Paw Withdrawal Thermal Latency Research Articles

Effect of dexmedetomidine on expression of c-fos protein in dorsal root ganglion neurons in a rat model of neuropathic pain

Objective To evaluate the effect of dexmedetomidine on the expression of c-fos protein in the dorsal root ganglion neurons in a rat model of neuropathic pain (NP).Methods Seventy-two adult male SpragueDawley rats,weighing 180-240 g,were randomly divided into 3 groups (n =24 each) using a random number table:sham operation group (group S),group NP,and dexmedetomidine group (group Dex).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.The right sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread in NP and Dex groups.In group Dex,dexmedetomidine 50μg/kg was injected intraperitoneally once a day starting from the end of operation until the animals were sacrificed.The equal volume of normal saline was given instead of dexmedetomidine in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before ligation (T0,baseline) and 3,7 and 14 days after ligation (T1-3).Eight animals were sacrificed after measurement of pain threshold at T13 and the dorsal root ganglions of the lumbar segments (L44) were removed for detection of c-fos expression (by immuno-histochemistry).Results Compared with group S,MWT was significantly decreased,TWL was shortened,and the expression of c-fos protein was up-regulated at T1-3 in NP and Dex groups.Compared with NP group,MWT was significantly increased,TWL was prolonged,and the expression of c-fos protein was down-regulated at T1-3 in Dex group.Conclusion Dexmedetomidine can inhibit upregulation of c-fos protein expression,thus attenuating NP in rats. Key words: Dexmedetomidine ; Neuralgia ; Ganglia, spinal ; Proto-oncogene proteins c-fos

Effect of curcumin on expression of MMP-2 and MMP-9 in spinal dorsal horn and dorsal root ganglion of rats with type 2 diabetic neuropathic pain

Objective To evaluate the effect of curcumin on the expression of MMP-2 and MMP-9 in the spinal dorsal horn and dorsal root ganglion (DRG) of rats with type 2 diabetic neuropathic pain (DNP).Methods Type 2 diabetes mellitus was induced by high-fat and high-sucrose diet and intraperitoneal streptozotocin 35 mg/kg,and confirmed by fasting blood glucose level ≥ 16.7 mmol/L in male Sprague-Dawley rats.Type 2 DNP was confirmed by the mechanical paw withdrawal threshold (MWT) and thermal paw withdraw latency (TWL) measured on day 14 after streptozotocin administration ＜ 80％ of the baseline value.The rats were then randomly divided into 3 groups (n =27 each):type 2 DNP group (group DNP); curcumin group (group Cur); solvent control group (group SC).In Cur and SC groups,curcumin 100 mg/kg and corn oil 4 ml/kg were injected intraperitonally,respectively,once a day for 14 consecutive days starting from day 14 after streptozotocin administration.Another 27 normal Sprague-Dawley male rats served as control group (group C) and were fed with normal forage.MWT and TWL were measured before type 2 DNP was induced,after type 2 DNP was induced,and at 3,7 and 14 days after curcumin injection(T1-5).The rats were sacrificed after MWT and TWL were measured at T3-5,and the lumbar segments of the spinal cord and DRG (L4-6) were removed for determination of the expression of MMP-2 and MMP-9 by Western blot.Results Compared with group C,MWT was significantly decreased and TWL was shortened,and MMP-2 and MMP-9 expression in the spinal dorsal horn and DRG was up-regulated in DNP,Cur and SC groups.Compared with DNP group,MWT was significantly increased and TWL was prolonged,and MMP-2 and MMP-9 expression in the spinal dorsal horn and DRG was down-regulated in Cur group,and no significant changes were found in the parameters mentioned above in SC group.Conclusion The mechanism by which curcumin attenuates type 2 DNP may be related to up-regulation of the expression of MMP-2 and MMP-9 in the spinal dorsal horns and DRG of rats. Key words: Curcumin ; Diabetes mellitus, type 2 ; Neuralgia; Spinal cord ; Ganglia, spinal Matrix metalloproteinase 2; Matrix metalloproteinase 9

Interleukin-1β in the surrounding incision tissues is involved in postoperative hyperalgesia in rats with nicotine withdrawal

