Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats.

In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCK(B) receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCK(B) receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20mg/kg). Intrathecal CI-988 (100, 200 and 500 microg) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCK(B) receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain.

Excitatory actions of GABA in the suprachiasmatic nucleus.

Neurons in the suprachiasmatic nucleus (SCN) are responsible for the generation of circadian oscillations, and understanding how these neurons communicate to form a functional circuit is a critical issue. The neurotransmitter GABA and its receptors are widely expressed in the SCN where they mediate cell-to-cell communication. Previous studies have raised the possibility that GABA can function as an excitatory transmitter in adult SCN neurons during the day, but this work is controversial. In the present study, we first tested the hypothesis that GABA can evoke excitatory responses during certain phases of the daily cycle by broadly sampling how SCN neurons respond to GABA using extracellular single-unit recording and gramicidin-perforated-patch recording techniques. We found that, although GABA inhibits most SCN neurons, some level of GABA-mediated excitation was present in both dorsal and ventral regions of the SCN, regardless of the time of day. These GABA-evoked excitatory responses were most common during the night in the dorsal SCN region. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor, bumetanide, prevented these excitatory responses. In individual neurons, the application of bumetanide was sufficient to change GABA-evoked excitation to inhibition. Calcium-imaging experiments also indicated that GABA-elicited calcium transients in SCN cells are highly dependent on the NKCC isoform 1 (NKCC1). Finally, Western blot analysis indicated that NKCC1 expression in the dorsal SCN is higher in the night. Together, this work indicates that GABA can play an excitatory role in communication between adult SCN neurons and that this excitation is critically dependent on NKCC1.

Long-term Follow-up of Cutaneous Hypersensitivity in Rats with a Spinal Cord Contusion.

Sometimes, spinal cord injury (SCI) results in various chronic neuropathic pain syndromes that occur diffusely below the level of the injury. It has been reported that behavioral signs of neuropathic pain are expressed in the animal models of contusive SCI. However, the observation period is relatively short considering the natural course of pain in human SCI patients. Therefore, this study was undertaken to examine the time course of mechanical and cold allodynia in the hindpaw after a spinal cord contusion in rats for a long period of time (30 weeks). The hindpaw withdrawal threshold to mechanical stimulation was applied to the plantar surface of the hindpaw, and the withdrawal frequency to the application of acetone was measured before and after a spinal contusion. The spinal cord contusion was produced by dropping a 10 g weight from a 6.25 and 12.5 mm height using a NYU impactor. After the injury, rats showed a decreased withdrawal threshold to von Frey stimulation, indicating the development of mechanical allodynia which persisted for 30 weeks. The withdrawal threshold between the two experimental groups was similar. The response frequencies to acetone increased after the SCI, but they were developed slowly. Cold allodynia persisted for 30 weeks in 12.5 mm group. The sham animals did not show any significant behavioral changes. These results provide behavioral evidence to indicate that the below-level pain was well developed and maintained in the contusion model for a long time, suggesting a model suitable for pain research, especially in the late stage of SCI or for long term effects of analgesic intervention.

The peripheral role of group I metabotropic glutamate receptors on nociceptive behaviors in rats with knee joint inflammation.

We examined whether the mGluR1 and mGluR5 were involved in development and maintenance of behavioral signs of non-evoked pain and secondary mechanical hyperalgesia induced by knee joint inflammation. Selective mGluR1 antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA: 50, 100, 200 microM/25 microl, n=10 per group) and selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP: 50, 100, 200 nM/25 microl, n=10 per group) was intra-articularly (i.a.) injected 30 min before and 4h after carrageenan injection and behavioral tests were conducted. In the pre-treatment, only a higher dose (200 nM) of MPEP significantly prevented the magnitude of weight load reduction, whereas AIDA (200 microM) and MEPE (50, 100 and 200 nM) significantly reduced the development of mechanical hyperalgesia compared to saline treated group. In the post-treatment, AIDA (200 microM) and MPEP at 100 and 200 nM partially reversed the reduction of weight load induced by carrageenan. MPEP significantly increased the withdrawal threshold to mechanical stimulation in a dose-dependent manner, whereas AIDA had significantly reversed the decreased the paw withdrawal threshold only at 200 microM. The present study demonstrated that i.a. MPEP, selective mGluR5 antagonist is more effective than selective mGluR1 antagonist, AIDA on non-evoked pain as well as mechanical hyperalgesia in both induction and maintenance phase in knee joint inflammation. It is suggested that peripheral mGlu5 receptors play a more prominent role in inflammatory pain including evoked and spontaneous pain. Thus, selective mGluR5 antagonist could be effective therapeutic tools in clinical setting.

