Chronic Constriction Injury Surgery Research Articles

(299) Therapeutic morphine prolongs neuropathic pain in Fischer 344 rats: a role for TLR4 and inflammasome signaling in the lumbar spinal cord

Despite their status as gold-standard therapeutic analgesics for neuropathic pain, opioids initiate microgliosis and proinflammatory cytokine release via Toll Like Receptor 4 (TLR4) in naïve animals, which enhances pro-nociceptive neuroexcitability. However, the behavioral and molecular impact of opioid-induced gliosis has not been defined in the presence of peripheral nerve injury, which also induces lumbar spinal gliosis per se. We hypothesized that sciatic chronic constriction injury (CCI)-allodynia would be enhanced by subsequent repeated morphine, in an inflammasome-dependent fashion involving TLR4, the purinergic receptor P2X7, and caspase-1, which facilitates release of mature interleukin (IL)-1b. Beginning 10 days after CCI surgery in male Fischer 344 rats, s.c. morphine (5 mg/kg b.i.d.) or equivolume saline was administered for 5 consecutive days. Compared to vehicle, morphine treatment significantly prolonged the duration of CCI-induced allodynia from 5 to 11 weeks (n=6/group; p<0.05). Continuous intrathecal infusion of inhibitors of TLR4 ([+]-naloxone; 60 mcg/h), P2X7 (Brilliant Blue G; 30 ng/h), or caspase-1 (ac-YVAD-cmk; 1 mcg/h) prevented morphine-prolonged CCI-allodynia when administered concomitantly with morphine, and, except for ac-YVAD-cmk, completely reversed established morphine-prolonged CCI-allodynia when administered 5 weeks after morphine dosing (n=6/group; p<0.05). Morphine treatment induced significant release of tumor necrosis factor (TNF)a and IL-1b (p<0.05) from lumbar dorsal spinal cords (p<0.05). Furthermore, a single intrathecal dose of soluble TNF receptor (100 mcg; Amgen), IL-1 receptor antagonist (100 mcg; Amgen) or IL-6 (10 mcg; K. Rice) transiently attenuated morphine-prolonged CCI-allodynia (n=6/group; p<0.05). These data suggest that morphine may paradoxically contribute to the incidence of chronic neuropathic pain due to an interaction between morphine and the products of nerve injury, resulting in sustained inflammasome signaling. These data further present an opportunity to pharmacologically inhibit paradoxical pain enhancement while retaining the analgesic properties of morphine, as each is mediated via different receptors and underlying mechanisms.

Hydrogen-Rich Saline Attenuated Neuropathic Pain by Reducing Oxidative Stress

Reactive oxygen species (ROS) are often associated with persistent pains such as neuropathic and inflammatory pain. Hydrogen gas can reduce ROS and alleviate cerebral, myocardial, and hepatic ischemia/reperfusion injuries. In the present study, we aim to investigate whether hydrogen-rich saline can reduce neuropathic pain in a rat model of chronic constriction injury (CCI). Thirty SD rats were randomly divided into three groups: sham group was administered sodium chloride by intrathecal injection (n=10); control groups underwent CCI surgery and were administered sodium chloride by intrathecal injection (n=10); vehicle group underwent CCI surgery and was administered hydrogen-rich saline by intrathecal injection (n=10). Drugs were administered in the dose of 100 ul/kg once a day at 0.5 hours before and 1-7 day after CCI surgery. The mechanical thresholds were tested at one day before and 3-14 day after CCI surgery. We found that hydrogen-rich saline significantly elevated the mechanical thresholds of neuropathic pain compared to vehicle (physiologic saline) control in CCI rats (p<0.05); it also decreased the levels of myeloperoxidase, maleic dialdehyde, and protein carbonyl in spinal cord by 7 days post-chronic constriction injury(p<0.05). In addition, hydrogen-rich saline also suppressed the expression of p38-mitogen-activated protein kinase (p38MAPK) and brain-derived neurotrophic factor (BDNF) in the spinal cord by 7 days post-chronic constriction injury (p<0.01, p<0.01, respectively), but had no effect on P2X4R (p>0.05), an ATP receptor. Intrathecal injection of hydrogen-rich saline can decrease oxidative stress and the expression of p38MAPK and BDNF that may contribute to the elevated threshold of neuropathic pain in rat CCI model.