Objective To explore the contribution of interleukin (IL)-1β in surrounding incision tissues to postoperative hyperalgesia in rats with nicotine withdrawal.Methods Ninety-six SD rats were randomly divided into Naive group (n=6),NT group (n=30),IP group (n=30) and NT+IP group (n=30).The rats in NT group,IP group and NT+IP group were divided into 5 subgroups by preoperative 2 h,postoperative 2,24,48 h and 72 h,six in each subgroup.Both paw mechanical withdrawal threshold (MWT) and paw thermal withdrawal latency (TWL) were determined at above time points for every rats.The hind paw edema was also observed.The level of IL-1β in the surrounding incisional tissues was measured by Elisa method.Results Compared with IP group,foot plantar MWT and TWL in NT+IP group were significantly reduced at postoperative 2,24,48 h and 72 h (P＜0.05),with most significant decrease at postoperative 48 h (P＜0.01).Compared with IP group for each time point,IL-1β level in NT+IP group was significantly increased (P＜0.01).Compared with preoperative 2 h (19.7± 1.9) ng/L,IL-1β level at all the time points in NT+IP group were significantly increased (P＜0.01).The IL-1β level began to rise at postoperative 2 h (62.2±3.7) ng/L,peaked at postoperative 48 h (129.5±6.3) ng/L,and then decreased at postoperative 72 h.Conclusions Nicotine withdrawal can cause postoperative incisional hyperalgesia and hind paw edema in rats.IL-1β is involved in postoperative incisional hyperalgesia in rats with nicotine abstinence. Key words: Nicotine; Dependence; Withdrawal; Hyperalgesia; Interleukin-1β

The effect of NR2B subunit palmitoylation at the spinal level after chronic dorsal root ganglia compression in rats.

The NR2B subunit (N-methyl-D-aspartate receptor 2B subunit) regulates the source of pain, and it participates in the formation of central sensitization. Palmitoylation was shown to be involved in the regulation of N-methyl-D-aspartate receptor internalization. In the present study, we investigated the effects of NR2B subunit palmitoylation in a chronic dorsal root ganglia compression (CCD) rat model. Paw mechanical withdrawal threshold and paw withdrawal thermal latency were used to assess mechanical allodynia and thermal hyperalgesia after a CCD operation and an intrathecal injection of the inhibitor of palmitoylation (2-bromopalmitate [2-BP]). The acyl-biotinyl exchange method, Western blotting, and coimmunoprecipitation were used to investigate the effects of pain processing and the expression of levels of NR2B palmitoylation and phosphorylation at the spinal level. CCD rats had long-lasting thermal hyperalgesia and mechanical allodynia, leading to upregulation of the level of NR2B palmitoylation and phosphorylation at the spinal level. An intrathecal treatment with 2-BP on day 14 after CCD surgery markedly improved pain behaviors and downregulated the expression of NR2B palmitoylation and phosphorylation. These data suggest that upregulated NR2B palmitoylation in CCD-induced neuropathic pain and intrathecal injection of 2-BP could reduce pain behaviors and NR2B phosphorylation. Our findings indicate that spinal NR2B palmitoylation is an important component of CCD-induced neuropathic pain, and it might be a potential target for chronic pain therapy.

Effects of curcumin on expression of receptor for advanced glycation end-products in spinal dorsal horn and dorsal root ganglion of rats with type 2 diabetic neuropathic pain

Objective To evaluate the effects of curcumin on the expression of receptor for advanced glycation end-products (RAGE) in the spinal dorsal horn and dorsal root ganglion (DRG) of the rats with type 2 diabetic neuropathic pain (DNP).Methods Male Sprague-Dawley rats,weighing 160-180 g,were used in this study.Type 2 diabetes mellitus was induced by high-fat and high-sucrose diet for 8 weeks and intraperitoneal streptozotocin (STZ) 35 mg/kg and confirmed by fasting blood glucose level ≥ 16.7 mmol/L 3 days later.Type 2 DNPwas confirmed by the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) measured on day 14 after STZ administration less than 80％ of the baseline value.Eighty-one rats with type 2 DNP were randomly divided into 3 groups (n =27 each) using a random number table:DNP group,DNP + curcumin group (DCur group),and DNP+ solvent group (group DSC).In DCur and DSC groups,curcumin 100 mg· kg-1 · d-1 and corn oil 4 ml · kg-1 · d-1 were injected intraperitonally,respectively,for 14 consecutive days starting from the day 14 after STZ administration.Another 27 normal male Sprague-Dawley rats served as control group (group C) and were fed with normal forage.MWT and TWL were measured before STZ injection,at day 14 after STZ injection,and on 3,7 and 14 days after curcumin injection.RAGE positive cells were determined by immuno-histochemistry and the expression of RAGE by Western blot in the spinal dorsal horn and DRG after MWT and TWL were measured on 3,7 and 14 days after curcumin injection.Results Compared with group C,MWT was significantly decreased and TWL was shortened at 14 days after STZ injection and each time point after curcumin injection,the rate of RAGE positive cells in the spinal dorsal horn and DRG was increased at each time point after curcumin injection,and the expression of RAGE was up-regulated in the spinal dorsal horn at each time point after curcumin injection and in the DRG at 7 and 14 days after curcumin injection in group DNP.Compared with group DNP,MWT was significantly increased and TWL was prolonged at each time point after curcumin injection,the rate of RAGE positive cells in the spinal dorsal horn and DRG was significantly decreased at 7 and 14 days after curcumin injection,and the expression of RAGE was down-regulated in the spinal dorsal horn at each time point after curcumin injection and in the DRG at 7 and 14 days after curcumin injection in group DCur,and no significant change was found in the parameters mentioned above in group DSC.Conclusion The mechanism by which curcumin attenuates DNP may be related to inhibition of up-regulated expression of RAGE in the spinal dorsal horn and DRG of rats with type 2 diabetes mellitus. Key words: Curcumin; Diabetes mellitus, type 2; Neuralgia; Spinal cord; Ganglia, spinal; Receptor for advanced glycation end-products

