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Objective:To evaluate the effect of α-ketoglutarate (AKG) on postoperative delirium (POD) in aged mice.Methods:Eighty male C57BL/6N mice, aged 18 months, weighing 30-35 g, were divided into 4 groups ( n=20 each) using a random number table method: control+ solvent group (group C), control+ AKG group (group C+ AKG), surgery+ solvent group (group S) and surgery+ AKG group (group S+ AKG). Dimethyl 2-oxoglutarate 0.6 mg/kg was injected intraperitoneally for 3 consecutive days before surgery in C+ AKG and S+ AKG groups, while the equal volume of normal saline was given in C and S groups.Exploratory laparotomy was performed under anesthesia with isoflurane to establish POD model.The behaviors of mice in each group were tested at 24 h before surgery and 6, 9 and 24 h after surgery using buried food test (the latency to eat food), open field test (total distance, latency to the center, time and freezing time spent in the center) and Y maze test (duration in the novel arm and the number of entries into the novel arm), respectively.Then the animals were sacrificed at 6 h after operation, hippocampal tissues were removed for determination of the expression of microglia-specific marker ionized calcium binding adaptor molecule-1 (Iba-1), the number of Iba-1 positive cells (using immunofluorescence staining), and the expression of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in hippocamapus (by Western blot). Results:Compared with group C, the latency to eat food at eath time point was significantly prolonged, latency to the center at 6 and 9 h after surgery was prolonged, time spent in the center at 6 and 9 h after surgery was shortened, freezing time at 6, 9 and 24 h after surgery was shortened, the number of entries into the novel arm at 6 and 9 h after surgery was decreased, duration in the novel arm at 6 h after surgery was shortened, the expression of Iba-1 was up-regulated, the number of Iba-1 positive cells was increased, and the expression of IL-1β and TNF-α in hippocampus was up-regulated in group S ( P<0.05), and no significant change was found in the behaviors indexes in group C+ AKG ( P>0.05). Compared with group S, the latency to eat food at each time point was significantly shortened, latency to the center at 9 h after surgery was shortened, time spent in the center at 6 and 9 h after surgery was prolonged, freezing time at 9 and 24 h after surgery was prolonged, the number of entries in the novel arm at 9 h after surgery was increased, the expression of Iba-1was down-regulated, the number of Iba-1 positive cells was decreased, and the expression of IL-1β and TNF-α in hippocampus was down-regulated in group S+ AKG ( P<0.05). Conclusion:AKG can alleviate POD, and the mechanism may be related to inhibiting the activation of microglia and and thus reducing inflammatory responses in aged mice.
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Objective:To explore the mechanism of Akt3 gene knockout on neuropathic pain induced by sciatic nerve ligation.Methods:Twelve Akt3 knockout mice with SPF grade C57BL/6 mice as background were randomly divided into Akt3 gene knockout + Sham group (Akt3 -/-+ Sham, n=6)and Akt3 gene knockout + CCI group(Akt3 -/-+ CCI, n=6). Twelve wild type C57BL/6 mice were randomly divided into wild + Sham group (WT + Sham, n=6) and wild + CCI group (WT + CCI, n=6) by random number table. Chronic sciatic nerve ligation was used to make neuropathic pain model in CCI group, and sham group was subjected to sham operation. The paw withdrawal mechanical threshold (PWMT) was measured 1 day before operation and 7 days after CCI. Three mice were randomly killed in Sham group 7 days after modeling, and 3 mice in CCI group were killed at 7 days and 14 days respectively.The L3-5 spinal cord segment was taken. Akt3, phosphorylated Akt (p-Akt), GSK3β and phosphorylated NR2B (p-NR2B) were detected by Western blot. SPSS 21.0 software was used for statistical analysis. Results:Group time interaction effects of PWMT, the expression of protein Akt3, p-Akt, GSK3β, p-NR2B in spinal cord were significantly different ( F=16.667, 269.899, 26.651, 572.998, 37.836, P<0.01). Compared with the Akt3 -/-+ Sham group, the PWMT in Akt3 -/-+ CCI group was significantly lower((0.34±0.20)g, (1.18±0.11)g, P<0.01), and the expression of p-Akt((0.90±0.08), (0.51±0.06), P<0.01), GSK3β((0.74±0.04), (0.29±0.02), P<0.01) and p-NR2B((0.96±0.11), (0.71±0.04), P<0.05) in spinal cord increased. Compared with the WT+ CCI group, the PWMT of the Akt3 -/-+ CCI group was obviously lower((0.34±0.20)g , (0.49±0.12)g, P<0.05), and the expression of p-Akt((0.90±0.08), (1.02±0.17), P<0.05)decreased, and the expression of GSK3β((0.74±0.04), (0.57±0.09), P<0.01) and p-NR2B((0.96±0.11), (0.91±0.08), P<0.05) increased. Conclusion:After Akt3 gene knockout, the aggravation of neuropathic pain after sciatic nerve ligation may be related with the change of Akt/GSK3β/NR2B expression.