Involvement of the spinal NALP1 inflammasome in neuropathic pain and aspirin-triggered-15-epi-lipoxin A4 induced analgesia

Neuroinflammation plays an important role in nerve-injury-induced neuropathic pain, but the explicit molecular mechanisms of neuroinflammation in neuropathic pain remain unclear. As one of the most critical inflammatory cytokines, interleukin-1β (IL-1β) has been regarded as broadly involved in the pathology of neuropathic pain. The inflammasome caspase-1 platform is one primary mechanism responsible for the maturation of IL-1β. Lipoxins, a type of endogenous anti-inflammatory lipid, have proved to be effective in relieving neuropathic pain behaviors. The present study was designed to examine whether the inflammasome caspase-1 IL-1β platform is involved in chronic constriction injury (CCI)-induced neuropathic pain and in lipoxin-induced analgesia. After rats were subjected to the CCI surgery, mature IL-1β was significantly increased in the ipsilateral spinal cord, and the inflammasome platform consisting of NALP1 (NAcht leucine-rich-repeat protein 1), caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) was also activated in spinal astrocytes and neurons, especially at the superficial laminae of the spinal dorsal horn; The aspirin-triggered-15-epi-lipoxin A4 (ATL), which shares the potent actions of the endogenous lipoxins, was administered to the CCI rats. Repeated intrathecal injection with ATL markedly attenuated the CCI-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1β maturation. These results suggested that spinal NALP1 inflammasome was involved in the CCI-induced neuropathic pain and that the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation.

Characterizing Activity and Muscle Atrophy Changes in Rats With Neuropathic Pain

The study of neuropathic pain has focused on changes within the nervous system, but little research has described systemic changes that may accompany neuropathic pain. As part of a larger project characterizing the metabolic, activity, and musculoskeletal changes associated with neuropathic pain, the objective of the current study was to characterize changes in spontaneous activity and skeletal muscle mass using an established animal model of neuropathic pain, the chronic constriction injury (CCI) model. Male Sprague-Dawley rats were used in this pre- and posttest quasi-experimental study. The experimental group (n = 13) received CCI surgery, while age- and weight-matched rats received sham surgery (SHAM; n = 5). Thermal testing verified the presence of neuropathic pain. Spontaneous cage activity was measured gravimetrically prior to and following CCI (n = 4). Animals were euthanized and skeletal muscle was dissected and weighed to determine muscle atrophy. Shorter foot withdrawal latency of the ipsilateral hind limb confirmed the presence of thermal hyperalgesia in CCI rats, a sign of neuropathic pain. Weight increased in both CCI and SHAM rats. Spontaneous activity decreased following CCI ligation. Muscles of the ipsilateral hind limb weighed significantly less than contralateral hind limb muscles in CCI rats 2 and 6 weeks after surgery. In addition, CCI rats had smaller ipsilateral hind limb muscles than SHAM rats. Neuropathic pain contributes to skeletal muscle atrophy and decreases in activity in rats.

Chronic Constriction Injury Induced Long-Term Changes in Spontaneous Membrane-Potential Oscillations in Anterior Cingulate Cortical Neurons in Vivo

