Hyperbaric oxygen therapy improves local microenvironment after spinal cord injury

Clinical studies have shown that hyperbaric oxygen therapy improves motor function in patients with spinal cord injury. In the present study, we explored the mechanisms associated with the recovery of neurological function after hyperbaric oxygen therapy in a rat model of spinal cord injury. We established an acute spinal cord injury model using a modification of the free-falling object method, and treated the animals with oxygen at 0.2 MPa for 45 minutes, 4 hours after injury. The treatment was administered four times per day, for 3 days. Compared with model rats that did not receive the treatment, rats exposed to hyperbaric oxygen had fewer apoptotic cells in spinal cord tissue, lower expression levels of aquaporin 4/9 mRNA and protein, and more NF-200 positive nerve fibers. Furthermore, they had smaller spinal cord cavities, rapid recovery of somatosensory and motor evoked potentials, and notably better recovery of hindlimb motor function than model rats. Our findings indicate that hyperbaric oxygen therapy reduces apoptosis, downregulates aquaporin 4/9 mRNA and protein expression in injured spinal cord tissue, improves the local microenvironment for nerve regeneration, and protects and repairs the spinal cord after injury.     

Temporal delimitation of the healing phases via monitoring of fracture callus stiffness in rats

The healing process consists of at least three phases: inflammatory, repair, and remodeling phase. Because callus stiffness correlates with the healing phases, it is suitable for evaluating the fracture healing process. Our aim was to develop a method which allows determination of callus stiffness in vivo, the healing time and the duration of the repair phase. The right femurs of 16 Wistar rats were osteotomized and stabilized with either more rigid or more flexible external fixation. Fixator deformation was measured using strain gauges during gait analysis. The strains were recalculated as the callus stiffness over the time course of healing, and the healing phases were identified based on stiffness thresholds. Our hypothesis was that stabilization with more flexible external fixation prolongs the repair phase, therefore resulting in an extended healing time. Confirming our hypothesis, the duration of the repair phase (rigid: approximately 15 days, flexible: approximately 41 days) and the healing time (rigid: approximately 27 days, flexible: approximately 62 days) were significantly longer for more flexible external fixation. Our method allows the quantitative detection of differences in the healing time and duration of the repair phase without multiple time‐point sacrifices, which reduces the number of animals in experimental studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1589–1595, 2014.     

大鼠髁突软骨细胞发育中相关信号通路的初步筛查

目的:通过建立的大鼠髁突软骨细胞发育过程中蛋白表达差异谱,分析可能参与的信号通路,为进一步认识髁突软骨生理性及病理性生长改建的信号调控机制提供相关信息。方法:收获出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色鉴定软骨细胞。提取各组软骨细胞总蛋白,采用i TRAQ标记定量蛋白,2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(MALDI-TOF-TOF),获取出生后大鼠髁突软骨细胞发育过程中差异蛋白的表达谱,所得数据用MASCOT软件处理,筛选样本之间有意义的差异蛋白,运用GO法及David软件进行KEGG信号通路分析。结果 :共鉴定137种具有可信度表达的蛋白,有44种蛋白参与至少27条KEGG信号通路,其中ECM-受体相互作用、焦点粘连、actin骨架调节、Ca2+信号通路、血管平滑肌收缩、Gn RH信号通路、肌醇三磷酸代谢、磷脂酰肌醇信号系统以及核糖体等信号通路具有统计学意义。结论:在大鼠髁突软骨发育中,各信号通路蛋白在时间、空间上差异性表达,共同形成复杂的信号传递网络。     

FGF-9参与调控小鼠上颌突间充质细胞体外成骨分化的研究

目的:研究成纤维细胞生长因子9(FGF-9)对体外培养的小鼠上颌突间充质细胞成骨分化的调控作用。方法 :体外培养E12.5(胚胎第12.5天)的小鼠上颌突间充质细胞,取第1代细胞进行成骨诱导,实验组加入FGF-9人重组蛋白,诱导培养1周后通过q PCR、免疫荧光和茜素红染色检测其成骨能力。结果:体外培养的E12.5小鼠上颌突间充质细胞表达FGF9和FGFR3。成骨诱导可促进上颌突间充质细胞表达成骨标记物ALP、Runx2和OCN;加入FGF-9人重组蛋白可降低成骨标记物ALP、Runx2和OCN的表达,减少钙结节形成。结论:体外培养时,FGF9参与负调控上颌突间充质细胞的成骨分化。     

