Objective To investigate the effect of hyperbaric oxygen on the expression of fractalkine (FKN) in the nerve tissues of rats with neuropathic pain (NP).Methods Thirty-two male Sprague-Dawley rats, weighing 250-280 g, aged 10-12 weeks, were divided into 4 groups (n=8 each) using the random number table: control group (group C), sham operation group (group S), group NP, and hyperbaric oxygen group (group H).NP was induced by chronic constriction injury (CCI) in anesthetized rats.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.Group H received hyperbaric oxygen therapy once a day for 5 consecutive days starting from day 1 after CCI.The rats were placed into the hyperbaric oxygen chamber, which was pressurised to 2 atmosphere absolute at a rate of 10 kPa/min, and maintained at this level for 60 min.The pressure was then decreased to the normal pressure at a rate of 10 kPa/min.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before CCI, and 3, 5, 7 and 14 days after CCI.After measurement of pain threshold at 3 and 7 days after CCI, 4 rats were selected and sacrificed.The sciatic nerve and lumbar segment of the spinal cord were removed for determination of the expression of FKN by Western blot.Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at each time point after CCI, the expression of FKN in the sciatic nerve at 3 days after CCI, and in the sciatic nerve and spinal cord at 3 and 7 days after CCI was upregulated in group NP (P＜0.05) , and no significant change was found in the parameters mentioned above in group S (P＞0.05).Compared with group NP, the MWT was significantly increased, and TWL was prolonged at each time point after CCI, and the expression of FKN in the sciatic nerve at 3 days after CCI, and in the sciatic nerve and spinal cord at 3 and 7 days after CCI was down-regulated in group H (P＜0.05).Conclusion The mechanism by which hyperbaric oxygen mitigates NP is related to inhibition of over-expression of FKN in the nerve tissues of rats.

Objective To evaluate the effects of tempol administered via different routes on neuropathic pain (NP) in rats.Methods Thirty-two male Sprague-Dawley rats,weighing 250-280 g,aged 8-10 weeks,were randomly divided into 4 groups (n=8 each) using a random number table:sham operation group (group S),group NP,intrathecal tempol group (group T1),and intraperitoneal tempol group (group T2).Neuropathic pain was induced by chronic constriction injury in chloral hydrate-anesthetized rats.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.The sciatic nerve was only exposed but not ligated in group S.After successful establishment of the model,a catheter was inserted at L4.5 interspace into the epidural space.In S and NP groups,0.9％ normal saline 20 μl was injected intrathecally,and 0.9％ normal saline 200 μl was injected intraperitoneally once a day for 7 consecutive days.In group T1,tempol 30 μg (in 20 μl of normal saline) was injected intrathecally,and 0.9％ normal saline 200 μl was injected intraperitoneally once a day for 7 consecutive days.In group T2,tempol 30 μg (in 200 μl of normal saline) was injected intraperitoneally,and 0.9％ normal saline 20 μl was injected intrathecally once a day for 7 consecutive days.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 3 days before operation,and at 1,3,5,7,10 and 14 days after operation.The animals were sacrificed after measurement of pain threshold at day 14 after operation.The lumbar segment of the spinal cord was removed to detect malondialdehyde (MDA) content and superoxide dismutase (SOD) activity by enzyme-linked immunosorbent assay.Results Compared with group S,the MWT was significantly decreased,and the TWL was shortened at each time point after operation,the content of MDA in the spinal cord was increased (P＜0.05),and no significant difference was detected in SOD activity in group NP (P＞0.05).Compared with group NP,the MWT was significantly increased at 5,7,10 and 14 days after operation,the TWL was prolonged at 1,3,5,7,10 and 14 days after operation,the content of MDA in the spinal cord was decreased,and the SOD activity was increased in group T1 (P＜0.05),and no significant change was found in the indexes mentioned above in group T2 (P＞0.05).Conclusion Intrathecal tempol can reduce NP in rats,and the mechanism is related to inhibition of lipid peroxidation in the spinal cord.