Background: Neuropathic pain induction by nerve injury has been shown by in vitro studies to be accompanied by synaptic strengthening in the anterior cingulate cortex (ACC) and has been shown by pharmacological studies in vivo to be prevented by blocking N-methyl-D-aspartate (NMDA) receptor-dependent ACC plasticity. These findings indicate that ACC neurons undergo nerve injury-induced synaptic modifications and further raise a new question about neuropathic pain-associated changes in neuronal activity in the ACC in vivo, particularly spontaneous neuronal oscillations – a process believed to be fundamental for many forms of brain function. Objective: In this study, we examined the change of spontaneous membrane-potential (MP) oscillations in the ACC in vivo in a neuropathic pain animal model of chronic constriction injury (CCI), which may account for neuropathic pain development, as well as pain hypersensitivity and spontaneous pain. Study Design: Experimental trial in rats. Methods: Neuropathic pain rats were produced by CCI surgery on the common sciatic nerve. Neuropathic pain-related behaviors were accessed by evoked responses to both mechanical and thermal stimuli, as well as spontaneous pain indicated by spontaneous foot lifting. In vivo wholecell recording was performed in both control and neuropathic pain rats under anaesthesia. MP and action-potential (AP) changes of layer II/III ACC pyramidal cells were measured in current-clamp mode. The level of anaesthesia was evaluated by monitoring respiratory and heart rates in some experiments. Results: Within 7 to 14 days after CCI surgery, the frequency of MP oscillations of ACC neurons was found to be significantly higher than that in control rats. Such an increase in oscillation frequency after surgery was not due to periphery transmission via the sciatic nerve subjected to CCI surgery and was indicated to be accounted for by neuronal modifications in the central nervous system. Furthermore, this increase was found to result in a higher overall level of MP excitation as well as an increase in spontaneous AP firing. Limitations: Our findings in MP and AP changes were obtained in anaesthetized brains; this issue remains to be further examined by using whole-cell recording in awake behaving animals. Conclusions: Neuropathic pain is accompanied by the increase in rates of spontaneous oscillations of ACC neurons. This change may be critical for neuropathic pain development, as well as pain hypersensitivity and spontaneous pain in neuropathic pain animals. Key Words: Neuropathic pain, anterior cingulate cortex, spontaneous activity, neuronal oscillation, chronic constriction injury

18. Repeated morphine enhances peripheral nerve injury-induced allodynia via central immune mechanisms

Glial activation occurs after morphine and peripheral nerve injury alone, but the behavioral and molecular impact in tandem is unknown. We predicted that sciatic chronic constriction injury (CCI)-allodynia would be enhanced by subsequent repeated morphine, in a Toll-like Receptor 4 [TLR4]-dependent fashion. Mild (1 sciatic constriction) and classic (4 sciatic constrictions) CCI was performed on male Sprague Dawley (SD) and Fischer 344 (F344) rats, respectively. Beginning 10–14 days after CCI surgery, s.c. morphine (5 mg/kg or equivolume saline b.i.d.) was administered for 5 consecutive days. This morphine protocol potentiated the magnitude of mild CCI-induced allodynia (von Frey test) in SD rats ( n = 10/group; p < 0.05), and prolonged the duration of classic CCI-induced allodynia in F344 rats ( n = 6/group; p < 0.05). The TLR4 antagonist (+)-naloxone was co-administered with morphine treatment via intrathecal osmotic minipump (60 μg/h), which completely abolished potentiated allodynia in SD rats ( n = 6/group; p < 0.05). A single intrathecal dose of interleukin-1 (IL-1) receptor antagonist (100 μg; Amgen) transiently restored thresholds to pre-surgery levels ( n = 4/group; p < 0.05). An interaction between CCI and morphine was present for GFAP expression at 21 days after the conclusion of morphine administration in SD rats ( n = 4/group; p < 0.01). These data indicate that morphine potentiation of allodynia is dependent on TLR4 and IL-1 signaling in the CNS, and that morphine and the products of nerve injury may interact, resulting in paradoxical enhancement of neuropathic pain.

