Evaluation of different procedure involved in the Transcranial Direct Current Stimulation (tDCS) technique experimental application

Introduction: The transcranial direct current stimulation (tDCS) is a non-invasive technique, which induces neuroplastic changes in the central nervous system of animals and humans. Furthermore, tDCS has been suggested as a therapeutic tool for pain management. The aim of this study was to standardize a non-invasive tDCS technique indexed by the nociceptive response of rats submitted to different conditions necessary to the tDCS application. Method: 60-day-old male Wistar rats (n=65), divided into 6 groups: control(C); non-active sham (NAS); active-sham (AS); active-sham restrained (ASR); non-active sham restrained (NASR); active tDCS treatment. Animals received treatment during 30 seconds (sham-active) or 20 minutes (restraint and tDCS)/8 days. Nociceptive threshold was assessed by Hot Plate test at baseline, immediately and 24h after the first session, immediately and 24h after the last session. Variance analysis of repeated measurements followed by Bonferroni was performed for intra-group comparison. Results: Physical restraint and 30 seconds stimulation (sham-tDCS) increased pain sensitivity (P≤0.05), and tDCS treatment was able to prevent the thermal hyperalgesia. Our original tDCS montage is similar to that used in the procedure with humans, because it is not an invasive technique. The electrodes are positioned on the head, and the animals are immobilized during the 20-minute treatment. As this procedure could involve behavior and neurochemical alterations due to stress induced by restriction (thus, it creates a research bias), we hypothesized that a 30-second electrical stimulus application (sham-tDCS) and the physical restriction used during tDCS treatment might alter nociceptive response in rats. Conclusion: There are methodological limitations in the present tDCS-technique. Although active-tDCS treatment is able to prevent these harmful effects, interference of these factors has to be considered during the results' analysis. Future adaptations of the tDCS-technique in rats are required to evaluate its therapeutic effects (AU)

Peripheral Cellular Mechanisms of Artemin-induced Thermal Hyperalgesia in Rats

In the present study, we investigated the role of peripheral ionotropic receptors in artemin-induced thermal hyperalgesia in the orofacial area. Male Sprague-Dawley rats weighting 230 to 280 g were used in the study. Under anesthesia, a polyethylene tube was implanted in the subcutaneous area of the vibrissa pad, which enabled drug-injection. After subcutaneous injection of artemin, changes in air-puff thresholds and head withdrawal latency time were evaluated. Subcutaneous injection of artemin (0.5 or 1 µg) produced significant thermal hyperalgesia in a dose-dependent manner. However, subcutaneous injection of artemin showed no effect on air-puff thresholds. IRTX (4 µg), a TRPV1 receptor antagonist, D-AP5 (40 or 80 µg), an NMDA receptor antagonist, or NBQX (20 or 40 µg), an AMPA receptor antagonist, was injected subcutaneously 10 min prior to the artemin injection. Pretreatment with IRTX and D-AP5 significantly inhibited the artemin-induced thermal hyperalgesia. In contrast, pretreatment with both doses of NBQX showed no effect on artemin-induced thermal hyperalgesia. Moreover, pretreatment with H-89, a PKA inhibitor, and chelerythrine, a PKC inhibitor, decreased the artemin-induced thermal hyperalgesia. These results suggested that artemin-induced thermal hyperalgesia is mediated by the sensitized peripheral TRPV1 and NMDA receptor via activation of protein kinases.

Differential Role of Central GABA Receptors in Nociception of Orofacial Area in Rats