Induction of total insensitivity to capsaicin and hypersensitivity to garlic extract in human by decreased expression of TRPV1.

TRPV1 is a cation channel which is activated by temperature (> or =42 degrees C) and capsaicin. In the present study, we found a person with total insensitivity to capsaicin and attempted to unravel its causes. The expression levels of TRPV1 protein and mRNA in the cells of the person's buccal mucosa were less than half of those in a normal subject. Sequential analysis of mRNA and genomic DNA revealed several point mutations mostly in the second intron of the person's TRPV1. Interestingly, the subject showed hypersensitivity to garlic extract, but TRPA1 (allicin receptor) level was normal. These results suggest that the decreased expression of TRPV1 may be related to a functional knock out in capsaicin sensation and hypersensitivity to allicin in humans.

Loss of spinal mu-opioid receptor is associated with mechanical allodynia in a rat model of peripheral neuropathy.

The present study investigated whether the loss of spinal mu-opioid receptors following peripheral nerve injury is related to mechanical allodynia. We compared the quantity of spinal mu-opioid receptor and the effect of its antagonists, such as naloxone and CTOP, on pain behaviors in two groups of rats that showed extremely different severity of mechanical allodynia 2 weeks following partial injury of tail-innervating nerves. One group (allodynic group) exhibited robust signs of mechanical allodynia after the nerve injury, whereas the other group (non-allodynic group) showed little allodynia despite having suffered the same nerve injury. In addition, we investigated the quantity of spinal mu-opioid receptor and the effect of its antagonists on pain behaviors after the rats had recovered from mechanical allodynia 16 weeks following nerve injury. Immunohistochemical and Western blot analyses at 2 weeks after nerve injury indicated that spinal mu-opioid receptor content was more reduced in the allodynic group compared to the non-allodynic group. Intraperitoneal naloxone (2 mg/kg, i.p.) and intrathecal CTOP (10 microg/rat, i.t.) administration dramatically induced mechanical allodynia in the non-allodynic group. However, as in naïve animals, neither the loss of spinal mu-opioid receptors nor antagonist-induced mechanical allodynia was observed in the rats that had recovered from mechanical allodynia. These results suggest that the loss of spinal mu-opioid receptors following peripheral nerve injury is related to mechanical allodynia.

Voltage-gated calcium channels play crucial roles in the glutamate-induced phase shifts of the rat suprachiasmatic circadian clock.

The resetting of the circadian clock based on photic cues delivered by the glutamatergic retinohypothalamic tract is an important process helping mammals to function adaptively to the daily light-dark cycle. To see if the photic resetting relies on voltage-gated Ca(2+) channels (VGCCs), we examined the effects of VGCC blockers on the glutamate-induced phase shifts of circadian firing activity rhythms of suprachiasmatic nucleus (SCN) neurons in hypothalamic slices. First, we found that a cocktail of amiloride, nimodipine and omega-conotoxin MVIIC (T-, L- and NPQ-type VGCC antagonists, respectively) completely blocked both phase delays and advances, which were, respectively, induced by glutamate application in early and late night. Next, we discovered that: (i) amiloride and another T-type VGCC antagonist, mibefradil, completely obstructed the delays without affecting the advances; (ii) nimodipine completely blocked the advances while having less impact on delays; and (iii) omega-conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole-cell recordings revealed that T-type Ca(2+) currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L-type Ca(2+) currents did not differ from early to late night in both regions. These results indicate that VGCCs play important roles in glutamate-induced phase shifts, T-type being more important for phase delays and L-type being so for phase advances. Moreover, the results point to the possibility that a nocturnal modulation of T-type Ca(2+) current in retinorecipient neurons is related to the differential involvement of T-type VGCC in phase delays and advances.

Dorsal column lesion reduces mechanical allodynia in the induction, but not the maintenance, phase in spinal hemisected rats.

