Proteomic analysis of the dorsal spinal cord in the mouse model of spared nerve injury-induced neuropathic pain

Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th-6th lumbar spinal cord in a mouse model of spared nerve injury（SNI）-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry（MS/MS）. After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain,and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.

High-frequency spinal cord stimulation produces longlasting analgesic effects by restoring lysosomal function and autophagic flux in the spinal dorsal horn

High-frequency spinal cord stimulation(HF-SCS) has been established as an effective therapy for neuropathic pain. However, the analgesic mechanisms involved in HF-SCS remain to be clarified. In our study, adult rat neuropathic pain was induced by spinal nerve ligation. Two days after modeling, the rats were subjected to 4 hours of HF-SCS(motor threshold 50%, frequency 10,000 Hz, and pulse width 0.024 ms) in the dorsal horn of the spinal cord. The results revealed that the tactile allodynia of spinal nerve-injured rats was markedly alleviated by HFSCS, and the effects were sustained for 3 hours after the stimulation had ceased. HF-SCS restored lysosomal function, increased the levels of lysosome-associated membrane protein 2(LAMP2) and the mature form of cathepsin D(matu-CTSD), and alleviated the abnormally elevated levels of microtubule-associated protein 1 A/B-light chain 3(LC3)-II and sequestosome 1(P62) in spinal nerve-injured rats. HF-SCS also mostly restored the immunoreactivity of LAMP2, which was localized in neurons in the superficial layers of the spinal dorsal horn in spinal nerve-injured rats. In addition, intraperitoneal administration of 15 mg/kg chloroquine for 60 minutes reversed the expression of the aforementioned proteins and shortened the timing of the analgesic effects of HF-SCS. These findings suggest that HF-SCS may exhibit longlasting analgesic effects on neuropathic pain in rats through improving lysosomal dysfunction and alleviating autophagic flux. This study was approved by the Laboratory Animal Ethics Committee of China Medical University, Shenyang, China(approval No. 2017 PS196 K) on March 1, 2017.

“Three Methods and Three Points” regulates p38 mitogen-activated protein kinase in the dorsal horn of the spinal cord in a rat model of sciatic nerve injury

Tuina is a traditional Chinese treatment for sensory disturbances caused by peripheral nerve injury and related diseases. Our previous studies showed that tuina regulates relevant regions and indices of the spinal dorsal horn using the Dian, Bo, and Rou method in Yinmen（BL37）, Yanglingquan（GB34）, and Weizhong（BL40）. Treatment prevents muscle atrophy, protects spinal cord neurons, and promotes sciatic nerve repair. The mechanisms of action of tuina for treating peripheral nerve injury remain poorly understood. This study established rat models of sciatic nerve injury using the crushing method. Rats received Chinese tuina in accordance with the principle of ＂Three Methods and Three Points,＂ once daily for 20 days. Tuina intervention reduced paw withdrawal latency and improved wet weight of the gastrocnemius muscle, as well as promoting morphological recovery of sciatic nerve fibers, Schwann cells, and axons. The protein expression levels of phospho-p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-1β also decreased. These findings indicate that ＂Three Methods and Three Points＂ promoted morphological recovery and improved behavior of rats with peripheral nerve injury.

Effect of propofol on glutamate-induced activation and related inflammatory cytokines of astrocytes from spinal cord dorsal horn