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Objective To investigate the expression of insulin-like growth factors-1(IGF-1)in ser-um and phosphorylated IGF-1 receptor in spinal cord in mouse model of bone cancer pain. Methods Sixty male C3H/HeJ mice weighed 18-22 g were randomly divided into Sham group(n=30)and Tumor group(n=30). The mice in Tumor group were inoculated with NCTC fibrosarcoma cells in the right femur bone marrow cavity. Paw withdrawl mechanical threshold(PWMT)and the number of spontaneous flinches(NSF)were measured on 1d before inoculation and on 4 d,7 d,10 d,14 d,21 d after inoculation(n=8). At each time point,the mice of each group were taken blood by removal eyeball and the samples of blood were obtained to detect the expression of IGF-1 by enzyme-linked immunosorbent assay(n=4). The mice after taken blood on 14 d after inoculation were perfused and the samples of spinal cord lumber(L3~5)segment were obtained to detect the expression of phosphorylated IGF-1 receptor by immunofluorescence assay(n=6). Results Com-pared with Sham group,PWMT was significantly decreased(P<0.05)and NSF was significantly increased(P<0.05)on 7~21 d after inoculation. Compared with baseline value and Sham group(baseline value(27.33± 0.52)pg/ml,Sham group(29.11±1.86)pg/ml,(24.51±3.61)pg/ml,(23.33±4.59)pg/ml,(25.29±2.99) pg/ml),the expression of IGF-1 in serum was significantly increased on 7~21 d after inoculation in Tumor group((39.76±3.92)pg/ml,(36.93±2.18)pg/ml,(38.85±2.40)pg/ml,(39.70±2.62)pg/ml). The mean fluorescence intensity of phosphorylated IGF-1 receptor was significantly higher on 14d after inoculation in Tumor group(2.40±0.11)compared with Sham group(0.05±0.01). Conclusion Expression of IGF-1 in serum and phosphorylated IGF-1 receptor in spinal cord were significantly increased in mice with bone cancer pain,and this change may be involved in the development and maintenance of bone cancer pain.
