Objective To investigate the effects of a highly selective protein kinase A (PKA) inhibitor, H89, injected intrathecally (IT) on hyperalgesia and phosphorylation of cAMP element binding protein(pCREB) in the dorsal horn neurons of the spinal cord induced by chronic constriction injury (CCI) to the sciatic nerve.Methods Fifty-eight adult female SD rats weighing 230-270 g were used in this study. CCI was produced by 4 loose ligatures place on the sciatic nerve of right hind leg at 1 mm interspace with 3-0 silk suture. The experiment was carried out in two parts. In part Ⅰ 28 animals were randomized to receive H89 1 (group H1), 2 (group H2 ) or 4 nmol (group H4) or 10 ?l of DMSO (the solvent) 10mmol?L-1 (control group) intrathecally (IT) (n = 7 each) 7 days after surgery. The paw-withdrawal latency following mechanical (MWL) and thermal stimulation (TWL) were recorded before (baseline) and 15, 30 and 60 min after drug administration. In part Ⅱ 24 rats were randomized to receive H89 1, 2 or 4 nmol or 10 ?l of DMSO 10 mmol?L-1 IT as in part Ⅰ (group H1, H2, H4 and control group, n = 6 each) . Another 6 animals received 10 ?l of DMSO 10 mmol?L IT 7 days after sham operation. The animals were killed 30 min after drug administration and lumbar (L4.5) segment of spinal cord was removed for determination of pCREB expression in the dorsal horn neurons of spinal cord using immuno-histochemical technique. Results MWL and TWL were significantly increased after drug administration in group H2(at 15min) and group H4(at 15 and 30 min) as compared to the baseline values(P

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

Objective To establish a drug-resistance cell line of human glioma with temozolomide ( TMZ) ,investigate its resistance mechanisms, and provide experimental evidence for optimal TMZ therapy. Methods A TMZ-resistant human glioma cell line,U251/TR,was established by stepwise exposure of human parental U251 cells to TMZ. Resistance index and cell viability were accessed by MTT assay. Western-Blot,RT-PCR,immunohistochemistry and immunofluorescence were used to detect MGMT expression for the analysis of resistance mechanism. Results A TMZ-resistant human glioma cell line,U251/TR,was developed after 8 months of stepwise induction with 0. 25-16. 00 μg·mL-1 TMZ. IC50 in U251/TR cells was approximately 7 times higher compared with that in U251 cells (P=0. 00 ). The MGMT expression was significantly increased in U251/TR cells compared with that in parental U251 cells (P=0. 00) . Conclusion A TMZ-resistant human glioma cell line,U251/TR,was established by stepwise exposure of human parental U251 cells to TMZ. The primary mechanism of TMZ resistance is associated with increased activity of MGMT.

ObjectiveTo design a weight-bearing pressure casting system for transtibial prostheses to make the total surface bearing socket and to control the stump and bearing during the casting process.MethodsThe weight-bearing pressure casting system was designed. 6 cases weared the socket by this method and traditional patellar tendon bearing socket by hand casting. Socket interface pressure was compared, and the effect of casting was assessed.ResultsCompared with patellar tendon bearing socket, the socket pressure in the socked by this method was distributed more equally throughout the transtibial residual limb during the casting process, and completed the stumps compression and stump-end pressure control.ConclusionWeight-bearing pressure casting system can make the total surface bearing socket, and improve the fit of the socket.

Objective To identify C0L7Al gene mutations in a family of recessive dystrophic epidermolysis bullosa (RDEB). Methods PCR and direct DNA sequencing were used to determine the mutation sites and types. PCR using allele-specific oligonucleotide primers was performed to further identify the pathogenic cause of this disease. Results The patient examined in this study was a compound heterozygote for a S48P missense mutation in exon 2 and a 3625del 11 PTC mutation in exon 27, which was a novel combination of COL7Al mutations in RDEB. Conclusion The missense mutation and the nonsense mutation in COL7Al gene are underlying causes of the Hallopeau-Siemens variant of RDEB.

Objective To explore a space craft precooling temperature at which excessive thermal stress on the crew member could be prevented or reduced in an overheated launch or reentry module. Method Five young male volunteers wearing a space suit participated in 25 tests at sea level.The space suit was either ventilated in a volume air flow rate of 100 L/min (STPD) with ambient air at temperatures (Ta) of 15℃,10℃,and 5℃,respectively,or not ventilated. Rectal (Tr),mean skin (Tsk) and mean body (Tb) temperatures were measured. Result At Ta 15℃,Tr decreased without significance (from 37.0±0.2℃to 36.7±0.3℃) in 120-min tests,whereas Tsk and Tb decreased significantly,and subjects had local cold strain whether the space suit was ventilated or not; while at Ta 10℃,Tr decreased from 37.0±0.3℃ to 36.3±0.3℃(P<0.05),subjects had a whole body cold strain,and both Tsk and Tb dropped continuously and significantly. Conclusion Ambient temperature 15℃,at which the thermal comfort states of crew was not significantly degraded,was acceptable after precooling in a space craft.