BACKGROUND： Astrocytes participate in central nervous system-mediated physiological or pathological processes, such as pain. Activated dorsal horn astrocytes from the spinal cord produce nerve active substances and proinflammatory cytokines, such as interleukin-lbeta （IL-1 β ）, IL-6, and tumor necrosis factor- α （TNF-α ）, which play important roles in pain transduction and regulation. OBJECTIVE： To investigate the effects of different doses of propofol on activation of cultured spinal cord dorsal horn astrocytes induced by glutamate, as well as changes in IL-1β, IL-6, and TNF- α, and 1L-10 （anti-inflammatory cytokine） expression in rats, and to explore the dose relationship of propofol. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Central Laboratory of Yunyang Medical College between March 2006 and December 2007. MATERIALS： Forty healthy, Wistar rats, aged 2-3 days, were selected. Propofol was provided by Zeneca, UK; glutamate by Sigma, USA; EPICS XL flow cytometry by Beckman culture, USA; rabbit-anti-mouse glial fibrillary acidic protein （GFAP） antibody kit and inflammatory cytokine detection kit were provided by Zhongshan Biotechnology Company Ltd., Beijing; multimedia color pathologic image analysis system was a product of Nikon, Japan. METHODS： Astrocytes were harvested from T11- L6 spinal cord dorsal horn of Wistar rats and incubated for 3 weeks. The cells were divided into seven groups, according to various treatment conditions： control group was cells cultured in Hank＇s buffered saline solution; intralipid group was cells cultured in intralipid （0.2 mL/L）; glutamate group was cells cultured with 100 u mol/L glutamate; propofol group was cells cultured with 250 u mol/L propofol; three glutamate plus propofol groups were cultured in 100 11 mol/L of glutamate, followed by 5, 25, and 250 u mol/L of propofol 10 minutes later. MAIN OUTCOME MEASURES： GFAP-labeled astrocytes were analyzed using a multimedia pathology imaging analysis system to detect area density （AD） and average optical density （AOD） of positive cells. The supernatant concentrations of IL-1β, TNF- α, IL-6, and IL-10 were determined using radioimmune assays. RESULTS： Compared with the control group, cells in the glutamate plus low-dose propofol group were activated and hypertrophic, and AD and AOD were significantly increased （P 〈 0.01 ）. Concentrations of IL-1β, TNF- α, and IL-6 were also significantly increased （P 〈 0.01）, while IL-10 levels remained unchanged （P 〉 0.05）, but still higher than the control and glutamate groups （P 〉 0.05）. Compared with the glutamate group, astrocyte activation was inhibited by moderate and high-dose propotol. In addition, with moderate and high-dose propofol, AD, AOD, IL-1β, TNF- α, and IL-6 concentrations were significantly decreased （P 〈 0.05-0.01）, and IL-10 levels were increased （P 〈 0.01 ）. CONCLUSION： Propofol can effectively inhibit glutamate-induced astrocyte activation in the spinal cord dorsal horn, significantly inhibit production of IL-1 β, TNF- α, and IL-6, and increase IL-10 synthesis and release in a dose-dependent manner.

The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

Up-regulation and time course of protein kinase C immunoreactivity during persistent inflammation of the rat spinal cord

BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain. OBJECTIVE： To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN： Randomized controlled animal experiment. SETTING： Institute of Basic Medical Science, Hebei Medical University MATERIALS： The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee. METHODS： Sprague-Dawley rats were divided randomly into control （n = 6） and experimental groups （n = 36）. Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES： Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, in the ipsi- and contralateral dorsal horn were investigated during different stages of inflammatory pain using immunohistochemistry. RESULTS： All 42 rats were included in the final analysis, without any loss. Pain reaction： consistent with previous findings, it was determined that a unilateral injection of formalin into the hind-paw resulted in significant edema and induced a series of nociceptive responses, such as licking, biting, or shaking the injected paw. The maximal inflammation change was observed 1 day after formalin injection and changes did not disappear until the day 7. Number of the PKC positive neurons： results demonstrated that the number of PKC immunoreactive neurons in the dorsal horn increased slightly after formalin injection at 1 hour, compared with the control group. PKC immunoreactivity was up-regulated at day 1, reduced at day 3, and appeared to recover at day 7. The number of PKC-positive neurons in the contralateral side was less than the ipsilateral side at each time sampled. Distribution of PKC immunoparticles over the neurons： PKC immunoreactivity was observed in the nucleus and cytoplasm, as well as on or near the membrane of neurons and synaptosomes in the spinal cord of the control group. PKC activated and translocated from nucleus to the membrane-associated site following formalin treatment. Significant changes were observed at 1 hour and 1 day. The intensity of staining was stronger in the ipsilateral side than the contralateral side at all time points following formalin injection （P 〈 0.01）, whereas the expression patterns of PKC immunoreactivity in the nuclei were very similar in the right and left hemispheres. CONCLUSION： PKC expression in the dorsal horn of the spinal cord peaked at 1 hour and 24 hours, and was very obvious at 24 hours. Protein kinase C expression in the spinal cord increased bilaterally, although it was greater in the ipsilateral hemisphere. In addition, PKC expression at the neuronal membrane and synaptosome was significantly increased. These results indicate that PKC expression is activated in the dorsal horn of the spinal cord during hyperalgesia.

