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1 Available online at Annals of Clinical & Laboratory Science, vol. 37, no. 4, Effects of Intrathecal Injection of Prednisolone Acetate on Expression of NR2B Subunit and nnos in Spinal Cord of Rats after Chronic Compression of Dorsal Root Ganglia Zheng-Liang Ma, 1 Wei Zhang, 1 Xiao-Ping Gu, 1 Wen-Sheng Yang, 2 and Yin-Ming Zeng 2 1 Department of Anesthesiology, Drum Tower Hospital, Nanjing University, Nanjing, China; 2 Jiangsu Institute of Anesthesiology, Xuzhou Medical College, Xuzhou, China Abstract. N-methyl-D-aspartate receptor subunit 2B (NR2B) and neuronal nitric oxide synthase (nnos) play important roles in the mechanism of neuropathic pain. To elucidate how glucocorticoids affect this mechanism, we studied the effects of intrathecal (it) injection of prednisolone acetate (PA) on a nociceptive stimulus and the changes of nnos and NR2B subunit expression in the spinal dorsal horn of Sprague Dawley rats following chronic compression of the dorsal root ganglia (CCD). Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured for 15 days postoperatively. An it injection of PA (2.0 mg/kg) every 3 days for postoperative days 1 to 15 inhibited the thermal hyperalgesia and tactile allodynia of CCD rats. Chronic compression of the dorsal root ganglia induced time-dependent upregulation of nnos and NR2B subunits of N-methyl-D-aspartate receptor within the spinal cord dorsal horn ipsilateral to CCD. Both upregulations were significantly diminished by it administration of PA (2.0 mg/kg), but not by lower doses of PA (0.5 or 1.0 mg/kg). The results suggest that PA upregulation of neuronal nitric oxide synthase and NR2B subunit expression in the spinal dorsal horn contributes to PA inhibition of hyperalgesia induced by chronic compression of dorsal root ganglia. Keywords: spinal dorsal horn, prednisolone, nnos, NR2B, neuropathic pain, spinal cord compression Introduction Chronic pain remains a major health care problem afflicting patients with low back pain and sciatica. Glucocorticoid (GC) injections have been used to treat neuropathic pain for over 40 years. The success of hormonal therapy was partly attributed to GC anti-inflammatory activity [1,2]. In recent years, GC receptors (GR) have been described in various neuronal populations of the spinal cord. In motor neurons of rats, acute GC treatment increases the activity of neurons, decreases neurofilament degradation, restores neuronal energy metabolism, and stimulates axonal regeneration, sprouting and neuronal survival [3]. Less information is available Address correspondence to Dr Zheng-Liang Ma, Department of Anesthesiology, Drum Tower Hospital, Medical Dept. of Nanjing University, Nanjing , Jiangsu Province, China; tel ; fax ; mazhengliang1964@yahoo.com.cn. about the effects of GC in sensory areas of the spinal cord, although GR are also present in dorsal horn neurons. In the present study, we examined the effects of GC in the superficial dorsal horn (laminae I III) areas, which are involved in the transmission of nociceptive inputs and the control of sympathetic outflow. The mechanisms of neuropathic pain involve changes in afferent and central spinal sensory relays, leading to neuronal hyperexcitability [4]. Central sensitization crucially involves spinal N-methyl-D-aspartate receptors (NMDAR) [5]. NMDAR antagonists have been shown to reverse neuropathic pain behaviors [6] and reduce dorsal horn neuronal hyper-responsiveness [7,8]. A recent study demonstrates that the NR2B subunit plays a key role in this process. Wei Guo et al [9] reported a prolonged increase of NR2B subunit in spinal dorsal horn neurons of rats caused by peripheral inflammation /07/ $ by the Association of Clinical Scientists, Inc.