The present study investigated the role of central GABA(A) and GABA(B) receptors in orofacial pain in rats. Experiments were conducted on Sprague-Dawley rats weighing between 230 and 280 g. Intracisternal catheterization was performed for intracisternal injection, under ketamine anesthesia. Complete Freund's Adjuvant (CFA)-induced thermal hyperalgesia and inferior alveolar nerve injury-induced mechanical allodynia were employed as orofacial pain models. Intracisternal administration of bicuculline, a GABA(A) receptor antagonist, produced mechanical allodynia in naive rats, but not thermal hyperalgesia. However, CGP35348, a GABA(B) receptor antagonist, did not show any pain behavior in naive rats. Intracisternal administration of muscimol, a GABA(A) receptor agonist, attenuated the thermal hyperalgesia and mechanical allodynia in rats with CFA treatment and inferior alveolar nerve injury, respectively. On the contrary, intracisternal administration of bicuculline also attenuated the mechanical allodynia in rats with inferior alveolar nerve injury. Intracisternal administration of baclofen, a GABA(B) receptor agonist, attenuated the thermal hyperalgesia and mechanical allodynia in rats with CFA treatment and inferior alveolar nerve injury, respectively. In contrast to GABA(A) receptor antagonist, intracisternal administration of CGP35348 did not affect either the thermal hyperalgesia or mechanical allodynia. Our current findings suggest that the GABA(A) receptor, but not the GABA(B) receptor, participates in pain processing under normal conditions. Intracisternal administration of GABA(A) receptor antagonist, but not GABA(B) receptor antagonist, produces paradoxical antinociception under pain conditions. These results suggest that central GABA has differential roles in the processing of orofacial pain, and the blockade of GABA(A) receptor provides new therapeutic targets for the treatment of chronic pain.

Participation of Central P2X7 Receptors in CFA-induced Inflammatory Pain in the Orofacial Area of Rats

We investigated the role of central P2X receptors in inflammatory pain transmission in the orofacial area in rats. Experiments were carried out using male Sprague-Dawley rats weighing 230-280g. Complete Freund's adjuvant (CFA, 40 microL) was applied subcutaneously to the vibrissa pad to produce inflammatory pain. The intracisternal administration of iso-PPADS tetrasodium salt, a non-selective P2X receptor antagonist, A317491 sodium salt hydrate, a P2X2/3 receptor antagonist, 5-BDBD, a P2X4 receptor antagonist, or A438079 hydrochloride, a P2X7 receptor antagonist, was performed 5 days after CFA injection. Subcutaneous injections of CFA produced increases in thermal hypersensitivity. Intracisternal injections of iso-PPADS (25 microg) or A438079 (25 or 50 microg) produced significant anti-hyperalgesic effects against thermal stimuli compared to the vehicle group. A317491 or 5-BDBD did not affect the head withdrawal latency times in rats showing an inflammatory response. Subcutaneous injections of CFA resulted in the up-regulation of OX-42, a microglia marker, and GFAP, an astrocyte marker, in the medullary dorsal horn. The intracisternal administration of A438079 reduced the numbers of activated microglia and astrocytes in the medullary dorsal horn. These results suggest that a blockade of the central P2X7 receptor produces antinociceptive effects, mediated by inhibition of glial cell function in the medullary dorsal horn. These data also indicate that central P2X7 receptors are potential targets for future therapeutic approaches to inflammatory pain in the orofacial area.

Effects of intrathecal injection rapamycin on neuropathic pain behaviors in mice / 中华行为医学与脑科学杂志

Objective To investigate the effects of continuously intrathecal injection rapamycin on neuro pathic pain behaviors in mice.Methods 48 male adult C57/BL6 mice received intrathecal catheter implantation successfully and without motor dysfunction and serious weight loss,were choosed and randomly divided into shamoperation group ( sham,n =24) and chronic constriction injury model group ( CCI,n =24 ).After operation,each group randomly divided into 3 group again.Group I did nothing,group Ⅱ intrathecally injected rapamycin 1 μg/5μlon day 1 to 6 after operation,group Ⅲ intrathecally injected 20％ DMSO 5μl on the same time ( sham,CCI,sham +R,sham + V,CCI + R,CCI + V,n =8 ).Bilateral mechanical paw withdrawal threshold (WMT) and thermal paw withdrawal latency(TWL) were tested on day 1 before CCI and day 1,3,5,7,10,14,17,21,28 after operation.Results Compared with sham group,both WMT and TWL (7d,MWT:( 1.02 ±0.12)g vs (0.42 ±0.12)g,F=51.01,P＜0.05;TWL:(7.03 ±0.71 )s vs (3.26 ±0.66)s,F=38.27,P＜0.05) were significantly decreased after CCI on the ipsilateral side.When intrathecally injected Rapamycin 1 μg/5 μl on day 1 ～6 after CCI,the mechanical allodynia relieved obviously ( 7 d,M WT:( 0.42 ± 0.18 ) g vs ( 0.86 ± 0.25 ) g,F =6.56,P ＜ 0.05 ),and at least continued to 10 d.On the contrary,the effects of rapamycin on thermal hyperalgesia just showed a trend of inhibition,there was no statistic meaning.In addition,the sham group and contralateral pain behaviors did not change (P＞ 0.05 ).Conclusion Rapamycin can relieve the neuropathic pain behaviors in mice after CCI,mainly the mechanical allodynia,but not thermal hyperalgesia.