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn

OBJECTIVE:To investigate whether the Chinese massage system,Tuina,exerts analgesic effects in a rat model of chronic constriction injury(CCI)by remodeling the synaptic structure in the spinal cord dorsal horn(SCDH).METHODS:Sixty-nine male Sprague–Dawley rats were randomly and evenly divided into the normal group,sham group,CCI group,CCI+Tuina group,CCI+MK-801[an N-methyl D-aspartate receptor subtype 2B(NR2B)antagonist]group,and CCI+MK-801+Tuina group.The neuropathic pain model was established using CCI with right sciatic nerve ligation.Tuina was administered 4 d after CCI surgery,using pressing manipulation for 10 min,once daily.Motor function was observed with the inclined plate test,and pain behaviors were observed by the Von Frey test and acetone spray test.At 19 d after surgery,the L3-L5 spinal cord segments were removed.Glutamate,interleukin 1β(IL-1β),and tumor necrosis factor-α(TNF-α)levels were detected by enzyme-linked immunosorbent assay.The protein expression levels of NR2B and postsynaptic density protein-95(PSD-95)were detected by Western blot,and the synaptic structure was observed by transmission electron microscopy(TEM).RESULTS:CCI reduced motor function and caused mechanical and cold allodynia in rats,increased glutamate concentration and TNF-αand IL-1βlevels,and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH.TEM revealed that the synaptic structure of SCDH neurons was altered.Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801(P<0.05 or<0.01).For the majority of experiments,no significant differences were found between the CCI+MK-801 and CCI+MK-801+Tuina groups.CONCLUSIONS:Chinese Tuina can alleviate pain by remodeling the synaptic structure,and NR2B and PSD-95 receptors in the SCDH may be among its targets.

Effect of pre-electroacupuncture on p38 and c-Fos expression in the spinal dorsal horn of rats suffering from visceral pain

Background Acupuncture is an effective way to relieve pain, but the mechanism by which electroacupuncture （EA） decreases the visceral pain state still remains unclear. This study aimed to evaluate the effects of pre-electroacupuncture on pain behaviors, p38 phosphorylation, and c-Fos protein and mRNA expression in both the colonic wall and spinal dorsal horn of rats suffering from visceral pain. This study also investigated the probable signaling regulatory mechanism of the analgesic effect induced by electroacupuncture. Methods All rats were randomized into the control （Con） group, the Con＋EA group, the visceral pain （VP） group, and VP＋EA group （n=8 for all groups）. The visceral pain model was established using 40 ul of 5% formalin solution injected into the colon of rats. EA was applied to the bilateral Jiaji acupoints for 20 minutes before application of visceral pain. Parameters for EA were set at a continuous wave （20 Hz） and intensity where the rats shook their whiskers but did not scrabble （≤1 mA）. The visceral pain score was recorded and the expressions of p38 and c-Fos protein were detected using Western blotting. Real-time quantitative PCR was also used to determine the expression of c-Fos mRNA. Results Rats in the VP group immediately presented with obvious visceral pain behaviors after being injected with formalin. p38 activity and c-Fos protein and mRNA expression in both the colonic wall and spinal dorsal horn were higher in the VP group than in the Con group （P 〈0.05）. By contrast, visceral pain behaviors were delayed in rats from the VP＋EA group. p38 activity and c-Fos protein and mRNA expression were lower in the VP＋EA group than that in the VP group （P〈0.01）. Conclusions Pre-electroacupuncture of the Jiaji acupoint has prophylactic analgesic effects on rats suffering from visceral pain. The p38 signal transduction pathway may be partly involved in the regulatory mechanism of this analgesic effect.

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu- puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and electroacu- puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es- pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high expres- sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi- cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro- pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re- sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.

Galanin and its receptor system promote the repair of injured sciatic nerves in diabetic rats

Various studies have reported that galanin can promote axonal regeneration of dorsal root ganglion neurons in vitro and inhibit neuropathic pain. However, little is known about its effects on diabetic peripheral neuropathy, and in vivo experimental data are lacking. We hypothesized that repeated applications of exogenous galanin over an extended time frame may also repair nerve damage in diabetic peripheral neuropathy, and relieve pain in vivo. We found that neuropathic pain occurred in streptozotocin-induced diabetic rats and was more severe after sciatic nerve pinch injury at 14 and 28 days than in diabetic sham-operated rats. Treatment with exogenous galanin alleviated the neuropathic pain and promoted sciatic nerve regeneration more effectively in diabetic rats than in non-diabetic rats after sciatic nerve pinch injury. This was accompanied by changes in the levels of endogenous galanin, and its receptors galanin receptor 1 and galanin receptor 2 in the dorsal root ganglia and the spinal dorsal horn when compared with nerve pinch normal rats. Our results show that application of exogenous galanin daily for 28 days can promote the regeneration of injured sciatic nerves, and alleviate neuropathic pain in diabetic rats.

