Weiya Ma

Morphological and pharmacological evidence for the role of peripheral prostaglandins in the pathogenesis of neuropathic pain.

Inflammatory mediators produced in the injured nerve have been proposed as contributing factors in the development of neuropathic pain. Prostaglandins (PGs) are probably included in these important inflammatory mediators. In the present study, 2 and 4 weeks following partial sciatic nerve ligation (PSNL), we observed a dramatic increase in the prostaglandin synthesizing enzyme cyclooxygenase (COX)2‐immunoreactive (IR) cell profiles in the injury site and adjacent region. Some of these COX2‐IR cells were identified as macrophages because they coexpressed ED1. None of these COX2‐IR cell profiles coexpressed the Schwann cell marker S100. In the contralateral sciatic nerve and sciatic nerve from normal rats, we failed to observe any of these COX‐IR cell profiles. We also observed COX1‐IR cell profiles (presumably Langerhans cells) in the epidermis of the footpad of both normal and PSNL rats. Interestingly, a greater number of COX1‐IR cell profiles were observed in the epidermis of the ipsilateral footpad of PSNL rats. Local injection of ketorolac, a nonselective COX inhibitor, into the ipsilateral plantar side or into the injury site of the sciatic nerve, effectively reversed the tactile allodynia induced by PSNL for > 5 days. Intraperitoneal or intramuscular injection of ketorolac had a similar but shorter antiallodynic effect. Intraplantar or peri‐neural injection of ketorolac dramatically suppressed the PSNL‐induced increase in the phosphorylation of a transcription factor cAMP response element binding protein (CREB) in the ipsilateral dorsal horn of L4 and L5 spinal cord of PSNL rats. Intraplantar or peri‐neural injection of ketorolac at the time of lesion did not prevent mechanical hypersensitivity but reduced it with a slow onset 3 weeks after lesion. Our data suggest that PSNL induces over‐production of PGs in peripheral tissues and that PGs probably sensitize nociceptors and are involved in central plasticity and sensitization at the spinal cord level, thus contributing to the maintenance of tactile allodynia.

Medium and large injured dorsal root ganglion cells increase TRPV-1, accompanied by increased α2C-adrenoceptor co-expression and functional inhibition by clonidine

Some electrophysiologic studies demonstrate new, excitatory α2‐adrenoceptors on peripheral nociceptors and their dorsal root ganglion (DRG) cell bodies after nerve injury, yet administration of α2‐adrenoceptor agonists at these sites reduces hypersensitivity rather than worsens it. Since TRPV‐1 expressing nociceptor afferents are important in many pain states, we examined the expression of this channel and its co‐expression with α2C‐adrenoceptors in injured DRG cell bodies and the ability of α2‐adrenoceptors to inhibit responses to stimulation. Rats underwent tight ligation of the left L5 and L6 spinal nerves, followed by behavioral testing, removal of L5 and L6 DRGs, and either immunostaining for TRPV‐1 channels and α2C‐adrenoceptors or intracellular calcium videomicroscopy in response to electrical field stimulation before and after perfusion with clonidine and capsaicin. Spinal nerve ligation produced tactile allodynia. In normal and sham controls, about one‐third of DRG neurons were TRPV‐1‐immunoreactive (IR), one half were α2C‐adrenoceptor‐IR and one‐fourth co‐expressed both. After nerve ligation there was a reduction in the number of small, strongly TRPV‐1‐IR or α2C‐adrenoceptor‐IR neurons, but an increase in medium and large, lightly stained cells and in their co‐expression. The proportion of clonidine inhibited cells which responded to capsaicin increased 5 fold after injury. We conclude that TRPV‐1 and α2C‐adrenoceptors are up‐regulated in some injured medium and large size neurons after nerve ligation. Increased co‐expression by immunocytochemistry, and increased proportion of cells inhibited by clonidine and expressing functional TRPV‐1 channels suggest that these cells may play an important role in the analgesic effects of α2‐adrenoceptor agonists in neuropathic pain.