The dorsal column-medial lemniscal (DC-ML) system is known to be a route of ascending input signals for mechanical allodynia following peripheral nerve injury. We examined whether the pain signals after spinal hemisection were transmitted via the DC-ML system in the induction and maintenance phases of the neuropathic pain. Under enflurane anesthesia, rats were subjected to spinal hemisection at T13 level and bilateral DC lesion was made at T8 level 1 day or 3 weeks after the hemisection. The DC lesion 1 day after the hemisection significantly reduced the mechanical, but not cold, allodynia, whereas the DC lesion 3 weeks after the hemisection did not change both mechanical and cold allodynia. These results suggest that the signals for mechanical allodynia following spinal hemisection should be transmitted via the DC-ML system in the induction, but not maintenance, phase.

Gabapentin relieves mechanical, warm and cold allodynia in a rat model of peripheral neuropathy.

Although recent studies demonstrated the relieving effect of gabapentin on neuropathic pain, the effect has not been sufficiently examined. In the present study, we investigated the effect of gabapentin on mechanical, warm and cold allodynia in a rat model of peripheral neuropathy. Under enflurane anesthesia, animals were subjected to the partial injury of the nerves innervating the tail. Behavioral tests for mechanical, cold and warm allodynia on the tail were performed by von Frey hair (2.0 g) stimulation, 4 and 40 degrees C water immersion, respectively. Intraperitoneal injection of gabapentin (30, 100, 300 mg/kg) significantly alleviated mechanical, warm and cold allodynia in a dose-dependent manner. Our results suggest that gabapentin is an effective agent against mechanical, warm and cold allodynia in a rat model of peripheral neuropathy.

Intraarticular pretreatment with ketamine and memantine could prevent arthritic pain: relevance to the decrease of spinal c-fos expression in rats.

To determine whether intraarticular pretreatment with N-methyl-D-aspartic (NMDA) receptor antagonist ketamine or memantine currently used in humans has prophylactic analgesia in arthritic pain, we examined the effects of their intraarticular injection before carrageenan injection into the knee joint on pain-related behavior and spinal c-Fos expression in rats. Injection of ketamine (0.2 mg and 1 mg) or memantine (0.1 mg, 0.2 mg, and 1 mg) into the knee joint, but not the abdominal cavity, immediately before carrageenan injection (2%, 40 microL) significantly prevented pain-related behavior. The intraarticular injection of ketamine (1 mg) or memantine (0.2 mg) also suppressed c-Fos expression in the laminae I-II and laminae V-VI at the L3-4 spinal level. Subsequent statistical analyses revealed that the degree of the spinal c-Fos expression was correlated with the extent of the pain-related behavior. These results suggest that peripheral administration of NMDA receptor antagonists has prophylactic analgesic effects in arthritic pain, which might be associated with the decrease of central nociceptive signaling. Because ketamine and memantine are currently used in humans and considered clinically safe, they may have therapeutic value in the treatment of joint pain.

The glutamatergic N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors in the joint contribute to the induction, but not maintenance, of arthritic pain in rats.

To determine whether both the N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the knee joint contribute to the induction and/or maintenance of arthritic pain, we examined the effects of intra-articular injection of NMDA receptor antagonist dizocilpine (MK-801) and non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) on the decrease in weight load induced by carrageenan injection into the knee joint cavity in rats. Injection of MK-801 (0.75 and 1.5 mM) and NBQX (0.25, 0.625 and 2.5 mM) immediately prior to carrageenan injection (2%, 40 microl) significantly prevented the pain-related behavior. However, injection of MK-801 (0.75 and 1.5 mM) and NBQX (0.625 and 2.5 mM) 5 h after carrageenan injection had no effect on pain-related behavior. These results suggest that both the NMDA and non-NMDA receptors in the knee joint are involved in the induction, but not maintenance, of arthritic pain.

Ascending pathways for mechanical allodynia in a rat model of neuropathic pain.

To determine what the routes by which mechanical allodynia is transmitted following peripheral nerve injury, we assessed the effects of the dorsal column (DC) lesion performed before and 2 weeks after the partial injury of nerves innervating the tail on mechanical allodynia. Ipsilateral DC lesion 2 weeks after neuropathic surgery significantly, but not completely, attenuated mechanical allodynia. In addition, the DC lesion before peripheral nerve injury did not prevent the generation of mechanical allodynia, which was completely blocked by subsequent contralateral hemisection of the spinal cord. However, unlike mechanical allodynia, DC lesion did not change thermal allodynia. These results suggest that the signals for mechanical allodynia following peripheral nerve injury are transmitted via the ipsilateral DC and the contralateral pathway(s).