Activation of mTOR in the spinal cord is required for pain hypersensitivity induced by chronic constriction injury in mice

BackgroundThe mammalian target of rapamycin (mTOR) is known to regulate cell growth, and it also participates in pain transmission as has been recently verified in inflammatory and neuropathic pain models. The targeting of mTOR represents a new strategy for the control of chronic pain. In the present study, we investigated the effect of mTOR in the expression of PSD95 and NR2B-PSD95 or GluA2-PSD95 interaction ratio in a chronic constriction injury (CCI) mice model. MethodsPaw mechanical withdrawal threshold (PMWT) and paw withdrawal thermal latency (PWTL) were respectively used to assess mechanical allodynia and thermal hyperalgesia after CCI operation and intrathecal injection of rapamycin. Western blot and co-immunoprecipitation were used to investigate the effects of rapamycin on the expression of PSD95 and interaction ratio of NR2B-PSD95 or GluA2-PSD95 in the spinal dorsal horn of mice. ResultsOur study demonstrated that the inhibition of spinal mTOR with intrathecal injections of rapamycin (1μg/5μL) for days 1–6 after CCI surgery led to an obvious decrease in CCI-induced neuropathic pain. Rapamycin significantly reduced the PMWT of CCI mice, whereas there was no significant effect on PWTL. The active form of the mTOR signaling pathway (p-mTOR, p-4EBP1 and p-p70S6k) at the spinal level remarkably increased in CCI mice, and rapamycin could inhibit this up-regulation. The increased expression of PSD95 and the interaction ratio of GluA2-PSD95 or NR2B-PSD95 could also be inhibited by intrathecal injection of rapamycin. ConclusionThese data suggest that the mTOR pathway is activated in the spinal dorsal horn in CCI-induced neuropathic pain, and the intrathecal injection of rapamycin can reduce mechanical allodynia. Our findings indicate that spinal mTOR is an important component of CCI-induced neuropathic pain, and mTOR may be a potential target for chronic pain therapy.

Involvement of Protein Kinase B/Akt in Analgesic Effect of Dexmedetomidine on Neuropathic Pain

Involvement of Protein Kinase B/Akt in Analgesic Effect of Dexmedetomidine on Neuropathic Pain

Effect of electroacupuncture on the pathomorphology of the sciatic nerve and the sensitization of P2X3 receptors in the dorsal root ganglion in rats with chronic constrictive injury

To explore the effect of electroacupuncture (EA) on the pathomorphology of the sciatic nerve and the role of P2X3 receptors in EA analgesia. The chronic constriction injury (CCI) model was adopted in this study. A total of 32 rats were randomly divided into four groups: sham CCI, CCI, CCI plus contralateral EA (CCI + conEA) and CCI plus ipsilateral EA (CCI + ipsEA). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. EA began at day 7 after the CCI operation and was applied to the Zusanli (ST 36) and Yanglingquan acupoints (GB 34). At day 14, the pathomorphologic changes of the operated sciatic nerve were demonstrated by hematoxylin and eosin staining. In addition, dorsal root ganglion (DRG) neurons isolated from rats were examined by electrophysiological recording to determine if the P2X3 receptor agonists, adenosine 5'-triphosphate disodium (ATP) and α,β-methylen-ATP (α,β-meATP) evoked inward currents. Pain thresholds in the CCI group were obviously decreased post CCI surgery (P<0.01). In the EA groups, thermal and mechanical threshold values were increased after the last EA treatment (P<0.05, P<0.01). There was no significant difference in light microscopic examination among the four groups (P>0.05). Current amplitude after application of ATP and α,β-meATP in DRG neurons were much larger in the CCI group compared to those obtained in sham CCI (P<0.05). ATP and α, β-meATP invoked amplitudes in the CCI + EA groups were reduced. There was no signififi cant difference between the CCI + conEA group and the CCI + ipsEA group (P>0.05). EA analgesia may be mediated by decreasing the response of P2X3 receptors to the agonists ATP and α,β-meATP in the DRG of rats with CCI. No pathological changes of the sciatic nerve of rats were observed after EA treatment.