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Objective To explore the effect of pre-treatment of subcutaneous injection of ketamine on remifentanil induced hyperalgesia and K+/Cl-cotransporter 2,KCC2) expression on spinal cord of rats.Methods60 male adult SD rats were randomly divided into five groups(n=12 in each group):control group (group C),the incision group(group I),the incision plus remifentanil group(group I+R),the incision plus ketamine group(group I+K) and the incision plus remifentanil and ketamine group(group I+R+K).Mechanical withdrawal threshold (MWT) was evaluated at 24 hours before incision(T0),2 hours,6 hours,24 hours and 48 hours after incision(T1~T4).The lumbar spinal cords of rats were taken out at T4 time point and the KCC2 detected was detected by immunofluorescence analysis and western blot analysis.ResultsCompared with group C(T1(14.5±1.7)g,T2(14.2±1.1)g,T3(13.9±1.8)g,T4(14.2±1.1)g),MWT of other groups at T1 (I(5.6±0.8)g,I+R(3.2±1.0)g,I+K(6.8±1.7)g,I+R+K(5.1±1.6)g),T2 (I(6.9±1.0)g,I+R(4.3±1.2)g,I+K(8.0±1.4)g,I+R+K(6.2±1.5)g),T3 (I(7.6±0.9)g,I+R(5.4±1.1)g,I+K(10.3±1.2)g,I+R+K(7.1±1.1)g),T4 (I(8.9±1.1)g,I+R(7.5±1.4)g,I+K(11.3±1.2)g,I+R+K(8.3±1.2)g)and the expression of KCC2 at T4 decreased (P<0.05).Compared with group I(T1(5.6±0.8)g,T2(6.9±1.0)g,T3(7.6±0.9)g,T4(8.9±1.1)g),MWT of group I+R (T1(3.2±1.0)g,T2(4.3±1.2)g,T3(5.4±1.1)g,T4(7.5±1.4)g) decreased at all time points after incision (T1~T4)(P<0.05) and the expression of KCC2 at T4 decreased significantly (P<0.05).Compared with group I(T1(5.6±0.8)g,T2(6.9±1.0)g,T3(7.6±0.9)g,T4(8.9±1.1)g),MWT of group I+K (T1(6.8±1.7)g,T2(8.0±1.4)g,T3(10.3±1.2)g,T4(11.3±1.2)g) increased at all time points after incision (T1~T4)(P<0.05) and the expression of KCC2 at T4 increased (P<0.05).Compared with group I+R(T1(3.2±1.0)g,T2(4.3±1.2)g,T3(5.4±1.1)g,T4(7.5±1.4)g),MWT of group I+R+K (T1(5.1±1.6)g,T2(6.2±1.5)g,T3(7.1±1.1)g,T4(8.3±1.2)g) increased at all time points after incision (T1~T4)(P<0.05) and the expression of KCC2 at T4 increased (P<0.05).ConclusionPre-treatment of subcutaneous injection of ketamine can reduce the hyperalgesia of rats induced by remifentanil and reduce the inhibition of KCC2 expression on dorsal horn of spinal cord.
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Objective To investigate the effects of Aktl gene knockout on pain behavior induced by chronic constriction injury model of sciatic nerve (CCI).Methods C57BL/6 male mice were randomly divided into Akt1 knockout group (KO group,n=12),wild type group(WT group,n=12).All mice were made model of CCI in the right sciatic nerve.Each mouse received tests of the paw withdrawal mechanical threshold (PWMT) and the paw withdrawal thermal latency(PWTL) at the times of 1d before and 1 d,3 d,5 d,7 d,10 d,14 d,17 d,21 d after surgery.Results For both KO group and WT group,the basic values of PMWT(right(0.89±0.15)g,(0.87±0.15)g; left(0.97±0.19) g,(1.05±0.14) g,P＞0.05) and PWTL(right (7.64±0.71) s,(7.56±0.68) s ;left: (7.67±0.6) s,(7.64±0.64) s,P＞0.05) showed no significantly statistical difference.Compared with WT group and the basic value,PWMT and PWTL were significantly decreased after surgery in KO group (P＜0.05).The PWMT and P WTL of the left paw in KO group and WT group had no obvious statistical difference (P＞0.05).However,the PWMT and PWTL of the right paw significantly decreased in the two groups compared with left paw.Conclusion h aggravates the neuropathic pain induced by CCI in mice when the Akt1 gene was knocked out.
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ObjectiveTo investigate the role of Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in neuropathic pain in mice.MethodsThirty-six male C57BL/6 mice,aged 7-8 months,weighing 20-25 g,were randomly divided into 2 groups:sham operation group (group.S,n =9) and chronic constrictive injury (CCI) group (n =27).CCI was produced by placing loosely constrictive ligatures around the common sciatic nerve.Six animals were taken from each group and paw withdrawal threshold to mechanical stimulation (PWMT)and paw withdrawal latency to thermal stimuli (PWTL) were measured on day 1 before operation and on day 1,3,5,7,10,14,17 and 21 after CCI.Three mice were sacrificed on day 3 after CCI in group S and on day 1,3,5,7,10,14 and 21 after CCI in group CCI.The L3-5 segment of the spinal cord was removed for determination of the expression of Akt,phospho-Akt and phospho-GSK-3β by Western blot.Results Compared with group S,PWMT was significantly decreased and PWTL was significantly shortened on day 1-21 after CCI,the expression of Akt was significantly up-regulated on day 7-21 after CCI,and the expression of phospho-Akt and phospho-GSK-3βwas significantly up-regulated on day 1-21 after CCI in group CCI ( P ＜ 0.05).ConclusionAkt/GSK-3β signaling pathway is involved in the development and maintenance of neuropathic pain in mice.