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice.

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. alpha2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4-L6 dorsal horn. Consistently, greater numbers of both TH- and DbetaH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by alpha2-adrenergic receptor agonists.

Role for both spinal cord COX-1 and COX-2 in maintenance of mechanical hypersensitivity following peripheral nerve injury

The effectiveness of non-steroidal anti-inflammatory drugs (NSAIDs) in treating neuropathic pain caused by nerve injury has been controversial. In the present study, 4 weeks following partial sciatic nerve ligation, a single intrathecal injection of the cyclooxygenase (COX)-1 preferring inhibitor ketorolac (50 microg) significantly attenuated tactile allodynia for 6 days. The COX-2 preferring inhibitor, NS-398 (60 microg) significantly reversed tactile allodynia 2 h following injection but this anti-allodynic effect did not last greater than 24 h. Surprisingly, the non-selective COX inhibitor, piroxicam (60 microg) was without effect. These data agree with previous studies suggesting that spinal prostaglandin synthesis is important in the maintenance of hypersensitivity states following nerve injury. They differ from results in other models by suggesting that both COX isoenzymes are important in this spinal process, and for the first time demonstrate a remarkably long duration of action from a single intrathecal injection of ketorolac. Inhibition of spinal COX may be an important mechanism of action in treating some patients with neuropathic pain following peripheral nerve injury.

Intrathecal injection of cAMP response element binding protein (CREB) antisense oligonucleotide attenuates tactile allodynia caused by partial sciatic nerve ligation.

The transcription factor cAMP responsive element binding protein (CREB) is important in regulating immediate-early genes and some late-effector genes involved in neuroplasticity in response to peripheral injury and stressful insults. Partial nerve injury elicited neuropathic pain is accompanied by increased phosphorylation of CREB in the ipsilateral spinal cord dorsal horn (Ma and Quirion, Pain 93 (2001) 295; Miletic et al., Pain 99 (2002) 493). The aim of this study is to determine whether increased phosphorylation of CREB in the dorsal horn contributes to the pathogenesis of neuropathic pain. Three weeks following partial sciatic nerve ligation (PSNL), daily intrathecal injection of antisense CREB oligodeoxynucleotide (ODN, 20 microg/day) for 5 days significantly attenuated tactile allodynia. The attenuation lasted for more than 4 days. Total CREB and phosphorylated CREB in both ipsilateral and contralateral dorsal horn neurons were dramatically reduced in antisense ODN injected PSNL rats 1 week after injection. The extent of reduction of total CREB and phosphorylated CREB containing cells in the dorsal horn ipsilateral to injury was greater than in the contralateral dorsal horn. These data suggest that phosphorylation of CREB is an important contributing event in the central plasticity of nerve injury and in the pathogenesis of neuropathic pain.

Perineural alpha(2A)-adrenoceptor activation inhibits spinal cord neuroplasticity and tactile allodynia after nerve injury.

BACKGROUND Nerve injury in animals increases alpha(2)-adrenoceptor expression in dorsal root ganglion cells and results in novel excitatory responses to their activation, perhaps leading to the phenomenon of sympathetically maintained pain. In contrast to this notion, peripheral alpha(2)-adrenoceptor stimulation fails to induce pain in patients with chronic pain. We hypothesized that alpha(2) adrenoceptors at the site of nerve injury play an inhibitory, not excitatory role. METHODS Partial sciatic nerve ligation was performed on rats, resulting in a reduction in withdrawal threshold to tactile stimulation. Animals received perineural injection at the injury site of clonidine, saline, or clonidine plus an alpha(2)-adrenergic antagonist, and withdrawal threshold was monitored. Immunohistochemistry was performed on the sciatic nerve ipsi- and contralateral to injury and on the spinal cord. RESULTS Clonidine reduced this hypersensitivity in a dose-dependent manner, and this was blocked by an alpha(2A)-preferring antagonist. Perineural clonidine injection had a slow onset (days) and prolonged duration (weeks). Systemic or intrathecal clonidine, or transient neural blockade with ropivacaine, had short lasting or no effect on hypersensitivity. alpha(2A)-adrenoceptor immunostaining was increased near the site of peripheral nerve injury, both in neurons and in immune cells (macrophages and T lymphocytes). Phosphorylated cAMP response element binding protein (pCREB) in lumbar spinal cord was increased ipsilateral to nerve injury, and this was reduced 1 week after perineural clonidine injection. CONCLUSIONS These data suggest that peripheral alpha(2) adrenoceptors are concentrated at the site of peripheral nerve injury, and their activation receptors produce long-lasting reductions in abnormal spinal cord gene activation and mechanical hypersensitivity.