Targeted liquid chromatography quadrupole ion trap mass spectrometry analysis of tachykinin related peptides reveals significant expression differences in a rat model of neuropathic pain

Animal models are widely used to perform basic scientific research in pain. The rodent chronic constriction injury (CCI) model is widely used to study neuropathic pain. Animals were tested prior and after CCI surgery using behavioral tests (von Frey filaments and Hargreaves test) to evaluate pain. The brain and the lumbar enlargement of the spinal cord were collected from neuropathic and normal animals. Tachykinin related peptides were analyzed by high performance liquid chromatography quadrupole ion trap mass spectrometry. Our results reveal that the β-tachykinin58–71, SP and SP3–11 up-regulation are closely related to pain behavior. The spinal β-tachykinin58–71, SP and SP3–11 concentrations were significantly up-regulated in neuropathic animals compared with normal animals (p<0.001; p<0.001 and p<0.05, respectively). In contrast, the spinal SP5–11 concentration in neuropathic animals revealed a significant down-regulation compared with normal animals (p<0.05). The brain β-tachykinin58–71 and SP concentrations were significantly up-regulated (p<0.05 and p<0.001, respectively). Interestingly, no significant concentration differences were observed in the spinal cord and brain for NKA, β-tachykinin58–70, SP1–7 and SP6–11 (p>0.05). The β-tachykinin58–71, SP and C-terminal SP metabolites could potentially serve as biomarkers in early drug discovery.

The role of HCN channels within the periaqueductal gray in neuropathic pain

Peripheral and spinal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a key role in neuropathic pain by regulating neuronal excitability. HCN channels are expressed in the ventral–lateral periaqueductal gray (vlPAG), a region that is important for pain modulation. However, the role of vlPAG HCN channels in neuropathic pain remains poorly understood. In the present study, we investigated the impact of changes to vlPAG HCN channels on neural activity in neuropathic pain. First, sciatic nerve chronic constriction injury (CCI) was established as a neuropathic pain model. Then, changes in HCN channels and their influence on vlPAG neuronal activity were detected. Our results indicate that after CCI surgery the following changes occur in vlPAG neurons: the expression of HCN1 and HCN2 channels is increased, the amplitude of the hyperpolarization-activated current (Ih) is augmented and its activation curve is shifted to more positive potentials and there is an increase in the frequency of action potential (AP) firing and spontaneous EPSCs that is attenuated by ZD7288, a HCN channel blocker. In addition, forskolin, which can elevate intracellular cAMP, mimics the CCI induced changes in neuronal excitability in the vlPAG. The effects of forskolin were also reversed by ZD7288. Taken together, the present data indicate an important role for HCN channels in the vlPAG in neuropathic pain.

Resveratrol Facilitates Pain Attenuation in a Rat Model of Neuropathic Pain Through the Activation of Spinal Sirt1

Little research has been conducted regarding the implications of Sirt1 (a classic III HDAC) in neuropathic pain. The aim of this study was to investigate the variation in the expressions of spinal Sirt1 and acetyl-histone H3 in a rat model of chronic constriction injury. A neuropathic pain model of chronic constriction injury (CCI) was established in a unilateral hind limb in Sprague-Dawley rats. Western blot analysis and immunohistochemistry revealed that Sirt1 (silent information regulator) expression decreased, whereas acetyl-histone H3 increased in the spinal cord following CCI surgery. The intrathecal administration of resveratrol, an activator of Sirt1, attenuated CCI-induced mechanical allodynia and thermal hyperalgesia, increased Sirt1 expression, and decreased acetyl-histone H3 in the spine. Resveratrol induced no obvious histopathological changes in the spinal cord. Our data provide new evidence for the contribution of spinal Sirt1 to the initiation and maintenance of neuropathic pain. The antinociceptive effects of resveratrol may be mediated through the activation of spinal Sirt1 in CCI rats.