Low-Frequency Electroacupuncture Alleviates Chronic Constrictive Injury-Induced Mechanical Allodynia by Inhibiting NR2B Upregulation in Ipsilateral Spinal Dorsal Horn in Rats

Objective: To study the effects of electroacupuncture(EA) in chronic constrictive injury(CCI) rat model and the expression of N-methyl-D-aspartate receptor type 2B(NR2B) in ipsilateral spinal dorsal horn in rats to explore the analgesic mechanisms of EA. Methods: According to the random number table, totally 180 rats were evenly divided into a sham group, a CCI group, and an EA group. CCI model was conducted with four4–0 chromic gut ligatures loosely ligated around the left sciatic nerve 1 cm above the trifurcation. Rats in the EA group received 2 Hz EA therapy bilaterally at acupoints of Zusanli(ST 36) and Sanyinjiao(SP 6) once daily(30 min/d) for 30 days after surgery. Paw withdrawal thresholds(PWTs) were measured on 0(baseline), 1, 3, 7, 15,30 days after surgery. Rats were sacri?ced on 0, 1, 3, 7, 15 and 30 days after surgery, and the L4–5 segments of spinal cord were removed to detect the expression of NR2B by immunohistochemistry and quantitative polymerase chain reaction. Results: PWTs in the CCI group were signi?cantly lower than the sham group at Day1–30 after surgery, and reached its lowest at Day 1(P<0.01). After EA treatment, the PWTs recovered rapidly and were signi?cantly higher than those in the CCI group on 3, 7, 15 and 30 days after surgery(P<0.01). The numbers of NR2B-immunoreactive cells of the CCI group signi?cantly increased after CCI surgery compared with the sham group(P<0.01). Compared with the CCI group, stimulation of EA markedly decreased the numbers of NR2B-immunoreactive cells at Day 3, 7, 15 and 30(P<0.05). In the sham group, NR2B mRNA was expressed at a low level. It increased after CCI surgery, which increased rapidly at Day 7(P<0.01) and reached its peak value at Day 15(P<0.01). After EA stimulation, relative quantity of NR2B mRNA expression was less than that in the CCI group at Day 15 and 30(P<0.05). Conclusions: Low frequency of EA had antinociceptive effect in CCI rat model. The analgesic effects of EA might be through the inhibition of NR2B.

Modulation of Pain and Itch by Spinal Glia

Chronic pain and itch are a pathological operation of the somatosensory system at the levels of primary sensory neurons, spinal cord and brain. Pain and itch are clearly distinct sensations, and recent studies have revealed the separate neuronal pathways that are involved in each sensation. However, the mechanisms by which these sensations turn into a pathological chronic state are poorly understood. A proposed mechanism underlying chronic pain and itch involves abnormal excitability in dorsal horn neurons in the spinal cord. Furthermore, an increasing body of evidence from models of chronic pain and itch has indicated that synaptic hyperexcitability in the spinal dorsal horn might not be a consequence simply of changes in neurons, but rather of multiple alterations in glial cells. Thus, understanding the key roles of glial cells may provide us with exciting insights into the mechanisms of chronicity of pain and itch, and lead to new targets for treating chronic pain and itch.

Upregulation of Spinal Voltage-Dependent Anion Channel 1 Contributes to Bone Cancer Pain Hypersensitivity in Rats

Voltage-dependent anion channel 1（VDAC1） is thought to contribute to the progression of tumor development. However, whether VDAC1 contributes to bone cancer pain remains unknown. In this study, we found that the expression of VDAC1 was upregulated in the L2–5 segments of the spinal dorsal horn at 2 and 3 weeks after injection of tumor cells into the tibial cavity. Intrathecal injection of a VDAC1 inhibitor significantly reversed the pain hypersensitivity and reduced the over-expression of Toll-like receptor 4（TLR4）. Intrathecal injection of minocycline, an inhibitor of microglia, also attenuated the pain hypersensitivity of rat models of bone cancer pain.These results suggest that VDAC1 plays a significant role in the development of complicated cancer pain, possibly by regulating the expression of TLR4.

Microglial Depletion does not Afect the Laterality of Mechanical Allodynia in Mice

Mechanical allodynia(MA),including punctate and dynamic forms,is a common and debilitating symptom suffered by millions of chronic pain patients.Some peripheral injuries result in the development of bilateral MA,while most injuries usually led to unilateral MA.To date,the control of such laterality remains poorly understood.Here,to study the role of microglia in the control of MA laterality,we used genetic strategies to deplete microglia and tested both dynamic and punctate forms of MA in mice.Surprisingly,the depletion of central microglia did not prevent the induction of bilateral dynamic and punctate MA.Moreover,in dorsal root ganglion-dorsal root-sagittal spinal cord slice preparations we recorded the low-threshold Aβ-fiber stimulation-evoked inputs and outputs of superficial dorsal horn neurons.Consistent with behavioral results,microglial depletion did not prevent the opening of bilateral gates for Aβpathways in the superficial dorsal horn.This study challenges the role of microglia in the control of MA laterality in mice.Future studies are needed to further understand whether the role of microglia in the control of MA laterality is etiology-or species-specific.

