Horn Of Rats Research Articles

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors*♦

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

Role of Stargazin in trafficking of GluRl-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn in rats with incisional pain

Objective To evaluate the role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in the spinal dorsal horn in rats with incisional pain.Methods Forty-five adult male Sprague-Dawley rats,aged 6-8 yr,weighing 280-300 g,were randomly divided into 5 groups (n =9 each):control group (group C),sham operation group (group S),incisional pain + normal saline group (group P),incisional pain + Stargazin small interference RNA (siRNA) group (group Ⅰ),and incisional pain + non-sense siRNA group (group N).In P,I and N groups,normal saline 10 μl,20 μmol/L siRNA and 20 μmol/L non-siRNA 10 μl were injected intrathecally,respectively,twice a day for 3 consecutive days.A 1 cm long incision was made in the plantar surface of right hindpaw.Cumulative pain score (CPS) and paw-withdrawal threshold to yon Frey stimuli (PWT) were measured at 3 h after incision.The animals were sacrificed after pain behavior assessment.Their lumbar segments of the spinal cord (L3-6) were removed.The expression of GluR1 and GluR2 in cell membrane and cytoplasm in spinal cord dorsal horn was determined by Western blot analysis.The co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was examined by co-immuno-precipitation.Results Compared with group C,the CPS was significantly increased,the PWT was decreased,the expression of GluR1 in cytoplasm was down-regulated and the expression of GluR1 in cell membrane was up-regulated in N and P groups,and the CPS was increasedin group Ⅰ (P ＜ 0.05 or 0.01).Compared with group P,the CPS was significantly decreased,the PWT was increased,the expression of GluR1 in cytoplasm was up-regulated,the expression of GluR1 in cell membrane was down-regulated,and the expression of Stargazin and co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was down-regulated in group Ⅰ (P ＜ 0.05 or 0.01).Conclusion Stargazin mediates trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn and is involved in the development of hyperalgesia in rats with incisional pain. Key words: Receptors, AMPA; Protein transport; Pain; Spinal cord; Stargazin

Effect of glial cell line-derived neurotrophic factor on Fos protein expression in the spinal dorsal horn of neuropathic pain rat model

Objective To study the effect of glial cell line-derived neurotrophic factor (GDNF)on Fos protein expression in the spinal dorsal horn of neuropathic pain rat model.Methods Adult Sprague-Dawley male rats were randomly divided into normal control group,sham operation group,spinal nerve ligation (SNL) group and SNL combined with treatment of GDNF group.Every group was divided into three sub-groups:SNL 3,7 and 14 days (n =10 in each group).Fos protein expression in the spinal dorsal horn was examined by using inununohistochemistry and Western blotting.Results Compared with normal control and sham operation groups,the Fos protein expression in the spinal dorsal horn of the rats in SNL group was increased significantly (1.00 ± 0.00,1.08 ± 0.12,and 4.62 ± 0.52 respectively).As compared with the rats in SNL group,there was an apparent decrease in GDNF treatment group three days later (1.82 ± 0.51).Conclusion Fos protein in the spinal dorsal horn of rats was involved in the mechanism of neuropathic pain.Intrathecal administration of GDNF could inhibit Fos protein expression in the spinal dorsal horn. Key words: Glial cell line-derived neurotrophic factor; Neuropathic pain; Fos protein

Expression of N-methyl-D-aspartate receptor subunit 2B containing N-methyl-D-aspartate receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain

Objective To investigate the role of N-methyl-D-aspartate receptor subunit 2B (NR2B) containing N-methyl-D-aspartate (NMDA) receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain.Methods Thirty male SD rats aged 7 weeks were randomly divided into 3 groups (n =10 each):control group (group C),H2S group (group S),and ketamine group (group K).Intraplantar administration of NaHS (H2S donor) 1 nmol (100 μl) for 5 days in group S and group K,and the equal volum (100 μl) of nodal saline (NS) in group C (once each day) was done.Then rats in group K were injected with ketamine 10 mg/kg (0.2 ml) intramuscularly for 7 days,and the equal volum of NS was injected in group C and group S (once each day).The paw withdrawal threshold (PWT) to mechanical stimulation was measured at first day before the first administration of NaHS/NS (Tc),20 min after the first administration of NaHS/NS (T1),20 min after the fifth administration of NaHS/NS (T2),and 20 min after the seventh administration of ketamine/NS (T3).The rats were sacrificed after the last PWT measurement.The lumber segment of the spinal cord was removed for determination of the expressions of NR2B subunit by using RT-PCR and Western blotting.Results The PWT at T1,T2 and T3 was (2.72 ± 1.17),(1.48 ± 0.57) and (2.40 ± 0.89) g respectively in group S,which was significantly lower than in group C,and the expressions of NR2B mRNA and protein in spinal dorsal horn was (0.75 ±0.07) and (1.36 ± 0.08) respectively,which was significantly higher than in group C.The PWT at T3 was (8.89 ±1.09)g in group K,which was higher than in group S,and the expression of NR2B mRNA and protein in spinal dorsal horn was (0.55 ± 0.05) and (1.02 ± 0.06) respectively,which was lower than in group S.Conclusion The upregnlation of NR2B subunit in spinal dorsal horn was one of meachanisms in hydrogen sulfide-induced pain and inhibition of NR2B expression may contribute to the antinociceptive effects of ketamine. Key words: Hydrogen sulfide; Ketamine; N-methyl-D-aspartate receptor subunit 2B; Spinal dorsal horn; Pain

Automatic System for Identification and Analysis of Burst

Although there are complex patterns of discharges exist in neurons, which are often clustered in bursts, the pattern of the burst discharge can substantially change with the alternation of the living environment of the neurons. Furthermore, there is a big difference in the physiological and pharmacological characteristics of various types of burst discharges. So it is necessary to analyze the information contained in various types of burst discharges. Here, an automatic system for identification and analysis of the neuron discharge based on plotting histograms of the logarithm of the interspike interval was designed. The system consists of neuron discharge collecting unit, neuron discharge processing unit, battery monitoring, real-time charging unit and bursts processing software. The system can identify the burst discharge from the neuron discharge without any omission and make statistical analysis. By using this device, the electrophysiological experiment that the spontaneous and evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats were recorded was smoothly completed. The result of statistical analysis indicated that the device can give the corresponding interspike interval aimed at various types of burst discharges and respectively identify the burst discharges in the different amplitude spikes, which provide a tool for further research on the biosensor and the neural communication.

Effect of intrathecal sufentanil and protein kinase C inhibitor on pain threshold and the expression of NMDA receptor/ CGRP in spinal dorsal horn in rats with neuropathic pain

To investigate the effect of intrathecal sufentanil and protein kinase C inhibitor on pain threshold and the expression of N-methyl-D-aspartate receaptors (NMDAR)/calcitonin generelated peptide (CGRP) in spinal dorsal horn in rats with neuropathic pain. Fifty-four healthy male Sprague-Dawley rats were randomly divided into 6 groups (9 in each group). The rats in the sham group(Group S) + spared nerve injury (SNI), SP+SNI, and P+SNI were intrathecally injected sufentanil (1 μg), sufentanil (1 μg) and chelerythrine chloride (11 μg), chelerythrine chloride (11 μg) followed by 10 μL normal saline once every day for 14 days postoperatively, respectively. Similarly, rats in the control group (Group C), the sham group (Group S), and SNI model group (Group SNI) were intrathecally injected 20 μL normal saline in the uniform interval. Pain behaviours were measured on Day 1 pre-surgery and on Day 1, 2, 7, and 14 after the intrathecal injection. The expressions of NMDAR and CGRP in the spinal dorsal horn of L5 segment were determined by immunohistochemistry on Day 2, 7, and 14 after the intrathecal injection. Compared with Group C and Group S, mechanical allodynia threshold in group SNI was decreased after the surgery (P<0.01), and expressions of NMDAR and CGRP immunoreactive soma in the spinal dorsal horn was significantly increased (P<0.01). Mechanical stimulation pain threshold was elevated in Group S+SNI, Group P+SNI, and Group SP+SNI compared with Group SNI (P<0.01), while expressions of NMDAR and CGRP immunoreactive soma in Group S+SNI, Group P +SNI, and Group SP+SNI were significantly decreased (P<0.05 or 0.01). Intrathecal administration of sulfentanil and protein kinase C inhibitor can provide significant antinociception in rats with neuropathic pain and obviously inhibit the upregulation of NMDAR and CGRP expressions in the spinal dorsal horn of SNI rat models.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Valproate upregulates glutamate transporters in rat spinal cord after peripheral nerve injury