Intrathecal injection of monocarboxylate transporter inhibitor α-cyano-4-hydroxy-cinnamate attenuates neuropathic pain in rats

Objective To investigate the effects of intrathecal injection of α-cyano-4-hydroxy-cinnamate(4-CIN) in rats with neuropathic pain induced by chronic constriction injury of sciatic nerve(CCI). Methods Eighteen male SD rats were divided randomly into 3 groups(n=6): sham group (group S), CCI model group (group C0) and 4-CIN group ( group C1). Group C0 and C1 were operated with the model of neuropathic pain induced by chronic constriction injury of sciatic nerve; and group S were treated as sham operated rats. Two days after operation, group C1 received intrathecal injection of 100 μmol 4-CIN dissolved in 10% DMSO 10 μl, while group C0 received intrathecal injection of 10% DMSO 10 μl only.The paw withdrawal thermal latency(PWTL) and paw withdrawal mechanical threshold(PWMT) were tested 1 d before operation and 1 d, 3 d, 7 d, 10 d, 14 d after operation(T0-5). Results The basic values of PWMT and PWTL before operation showed no statistically significant differences among the three groups.On T1-5, the PWMT((11.71±2.81)g, (9.76±1.00)g, (8.22±1.33)g, (6.50±1.48)g, (4.67±1.03)g) and PWTL((11.36±2.18)s, (11.60±2.54)s, (8.54±1.42)s, (7.59±1.00)s, (6.88±0.42)s) in group C0 were significantly lower than those in group S (P 0.05). Conclusion Intrathecal administration of 4-CIN can attenuate neuropathic pain in rats induced by CCI. Key words: Monocarboxylate transporter; Inhibitor 4-CIN; Neuropathic pain; Spinal cord

Changes in levels of motilin in duodenum in a rat model of incisional pain

Objective To evaluate the changes in the levels of motilin in the duodenum in a rat model of incisional pain.Methods Eighty-four healthy male Sprague-Dawley rats,aged 6-8 months,weighing 180-220 g,were randomized into 2 groups (n =42 each) using a random number table:control group (group C) and incisional pain group (group P).The animals were anesthetized with sevoflurane.In group P,a 1 cm long incision was made in the plantar surface of right hindpaw.Six rats were chosen from each group and mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before operation (T0) and 1,6,24,48 and 72 h after operation (T1-5).Six rats were chosen from each group at T0-5 and sacrificed and the duodenal mucosal tissue was prepared for measurement of motilin levels by ELISA.Pearson linear correlate analysis was performed between the motilin level and pain threshold at each time point in group P.Results Compared with group C,MWT was significantly decreased,TWL was shortened,and motilin levels were significantly increased at T1-4,and no significant change was found at T0 and T5 in group P.The motilin levels were negatively correlated with MWT (r =-0.8 910) and TWL (r =-0.8 463) in group P.Conclusion Incisional pain can promote the secretion of motilin in the duodenum. Key words: Pain, postoperative ; Motilin; Duodenum

Effect of intraperitoneal WSLP/NR2B siRNA compound on neuropathic pain in rats

Objective To evaluate the effect of intraperitoneal water soluble lipopolymer (WSLP)/ N-methyl-D-aspartate receptor subunit 2B (NR2B) siRNA compound on the neuropathic pain (NP) in rats.Methods Eighty-four healthy male Sprague-Dawley rats,aged 6 weeks,weighing 180-220 g,were randomly divided into 7 groups (n =12 each) using a random number table:control group (group C),sham operation group (group S),NP group,WSLP/NR2B siRNA group (siWSLP group),WSLP/negative control siRNA group (ncWSLP group),PEI/NR2B siRNA group (PEI group) and WSLP group (WSLP group).NP was produced by ligation of the left L5 spinal nerve.In group S,the left L5 spinal nerve was only exposed,but not ligated.In group C,the rats underwent no treatment.Groups siWSLP,ncWSLP,PEI and WSLP received single intraperitoneal injection of WSLP/NR2B siRNA,WSLP/negative control siRNA,PEI/NR2B siRNA and WSLP compound 2 ml,respectively,at 10 days after NP.At 1 day before operation,7 days after operation,and 3,7,14 and 21 days after intraperitoneal injection,6 rats in each group were chosen randomly to measure mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL).At 3 days after intraperitoneal injection,the left 6 rats in each group were sacrificed and the spinal cord was removed for detection of NP2B mRNA expression (using PCR) and NR2B expression (by Western blot).Results Compared with group C,MWT was significantly decreased,and TWL was shortened on 7 days after operation and 3,7 and 21 days after intraperitoneal injection,and the expression of NR2B mRNA and protein was down-regulated on 3 days after administration in the other groups.Compared with group NP,MWT was significantly increased,and TWL was prolonged on day 3 and 7 after intraperitoneal injection,and the expression of NR2B mRNA and protein was down-regulated on day 3 after administration in siWSLP group.Conclusion Intraperitoneal WSLP/NR2B siRNA compound can effectively relieve the NP in rats. Key words: Gene therapy; Transfection ; RNA, small interfering; Receptors, N-methyl-D-aspartate ; Neuralgia; Injections,intraperitoneal