Objective To evaluate the effect of B-vitamins(B1,B6,B12)on diabetic neuropathic pain (DNP)in rats.Methods 104 male SD rals weighing 200-230 g were randomly divided into 13 groups(n=8 each):group Ⅰ control(group C);group Ⅱ DNP;group Ⅲ DNP+ normal saline(solvent of vitamins,group NS);group Ⅳ,Ⅴ,Ⅵ DNP+vitamin B1 10,33 or 100mg/kg,kg(group B1 10,group B133,group B1 100);group Ⅶ,Ⅷ,Ⅺ DNP+vitamin B6 10,33 or 100 mg/kg(group B6 10,group B633,group B6100);group Ⅹ,Ⅲ,ⅫDNP+vitamin B12 0.5,1.5 or 4.5 mg/kg (group B12 0.5,group B121.5,group B124.5)and group ⅩⅢ DNP+vitamin B1 10/B6 33/B12 1.5 mg/kg(group VBC).Diabetes was induced with intraperitoneal(IP) streptozocin mg/kg in group Ⅱ-ⅩⅢ.B-vitamins were give.IP once a day for 14 consecutive days starting from 14 d after IP streptozocin in group Ⅳ-ⅩⅢ.Venous blood samples were taken before(baseline)and 3 d after IP streptozocin for determination of blood glucose level. Successful induction of diabetes was defined as blood glucose > 14.6 mmol/L. Mechanical paw withdrawal threshold to yon Frey stimuli (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured 2 days before and 14 days after IP streptozocin and on the 1, 3, 7, 14 days of B-vitamin administration. Animals with pain threshold measured at 14 days after IP streptozocin decreasing by less than 15% of the baseline were excluded from the study. The animals were sacrificed after the last pain threshold measurement and L4,5 lumbar segment of the spinal cord and dorsal root ganglions (DRG) were removed for determination of p-CREB expression using immuno-histuchemistry. Results MWT was significantly lower and TWL was significantly shorter and the expression of p-CREB was significantly higher in the other groups than in group C. B-vitamin administration significantly reduced thermal and mechanical hyperalgesia induced by diabetes and down-regulated the expression of p-CREB in a dose-dependent manner as compared with group DNP. The inhibitory effect of vitamin B complex against thermal and mechanical hyperalgesia was significantly stronger and the expression of p-CREB was significantly lower in group VBC as compared with group B110, group B633 and group B121 .5 respectively. Conclusion B-vitamains can attenuate DNP through inhibition of phospberylation of CREB in the spinal dorsal horn and DRG.

Objective To explore the features of leukoencephalopathy with cerebral calcifications and cysts (LCC) in clinic, radiology and pathology in order to improve skills in diagnosis. Methods Two female patients had CT and/or MRI scan, and case 2 had contrast enhancement MRI scan additionally. Both cases had blood biochemistry examinations including calcium, phosphorus and alkaline phosphatase et al. Case 2 had lumbar puncture and cerebrospinal fluid test. The major cystic lesion was surgically removed in both patients and offered a histopathological examination. Results CT scan reveaed diffuse calcifications in the bilateral basal ganglia, white matter of frontal lobe and/or dentate nuclei of cerebellum in both cases, and major cystic lesion in right frontal lobe (case 1) and the left parietal lobe (case 2). The rim of enhancement was observed in cystic lesion on MRI. Histopatbological examination revealed angiomatous rearrangements of the microvessels with fibroid, hyaline degeneration and haemosiderin deposits, brain tissue associated with areas of demyelinization, some Rosenthal fibers, gliosis, calcium deposits and hemorrhage, fibrinoid necrosis occurs in partial vessels associated with thrombogenesis and stenosis as changes in arteriolitis. Blood biochemistry examination showed normal. Cerebrospinal fluid test in case 2 showed increased intracranial pressure(350 mm H_2O,I mm H_2O =0. 0098 kPa). Conclusions The onset of LCC varies and occurs from early infancy to adult. The asymmetrical calcification is characteristic in LCC. Hemorrhage could be involved in the pathogenesis of cystic formation. LCC is characterized by a cerebral obliterative microangiopathy, both demyelinization and the edematous changes could probably result in white matter abnormalities on neuroimaging.

Objective To study the effect of different approach on myocardial contractile force, angiogenesis and collagen renovation in acute myocardial infarct site by implantation of bone marrow mesenchymal stem cells (MSCs). Methods 32 Gui-zbou Xiang porcine were randomly divided into control group (group C), intra-coronary arterial injection group (group A) ,topical injection group (group T) and intra-coronary arterial + topical injection group( group A + T). 3 ml bone marrow was extracted. MSCs were cultured according to the methods of Wakitani's. After being co-cultured with 5-azacytidine for 24 hours, these cells were labelled with bromodeoxyuridine ( BrdU ). Autologus MSCs were implanted into the acute myocardial infarct site via the distal segment of the ligated left anterior descending artery (LAD) ,topical injection and both. Specimens and muscle strip were harvested in 3 and 6 weeks after MSCs implantation for microvessel count, P Ⅲ NP activity and contractile force measurement. Results The contractile force(48.6±5.9) %, (42.1±6.2 ) %, ( 56.9±5.1 ) %, microvessel count ( 19.6±4.3,17.1±4.0, 23.2±5.5 ) and collagen renovation (4.6±0.5 ) μg/L, ( 5.9±0.7 ) μg/L, ( 3.9±0.3 ) μg/L in the cell implant groups was better than that in the control group(37.9±5.4)% ,13.2±3.8,(8.7±0.8) μg/L,(P <0.01) ,espe-cially in group A + T. Conclusions Implantation of MSCs through intra-coronary arterial plus topical injection of MSCs maybe an ideal approach for infracted myocardium.