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of chronic oral administration or intrathecal infusion of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate–aspartate transporter(GLAST) in the spinal dorsal horn in rats after L5–L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn.

CCN3: the-pain-killer inside me

There is increasing evidence that metalloproteinases are involved in neuropathic pain [Dev et al., Expert Opin Investig Drugs 19:455-468 2010] Hence, the identification of molecules that can regulate MMP9 and MMP2 is warranted. In a recent publication, Kular et al. (2012) claim that CCN3 functions to decrease inflammatory pain via the regulation of two metalloproteinases, MMP2 and MMP9, in response to experimentally-induced inflammation. Their conclusion is based on the following observations : i) the expression of CCN3 was reduced following induction of pain by subcutaneous injection of complete Freund's adjuvent in rat's paw, ii) an inhibition of MMP9 decreased CFA-associated mechanical allodynia, iii) inhibition of CCN3 expression by siRNA led to an upregulation of MMP2 in the dorsal horn of the spinal cord (DHSC) and MMP9 in the dorsal root ganglia (DRG), iv) a partial effect of CCN3 on CFA-induced expression of MMP9 and MMP2 in DRG and DHSC following intrathecal injection of CCN3. Unfortunately, the conclusion of this study is weakened by the lack of experimental evidence showing a direct relationship between the expression of CCN3 and MMPs. Furthermore, several results contained in this manuscript only confirm data that were previously established by others. Owing to the wide range of activities which have been attributed to CCN3 (Perbal, Mol Pathol 54:57-79 2001, Brigstock, J Endocrinol 178:169-175 2003, Perbal, Lancet 363(9402):62-64 2004, Perbal, Cell Commun Signal 4:6 2006, Holbourn et al. Trends Biochem Sci. 33:461-473 2008, Leask and Abraham, J Cell Sci 119:4803-4810 2006, Jun and Lau, Nat Rev Drug Discov 10:945-963 2011, Rachfal and Brigstock, Vitam Horm 70:69-103 2005), the mechanisms underlying the potential role of CCN3 in the expression of these MMPs in the context of inflammatory pain must be thoroughly studied before a meaningful conclusion can be reached. Indeed, Kular et al. description of variations in CCN3, MMP9 and MMP2 levels occurring simultaneously is not sufficient to draw a functional relationship between these three proteins. It should be noted that the expression of CCN3 was already reported to repress MMP9 (Benini et al., Oncogene 24:4349-4361 2005, Fukunaga-Kalabis et al., Oncogene 27:2552-2560 2008) and the roles of CCN3 in inflammatory processes has been extensively documented in the past few years (Bleau et al., Front Biosci 10:998-1009 2005, Lin et al., J Biol Chem 280:8229-8237 2005, Perbal, Cell Commun Signal 4:6 2006, Hughes et al., Diabetologia 50:1089-1098 2007, Lin et al., J Cell Commun Signal 4:141-153 2010, Pasmant et al., J Neuropathol Exp Neurol 69:60-69 2010, Shimoyama et al., Thromb Vasc Biol 30:675-682 2010, Lemaire et al., J Invest Dermatol 130:2517 2010, Chen and Lau, J Cell Commun Signal 4:63-69 2010, Le Dréau et al., Glia 58:1510-1521 2010, Rittié et al. J Cell Commun Signal 5:69-80 2011, Janune et al., J Cell Commun Signal 5:167-171 2011). In addition, the expression of CCN3 in the neurons of dorsal root ganglia and dorsal horn of the spinal horn in rat and human has also been documented (Su et al., C R Acad Sci III 321:883-892 1998, Mol Pathol 54:184-191 2001, Kocialkowski et al., Anat Embryol (Berl) 203:417-427 2001). Implication of CCN3 in cognitive functions (Su et al., Sheng Li Xue Bao 52:290-294 2000) and the possible involvement of CCN3 in the regulation of pain was already suggested almost a decade ago (Perbal, Expert Rev Mol Diagn 3:597-604 2003, Perbal et al., Mol Pathol 56:80-85 2003) with the demonstration of cell-specific effects of CCN3 on intracellular calcium stores and inhibition of anionic channels by CCN3 (Li et al., Mol Pathol 55:250-261 2002, Lombet et al., Cell Commun Signal 1:1 2003, Perbal, Expert Rev Mol Diagn 3:597-604 2003, Perbal et al., Mol Pathol 56:80-85 2003). Aside from these general aspects, and in the light of the potential participation of CCN3 in the whole process of pain sensing, the reader would have appreciated the discussion in this manuscript not being essentially a flat summary of the data presented, but a more thorough discussion of the possible role for CCN3 in the regulation of MMPs and its significance in the context of the wide biological functions of CCN3.