Role of histone deacetylase in spinal cord in maintenance of neuropathic pain in rats

Objective To evaluate the role of histone deacetylase in the spinal cord in the maintenance of neuropathic pain (NP) in rats.Methods Twenty-seven male Sprague-Dawley rats,weighing 230-270 g,were randomly divided into 3 groups (n =9 each) using a random number table:sham operation group (group S),group NP,and NP + intrathecal Trichostatin A (TSA) group (group T).NP was induced by chronic constrictive injury.At 7 days after operation,5％ DMSO,5％DMSO and TSA 10 μg (10 μl) were injected intrathecally once a day for 3 consecutive days in S,NP and T groups,respectively.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 3,7,10,14 and 21 days after operation (T0-5).Results Compared with group S,MWT was significantly decreased and TWL was shortened at T2-5 in NP and T groups.Compared with group NP,MWT was significantly increased and TWL was prolonged at T3,4 in group T.Conclusion Histone deacetylase in the spinal cord is involved in the maintenance of neuropathic pain in rats. Key words: Histone deacetylases; Neuralgia

Neurotrophic factor derived from spinal cord glial cell line in rats with neuropathic pain involved in the modulation of dopamine D2 receptor agonists hyperalgesia

Objective To study the effects of intrathecal injection of dopamine D2 receptor agonist quinpirole on expression of glial cell line-derived neurotrophic factor in the spinal cord in rats with chronic constrictive injury (CCI) and explore its possible mechanism of mediating antinociception.Methods In this study,models were established CCI in male Sprague-Dawley rats.The experiment was performed as 2 parts.In part one,30 CCI rats were randomly divided into 5 groups (n=6):CCI+saline (group NS),CCI+quinpirole 0.1 μg (group Q0.1),CCI+quinpirole 1 μg (group Q1),CCI+quinpirole 5 μg (group Q5),CCI+quinpirole 10μg (group Q 10).The drugs were injected intrathecally on day 7 after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured before and at 0.5,1,2,4,8 h and 16 h after intrathecal injection.In part two 54 CCI rats were randomly divided into 3 groups (group Q5,Q10 and NS,n=18).Group Q5 and Q10 were sacrificed at 0.5,1,2,4,8,16 h after intrathecal quinpirole 5,10 μg on day 7 after CCI.Group NS,a single intrathecal injection of saline 10 μl.Group M,another 6 normal rats as model control group.Another 6 normal rats as control group C.The expression of glial cell linederived neurotrophic fact (GDNF) in the spinal cord was determined by Western blot.Results Part one:compared with group NS,the PWMT and PWTL of group Q0.1 at each time point after injection had no statistically significant difference (P＞0.05).The PWMT and PWTL of 2 h of group Q1 [(4.3±1.5) g,(13.2±1.6) s].1,2,4 h of group Q5 [(4.7±1.6),(5.3±1.6),(4.7±2.1) g,(14.0±1.7),(15.2±1.5),(13.4±1.6) s] and Q10 [(6.0±1.3),(7.3±1.0),(5.3±2.1) g,(15.3±1.8),(17.5±1.2),(14.9±1.7) s] after drug administration were significantly promoted (P＜0.05).Part two:compared with group C,the expression of GDNF was significantly increased in group M (0.95±0.09)(P＜0.05).Compared with group M and NS,group Q5 [(1.47±0.12),(1.24±0.05)] and Q10[(1.63±0.08),(1.27±0.06)] significantly increased the expression of GDNF (P＜0.05),and it can last until 4 h after injection at least.Conclusions Intrathecal injection of quinpirole can significantly increase the level of GDNF expression in the spinal cord.At the same time,the pain behavior was improved.The upregulation of GNDF expression is involved in the modulation of dopamine D2 receptor agonist on neuropathic pain. Key words: Neuropathic pain; Dopamine agonist; Spinal; Glial cell line-derived neurotrophic fact; Intrathecal injection