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Objective To investigate the effects of repeated intrathecal cyclic AMP response elementbinding protein (CREB) antisense oligodeoxynucleotide (ODN) on the expression of NR2A in spinal cord in mice with neuropathic pain produced by chronic constrictive injury of the sciatic nerve (CCI).Methods Forty C57BL/6 male mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups ( n =10 each):normal saline group (group NS),CREB sense ODN group (group S),CREB missense ODN group (group M),and CREB antisense ODN group (group A).In groups NS,S,M and A,normal saline 5μl,sense ODN 5 μg/5 μl,missense ODN 5 μg/5 μl and antisense ODN 5 μg/5 μl were injected intrathecally once a day for 6 days,starting from the 1st day after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured on day 1 before CCI and on day 1,3,5 and 7 after CCI.Five mice from each group were sacrificed on day 7 and 14 after CCI and the lumbar segment of the spinal cord (L3-5 )was removed for determination of NR2A expression using Western blot and RT-PCR.Results Compared with the baseline value,no significant change was found in PWMT and PWTL on day 1-7 after CCI in group A ( P ＞0.05),while PWMT and PWTL were significantly decreased on day 1-7 after CCI in groups NS,S and M (P ＜0.05).Compared with groups NS,S and M,the expression of NR2A mRNA and protein was significantly downregulated on day 7 and 14 after CCI in group A ( P ＜ 0.05).The expression of NR2A mRNA and protein was significantly up-regulated on day 14 after CCI compared with that on day 7 after CCI in all the groups.Conclusion Intrathecal CREB antisense ODN during the development of neuropathic pain can attenuate neuropathic pain and inhibition of the expression of NR2A in mouse spinal cord may be involved in the mechanism.
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ObjectiveTo investigate the effects of Akt3 gene knockout on neuropathic pain behaviors induced by chronic constriction injury of sciatic nerve (CCI).MethodsExperiment was divided into two groups:Akt3 knockout group (Akt3-/-,n =12),wild type group (WT,n =12 ).Randomly numbered,the right sciatic nerve of mice were received the operation of chronic constriction injury.Paw withdrawal mechanical threshold (PWMT)and paw withdrawal thermal latency (PWTL) were tested on day 1 before operation and day 1,3,5,7,10,14,17,21 afterCCI.ResultsThe basic values of PWMT(right:(1.09±0.20)g,(1.17±0.22)g;left:(1.17±0.15)g,(1.22±0.23)g,P＞0.05) andPWTL(right:(6.18±1.11)s,(6.20±1.25)s;left:(5.82±0.91)s,(5.92± 1.71 ) s,P ＞ 0.05 ) had no statistically significant differences between two groups.On day 1 after operation,compared with basic values,the PWMT and PWTL of the right paw in both Akt3-/- group and WT group decreased significantly (P ＜ 0.05 ),and at least lasted up to day 21.The PWMT( 3d:(0.42 ± 0.22 ) g,(0.72 ± 0.36) g ; 17d:(0.29 ±0.15)g,(0.49 ±0.19) g;21d:(0.27 ±0.18)g,(0.56 ±0.15)g,P＜0.05) and PWTL(5d:(2.43 ±0.68)s,(3.13±0.52)s;17d:(2.43±1.26)s,(3.84±1.29)s ;21d:(2.14±1.23)s,(4.07±1.26)s,P＜0.05 ) of the right paw in Akt3-/- group was significantly lower than those in WT group.The PWMT and PWTL of the left paw in Akt3-/- group and WT group had no obvious differences (P ＞ 0.05 ). However.compared to left paw,the PWMT and PWTL of the right paw of the two groups were obviously lower (P ＜ 0.05 ).ConclusionThe neuropathic pain induced by CCI increased in Akt3 gene knockout mice.