Electroacupuncture and Lumbar Transplant of GDNF-Secreting Fibroblasts Synergistically Attenuate Hyperalgesia after Sciatic Nerve Constriction

Electroacupuncture (EA) has been shown to induce potent analgesic effects on neuropathic pain in both patients and rodents. Cell therapy to release antinociceptive agents near the pain processing centers of the spinal cord is a promising next step in the development of treatment modalities. This study investigated the effects of the combination of EA and cell therapy by glial cell line-derived neurotrophic factor (GDNF) on neuropathic pain in rats. The hyperalgesic state was induced by chronic constriction injury (CCI) of the sciatic nerve and fibroblasts genetically modified to secrete bioactive GDNF (FBs-GDNF) were used for cell therapy. Fifty-eight rats with neuropathic pain were randomly divided into five groups (CCI+PBS, n = 11; CCI+FBs-GDNF, n = 12; CCI+EA+PBS, n = 11; CCI+EA+FBs-pLNCX2, n = 12; CCI+EA+FBs-GDNF, n = 12). On the 7th day after CCI, the rats received intrathecal transplantation of FBs-GDNF or control fibroblasts (FBs-pLNCX2). In the meantime, EA was administered once every other day from the 7th day after CCI surgery for 21 days. The paw withdrawal latency (PWL) to radiant heat was measured every other day. The results showed that the ipsilateral PWL of the rats from all three EA treatment groups significantly increased starting on the 12th day compared with the PBS control group. Strikingly, the group which received EA treatment and FBs-GDNF transplantation (CCI+EA+FBs-GDNF) showed a significantly decreased thermal hyperalgesia after 2 weeks post CCI surgery compared with the groups which received EA treatment and FBs-pLNCX2 transplantation (CCI+EA+FBs-pLNCX2) or PBS (CCI+EA+PBS) as well as the FBs-GDNF transplantation group without EA treatment (CCI+FBs-GDNF). Our data suggest that EA and cell therapy can synergistically attenuate hyperalgesia in neuropathic pain rats.

Temporal distribution of p300/CBP immunoreactivity in the adult rat spinal dorsal horn following chronic constriction injury (CCI).

p300 and its homolog cyclic AMP response element binding protein (CBP) are coactivators that were identified to participate in many biological processes including neural development and cognition. Their roles within the rodent spinal cord have not been reported systematically; in this study, their spatiotemporal distribution in the spinal cord of adult rat following chronic constriction injury (CCI) was studied. p300 and CBP expressed predominantly in nuclei in the gray matter of rat spinal cord. Rats undergoing CCI surgery showed increased p300/CBP immunoreactivity (IR) compared with normal control and sham-operated rats. The number of IR cells reached the peak at day 14 following CCI compared with those on day 3, 7, and 21, accompanied with significant behavioral changes of neuropathic pain. Cell-type determination by immunofluorescence at day 14 following CCI revealed that p300 and CBP expressed in neurons, but not in astrocytes or microglial cells. These results suggest that p300 and CBP are probably involved in the maintenance of neuropathic pain on spinal cord level. Furthermore, p300 and CBP may serve as a sensor only in neurons but not in astrocytes or microglia cells in the adult rat spinal cord.

Preventive and curative effects of radon inhalation on chronic constriction injury‐induced neuropathic pain in mice

Radon therapy is clinically useful for the treatment of pain-related diseases. However, there have been no studies regarding the effects of radon inhalation on neuropathic pain. In this study, we aimed to determine whether radon inhalation actually induced a remission of neuropathic pain and improved the quality of life. First, we investigated the antinociceptive effects of radon inhalation in the chronic constriction injury (CCI) model of neuropathic pain. We evaluated pain behaviour in mice before and after CCI surgery, using von Frey test. Pretreated mice received CCI surgery immediately after 24-h inhalation of radon at background (BG) concentration (c. 19 Bq/m(3) ), or at a concentration of 1000 or 2000 Bq/m(3) , and post-treated mice inhaled similar levels of radon 2 days after CCI surgery. CCI surgery induced mechanical allodynia and hyperalgesia on a plantar surface of mice, as assessed using von Frey test, and 2000 Bq/m(3) radon inhalation alleviated hyperalgesic conditions 22-37% compared to BG level concentration. Concurrently, CCI surgery increased norepinephrine (NE), tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO) concentrations in plasma, and leukocyte migration in paws. Furthermore, CCI-induced neuropathy reduced superoxide dismutase (SOD) activity. Treatment with radon inhalation, specifically at a concentration of 2000 Bq/m(3) , produced antinociceptive effects, i.e., lowered plasma TNF-α, NE and NO levels and restored SOD activity, as well as pain-related behaviour. This study showed that inhalation of 2000 Bq/m(3) radon prevented and alleviated CCI-induced neuropathic pain in mice.