Effects of morphine on mechanical allodynia in a rat model of central neuropathic pain.

We examined whether morphine reduced the behavioral signs of neuropathic pain below level induced by T13 spinal hemisection in rats. In order to examine the effect of morphine on the mechanical allodynia, morphine alone, morphine with naloxone and saline were administered intraperitoneally and intrathecally and behavioral tests were conducted. In systemic injection, mechanical allodynia was reduced only when a higher concentration of morphine (5 mg/kg) was used. Intrathecally injected morphine (0.5, 1, 2, 5 microg) reduced mechanical allodynia dose-dependently. It is suggested that systemic morphine has limited effect on mechanical allodynia but direct spinal administration of morphine is more effective in controlling central pain following spinal cord injury.

Cold and mechanical allodynia in both hindpaws and tail following thoracic spinal cord hemisection in rats: time courses and their correlates.

We assessed (1) the time courses of cold and mechanical allodynia in both hindpaws and the tail, and (2) the relationship of the allodynia signs between different sites following spinal cord hemisection. Under enflurane anesthesia, rats were subjected to spinal hemisection at T13. The hemisected rats exhibited a significant increase in mechanical and cold allodynia signs of both hindpaws and the tail for 22-26 weeks postoperatively. In addition, mechanical allodynia signs were significantly correlated not only between the ipsilateral and the contralateral hindpaws, but also between the hindpaws and the tail. These results suggested that cold and mechanical allodynia developed extensively and lasted for a long time following spinal cord hemisection, and mechanical allodynia shown at different sites may be induced at least in part by common generating mechanisms.

Increases in spinal vasoactive intestinal polypeptide and neuropeptide Y are not sufficient for the genesis of neuropathic pain in rats.

We tested the hypothesis that increases in the spinal levels of vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were related to the development of neuropathic pain. To this aim, we compared two groups of rats. One group showed well-developed neuropathic pain in the tail following unilateral transection of the inferior and superior caudal trunks between the S1 and S2 spinal nerves, and the other group showed poorly-developed neuropathic pain despite the same nerve injury. The increases in immunoreactivity of VIP and NPY in the S1 dorsal horn (injured segment) were not significantly different between the two groups. These results suggested that increases in the spinal levels of VIP and NPY after peripheral nerve injury were not sufficient for the development of neuropathic pain.

Local neurokinin-1 receptor in the knee joint contributes to the induction, but not maintenance, of arthritic pain in the rat.

Substance P is known to exert various pro-inflammatory effects that are mediated by neurokinin-1 (NK-1) receptor in peripheral tissues. This study examined the effect of the NK-1 receptor antagonist cis-2-[diphenylmethyl]-N-[(2-iodophenyl)-1-azabicyclo[2.2.2]octan-3-amine] (L-703,606) on nociceptive response following carrageenan injection (2%, 50 microl) into the knee joint cavity of the right hind leg. L-703,606 injection (0.1 or 1 mM, 50 microl) into the same joint cavity immediately before the carrageenan injection significantly reduced the nociceptive response. However, antagonist treatment at 5 h after carrageenan injection was ineffective in alleviating nociception. Neither intraperitoneal injection of the antagonist (1 mM, 50 microl) immediately before the carrageenan injection was effective. These results suggest that local NK-1 receptor contributes to the induction, but not maintenance, of arthritic pain.

A mouse model for peripheral neuropathy produced by a partial injury of the nerve supplying the tail.

We attempted to develop a mouse model for peripheral neuropathy by a partial injury of the nerve supplying the tail. Under enflurane anesthesia, the unilateral superior caudal trunk was resected between the S3 and S4 spinal nerves. Tests for thermal allodynia were conducted by immersing the tail into 4 or 38 degrees C water. The mechanical allodynia was assessed by stimulating the tail with a von Frey hair (1.96 mN, 0.2 g). After the nerve injury, the experimental animals had shorter tail withdrawal latencies to cold and warm water immersion than the presurgical latency, and exhibited an increase in tail response to von Frey stimulation. We interpret these abnormal sensitivities as the signs of mechanical, cold and warm allodynia following the superior caudal trunk injury in the mouse.