2 350 Annals of Clinical & Laboratory Science, vol. 37, no. 4, 2007 Another response to neuronal activation in the sensory spinal cord areas is the expression of nitric oxide synthase (NOS). NOS can be activated by NMDA receptor post-synaptically, and reports indicate that NO-producing neurons maintain and facilitate hyperalgesia. NOS activity is up-regulated in the laminae I III following peripheral inflammation, axotomy, and spinal cord lesions [10]. Recent data suggest that nnos may be the most important subgroup of NOS and a close relationship between nnos and neuropathic pain has been demonstrated by many previous studies. Although there is no information about GC effects on NOS in the spinal cord, data from other systems show that GC down-regulates this enzyme [11]. Thus, it is possible that nnos constitutes an important target of GC action during neuropathic pain. The objectives of this investigation were (a) to determine changes in NR2B and nnos at several time points after chronic compression of the dorsal root ganglion (CCD), and (b) to asses the responses of these parameters to administration of prednisolone acetate (PA), in order to elucidate the antinociceptive mechanisms of PA at the spinal level. To our knowledge, the modulation of NR2B and nnos by GC has not previously been explored in CCD rats. Materials and Methods Commercial sources of reagents used in this research were as follows: prednisolone acetate, Sigma-Aldrich Co., Shanghai, China; nnos and NR2B primary antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA; PBS buffer, ABC kit, and other reagents, Boster Co., Wuhan, China. Sprague- Dawley rats (250 to 300 g, male) were provided by the Experimental Animal Center of Xuzhou Medical College and were kept with a 12 hr/12 hr light-dark cycle. All experiments were approved by the Animal Care and Use Committee of Xuzhou Medical College. Implantation of intrathecal catheter. Rats were implanted with catheters by the method of Yaksh and Rudy [12]. In brief, each rats was anesthetized with pentobarbital sodium (30 mg/kg, ip), the occipital muscles were bluntly separated, and the cisternal membrane was exposed. A polyethylene catheter (PE-10) was inserted via an incision in the cisterna magna and advanced cm caudally to the level of the lumbar enlargement. After the rat had recovered from anesthesia, correct intrathecal placement was confirmed by injection of 10 μl of 2% lidocaine through the catheter. The catheter was judged to be intrathecal if paralysis and dragging of bilateral hind limbs occurred within 30 sec after this injection. Rats with signs of motor dysfunction were excluded from the experiment. The rats were housed individually after surgery and allowed to recover 5 days before the CCD test. Experimental groups. The rats were assigned to 2 groups: 7 day and 15 day. Each group was sub-divided into 5 sub-groups as follows: (a) (sham group): rats without CCD + it injection of artificial cerebrospinal fluid (ACSF) (20 μl); (b) (ACSF group): rats with CCD + it injection of ACSF (20 μl); (c) (PA 0.5 mg/kg group): rats with CCD + it injection of PA, 0.5 mg/kg; (d) (PA 1.0 mg/kg group): rats with CCD + it injection of PA, 1.0 mg/kg; (e) (PA 2.0 mg/kg group): rats with CCD + it injection of PA, 2.0 mg/kg. The it injections were given every 3 days postoperatively. For the PA injection groups, the catheters were rinsed with 5 μl of ACSF. Chronic compression of dorsal root ganglia (CCD). Chronic compression of dorsal root ganglia was performed under anesthesia with pentobarbital sodium (30 mg/kg, ip). The right L4 and L5 intervertebral foramina were exposed and a stainless steel wire (4 mm long, 0.7 mm diameter) was inserted in each to provide stable compression of the L4 and L5 dorsal root ganglion [13]. Sham surgery was performed by exposing the right L4 and L5 intervertebral foramina without compression. Behavioral studies. Mechanical allodynia was assessed by use of von Frey filaments (Stoelting Co., Wood Dale, IL, USA). Rats were placed in individual plastic boxes ( cm) on a metal mesh floor and allowed to acclimatize for 30 min. The filaments were presented, in ascending order of strength, perpendicular to the plantar surface with sufficient force to cause slight bending against the paw and held for 6 to 8 sec. Brisk withdrawal or paw flinching were considered as positive responses. The paw withdrawal mechanical threshold (PWMT) was determined by sequentially increasing and decreasing the stimulus strength (the up-and-down method), and the data were analyzed using the nonparametric method of Dixon, as described by Chaplan et al [14]. Thermal hyperalgesia [15] was assessed with the paw withdrawal thermal latency (PWTL) to radiant heat according to the protocol of Hargreaves et al [15]. Rats were placed in clear plastic cages on an elevated glass plate and allowed to acclimatize for 30 min before testing. A radiant thermal stimulator (BME410A, Institute of Biological Medicine, Academy of Medical Science, China) was focused onto the plantar surface of the hindpaw through the glass plate. The nociceptive endpoints in the radiant heat test were the characteristic lifting or licking of the hindpaw, and the time to the endpoint was considered the PWTL [16]. To avoid tissue damage, a cut-off time of 30 sec was used [17]. There were 5 trials per rat and 5 min intervals between trials. The mean PWTL was obtained from the final 3 stimuli [18].