Involvement of NF-κB and the CX3CR1 Signaling Network in Mechanical Allodynia Induced by Tetanic Sciatic Stimulation

Tetanic stimulation of the sciatic nerve （TSS） triggers long-term potentiation in the dorsal horn of the spinal cord and long-lasting pain hypersensitivity. CX3CL1- CX3CR1 signaling is an important pathway in neuronal- microglial activation. Nuclear factor nB （NF-KB） is a key signal transduction molecule that regulates neuroinflamma- tion and neuropathic pain. Here, we set out to determine whether and how NF-~B and CX3CR1 are involved in the mechanism underlying the pathological changes induced by TSS. After unilateral TSS, significant bilateral mechanical allodynia was induced, as assessed by the von Frey test. The expression of phosphorylated NF-nB （pNF-nB） and CX3CR1 was significantly up-regulated in the bilateral dorsal horn. Immunofluorescence staining demonstrated that pNF-κB and NeuN co-existed, implying that the NF-κB pathway is predominantly activated in neurons following TSS. Administration of either the NF-κB inhibitor ammo- nium pyrrolidine dithiocarbamate or a CX3CR1-neutralizing antibody blocked the development and maintenance of neuropathic pain. In addition, blockade of NF-κB down- regulated the expression of CX3CL1-CX3CR1 signaling,and conversely the CX3CRl-neutralizing antibody also down-regulated pNF-rd3. These findings suggest an involvement of NF-κB and the CX3CR1 signaling network in the development and maintenance of TSS-induced mechanical allodynia. Our work suggests the potential clinical application of NF-κB inhibitors or CX3CR 1-neutralizing antibodies in treating pathological pain.

Population coding of somatic sensations

The somatic sensory system in^udes a variety of sensory modalities, such as touch, pain, itch, and temperature sensitivity. The coding of these modalities appears to be best explained by the population-coding theory, which is com- posed of the following features. First, an individual somatic sensory afferent is connected with a specific neural circuit or network （for simplicity, a sensory-labeled line）, whose isolated activation is sufficient to generate one specific sensation under normal conditions. Second, labeled lines are interconnected through local excitatory and inhibitory interneurons. As a result, activation of one labeled line could modulate, or provide gate control of, another labeled line. Third, most sensory fibers are polymodal, such that a given stimulus placed onto the skin often activates two or multiple sensory-labeled lines; crosstalk among them is needed to generate one dominant sensation. Fourth and under pathological conditions, a disruption of the antagonistic interaction among labeled lines could open normally masked neuronal pathways, and allow a given sensory stimulus to evoke a new sensation, such as pain evoked by innocuous mechanical or thermal stimuli and itch evoked by painful stimuli. As a result of this, some sensory fibers operate along distinct labeled lines under normal versus pathological conditions. Thus, a better understanding of the neural network underlying labeled line crosstalk may provide new strategies to treat chronic pain and itch.

Gabapentinoid Insensitivity after Repeated Administration is Associated with Down-Regulation of theα2δ-1 Subunit in Rats with Central Post-Stroke Pain Hypersensitivity

The α2δ-1 subunit of the voltage-gated Ca2＋ channel （VGCC） is a molecular target of gabapentin （GBP）, which has been used as a first-line drug for the relief of neuropathic pain. GBP exerts its anti-nociceptive effects by disrupting trafficking of the α2δ-1 subunit to the presynaptic membrane, resulting in decreased neurotrans- mitter release. We previously showed that GBP has an anti- allodynic effect in the first two weeks; but this is followed by insensitivity in the later stage after repeated adminis- tration in a rat model of central post-stroke pain （CPSP） hypersensitivity induced by intra-thalamic hemorrhage. To explore the mechanisms underlying GBP insensitivity, the cellular localization and time-course of expression of the α2δ-1 subunit in both the thalamus and spinal dorsal horn were studied in the same model. We found that the α2δ-1 subunit was mostly localized in neurons, but not astrocytes and microglia. The level of α2δ-1 protein increased in the first two weeks after injury but then decreased in the third week, when GBP insensitivity occurred. Furthermore, the c^2g-1 down-regulation was likely caused by later neuronal loss in the injured thalamus through a mechanism other than apoptosis. In summary, the present results suggest that the GBP receptor ~2^-1 is mainly expressed in thalamic neurons in which it is up-regulated in the early stage of CPSP but this is followed by dramatic down-regulation, which is likely associated with GBP insensitivity after long-term use.