Changes of Mu-opioid receptor and neuron-restrictive silencer factor in periaquductal gray in mouse models of remifentanil-induced postoperative hyperalgesia

To determine the changes of Mu-opioid receptor (Mor) and neuron-restrictive silencer factor (NRSF) in periaquductal gray (PAG) in mouse models of remifentanil-induced postoperative hyperalgesia. Thirty-two Kun-Ming mice were randomly divided into 4 groups (8 mice in each group): Group C (mice underwent a sham procedure and saline was infused subcutaneously over a period of 30 min), Group I (mice underwent a surgical incision and the same volume of saline), Group R (mice underwent a sham procedure and remifentanil was infused subcutaneously at the moment of surgical incision over a period of 30 min), and group IR (mice underwent a surgical incision and remifentanil). Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) tests were performed 24 h before the operation and 2, 6, 24, and 48 h after the operation. The specimens were collected after behavioral testings at 48 h. The expressions of Mor and NRSF in mice's PAG neurons were determined by Western blot. Mechanical allodynia and thermal hyperalgesia developed in Group I, R and IR (P<0.01). Intraoperative infusion of remifentanil enhanced mechanical allodynia and thermal hyperalgesia in mice with planta incision (P<0.01). In Group R and Group IR, the expression of Mor was significantly lower (P<0.01) and NRSF was significantly higher (P<0.01) when compared with Group C and Group I. Intraoperative infusion of remifentanil induces postoperative hyperalgesia in mouse models, accompanied with decreased expressions of Mor and increased of NRSF level in PAG neurons, which may be involved in remifentanil induced hyperalgesia.

Relationship between function of spinal NMDA receptors and delta opioid receptors in a rat model of incisional pain-remifentanil-induced hyperalgesia

Objective To investigate the relationship between the function of spinal NMDA receptors and δ opioid receptors in a rat model of incisional pain-remifentanil-induced hyperalgesia.Methods Thirty male Sprague-Dawley rats,aged 2-3 months,weighing 260-280 g,in which intrathecal catheters were successfully implanted or in which the catheter was successfully placed via the caudal vein,were randomly divided into 3 groups (n =10 each) uisng a random number table:control gourp (group C),remifentanil + incisional pain group (group RI),and δ opioid receptor inhibitor naltrindole group (group NI).A 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the hindpaw in chloral hydrate-anesthetized rats.In group C,normal saline 0.1 ml· kg-1 · min-1 was infused via the caudal vein for 60 min.In group RI,remifentanil 1.0 μg· kg-1 · min-1 was infused via the caudal vein for 60 min,and the model of incisional pain was established immediately at the beginning of infusion.In group NI,naltrindole 30 nmol (10 μl) was administrated intrathecally,then remifentanil was infused for 60 min at a rate of 1.0 μg· kg-1 · min-1 and the model of incisional pain was established immediately at the beginning of infusion.At 24 h before normal saline or remifentanil infusion and 2,6,24 and 48 h after the end of infusion (T0-4),mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were sacrificed after the last behavioral test.L4-6 segments of the spinal cords were removed for determination of NMDA receptor expression in total and membrane protein using Western blot analysis.The ratio of NMDA receptor expression in membrane protein to that in total protein (m/t ratio) was calculated.Results Compared with the basline value at T0,MWT was significantly decreased,and TWL was shortened at T1-4 in RI and NI groups.MWT was gradualy decreased,and TWL was gradualy shortened at T1-3 and no significant difference was found between MWT and TWL at T3 and T4 in RI and NI groups.Compared with group C,MWT was significantly decreased,and TWL was shortened at T1-4,the expression of NR1-and NR2B-containing NMDA receptors in total and membrane protein was down-regulated,and m/t ratio was decreased in group NI.Conclusion Activation of δ opioid receptors can enhance the function of NR1-and NR2B-containing NMDA receptors,which may be involved in the development of incisional pain-remifentanil-induced hyperalgesia in rats. Key words: Piperidines; Hyperalgesia; Receptors, opioid, delta; Receptors, N-methyl-D-aspartate ; Spinal cord

Effects of curcumin on activity of NR2B and NR1 in spinal dorsal horn in a rat model of diabetic neuropathic pain