3 Prednisolone effect on nociceptive expression of NR2B and nnos in rat spinal cord 351 Slices were fixed on microscope slides coated with gelatin. A computer-assisted image analysis system (Leica) was used to determine the number of positive neurons. Six random sections from each animal were observed at 400 magnification with an Olympus optical microscope equipped with an Olympus DP11 camera. For each rat, the mean number of cell counts in superficial lamina (I III) of the spinal cord was calculated by averaging the number of stained cells from 6 sections. Fig. 1. Effect of prednisolone acetate (PA) on the paw withdrawal mechanical threshold with Von Frey filaments in CCD rats (n = 10 rats/group; mean ± SD; *p <0.05, **p <0.01 vs ACSF group; #p <0.05, ##p <0.01 vs baseline data). Immunohistochemical staining. Immunohistochemistry was used to detect nnos and NR2B immunoreactivities in the spinal cord. Rats anesthetized with pentobarbital sodium (50 mg/kg, ip) were perfused through the ascending aorta with 0.9% NaCl, followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, ph 7.4). The lumbosacral spinal cords were carefully dissected out and stored in the same fixative for 3 hr (4 C). After fixation, spinal cord segments were cryoprotected in 20% sucrose with the same phosphate buffer and stored overnight at 4 C. Transverse frozen sections (40 μm) were cut with a cryostat maintained at -28 C. Slices were placed in 0.3 % triton PBS for 30 min. Tissue sections were sequentially exposed to 10% goat serum for 10 min at room temperature and then to the primary antibody (NR2B, 1:1000; nnos, 1:500) dilution at 4 C for 24 hr. Sections were washed twice with PBS and incubated in goat anti-rabbit serum (1:200) for 1 hr. This step was followed by three washes in PBS and staining using the ABC kit. The DAB chromogenic reaction was monitored carefully for about 5 min and PBS was used to stop the reaction in time. Fig. 2. Effect of prednisolone acetate (PA) on the paw withdrawal thermal latency in CCD rats (n = 10 rats/group; mean ± SD; *p <0.05, **p <0.01 vs ACSF group; #p <0.05 ##p <0.01 vs baseline data). Statistical analysis. The data were expressed as means ± SD and analyzed by ANOVA, followed either by a multiple comparison test or by an unpaired t-test. Values of p <0.05 were considered statistically significant. Results Effect of intrathecal injection of PA on the paw withdrawal mechanical threshold. Compared to the baseline before operation, the PWMT of all groups (except the sham group) showed downregulation at 1 and 3 days after CCD (p <0.01) (Fig. 1). There were no significant differences between the CCD groups at these times. The PWMT of the PA 2.0 mg/kg group (but not the PA 0.5 mg/kg or PA 1.0 mg/kg groups) showed upregulation compared to the ASCF group from days 5 to 15 postoperatively (p <0.05 (Fig. 1). Effect of intrathecal injection of PA on paw withdrawal thermal latency. Intrathecal injection of ASCF did not affect the paw withdrawal thermal latency (PWMT) in rats of the sham group. The PWMT in rats of the other groups showed downregulation compared to baseline at 1 and 3 days after CCD (p <0.01) (Fig. 2). Intrathecal injection of PA 2.0 mg/kg (but not PA 0.5 mg/kg or PA 1.0 mg/kg) prolonged the PWMT compared to the ASCF group from days 5 to 15 after the operation (p <0.01) (Fig. 2). Effect of PA on nnos immunohistochemical staining. The effect of PA treatment on nnos expression was determined in the superficial dorsal horn laminae I-III of the lumbar enlargement, which are areas that are involved in the transmission of nociceptive inputs and control of sympathetic outflow [19]. In agreement with previous reports, constitutive nnos expression was weak to absent in the sham group. The expression of nnos in the superficial dorsal horn of the ACSF, PA 0.5 mg/kg,