Neuroendocrine Mechanisms of Acupuncture

Neuroendocrine Mechanisms of Acupuncture

Effect of Electroacupuncture on Activation of p38MAPK in Spinal Dorsal Horn in Rats with Complete Freund's Adjuvant-Induced Inflammatory Pain

Activation of mitogen-activated protein kinases (MAPKs), especially p38 MAPK, plays an important role in the development of central sensitization related to persistent inflammatory pain. Electroacupuncture (EA) is well known to relieve persistent inflammatory pain. However, little is known about relationship between EA and p38 MAPK. Inflammatory pain rat model was induced by intraplantar injection of complete Freund's adjuvant (CFA). Male adult SD rats were randomly divided into the saline group, CFA group, and CFA + EA group. EA (constant saquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1 to 2 mA) was applied to bilateral “Zusanli” (ST 36) and “Kunlun” acupoints (BL 60) for 30 min, once per day. The paw edema and paw withdrawal threshold (PWT) were measured at preinjection and days postinjection 1, 3, and 14. Spinal p-p38MAPK- immunoreactivty (p-p38MAPK-IR) cells were detected by immunohistochemistry at postinjection day 3 and 14. EA significantly inhibited paw edema at postinjection days 14 and increased PWT at postinjection days 3 and 14. Moreover, the increasing number of spinal p-p38MAPK-IR cells which was induced by CFA injection was suppressed by EA stimulation. These results indicate that anti-inflammatory and analgesic effect of EA might be associated with its inhibition of spinal p38 MAPK activation and thereby provide a potential mechanism for the treatment of inflammatory pain by EA.

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor

Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.

Up-regulation of spinal glutamate transporters contributes to anti-hypersensitive effects of valproate in rats after peripheral nerve injury

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate–aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5–L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters.

Effect of low-load eccentric training before high-intensity eccentric exercise on delayed onset muscle soreness, revealed by c-fos expression in the spinal dorsal horn in rats

BackgroundDelayed onset muscle soreness (DOMS) occurs after unaccustomed exercise and is particularly associated with high-intensity eccentric exercise. Eccentric exercise is reported to have a protective effect against DOMS caused by...

Changes in expression of glutamate-aspartate trausporter in spinal dorsal horn in rats with inflammatory pain and chronic morphine tolerance

Objective To investigate the changes in the expression of glutamate-aspartste transporter in spinal dorsal horn in rats with inflammatory pain and chronic morphine tolerance. Methods Thirty healthy male SD rats in which intrathecal (IT) catheters were successfully placed without complications were randomized into 3 groups ( n = 10 each): normal saline group ( group NS), arthritis group ( group A), and arthritis + morphine group (group AM). NS 50 μl was injected into the ankle joint of the left hindlimb in group NS, while complete Freund's adjuvant was injected in the other two groups instead. After 3 days, group NS and A received IT NS 10 μl twice a day for 7 consecutive days, group AM IT morphine 10 μg (10 μl) twice a day for 7 consecutive days. Mechanical pain threshold (MPT) was measured before IT administration and at day 2, 4, 6 and 8 after IT administration (T0-4). The animals were sacrificed after the last MPT measurement. The spinal cords were isolated for determination of GLAST expression in spinal dorsal horn. Results Compared with group NS, MPT was significantly decreased in the other groups and GLAST expression was down-regulated in group AM (P ＜ 0.05). Compared with group A, no significant change was found in MPT at T3,4 (P ＞ 0.05), while GLAST expression was down-regulated in group AM ( P ＜ 0.05). Conclusion The development of chronic morphine tolerance is related to the decrease in the function of GLAST in spinal dorsal horn in rats with inflammatory pain. Key words: Glutamic acid; Amino acid transport systems; Inflammation; Drug tolerance; Morphine