Objective To evaluate the relationship between annexin 1 (ANXA1) and the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxiu.Methods The intestinal epithelial cells at the logarithmic growth phase were seeded in culture palates and randomly divided into 4 groups (n =36 each) using a random number table:control group (group C),cell injury group (group I),ANXA1 overexpression group (group OE),and ANXA1 silencing group (group S).Lentivirus with ANXA1 overexpression and silencing was transfected into intestinal epithelial cells to construct a stable cell line.In I,OE and S groups,endotoxin was added with the final concentration of 100 μg/ml,and the cells were then incubated for 24 h to establish the cell injury model.The culture medium was changed,and the cells were then incubated for 24 h in group C.The cell apoptosis was detected by flow cytometry,the cell permeability was determined by Transwell assay,and the cell viability was evaluated by methyl thiazolyl tetrazolium assay.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in I,OE and S groups (P＜0.05).Compared with group Ⅰ,the apoptosis rate was significantly decreased,the cell permeability and viability were significantly increased in group OE,and the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in group S (P＜0.05).Conclusion ANXA1 is involved in the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxin.

Objective To evaluate the relationship between extracellular signal-regulated kinase (ERK)and ketamine-induced apoptosis in rat hippocampal neurons.Methods Sprague-Dawley rats at 18 days of gestation were anesthetized.The fetal rats were obtained under the sterile condition and decapitated.The hippocampal neurons were isolated and primarily cultured for 5 days,and were seeded in 6-well plates (2 ml/well) or in 96-well plates (100μl/well) at a density of 5 × 105/ml.The cells were randomly divided into 4 groups (n =18 each):control group (group C),fibroblast growth factor (FGF-2,an ERK agonist) group (group F),ketamine group (group K) and FGF-2 + ketamine group (group FK).The cells were cultured in the plain culture medium in group C.FGF-2 50 ng/ml was added to the culture medium in group F.Ketamine was added to the culture medium in group K.FGF-2 50 ng/ml was added to the culture medium at 20 min before ketamine 100 μmol/L was added in group FK.The phosphorylation of ERK in hippocampal neurons was detected by Western blot at 10 min after treatment.At 24 h after treatment,the neuronal apoptosis was detected by Hoechst33342/PI staining,and the cell survival rate was detected by MTT assay.The apoptosis rate was calculated.Results Compared with group C,the phosphorylation of ERK in hippocampal neurons and the cell survival rate was significantly decreased and the apoptosis rate was increased in K and FK groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between F and C groups (P ＞ 0.05).The phosphorylation of ERK in hippocampal neurons and the cell survival rat was significantly higher and the apoptosis rate was lower in group FK than in group K (P ＜0.05).Conclusion Ketamine induces apoptosis in rat hippocampal neurons by inhibiting activation of ERK in hippocampal neurons.

PURPOSE: To investigate the effects of hyperbaric oxygen (HBO) pretreatment on cognitive decline and neuronal damage in an Alzheimer’s disease (AD) rat model. MATERIALS AND METHODS: Rats were divided into three groups: normal saline (NS), AD, and HBO+AD. In the AD group, amyloid β peptide (Aβ)₁₋₄₀ was injected into the hippocampal CA1 region of the brain. NS rats received NS injection. In the HBO+AD group, rats received 5 days of daily HBO therapy following Aβ₁₋₄₀ injection. Learning and memory capabilities were examined using the Morris water maze task. Neuronal damage and astrocyte activation were evaluated by hematoxylin-eosin staining and immunohistochemistry, respectively. Dendritic spine density was determined by Golgi-Cox staining. Tumor necrosis factor-α, interleukin-1β, and interleukin-10 production was assessed by enzyme-linked immunosorbent assay. Neuron apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Protein expression was examined by western blotting. RESULTS: Learning and memory dysfunction was ameliorated in the HBO+AD group, as shown by significantly lower swimming distances and escape latency, compared to the AD group. Lower rates of neuronal damage, astrocyte activation, dendritic spine loss, and hippocampal neuron apoptosis were seen in the HBO+AD than in the AD group. A lower rate of hippocampal p38 mitogen-activated protein kinase (MAPK) phosphorylation was observed in the HBO+AD than in the AD group. CONCLUSION: HBO pretreatment improves cognition and reduces hippocampal damage via p38 MAPK in AD rats.
Alzheimer Disease/*therapy ; Amyloid beta-Peptides/*administration & dosage ; Animals ; Apoptosis ; *Cognition/drug effects ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Hippocampus/*enzymology ; *Hyperbaric Oxygenation ; In Situ Nick-End Labeling ; Interleukin-10/biosynthesis ; Interleukin-1beta/biosynthesis ; Learning/drug effects ; Male ; Memory/drug effects ; Neurons ; Peptide Fragments/*administration & dosage ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride/administration & dosage ; Tumor Necrosis Factor-alpha/biosynthesis ; p38 Mitogen-Activated Protein Kinases/*metabolism