4 352 Annals of Clinical & Laboratory Science, vol. 37, no. 4, 2007 Fig. 3. Effect of prednisolone acetate (PA) on the number of nnos positive neurons in spinal dorsal horn ( n = 6 rats/ group; mean ± SD; *p <0.05, **p <0.01 vs sham group; #p <0.05 vs ACSF group). Fig. 5 Effect of prednisolone acetate (PA) on the number of NR2B positive neurons in spinal dorsal horn (n = 6 rats/ group; mean ± SD; **p <0.01 vs sham group; ##p <0.01 vs ACSF group). and 1.0 mg/kg groups was increased significantly compared to the sham group (p <0.01) (Fig. 3). The expression of nnos in the PA 2.0 mg/kg group was greatly decreased compared to the ACSF group, both on day 7 and day 14 postoperatively (p <0.05) (Fig. 3). Typical photomicrographs of the nnos immunoreactive cells in the sham, ACSF, and PA 2.0 mg/kg groups are shown in Fig. 4. Effect of PA on NR2B immunohistochemical staining. The NR2B-positive neurons were greatly increased in the superficial dorsal horn of 3 groups (ACSF, PA 0.5 mg/kg, PA 1.0 mg/kg) compared to the sham group (p <0.01) both on days 7 and 14 postoperation; there were no significant differences among these 3 groups (Fig. 5). Compared to the ACSF group, the NR2B-positive neurons of the PA 2.0 mg/kg group were decreased on day 7 and day 14 postoperatively (p <0.01) (Fig. 5). Typical photomicrographs of NR2B immunoreactive cells in the sham, ACSF, and PA 2.0 mg/kg groups are shown in Fig. 6. Fig. 4. Typical photomicrographs (original magnification:100x) showing nnos immunoreactive cells in the superficial dorsal horn (laminae I III). In sham-operated rats only a few stained cells were observed (panel 1), whereas many nnos immunopositive cells appeared in ACSF group rats (panel 2). The PA 2.0 mg/kg treated rats showed remarkably inhibited staining intensity compared to the ACSF group (panel 3).

5 Prednisolone effect on nociceptive expression of NR2B and nnos in rat spinal cord 353 Fig. 6. Typical photomicrographs (original magnification 100x) representing NR2B histochemistry staining of neurons in the dorsal horn (laminae I III). Seven days after sham operation, a moderate basal staining intensity was obtained in sham operated rats (panel 1), which was substantially increased in ACSF group rats (panel 2), and PA 2.0 mg/kg remarkably inhibited the increased expression in the spinal cord (panel 3). Discussion Peripheral nerve injury leads to central sensitisation, where increased excitability of neurons in the spinal dorsal horn leads to chronic hyperalgesia and allodynia. In this study we describe effects of PA on behavior and on NR2B and nnos expression in the spinal dorsal horn lamina I III of CCD rats. In agreement with previous studies, the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of CCD rats with artificial cerebrospinal fluid (ACSF) treament were significantly lower than the sham group [13]. Our results show these behavioral changes can last a long time (15 days). Intrathecal injection of PA (2.0 mg/kg) every 3 days reversed the neuropathic pain behaviors. We found a virtual abolition of thermal hyperalgesia and marked attenuation of mechanical allodynia in the PA 2.0 mg/kg group, compared to the ACSF group. This did not happen in the PA 0.5 mg/kg or PA 1.0 mg/ kg groups. Thus the effect of prednisolone acetate is dependent on the dose given intrathecally. For the expression of NR2B, low levels of NR2B immunoreactive neurons were found in spinal cord within laminae I III of the shamoperated animals. However, a high number of NR2B immunoreactive neurons appeared in these laminae of rats with ACSF treament. As previous data show, the NR2B subunits are important candidates in the activation of NMDA receptors, which play a pivotal role in injury-induced sensitization in the spinal cord. Immunoblotting and immunohistochemical studies show an alteration in expression levels of NR2B in animals with established neuropathic pain [20]. Recent evidence suggests that NR2B protein shows a distribution associated both with small diameter fibres in laminae I and II, which are postulated to be of primary afferent origin [21], and post-synaptically on dorsal horn neurons [22]. This study shows that the expression of NR2B subtypes in the spinal dorsal horn changes radically over the time of neuropathic pain in CCD rats and that the increased expression is reversed by it injection of PA (2.0 mg/kg). A close correlation between nnos and neuropathic pain has been documented [23]. Some reports indicate that NO-producing neurons maintain and facilitate hyperalgesia [24]. In our studies, CCD also produced a unilateral increase in the level of nnos positive neurons in the superficial layers within laminae I III. Strong nociceptive input derived from spinal cord hemisection, root avulsion, hindpaw inflammation, and axotomy produces robust increases in NOS immunoreactivity. These stimuli caused at most a transient increase for a few hours, followed by a decrease