Dose and administration-period play a key role in the effect of ceftriaxone on neuropathic pain in CCI-operated rats

Abstract Introduction An unmet medical need for more effective therapies of neuropathic pain exits. Here modulation of the glutaminergic system represents an unexplored possibility. Down-regulation ofglutamate transporters potentiates pain transmission by delaying the removal of glutamate from the synapse. In the spinal cord, glutamate transporter 1 (GLT–1) is responsible for more than 90% of the glutamate uptake. Ceftriaxone, a β-lactam, is believed to induce the expression of GLT–1 through the transcriptional factor (NF-ºB) pathway, which results in induced promoter activity and thereby increased synthesis of GLT-1 protein. Objectives To evaluate the analgesic effect of ceftriaxone in the Chronic Constriction Injury (CCI) rat model of neuropathic pain and to investigate the pharmacodynamics of ceftriaxone in a chronic dosing regime. Methods In CCI rats, mechanical and thermal hypersensitivity, were determined with von Frey filaments and Hargreaves test, respectively. Groups of rats received ceftriaxone (200, 300 or 400 mg/kg, i.p.) once daily in 7–19 days and the control groups received vehicle. Results From a total of 24 CCI operated rats, 16 rats developed both mechanical (withdrawal threshold ≤3g) and thermal hypersensitivity (latency threshold ≤13 s.). Ceftriaxone alleviated mechanical allodynia and thermal hyperalgesia in CCI operated. Daily dosing of ceftriaxone 200, 300 and 400 mg/kg reached the same withdrawal threshold levels as before the CCI surgery, after 18, 12 and 7 days, respectively. This indicates that the dynamic effect of ceftriaxone is not only dependent of the dose, but also the duration of administration. Thus, it seems that dose exposure above a certain threshold is necessary to induce protein synthesis. Conclusion The CCI model is a useful model to evaluate the anti-nociceptive effects of ceftriaxone. Increased dose do not only elevate effect magnitude but also the rate of with which the effect appears.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Automated measurement of spontaneous pain‐associated limb movement and drug efficacy evaluation in a rat model of neuropathic pain

The withdrawal response elicited by a nociceptive stimulus, i.e., evoked pain measure, is commonly used as an efficacy endpoint in neuropathic pain animal models. It, however, has several limitations, which highlight the importance of examining spontaneous pain. The present study describes an automated method for measuring spontaneous pain behaviour in a rat model of neuropathic pain caused by chronic constriction injury (CCI) of sciatic nerve. After CCI surgery, a small magnet was implanted into the operated limb. The rat was placed in a test chamber that was surrounded by wire coil. Limb movements, including lifting/guarding, flinching/shaking, licking and walking in the operated limb, caused changes in the electromagnetic field, including a change in voltage and transformed into a signal via an amplifier. CCI rats consistently showed more frequent limb movement than sham rats. There was no significant correlation between the frequency of spontaneous pain behaviour and the evoked pain symptoms. Treatment with duloxetine (30 mg/kg p.o.) and amitriptyline (30 and 100 mg/kg p.o.) significantly reduced this frequency. Pregabalin at 30 mg/kg p.o. tended to reduce the frequency, and diclofenac up to 10 mg/kg p.o. had no effect. A non-subjective automated method for measuring spontaneous pain behaviour in an animal model of neuropathic pain was established. It is expected that the current system will greatly enhance the analysis of spontaneous pain-related behaviour, which is a predominant symptom in patients with neuropathic pain. The current system may also be valuable in the screening of potential analgesic treatments.

