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Objective To investigate the effects of midazolam on extracellular signal-regulated kinase 1 (ERK1),ERK2 and cyclic AMP response element binding protein (CREB) phosphorylation in hippocampal in rats.Methods Eighty male SD rats weighing 250-300 g were randomly divided into 2 groups ( n = 40) : group control (group C) and group midazolam (group M).The animals underwent a continuous multi-trial inhibitory avoidance training .The times of trial needed for each animal to attain the learning criterion ( 100 s) were recorded.Each animal was given intraperitoneal midazolam 3 mg/kg or normal saline 2 ml/kg at 15 min before training.The memory retention was tested at 0.5,1,2 and 24 h (n = 8,at each time point)after the training session and the memory latency was recorded.The animals were sacrificed 15 min after administration (T0) and after the memory testing (T1-4) and hippocampns was obtained for determination of phosphorylated ERK1 (p-ERK1),p-ERK2 and p-CREB expression.Results Compared with group C,the times of trial to attain the learning criterion were significantly increased,memory latency shortened at T2-4,ERK1 phosphorylation decreased at T0,3.4 while ERK2 and CREB phosphorylation decreased at T0-4.Conclusion Midazolam can inhibit ERK1,ERK2 and CREB phosphorylation in hippocampal in rats.
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Objective To prepare the survival motor neuron(SMN)polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy(SMA).Methods A prokaryotic expressional plasmid named pET-28? (+)/SMN was constructed and SMN-His fusion protein was induced.The fusion protein was used to immunize New Zealadd rabbits to prepare SMN polyclonal antibody.A eukaryotic expressional plasmid named pcDNA3.1/myc-HisB-SMN was constructed and used to transfect CHO cells.Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ, 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis.Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients.Results Correct pET-28a(+)/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a(+)/SMN.Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein.SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus.The results of Western-blotting were as follows:the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase(GAPDH)band density (SMN/GAPDH)is 0.619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type Ⅲ and Ⅱ were 0.347 and 0.340 respectively, which were lower than that of normal controls.However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0.079, which was quite lower than that of normal controls.Conclusions The rabbit anti-human full-length SMN polyclonal antibody is of high specificity and sensitivity, which makes the basis for the research of SMN function and SMA pathogenesis.There may be a correlation between the SMN level in skeletal muscle and the severity of disease.
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Objective To investigate the changes in CREB_1 proteins in five brain regions of rats with morphine addiction and withdrawal with the technique of immunohistochemistry. Methods Thirty six adult male Sprague-Dawley rats were randomly divided into six groups (n=6 for each), i.e. acute morphine dependent group, acute abstinence group, acute control group, chronic morphine dependent group, chronic abstinence group and chronic control group. Animals in dependent groups and abstinence groups were administered with morphine by intraperitoneal injection till morphine dependent models were established. The rats in abstinence groups withdrawal syndromes were induced with naloxone 5mg/kg for 30min. The rats in control groups were injected with saline. All rats were sacrificed by decapitation. The coronal sections of discrete brain regions (hypothalamus, nucleus accumbens, prefrontal cortex, locus coeruleus, hippocampus) were obtained. The relative concentrations of CREB_1 protein were determined with immunohistochemistry. Results In acute morphine dependent and abstinence groups, CREB_1 protein decreased significantly compared with the acute control group in locus coeruleus (P0.05). Conclusion The morphine-induced CREB_1 protein changes may reflect differential G protein-cAMP-CREB signal transduction pathways in morphine dependent and abstinence rats.
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0.05) . The CREB-1 protein expression in cortex and hippocampus was significantly up-regulated while that in nucleus accumbens was significantly down-regulated in chronic morphine dependence and abstinence group (groupⅢandⅣ) as compared with control group. The CREB-1 protein expression in nucleus accumbens in groupⅣwas significantly lower than that in groupⅢ. Conclusion Acute morphine dependence and abstinence do not significantly affect CREB-1 protein expression in the brain. The changes in CREB-1 protein expression are different in different brain regions in chronic morphine dependence and abstinence rats.
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Objective To determine whether chronic constriction injury (CCI) to sciatic nerve is associated with changes in the phosphorylation of CREB in dorsal root ganglia ( DRG) and superficial dorsal horn neurons of the spinal cord.Methods Thirty-two adult female SD rats weighing 230-270 g were randomly divided into 4 groups (n = 8 each): Ⅰ blank control;Ⅱ sham operation; Ⅲ CCI 2w and Ⅳ CCI 4w. The animals were anesthetized with intraperitoneal pentobarbital 40 mg?kg-1. Right sciatic nerve was exposed and 4 ligatures were placed on the right sciatic nerve at 1 mm interspace with 3-0 silk suture. Paw withdrawal threshold to mechanical stimulation (von Frey filament) applied to plantar surface ( MWT) and paw withdrawal latency to thermal stimulation (radiant heat) (TWL) were measured before operation (baseline) and 14 days (group Ⅰ,Ⅱ and Ⅲ) or 28 days (group Ⅳ) after nerve ligation. The animals were killed the next day and the L4,5 segment of the spinal cord and L5 dorsal root ganglion were removed for determination of expression of phosphorylated-CREB-immuno-reaction(pCREB-IR) using immuno-histochemistry. The pCREB-IR cells both in DRG and superficial dorsal horn neurons were quantified and analyzed. Results The animals developed mechanical and thermal hyperalgesia on the 14th day after CCI (in group CCI 2w) . The hyperalgesia was greatly attenuated on the 28th day after CCI. Interestingly enough the animals in sham operation group (Ⅱ) also developed mechanical hyperalgesia to some extent on the 14th day after operation. The number of pCREB-IR cells was significantly increased in the ipsilateral L5 DRG and superficial dorsal horn in group Ⅲ(CCI 2w) as compared to sham operation group ( P