Objective To evaluate the effects of curcumin on the activity of NR2B and NR1 in the spinal dorsal horn in a rat model of diabetic neuropathic pain (DNP).Methods Diabetes mellitus was induced by high-sucrose and high-fat diet and intraperitoneal streptozotocin 35 mg/kg,then confirmed by fasting blood glucose level ≥ 16.7 mmol/L 3 days later in male Sprague-Dawley rats.DNP was confirmed by the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) measured on day 14 after streptozotocin administration ＜ 80％ of the baseline value.The rats were then randomly divided into 3 groups (n =27 each) using a random number table:DNP,DNP+ curcumin group (group DCur)and DNP + solvent control group (group DSC).Curcumin 100 mg· kg-1 · d-1 and corn oil 4 ml· kg-1 · d-1 were injected intraperitoneally for 14 consecutive days starting from 14 days after administration of streptozotocin in DCur and DSC groups,respectively.Another 27 normal male Sprague-Dawley rats served as control group (group C) and were fed with normal forage.At 3,7 and 14 days after curcumin injection,MWT and TWL were measured and the lumbar segments (L4-6) of the spinal cord were removed.The expression of pTyr1472-NR2B and pSer896-NR1 in the spinal dorsal horn was determined by immunohistochemistry and Western blot.Results Compared with group C,MWT was significantly decreased,TWL was shortened,and the expression of pTyr1472-NR2B was up-regulated at each time point in group DNP.Compared with group DNP,MWT was significantly increased,and TWL was prolonged at 7 days after curcumin injection,and the expression of pTyr1472-NR2B was down-regulated at 3 days after curcumin injection in group DCur.There was no significant difference in each parameter between DNP and DSC groups,and in the expression of pSer896-NR1 between the four groups.Conclusion The mechanism by which curcumin mitigates neuropathic pain in type 2 diabetic rats may be related to inhibition of up-regulation of pTyr1472-NR2B expression,while unrelated to pSer896-NR1 expression in the spinal dorsal horn. Key words: Curcumin; Diabetes mellitus, type 2; Neuralgia; Spinal cord; pTyr1472-NR2B; pSer896-NR1

Release of L-EK in cerebrospinal fluid induced by intrathecal HEK293 cells modified with human preproenkephalin gene and the analgesic efficacy of L-EK for bone cancer pain

Objective To evaluate the release of leucine-enkephalin (L-EK) in the cerebrospinal fluid induced by intrathecal human embryonic kidney (HEK293) cells modified with human preproenkephalin (hPPE)gene and the analgesic efficacy of L-EK for bone cancer pain.Methods Forty CIBP female Sprague-Dawley rats were randomized into transplantation (CIBP+ hPPE/HEK293,n =20) and control (CIBP + HEK293,n =20)groups using a random number table.At 1 day before inoculation of cancer cells (T1,baseline) and 8,15,21,25,32 and 35 days after inoculation (T2-7),thermal paw withdrawal latency (TWL) was measured,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were recorded.After observation at T4,10 rats were chosen from each group and sacrificed and the cerebrospinal fluid of rats was collected in an ice bath for detection of hPPE expression using radioimmunoassay.Results Compared with control group,TWL was significantly prolonged,the concentration of L-EK in the cerebrospinal fluid was increased,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were decreased at T4-7 in transplantation group.Conclusion Intrathecal HEK293 cells modified with hPPE gene can continuously secrete L-EK and mitigate bone cancer pain. Key words: Cancer pain ; Cell analgesia ; Intrathecal puncture ; Gene-modified ; Determination of pain

Effects of intrathecal injection of different dose of protein kinase B inhibitor IV on hyperalgesia induced by remifentanil

Objective To investigate the role and mechanism of protein kinase B (Akt) in opioid-induced hyperalgesia (OIH) with an incisional pain model.Methods Sixty C57BL/6 male mice were randomly divided into 5 groups (n=12):incision pain+ dimethyl sulfoxide(DMSO) group(group Ⅰ),incision pain+remifentanil+DMSO group(group R),Akt inhibitor Ⅳ 0.08 μg/10 μl group(group A1),Akt inhibitor Ⅳ 0.16 μg/10 μl group(group A2) and Akt inhibitor Ⅳ 0.32 μg/10 μl group(group A3).DMSO was the solvent of Akt inhibitor Ⅳ.Incisional pain model was made in the right paw of all the mice.In group R and group A1,A2,A3,subcutaneous remifentanil(0.04 mg/kg) was infused for 30 min while the surgery was being performed and intrathecal 10 μl DMSO(10％) with corresponding concentration of the Akt inhibitor Ⅳ 10 μl was injected respectively.The paw mechanical withdrawal threshold (PMWT) and the paw withdrawal thermal latency(PWTL) was tested at 1 d(T0) before and 6 h(T1),1(T2),2(T3),3(T4),5(T5),7 d(T6) after surgery.Results Compared with group Ⅰ and the baseline value,PMWT and PWTL were significantly decreased after surgery except 7 d in group R and all group A(P＜0.05).Compared with group R,PMWT[(5.03±0.62),(6.10±0.86),(5.92±0.88),(6.01±1.02) g.(4.07±0.79),(4.73±0.48),(4.77±0.59),(4.86±0.56) g.(5.05±0.75),(5.99±0.63),(5.99±0.71),(6.00±0.81) g] and PWTL[(0.48±0.06),(0.60±0.08),(0.61±0.07),(0.58±0.04) s.(0.38±0.07),(0.50±0.08),(0.48±0.06),(0.45±0.08) s.(0.37±0.09),(0.52±0.09),(0.49±0.12),(0.58±0.21) s] were significantly increased after surgery except 7 d in groups A1,A2,A3 (P＜0.05).While there was no significant difference among group A1,A2 and A3 (P＞0.05).Conclusions Intrathecal injection of different doses of Akt inhibitor Ⅳ effectively alleviates hyperalgesia induced by remifentanil in non-dose-dependent way. Key words: Protein kinase B; Protein kinase B inhibitor Ⅳ; Remifentanil; Hyperalgesia