Activation of Spinalα2-Adrenoceptors Using Diluted Bee Venom Stimulation Reduces Cold Allodynia in Neuropathic Pain Rats

Cold allodynia is an important distinctive feature of neuropathic pain. The present study examined whether single or repetitive treatment of diluted bee venom (DBV) reduced cold allodynia in sciatic nerve chronic constriction injury (CCI) rats and whether these effects were mediated by spinal adrenergic receptors. Single injection of DBV (0.25 or 2.5 mg/kg) was performed into Zusanli acupoint 2 weeks post CCI, and repetitive DBV (0.25 mg/kg) was injected for 2 weeks beginning on day 15 after CCI surgery. Single treatment of DBV at a low dose (0.25 mg/kg) did not produce any anticold allodynic effect, while a high dose of DBV (2.5 mg/kg) significantly reduced cold allodynia. Moreover, this effect of high-dose DBV was completely blocked by intrathecal pretreatment of idazoxan (α2-adrenoceptor antagonist), but not prazosin (α1-adrenoceptor antagonist) or propranolol (nonselective β-adrenoceptor antagonist). In addition, coadministration of low-dose DBV (0.25 mg/kg) and intrathecal clonidine (α2-adrenoceptor agonist) synergically reduced cold allodynia. On the other hand, repetitive treatments of low-dose DBV showing no motor deficit remarkably suppressed cold allodynia from 7 days after DBV treatment. This effect was also reversed by intrathecal idazoxan injection. These findings demonstrated that single or repetitive stimulation of DBV could alleviate CCI-induced cold allodynia via activation of spinal α2-adrenoceptor.

Biphasic effects of chronic intrathecal gabapentin administration on the expression of protein kinase C gamma in the spinal cord of neuropathic pain rats

Objectives Patients suffering from neuropathic pain are difficult to treat and many methods are used to resolve this issue. In this study, we used a model of neuropathic pain comprising rats with chronic constriction injury (CCI) on the left sciatic nerve to investigate the chronic effect of gabapentin via intrathecal administration. We also observed the expression of dorsal spinal protein kinase C gamma subunit (PKCγ) and other pain-related molecules in the spinal area which included cyclooxygenase 2 (COX2), c-Fos and cyclic AMP-dependent transcription factor (ATF3) in the neuropathic pain animals. Methods Male Sprague-Dawley (SD) rats (250–380 g) were randomly assigned to four groups, i.e., control, gabapentin (Gaba), MK801, and gabapentin plus MK801 (Gaba+M) groups. A PE-5 catheter was inserted into the lumbar spine area via the cervical spine area. CCI was performed the following day after the intrathecal catheter implant surgery. Gabapentin (1.05 μmol/day) was then given the following day after CCI surgery. Intrathecal gabapentin was administrated for 14 consecutive days. Pain-related behavior was assessed every 2 days thereafter by measuring the latency of foot withdrawal elicited by noxious radiant heat or Von Frey microfilament applied to the hind-paw plantar surface. MK801 (30 μg/day), an N-methyl-D-aspartate (NMDA) receptor blocker, was also added for potential effect. The tissue of dorsal horn of the lumbar spine was harvested on the 14 th day for the expression of COX2, c-Fos, ATF3 and PKCγ with Western blotting, and positive finding protein was then checked on 7 th day for further evaluation. Results The beneficial effect of intrathecal gabapentin of statistic significance on thermal duration and mechanical microfilament appeared after 7-day and 11-day consecutive treatment, respectively. Furthermore, the NMDA receptor blocker also potentiated the effect on the behavior of thermal and mechanical stimulations. Gabapentin had no effect on the expression of COX2, c-Fos and ATF3. Interestingly, the expression of PKCγ in the spinal cord was initially inhibited by gabapentin on the 7 th day but was potentiated on the 14 th day. Conclusions Our results indicate that chronic intrathecal gabapentin has beneficial effects on the behaviors of both thermal and mechanical stimulations in the neuropathic pain animals and the NMDA blocker can potentiate this effect. Furthermore, gabapentin has biphasic effect on the expression of PKCγ in the spinal cord on Day 7 and Day 14 for the model rats with CCI.