Effect of intraperitoneal injection of thalidomide on pain behaviors in a mouse model of bone cancer pain / 中华行为医学与脑科学杂志

Objective To investigate the effect of intraperitoneal injection of thalidomide on pain behaviors in a mouse model of bone cancer pain. Methods 36 male C3H/HeJ mice were divided randomly into tumor group (n= 18) and sham group (n= 18) ,six mice from each group were chosen to examine the time course of changes in behavior after tumor cells inoculated to the bone. 2 × 105 osteosarcoma NCTC 2472 cells were implanted into the intramedullary space of the right femurs of mice to induce ongoing bone cancer related pain behaviors. The sham group was inoculated by α-MEM without any cells. On the day before inoculation,the tumor mice were divided randomly into tumor + thalidomide group and tumor + vehicle group. The sham group mice were further divided randomly into sham + thalidomide group and sham + vehicle group. Pain ethology indexes such as paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were observed on 1 d before inoculation and on 3 d ,5 d ,7 d, 10 d, 14 d after inoculation. Results ( 1 ) At day 7 after the operation, compared with sham mice ( 1. 70 ± 0. 33 ) g, PWMT of tumor mice decreased to ( 1.07 ± 0. 30) g (P ＜ 0. 05 ). At day 10, PWTL shortened to ( 12.60 ± 1.69 ) s (P ＜ 0. 05 ) compared with sham mice ( 17.70 ± 1.54 ) s. And the pain behaviors of tumor mice were aggravated along with the development of cancer pain. (2) At day 7 after the operation, compared with tumor + vehicle group ( 1. 07 ± 0.39 ) g, PWMT of tumor + thalidomide group increased to ( 1. 53 ± 0. 39 ) g (P ＜0.05). At day 10, PWTL extended to ( 16.48 ± 1.13 ) s compared with sham mice ( 12.64 ± 1. 56) s (P ＜0. 05 ). Conclusion Intraperitoneal injection of thalidomide can efficiently relieve mechanical hyperalgia and thermal hyperalgia in a mouse model of bone cancer pain.

Effect of intrathecal glycine and related amino acids on the allodynia and hyperalgesic action of strychnine or bicuculline in mice / 대한마취과학회지

BACKGROUND: The intrathecal (IT) administration of glycine or GABAA receptor antagonist result in a touch evoked allodynia through disinhibition in the spinal cord. Glycine is an inhibitory neurotransmitter that appears to be important in sensory processing in the spinal cord. This study was aimed to evaluate the effect of glycine-related amino acids on antagonizing the effects of IT strychnine (STR) or bicuculline (BIC) when each amino acid was administered in combination with STR or BIC. METHODS: A total of 174 male ICR mice were randomized to receive an IT injection of equimolar dose of glycine, betaine, beta-alanine, or taurine in combination with STR or BIC. Agitation in response to innocuous stimulation with a von Frey filament after IT injection was assessed. The pain index in hot-plate test were observed after it injection. The effect of it muscimol in combination with str or bic were also observed. RESULTS: The allodynia induced by STR was relieved by high dose of glycine or betaine. But, allodynia induced by BIC was not relieved by any amino acid. Whereas the STR-induced thermal hyperalgesia was only relieved by high dose of taurine at 120 min after IT injection, the BIC-induced one was relieved by not only high dose of taurine at 120 min but also low dose of glycine or betaine at 60 min after IT injection. The BIC-induced allodynia and thermal hyperalgesia was relieved by IT muscimol. CONCLUSIONS: This study suggests that IT glycine and related amino acids can reduce the allodynic and hyperalgesic action of STR or BIC in mice.