核因子-kB、肿瘤坏死因子-a在糖尿病大鼠膀胱的表达及相互关系的研究

【目的】观察糖尿病大鼠膀胱组织的病理改变，检测核因子-kBp65、肿瘤坏死因子-a表达的变化，并探讨两者关系。【方法】采用右下腹腔内一次性注射链脲佐菌素60mg／kg诱导Wistar大鼠糖尿病模型26只，分别于成模后1、3、6个月观察膀胱组织的病理改变，检测核因子-kBp65、肿瘤坏死因子-a表达变化。【结果】随糖尿病病程延长，膀胱组织发生病理改变，核因子-kBp65、肿瘤坏死因子-a表达显著增强，两者呈正相关。【结论】核因子-kBp65、肿瘤坏死因子-a与糖尿病膀胱的病理、功能改变相关。     

虎潜丸对去势大鼠骨密度及BMP-2水平影响的研究

目的：探讨虎潜丸干预对去势大鼠骨密度及骨组织中BMP－2水平的影响。方法6个月龄的40只雌性SD大鼠随机分为手术模型组、雌激素组、虎潜丸组和假手术组。确定造模成功后,虎潜丸组给予虎潜丸煎剂浓缩液灌胃,每天一次;雌激素组给予尼尔雌醇悬浊液灌胃,每周一次;手术模型组和假手术组给予蒸馏水灌胃,每天一次。灌胃治疗12周后测量各组大鼠的骨密度及骨组织中BMP－2含量。结果虎潜丸组及雌激素组与手术模型组相比较,全身骨密度和腰椎骨密度都得到了一定提高,差异具有统计学意义（P＜0．05）;骨组织中BMP－2的水平也都有显著升高,差异具有统计学意义（P＜0．05）。结论虎潜丸对切除卵巢后所致的骨质疏松症有明显的治疗作用,其机制可能是在一定程度地提高了组织中BMP－2的水平,从而抑制骨组织的吸收,促进骨组织的形成。     

胆红素脑病仔鼠脑组织S-100的mRNA及其蛋白变化研究

目的：观察胆红素脑病仔鼠脑组织S-100蛋白（S-100）的mRNA及其蛋白变化．探讨其对胆红素脑病的诊断价值和其分子生物学机制。方法：采用新生7d龄清洁级Wistar大鼠腹腔注射胆红索200mg／kg制备胆红素脑病模型。应用逆转录-聚合酶链反应（RT—PCR）技术动态观察胆红素脑病仔鼠不同时间点脑组织S-100 mRNA表达变化；应用免疫组织化学方法动态观察不同时间点脑组织S-100蛋白表达变化。结果：实验组脑组织S-100 mRNA表达12h明显升高．48h达峰值．至96h与对照组差异无显著性意义：造模后12h海马区脑组织S-100阳性表达明碌升高，48h达峰值，至96h与对照组无显著性差异；皮质区6h明显升高。48h达峰值，至96h与对照组差异无显著性意义；丘脑区6h明显升高，72h下降，至96h与对照组无显著性差异；苍白球区6h明显升高，48h达峰值，72h下降，至96h与对照组比较仍有显著性差异。结论：S-100mRNA的变化同S-100蛋白的变化趋势一致，是判定胆红素脑病的可靠神经生化指标。     

早期“强制性使用”运动疗法对局灶性脑梗死大鼠行为学评分及神经生长因子的影响

目的：探讨脑卒中后早期强制性使用单侧肢体对局灶性脑梗死大鼠行为学评分及大脑皮质梗死周围（NGF）表达的影响。方法：健康雄性Sprague-Dawley（SD）大鼠,用大鼠大脑中动脉线栓法（MCAO）建立永久性缺血模型,按照Zea-Longa的方法对动物的行为缺陷进行评分,选取1—3分者60只,将60只大鼠随机分为2组,健侧前肢固定组（A组）30只,未固定组（B组）30只,7d时解除固定,再采用随机区组法分为14d时处死组和21d处死组,各组动物在7d时和处死前做神经功能评分（Longa评分）,采用免疫组织化学（SP）方法观察NGF蛋白的表达;采用（POD法）原位杂交方法检测NGF mRNA表达。结果：固定组与未固定组相比,早期“强制性使用”运动疗法可以改善14d及21d的行为学评分,有显著性意义（P〈0.05）;在梗死周围皮质NGF蛋白和NGF mRNA的表达,固定组14d时表达均明显高于非固定组（P〈0.05）,而在21d时无显著性意义。结论：早期“强制性使用”运动疗法可以改善神经功能的恢复,从基因水平来提高NGF的表达可能为其发挥作用的机制之一。