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Objective To investigate the effects of continuously intrathecal injection rapamycin on neuro pathic pain behaviors in mice.Methods 48 male adult C57/BL6 mice received intrathecal catheter implantation successfully and without motor dysfunction and serious weight loss,were choosed and randomly divided into shamoperation group ( sham,n =24) and chronic constriction injury model group ( CCI,n =24 ).After operation,each group randomly divided into 3 group again.Group I did nothing,group Ⅱ intrathecally injected rapamycin 1 μg/5μlon day 1 to 6 after operation,group Ⅲ intrathecally injected 20％ DMSO 5μl on the same time ( sham,CCI,sham +R,sham + V,CCI + R,CCI + V,n =8 ).Bilateral mechanical paw withdrawal threshold (WMT) and thermal paw withdrawal latency(TWL) were tested on day 1 before CCI and day 1,3,5,7,10,14,17,21,28 after operation.Results Compared with sham group,both WMT and TWL (7d,MWT:( 1.02 ±0.12)g vs (0.42 ±0.12)g,F=51.01,P＜0.05;TWL:(7.03 ±0.71 )s vs (3.26 ±0.66)s,F=38.27,P＜0.05) were significantly decreased after CCI on the ipsilateral side.When intrathecally injected Rapamycin 1 μg/5 μl on day 1 ～6 after CCI,the mechanical allodynia relieved obviously ( 7 d,M WT:( 0.42 ± 0.18 ) g vs ( 0.86 ± 0.25 ) g,F =6.56,P ＜ 0.05 ),and at least continued to 10 d.On the contrary,the effects of rapamycin on thermal hyperalgesia just showed a trend of inhibition,there was no statistic meaning.In addition,the sham group and contralateral pain behaviors did not change (P＞ 0.05 ).Conclusion Rapamycin can relieve the neuropathic pain behaviors in mice after CCI,mainly the mechanical allodynia,but not thermal hyperalgesia.
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Objective To investigate the effects of intrathecally cyclic AMP response element-binding protein(CREB) antisense oligodeoxynucleotide (ODN) on neuropathic pain behaviors.Methods Using mouse model of neuropathic pain induced by chronic constriction injury of sciatic nerve (CCI),24 male C57BL/6 mice successfully received intrathecal catheter implantation and without motor dysfunction were randomly divided into 4groups(n=6):Saline group(NS),CREB sense ODN group(S),CREB missense ODN group(M),CREB antisense ODN group(A).Mice in NS,S,M and A were intrathecally treated with Saline 5μ l,Sense ODN 5μg/5μl,Missense ODN 5μg/5μl and Antisense ODN 5μg/Sμl once daily on day 1 ～6 after CCI respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency(PWTL) were tested on day 1 before CCI and day 1,3,5,7,10,14,17,21 after CC(I).Results Mice in A group maintained the pain thresholds in the baseline and lasted at least 7 days after CCI ( 7 d,PWMT:( 0.81 ± 0.20 ) g vs ( 1.00 ± 0.19 ) g,P ＞ 0.05 ;PWTL:(5.96 ± 0.69) s vs (6.93 ± 1.08 ) s,P ＞ 0.05 ).The withdrawal thresholds in the ipsilateral hind paws of the mouse were significantly lower than baseline in A group on day 10 after CCI( 10 d,PWMT:(0.56 ±0.19)g vs (1.00±0.19)g,P＜0.05; PWTL:(3.93 ±0.28)s vs (6.93 ± 1.08)s,P＜0.05).Compared with NS group ( 10 d,PWMT:(0.56 ±0.19)g vs (0.37 ±0.08)g,P＜0.05; PWTL:(3.93 ±0.28)s vs (3.14 ±0.45)s,P＜0.05),S group,M group,the withdrawal thresholds of A group was significantly elevated on day 10 after CCI.These effects lasted up to at least day 21 after CCI.Conclusion Intrathecally treated with CREB antisense ODN in the development of neuropathic pain induced by CCI completely improved pain behaviors during the course of injection,and the effects of relief pain lasted at least 15d after no injection.