Spinal noradrenergic activation mediates allodynia reduction from an allosteric adenosine modulator in a rat model of neuropathic pain

&NA; Activation of adenosine A1 receptors by endogenous adenosine or synthetic agonists produces anti‐nociception in animal models of acute pain and also reduces hypersensitivity in models of inflammatory and nerve‐injury pain. Allosteric adenosine modulators facilitate adenosine agonist binding to the A1 receptor. The purpose of the current study was to examine the effect, mechanisms of action, and interaction with noradrenergic systems of intrathecal (i.t.) or oral administration of the allosteric adenosine modulator T62 in a rat model of neuropathic pain. A spinal nerve ligation rat model (SNL; ligation of left L5 and L6 spinal nerve roots) was used. One week after SNL surgery, an i.t. catheter was inserted. Withdrawal threshold to mechanical stimulation of the left hind paw was determined before and after surgery, confirming mechanical hypersensitivity. Oral or i.t. T62 reduced hypersensitivity induced by SNL. The effects of i.t. T62 were inhibited by i.t. injection of an A1 receptor antagonist and by an &agr;2‐adrenergic antagonist but not by an A2 adenosine receptor antagonist. Anti‐dopamine &bgr; hydroxylase (D&bgr;H)‐saporin treatment reduce spinal norepinephrine content by 97%, accompanied by an almost complete loss of D&bgr;H immunoreactive axons in the spinal dorsal horn and neurons in the locus coeruleus. The effect of T62 was completely lost in animals treated with anti‐D&bgr;H‐saporin. These data support the hypothesis that activation of the A1 receptor by the allosteric modulator, T62, produces anti‐nociception via spinal noradrenergic activation.

Four PGE2 EP receptors are up-regulated in injured nerve following partial sciatic nerve ligation.

We previously reported that cyclooxygenase 2 (COX2) is up-regulated in macrophages in injured nerve of rats with partial sciatic nerve ligation (PSNL) and that local injection of the COX inhibitor ketorolac reversed tactile allodynia (Eur. J. Neurosci. 15: 1037-1047, 2002). These findings suggest that prostaglandins (PGs) are overproduced in injured nerve and are involved in the pathogenesis of neuropathic pain. In this study, we examined whether overproduced PGs alter the expression of PGE2 receptors, EP1-EP4, in injured nerve of PSNL rats. We found that cell profiles immunoreactive (IR) for four EP receptors, EP1, EP2, EP3, and EP4, are dramatically increased in injured nerve 2 and 4 weeks after PSNL. EP4-IR cells were the most abundant among these receptor-expressing cells. Immunoreactivities of all four EP receptors were localized to the cell nucleus. These EP-IR cells were never found in uninjured nerve. More than 80% EP1- and about 30% EP4-IR cells were identified as infiltrating macrophages since they coexpressed ED1. Only 3% EP2- and 6% EP3-IR cells coexpressed ED1. These findings suggest that majority of EP2-, EP3-, and EP4-IR cells are other types of inflammatory cells than macrophages. About 48% of macrophages expressed EP1 and 45% expressed EP4. Only 3 and 6% of macrophages, respectively, expressed EP2 and EP3. Perineural injection of ketorolac reversed tactile allodynia and suppressed the up-regulation of EP1 and EP4, but not the recruitment of ED1-IR marcrophages, in injured nerve. Our data suggest that following PSNL, PGE2 is one of the possible PGs overproduced in injured nerve and PG overproduction is involved in the up-regulation of EP receptors in injured nerve.

