Beiche, F Brune, K Geisslinger, G Goppelt-Struebe, M
Zhu X, Conklin D, Eisenach JC: Cyclooxygenase-1 in the spinal cord plays an important role in postoperative pain. Pain 2003; 104: 15–23Zhu, X Conklin, D Eisenach, JC
Lashbrook JM, Ossipov MH, Hunter JC, Raffa RB, Tallarida RJ, Porreca F: Synergistic antiallodynic effects of spinal morphine with ketorolac and selective. Pain 1999; 82: 65–72Lashbrook, JM Ossipov, MH Hunter, JC Raffa, RB Tallarida, RJ Porreca, F
Shortland P, Fitzgerald M: Neonatal sciatic nerve section results in a rearrangement of the central terminals of saphenous and axotomized sciatic nerve afferents in the dorsal horn of the spinal cord of the adult rat. Eur J Neurosci 1994; 6: 75–86Shortland, P Fitzgerald, M
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D  ) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar  :A, D  , 200 μm; B, C, E  , and F  , 20 μm.
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D 
	) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E 
	) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F 
	) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar 
	:A, D 
	, 200 μm; B, C, E 
	, and F 
	, 20 μm.
Fig. 1. Cyclooxygenase-1 (COX-1) immunoreactivity in spinal cords of normal rats and those that underwent partial sciatic nerve transsection (PSNT) 4 weeks after surgery. (A, D  ) Low magnification of COX-1 staining in spinal cords of normal and PSNT rats. (B, E  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of normal rat. (C, F  ) High magnification of COX-1 staining in superficial and deep spinal dorsal horn of PSNT rat. Scale bar  :A, D  , 200 μm; B, C, E  , and F  , 20 μm.
×
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B  ) Sham-operated and SNL rats 4 h after surgery. (C, D  ) Sham-operated and SNL rats 4 days after surgery. (E, F  ) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole  in the ventral white matter  . The area of quantification in the superficial and deep laminae is indicated by the upper  and lower rectangles  in F  . Scale bar  , 200 μm.
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B 
	) Sham-operated and SNL rats 4 h after surgery. (C, D 
	) Sham-operated and SNL rats 4 days after surgery. (E, F 
	) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole 
	in the ventral white matter 
	. The area of quantification in the superficial and deep laminae is indicated by the upper 
	and lower rectangles 
	in F 
	. Scale bar 
	, 200 μm.
Fig. 2. Cyclooxygenase-1 (COX-1) immunoreactivity change over time in the spinal cords from sham-operated rats and those that underwent L5–L6 spinal nerve ligation (SNL). (A, B  ) Sham-operated and SNL rats 4 h after surgery. (C, D  ) Sham-operated and SNL rats 4 days after surgery. (E, F  ) Sham-operated and SNL rats 2 weeks after surgery. Contralateral sides are marked by a hole  in the ventral white matter  . The area of quantification in the superficial and deep laminae is indicated by the upper  and lower rectangles  in F  . Scale bar  , 200 μm.
×
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar  ) and deep laminae (IV, V, and VI) (open bar  ) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar  represents the mean, and error bars  indicate the SEM. Significant differences between the each time point and baseline are indicated by **P  < 0.01, ***P  < 0.001 (one-way ANOVA) (n = 3 in each group).
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar 
	) and deep laminae (IV, V, and VI) (open bar 
	) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar 
	represents the mean, and error bars 
	indicate the SEM. Significant differences between the each time point and baseline are indicated by **P 
	< 0.01, ***P 
	< 0.001 (one-way ANOVA) (n = 3 in each group).
Fig. 3. Quantification of cyclooxygenase-1 (COX-1) immunoreactivity in the superficial laminae (I, II, and III) (filled bar  ) and deep laminae (IV, V, and VI) (open bar  ) in the ipsilateral dorsal horn of L5–L6 spinal nerve ligation rats over time. Each bar  represents the mean, and error bars  indicate the SEM. Significant differences between the each time point and baseline are indicated by **P  < 0.01, ***P  < 0.001 (one-way ANOVA) (n = 3 in each group).
×
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar  , 20 μm.
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F 
	) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar 
	, 20 μm.
Fig. 4. High magnification of cyclooxygenase-1 (COX-1) immunoreactivity over time in the ipsilateral spinal dorsal horn of rats that underwent L5–L6 spinal nerve ligation. (A, B  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 h after surgery. (C, D  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 4 days after surgery. (E, F  ) Superficial and deep laminae of the ipsilateral spinal dorsal horn 2 weeks after surgery. Scale bar  , 20 μm.
×