Effects of lactoferrin on activity of PKG in spinal dorsal horn in a rat model of neuropathic pain

Objective To investigate the effects of lactoferrin on activity of PKG in spinal dorsal horn in a rat model of neuropathic pain(NP).Methods Thirty-two male SD rats weighing 200-250 g were randomly divided into4 groups(n = 8 each): sham operation group(group S),NP group,lactoferrin group and KT5823(an inhibitor of PKG)group.Neuropathic pain was produced by placing loosely constrictive ligatures around the common sciatic nerve in group NP,lactoferrin and KT5823,while the sciatic nerve was only exposed but not ligated in groupS.In group S and NP,normal saline 10 μl + 50% dimethyl sulfoxide(DMSO)10 μl were injected intrathecally.Lactoferrin 100 μg + 50% DMSO 10 μl were given intrathecally in group lactoferrin.Lactoferrin 100 μg + KT5823 10 μl were given intrathecally in group KT5823.The paw withdrawal latency(PWL)to a thermal nociceptive stimulus was measured every 30 min within 180 min after administration.The rats were then sacrificed and the spinal cord was removed.The activity of PKG in the spinal dorsal horn was determined by immunofluorescence.Results Compared with group NP and KT5823,the PWL was significantly prolonged after administration in group lactoferrin and the PKG activity was significantly increased in group lactoferrin(P ＜ 0.05).There was no significant difference in the parameters mentioned above between group NP and group KT5823(P ＞ 0.05).Conclusion Lactoferrin reduces NP by inhibiting the activity of PKG in spinal dorsal horn in rats. Key words: Lactoferrin; Neuralgia; Cyclic GMP-dependent protein kinases

Catechol- O-methyltransferase (COMT) inhibition reduces spinal nociceptive activity

Several variants of the catechol- O-methyltransferase (COMT) gene have recently been linked to pain sensitivity. In the present study, electrophysiological field potential recordings from the dorsal horn in rats were used to examine the spinal effect of reduced COMT activity. The data demonstrated that 30 mg/kg of the COMT inhibitor OR 486 reduced spinal nociceptive responses to painful stimuli ( p ≤ 0.01, OR 486 vs. vehicle) and attenuated the expression of spinal long-term potentiation (LTP), an often studied model for central sensitization ( p ≤ 0.01, HFS vs. HFS + OR 486). Our findings suggest that low COMT activity may have an antinociceptive effect in the spinal cord.

Neuroprotetive Effect of Curcuma longa Alcoholic Extract on Peripheral Nerves Degeneration after Sciatic Nerve Compression in Rats

Summary There are extensive evidences those axonal processes of the nervous system (peripheral and/or central) may degenerate after nerve injuries. Wallerian degeneration and choromatolysis are the most conspicuous phenomena that occur in response to injuries. In this research the effect of root alcoholic extract of Salvia staminea on neuronal density of motoneurons in spinal cord anterior horn in rats was studied. Forty adult male wistar rats were used and divided to five groups (control, compression, three experimental groups). In compression and experimental groups right sciatic nerve were highly compressed for 60 s, assigned to experimental groups (Compression + alcoholic extract of salvia root injections (25 ,50,75 mg/kg -1 ,

Effect of intrathecal administration of a mixture of butorphanol and ketamine on cAMP-PKA-CREB signal transduction pathway in spinal dorsal horn of rats with inflammatory pain