Uterine cervical distension induces cFos expression in deep dorsal horn neurons of the rat spinal cord.

Background Uterine cervical distension underlies labor pain, yet its neurophysiology and pharmacology of inhibition remain unexplored. The authors examined uterine cervical distension-evoked cFos immunoreactivity in rat spinal cords, and the inhibitory effect of spinal cyclo-oxygenase inhibition on cFos expression. Methods Female rats were anesthetized with halothane, and pairs of metal rods were inserted in each cervical os through a mid-line laparotomy. A submaximal distension force (75 g) was applied for either 30 or 60 min, or, in control animals, no force was applied. Other animals received cervical lidocaine infiltration prior to uterine cervical distension. At the end of the experiments, the spinal cord at T12 to L2 levels was harvested and immunostained for cFos protein. Other animals received intrathecal ketorolac (0, 5, 25, and 50 &mgr;g; n = 5–6 for each group) prior to uterine cervical distension. Results Uterine cervical distension significantly increased cFos immunoreactivity in the spinal cord from T12 to L2, with most cFos expression in the deep dorsal and central canal regions. Surgical preparation alone without uterine cervical distension resulted in minimal cFos expression, primarily in the superficial dorsal horn. Uterine cervical distension-evoked cFos expression was prevented by prior infiltration of lidocaine into the cervix. Intrathecal ketorolac produced a dose-dependent inhibition of uterine cervical distension-induced cFos expression. Conclusion The present study demonstrates that uterine cervical distension results in a similar pattern of spinal cord neuronal activation as seen with other noxious visceral stimuli. The inhibition of cFos expression by intrathecal ketorolac suggests that spinal cyclo-oxygenase plays a role in uterine cervical distension-induced nociception.

Intrathecal lidocaine reverses tactile allodynia caused by nerve injuries and potentiates the antiallodynic effect of the COX inhibitor ketorolac.

Background Systemic lidocaine and other local anesthetics reduce hypersensitivity states induced by both acute inflammation and peripheral nerve injury in animals and produce analgesia in some patients with chronic pain. The mechanisms underlying the antiallodynic effect of systemic lidocaine are unclear, although most focus is on peripheral mechanisms. Central mechanisms, particularly at the spinal dorsal horn level, are less known. In this study, the authors aimed to determine whether intrathecal lidocaine has an antiallodynic effect on established mechanical allodynia in two well-characterized neuropathic pain rat models: partial sciatic nerve ligation (PSNL) and spinal nerve ligation (SNL). Methods Lidocaine (100–300 &mgr;g) was intrathecally injected in PSNL and SNL rats. The withdrawal threshold of both hind paws in response to mechanical stimulation was measured using a series of calibrated von Frey filaments. Results This single injection reduced ongoing tactile allodynia in PSNL and SNL rats. The antiallodynic effect of intrathecal lidocaine lasted longer in PSNL (> 3 days) than in SNL rats (< 3 days). Intraperitoneal lidocaine (300 &mgr;g) had no effect on tactile allodynia in PSNL rats. In SNL rats, prior intrathecal lidocaine (200 and 300 &mgr;g) potentiated the antiallodynic effect of intrathecal ketorolac, a nonselective cyclooxygenase inhibitor. Intrathecal ketorolac alone had no antiallodynic effect on SNL rats. However, prior intrathecal lidocaine (100 &mgr;g) failed to potentiate the antiallodynic effect of intrathecal ketorolac. Conclusion The authors’ data suggest that intrathecal lidocaine possibly suppressed the hyperexcitability of the dorsal horn neurons and likely interacted with eicosanoid systems in the spinal dorsal horn.