Objective:To evaluate the effect of c-CBL overexpression on activation of astrocytes in the spinal cord of rats with neuropathic pain and the relationship with Kindlin-1.Methods:Eighteen clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (S group), neuropathic pain group (NP group) and c-CBL overexpression group (c-CBL group). The model of neuropathic pain was developed by chronic compression of the sciatic nerve in anesthetized rats.On 1 day before operation, c-CBL overexpression vector 10 μl was intrathecally injected in group c-CBL, while blank vehicle 10 μl was intrathecally injected in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on 1 day before operation (T 0) and 1, 4 and 7 days after operation (T 1-3). The rats were sacrificed by decapitation after measurement of the pain threshold at T 3, and the spinal cord of L 4-6 was taken for determination of the expression of Kindlin-1, glial fibrillary acidic protein (GFAP), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) (by Western blot) and co-expression of Kindlin-1 with c-CBL (by co-immunoprecipitation). Results:Compared with group S, the MWT was significantly decreased and TWL was shortened at T 2, 3 in group NP and group c-CBL, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was significantly up-regulated in group NP, and the expression of c-CBL was significantly up-regulated in group c-CBL ( P<0.05). Compared with group NP, the MWT was significantly increased and TWL was prolonged at T 2, 3, the expression of Kindlin-1, GFAP, TNF-α and IL-1β was down-regulated, and the expression of c-CBL was up-regulated in group c-CBL ( P<0.05). The results of co-immunoprecipitation showed that there was a protein interaction and co-expression relationship between Kindlin-1 and c-CBL in group NP, and the co-expression of Kindlin-1 with c-CBL was enhanced in group c-CBL when compared with group NP. Conclusions:The overexpression of c-CBL can inhibit activation of astrocytes by down-regulating the expression of Kindlin-1 in the spinal cord, thus reducing inflammatory responses and relieving neuropathic pain in rats.

Objective To evaluate the role of spinal kindlin-1 in neuropathic pain in rats and the relationship with Wnt3a.Methods Eighteen clean-grade healthy male Sprague-Dawley rats,weighing 250-280 g,aged 10-12 weeks,were divided into 3 groups (n =6 each) using a random number table:sham operation group (group S),neuropathic pain group (group NP) and kindlin-1 inhibitor group (group K).Neuropathic pain was induced by chronic compression of the sciatic nerve.The sciatic nerve was only exposed but not ligated in group S.In group K,shRNA was intrathecally injected at 21 days before operation to inhibit the expression of kindlin-1.Vector virus was intrathecally injected at 21 days before operation in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 1,4,7,10 and 13 days after operation.Rats were sacrificed at 13 days after measurement of pain threshold and the spinal cord was removed for determination of the expression of kindlin-1 and Wnt3a (by Western blot) and expression of Wnt3a mRNA (by real-time polymerase chain reaction).Results Compared with group S,the MWT was significantly decreased and the TWL was shortened at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was up-regulated in group NP (P＜0.05).Compared with group NP,the MWT was significantly increased and the TWL was prolonged at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was down-regulated in group K (P＜0.05).Conclusion Kindlin-1 is involved in the development of neuropathic pain by up-regulating the expression of Wnt3a in rats.