The Long-Lasting Pattern of the Neuropathic Radicular Pain in An Autologous Nucleus Pulposus Model of Rat

OBJECTIVE: To observe the long-lasting changes of pain progression with time course in an autologous nucleus pulposus model of rat. METHOD: The subjects were 25 Sprague-Dawley (Sprague- Dawley, 250 gm) male rats. They were randomly assigned into either the sham or experimental group. In the experimental group (n=15), autologous nucleus pulposus was harvested from the coccygeal intervertebral disc of the rat and this was grafted on the left L5 dorsal root ganglion. In the sham group (n=10), the left L4 and L5 nerve roots were exposed by laminectomy, but the nucleus pulposus was not grafted. All the rats were evaluated for mechanical allodynia and thermal hyperalgesia at 2 days before surgery, and on days 1, 5, 10, 20, 30, 40 and 50 after surgery. The morphological changes of the spinal nerves were assessed by toluidine blue staining on days 5 after surgery. RESULTS: In the ipsilateral hindpaw of the experimental group, there was a dramatic drop of the mechanical withdrawal threshold and the thermal withdrawal latency on day 1 after surgery, which was maintained at day 50 after surgery. In morphological study, pathological findings such as swelling of the myelin sheath, demyelination, swelling and degeneration of the axoplasm were observed in the spinal nerve at day 5 after surgery. CONCLUSION: The long-lasting pattern of neuropathic radicular pain shown in a rat model of lumbar disc herniations is helpful to understand the natural history of neuropathic radicular pain due to ruptured nucleus pulposus.

Protective effect of adenosine in diabetic neuropathic pain is mediated through adenosine A1-receptors.

Diabetic neuropathic pain is generally considered to be one of the most troublesome complications affecting diabetic patients and current therapy provides inadequate pain relief. In the present study, the effect of adenosine was investigated in a model of diabetic neuropathic pain. Diabetes was induced by streptozotocin (65 mg/kg, ip) in male Sprague Dawley rats and subjected to thermal (cold and hot) and chemical (formalin) stimuli. Diabetic rats developed hyperalgesia by the end of six weeks in thermal and chemical stimuli test. Adenosine (100, 200 and 500 mg/kg, ip) produced significant reversal of responses to thermal and chemical stimuli in diabetic rats. 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX 1 mg/kg, ip), an adenosine A1-receptor antagonist, but not 3,7-dimethyl-l-propargylxanthine (DMPX 1 mg/kg, ip), an adenosine A2A-receptor antagonist, reversed the protective effect of adenosine. These results indicate that adenosine is an effective analgesics in a model of diabetic neuropathy, and the protection produced by adenosine is via stimulation of adenosine A1-receptors.

Clinical Features of Central Neuropathic Pain after Contusive Spinal Cord Injury in Rats

OBJECTIVE: To investigate the characteristics of the central neuropathic pain (CNP) after contusive spinal cord injury in rats. METHOD: Twenty Sprague-Dawley rats (300+/-50 g, male) undergone the free-drop contusion injury from the drop-height of 2.5 cm at T10 cord (n=20) and ten rats undergone sham operation (n=10) were subjected to the neurobehavioral analyses by the Basso Beattie Bresnahan (BBB) locomotor rating scales, Touch test(TM) sensory evaluator (TTSE, North Coast Medical Inc., Canada) and Plantar test(R)(Ugo Basile, Italy) after contusion at the 1(st), 3(rd), 5(th), 7(th), 14(th), 21(st) and 28(th) day. RESULTS: The scores of BBB scales were the lowest at the 1st day and then slowly increased to spontaneous recovery state, but they were significantly lower than those of control group (p<0.05). The thresholds of mechanical allodynia were significantly increased just after cord contusion, but progressed to decline, and significantly decreased after the 21(st) day (p<0.05). The latencies of thermal hyperalgesia were delayed just after cord contusion, but significantly shorter than those of the control group after the 7(th) day (p<0.05). CONCLUSION: These results would be helpful for the study of the CNP after contusive spinal cord injury in rats.