Effects of hyperbaric oxygen treatment on expression of nerve growth factor in spinal cord of rats with neuropathic pain

Objective To evaluate the effect of hyperbaric oxygen (HBO) treatment on the expression of nerve growth factor (NGF) in the spinal cord of rats with neuropathic pain (NP).Methods Thirty healthy male Sprague-Dawley rats,aged 8-10 weeks,weighing 270-300 g,were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (group S),group NP,pure oxygen group (group O),treatment with HBO at 2.5 atmosphere absolute group (group H2.5) and treatment with HBO at 3.0 atmosphere absolute group (group H3.0).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.NP was induced by chronic constrictive injury.The left sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1-mm intervals with 4-0 silk thread.HBO treatment was performed with the corresponding atmosphere absolute once a day for 7 consecutive days starting from 1 day after operation in H2.5 and H3.0 groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 3,5,7 and 14 days after operation.The animals were sacrificed after the last measurement of pain threshold,and the lumbar segments of the spinal cord were removed to detect the expression of NGF by immuno-histochemistry and Western blot analysis.Results Compared with group S,MWT was significantly decreased,TWL was shortened,and the expression of NGF was up-regulated at each time point after operation in NP and O groups.Compared with group NP,MWT was significantly increased,TWL was prolonged,and the expression of NGF was up-regulated at each time point after operation in H2.5 and H3.0 groups,and no significant change was found in MWT,TWL and expression of NGF in group O.Compared with group H2.5,the expression of NGF was significantly down-regulated,and no significant change was found in MWT and TWL in group H3.0.Conclusion The mechanism by which HBO treatment mitigates NP is related to up-regulation of the expression of NGF in the spinal cord of rats. Key words: Hyperbaric oxygen; Neuralgia; Nerve growth factor; Spinal cord

Role of glycogen synthase kinase-3β in expression and trafficking of GluR1-and GluR2-containing AMPA receptors in spinal dorsal horns of rats with remifentanil-induced hyperalgesia

Objective To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the expression and trafficking of GluR1-and GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia.Methods Twenty-four male Sprague-Dawley rats,weighing 240-260 g,aged 2-3 months,were randomly divided into 3 groups (n =8 each) using a random number table:control gourp (group C),remifentanil group (group R),and remifentanil + TDZD-8 (GSK-3β inhibitor) group (group R + T).In group R,remifentanil was infused intravenously at 1.0 μg· kg-1 ·min-1 for 1 h.In group R + T,TDZD-8 1 mg/kg was injected intravenously and then remifentanil was infused intravenously at 1.0 μg· kg-1 · min-1 for 1 h.In group C,normal saline was infused intravenously at 0.1 ml· kg-1 · min-1 for 1 h.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before remifentanil infusion and 2,6,24 and 48 h after remifentanil infusion.The rats were sacrificed after the last behavioral test,and L4.6 segments of the spinal cord were removed to determine the expression of AMPA receptor GluR1 and GluR2 in membrane and total protein in the spinal dorsal horn by Western blot.The ratio of GluR1 expression to GluR2 expression (GluR1/GluR2) in membrane protein was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of GluR1 in membrane and total protein was up-regualted,the expression of GluR2 in membrane and total protein was down-regulated,and GluR1/GluR2 in membrane protein was increased in group R.Compared with group R,MWT was significantly increased,TWL was prolonged,the expression of GluR1 in membrane and total protein was down-regulated,the expression of GluR2 in membrane and total protein was upregulated,and GluR1/GluR2 in membrane protein was decreased in group R + T.Conclusion GSK-3β mediates up-regulated expression and insertion of GluR1-containing AMPA receptors and down-regulated expression and internalization of GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia. Key words: Glycogen synthesis kinase 3 ; Receptors, AMPA ; Piperidines ; Hyperalgesia ; Spinal cord

Dopamine D3 receptor dysfunction prevents anti-nociceptive effects of morphine in the spinal cord