6 354 Annals of Clinical & Laboratory Science, vol. 37, no. 4, 2007 persisting over many days [10]. However, in our study, the increased nnos positive neurons in superficial layers within laminae I III of the ACSF group at 7 and 14 days after CCD may be the result of de novo synthesis of the enzyme and its transport from the dorsal root ganglia to the spinal cord, although up-regulation of the enzyme activity in local interneurons is also possible [25]. Our data demonstrate that PA exerts an inhibitory effect on the number of NR2B and nnos positive neurons in lamina I III, elevated as a consequence of CCD. In this context, both of these molecules have been implicated in nociceptive transmission, because their inhibition resulted in a diminished response to pain. For the decrease of NR2B positive neurons, in molecular terms, GC action upon NR2B may be related to steroid antiinflammatory and immunosuppressive effects, mainly achieved by transcriptional interference. As to nnos, both natural and synthetic GC exert a negative regulation of this enzyme in brain and spinal cord motoneurons [26,27]. Dexamethasone (DEX) can also reduce apoptosis in contused spinal cord [28]. The inhibition was also found in macrophages in an in vitro study [29]. Interestingly, our data show that both NR2B and nnos are inhibited by PA. Reuss and Reuss [30] reported that the NMDA receptor activates NOS postsynaptically and that NOS produces NO release to pre-synaptically up-regulate the function of the NMDA receptor. Thus the NMDA receptor and NOS comprise a local circuit that amplifies the signal of nociceptive transmission. If sustained production of these factors after CCD is required for maintenance of experimentally-produced hyperalgesia, and if their lesion-induced increase is likely to cause persistence of pain, blockade of this circuit by GC treatment would likely reduce pain excitatory neurotransmission at the spinal cord level. In clinical studies, GC has a good effect in curing intervertebral disk hernia through extension of inflammatory reaction and hydropsia ambient to the nerve root. Recent data show that epidural injection of PA can relieve the pain of patients after a spinal operation. All of these findings indicate GC has analgesic effects at the spinal level. Our results indicate that CCD can induce long-lasting NR2B and nnos up-regulation in the spinal dorsal horn. This up-regulation may contribute to nociceptor activity-induced spinal plasticity and development of central sensitization and persistent pain. Furthermore, intrathecal injection of PA 2.0 mg/kg remarkably inhibited CCD-induced mechanical allodynia and thermal hyperalgesia and inhibited the increases of NR2B and nnos expression in the spinal cord. These results suggest that activation of the NMDA-NOS pathway contributes to the development of neuropathic pain induced by CCD. The analgesia of intrathecal administration of PA may be mediated by the interruption of this pathway. Acknowledgement This research was supported by Grant 02KJB from the Department of Education Foundation of Jiangsu Province, China. References 1. Bartholdi D, Schwab ME. Methylprednisolone inhibits early inflammatory processes but not ischemic cell death after experimental spinal cord lesion in the rat. Brain Res 1995;672: Cronstein BN, Kimmel SC, Levin RI, Martiniuk F, Weissmann G. Corticosteroids are transcriptional regulators of acute inflammation. Trans Assoc Am Phys 1992;105: Brandoli C, Shi B, Pflug B, Andrews P, Wrathall JR, Mochetti II. Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord. Mol Brain Res 2001;2001: Kohama I, Ishikawa K, Kocsis JD. Synaptic reorganization in the substantia gelatinosa after peripheral nerve neuroma formation: aberrant innervation of lamina II neurons by a-beta afferents. J Neurosci 2000;20: Sotgiu ML, Biella G. Differential effects of MK-801, a N-methyl-D-aspartate non-competitive antagonist, on the dorsal horn neuron hyperactivity and hyperexcitability in neuropathic rats. Neurosci Lett 2000;283: Garry EM, Moss A, Delaney A, O Neill F, Blakemore J, Bowen J, Husi H, Mitchell R, Grant SG, Fleetwood- Walker SM. Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/cam kinase II interactions are disrupted in PSD-95 mutant mice. Curr Biol 2003;13: Suzuki R, Matthews EA, Dickenson AH. Comparison of the effects of MK-801, ketamine and memantine on responses of spinal dorsal horn neurons in a rat model of mononeuropathy. Pain 2001;91:

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