Objective To investigate the effect of intrathecal administration of a mixture of butorphanol and ketamine on cAMP-PKA-CREB signal transductian pathway in the spinal dorsal ham of the rats with inflammatory pain. Methods Twenty-four male SD rats, weighing 240-280 g,in which intrathecal catheters were successfully placed, were divided into 4 groups randomly (n = 6 each): inflammatory pain group (group IP), butorphanol group (group B), ketamine group (group K), and butorphanol + ketamine group (group BK). The inflammatory pain was induced by injection of 5% formalin 50 μl into the plantar surface of left hind paw. Normal saline 10 μl, butorphannl 12.5 μg, ketamine 50 μg, and a mixture of butorphanol 12.5 μg and ketamine 50 μg was injected intrathecally 30 min before subcutaneous injection of formalin in group IP, B, K and BK respectively.Pain intensity score (PIS) was used to assess pain behavior every 5 min within an hour after subcutaneous injection of formalin. The animals were killed at 2 h after subcutaneous injection of formalin, and the L5 segment of the spinal cord was removed for determination of protein kinase A (PKA) and phosphorylated cAMP response element binding protein (p-CREB) expression using immunohistochemistry. Results Fonnahn administration induced pain behaviour expressed as two phases. PIS scores, PKA and p-CBEB expression, and staining scores were significantly lower during the fast and second phases in group BK than in group IP (P 0.05). Conclusion lntrathecal injection of a mixture of butorphanol and ketamine can reduce inflammatory pain in rats, and the mechanism may be related to the cAMP-PKA-CREB signal transduction pathway. Key words: Butorphanol; Ketamine; Cyclic AMP; Cyclic AMP-dependent protein kinnses; Cyclic AMP response element-binding protein; Pain; Injections, spinal

Relationship between abnormal complement activation in the spinal dorsal horn and neuropathologic pain in rats

Objective To observe the change of complement 3 in dorsal horn of spinal cord and to investigate the role of ab-normal activation of complement protein in pathogenesis of NPP. Methods This study comprises two parts. In part 1, 84 healthy male Sprague-Dawley rats were divided randomly into seven groups:normal control group, sham I d group, sham 3 d group, sham 7 d group, CCI 1 d group, CCI 3 d group, CCI 7 d group (n=12). The mechanical pain threshold were measured in different time ac-cording to the group, while the mRNA and protein of Complement 3 were determined by RT-PCR, immunoturbidimetry and immuno-histochemistry. In part 2, 48 healthy male Sprague-Dawley rats were divided randomly into sham operation + saline group, CCI + saline group, CCI + CVF group(continual injection CVF group) and CCI + saline + CVF group(single injection CVF group)(n=12). Pain thresholds by mechanical stimulation were recorded at preoperation and 1, 3, 7, 14 d after operation in each group. Comple-ment 3 expression in the spinal dorsal horn was determined immunohistochemically to confirm the efficacy of CVF. The SOD activity and MDA content in the spinal cord homogenate were determined, and intracellular changes of spinal dorsal horn neurons were ob-served under electronmicroscopy. Results The expression of mRNA and protein of complement 3 in spinal dorsal horn increased at 1, 3, 7 d after CCI, but those were not shown in rats in sham operation group and normal control group. The pearson analysis demonstrated that there was an initate relation between complement 3 content and pain threshold. Hyperalgesia was obviously alleviat-ed in CCI + saline + CVF group after administration of CVF, and the threshold revived following attenuation of the role of CVF. Hy-peralgesia was not evoked after sciatic nerve ligation in CCI + CVF group. Compared with CCI + saline group, the SOl) activity in-creased sinificianfly and MDA content decreased obviously in CCI + CVF group (P<0.05). Electronmicroscopy demonstrated mito-chondrial swelling and cytomembrane breakdown of spinal dorsal horn neurons in CCI + saline group while mitochondrial swelling was mitigated in spinal dorsal horn neurons in CCI + CVF group. Conclusion Abnormal complement activation occurred in the spinal dorsal horn of rats with peripheral nerve injury induced NPP and participated in the development of hyperalgesia. Key words: Neuropathic pain; Chronic constriction injury; Hyperalgesia; Complement 3 ; Cobra venom factor