Dopamine (DA) modulates spinal reflexes, including nociceptive reflexes, in part via the D3 receptor subtype. We have previously shown that mice lacking the functional D3 receptor (D3KO) exhibit decreased paw withdrawal latencies from painful thermal stimuli. Altering the DA system in the CNS, including D1 and D3 receptor systems, reduces the ability of opioids to provide analgesia. Here, we tested if the increased pain sensitivity in D3KO might result from a modified μ-opioid receptor (MOR) function at the spinal cord level. As D1 and D3 receptor subtypes have competing cellular effects and can form heterodimers, we tested if the changes in MOR function may be mediated in D3KO through the functionally intact D1 receptor system. We assessed thermal paw withdrawal latencies in D3KO and wild type (WT) mice before and after systemic treatment with morphine, determined MOR and phosphorylated MOR (p-MOR) protein expression levels in lumbar spinal cords, and tested the functional effects of DA and MOR receptor agonists in the isolated spinal cord. In vivo, a single morphine administration (2 mg/kg) increased withdrawal latencies in WT but not D3KO, and these differential effects were mimicked in vitro, where morphine modulated spinal reflex amplitudes (SRAs) in WT but not D3KO. Total MOR protein expression levels were similar between WT and D3KO, but the ratio of pMOR/total MOR was higher in D3KO. Blocking D3 receptors in the isolated WT cord precluded morphine's inhibitory effects observed under control conditions. Lastly, we observed an increase in D1 receptor protein expression in the lumbar spinal cord of D3KO. Our data suggest that the D3 receptor modulates the MOR system in the spinal cord, and that a dysfunction of the D3 receptor can induce a morphine-resistant state. We propose that the D3KO mouse may serve as a model to study the onset of morphine resistance at the spinal cord level, the primary processing site of the nociceptive pathway.

Neonatal Bee Venom Exposure Induces Sensory Modality-Specific Enhancement of Nociceptive Response in Adult Rats

Previous studies have shown that inflammatory pain at the neonatal stage can produce long-term structural and functional changes in nociceptive pathways, resulting in altered pain perception in adulthood. However, the exact pattern of altered nociceptive response and associated neurochemical changes in the spinal cord in this process is unclear. In this study, we used an experimental paradigm in which each rat first received intraplantar bee venom (BV) or saline injection on postnatal day 1, 4, 7, 14, 21, or 28. This was followed 2 months later by a second intraplantar bee venom injection in the same rats to examine the difference in nociceptive responses. We found that neonatal inflammatory pain induced by the first BV injection significantly reduced baseline paw withdrawal mechanical threshold, but not baseline paw withdrawal thermal latency, when rats were examined 2 months from the first BV injection. Neonatal inflammatory pain also exacerbated mechanical, but not thermal, hyperalgesia in response to the second BV injection in these same rats. Rats exposed to neonatal inflammation also showed up-regulation of spinal NGF, TrkA receptor, BDNF, TrkB receptor, IL-1β, and COX-2 expression following the second BV injection, especially with prior BV exposure on postnatal day 21 or 28. These results indicate that neonatal inflammation produces sensory modality-specific changes in nociceptive behavior and alters neurochemistry in the spinal cord of adult rats. These results also suggest that a prior history of inflammatory pain during the developmental period might have an impact on clinical pain in highly susceptible adult patients.

Effects of intrathecal opioids combined with low-dose naloxone on motilin and its receptor in a rat model of postoperative pain

AimsTo investigate the effects of intrathecal morphine and fentanyl combined with low-dose naloxone on the expression of motilin and its receptor in a rat model of postoperative pain. Main methodsAn intrathecal catheter was implanted, and saline, opioids (morphine and fentanyl) and naloxone were intrathecally administered 7days later. An incisional pain model was established to induce pain behaviors in rats by unilateral plantar incision. Thermal hyperalgesia and mechanical allodynia were measured by using a radiant heat and electronic Von Frey filament, respectively. The expression of motilin in the hippocampus, stomach, duodenum, and plasma was determined by ELISA; and the expression of motilin receptor in the hippocampus was detected by Western blot assay. Key findingsMotilin and its receptor were detected in the hippocampus. Acute incisional pain increased the motilin expression in the hippocampus and duodenum, while decreasing its expression in the gastric body and plasma. Postoperative analgesia with morphine+fentanyl upregulated the expression of motilin in the hippocampus; however, motilin was downregulated in peripheral sites. Naloxone at 1ng/kg restored motilin to baseline levels. Acute pain, morphine+fentanyl, and naloxone all induced the expression of motilin receptor in the hippocampus. SignificanceAcute pain, postoperative analgesia with opioids, and naloxone significantly impacted the expression of hippocampal and peripheral motilin. Variation trends in all sites were not identical. Intrathecal injection of low-dose naloxone upregulated paw withdrawal thermal latency and enhanced the analgesic effects of opioids. The findings presented here provide a new basis for central and peripheral regulations in GI motility, clinical postoperative analgesia, and management of analgesic complications.