脊柱微创手术对骨质疏松性椎体骨折的疗效及安全性分析

目的：探究脊柱微创手术对骨质疏松性椎体骨折的疗效及安全性。方法：选取我院骨科于2008年3月-2011年3月收治的72例骨质疏松性椎体压缩骨折进行分组治疗,观察组（n=36）接受脊柱微创手术,对照组（n=36）接受传统保守治疗,对比两组患者术后生活情况、疗效及观察组术后并发症。结果：两组患者治疗前压缩椎体高度/病椎上下椎体高度和之半、椎体后凸Cobb角及骨块侵占椎管比率无明显统计学差异（P〉0.05）,治疗后上述指标均显著改善,观察组改善程度较对照组更为明显（P〈0.05）;观察组治疗优良率91.7%,对照组为58.3%,其中6例患者因症状加重转为手术治疗,观察组治疗效果明显优于对照组（P〈0.05）;观察组患者术后未出现内植物松动、断裂、形成假关节、截瘫等并发症,钉棒内固定系统坚强可靠;两组患者均获得有效随访,平均随访时间（9.7±2.5）个月,观察组平均下地时间（4.1±1.7）d,肌力恢复时间（3.7±0.8）周,Frankel神经功能均恢复E级;对照组平均下地时间（7.4±3.0）d,肌力恢复时间（5.9±1.4）周,Frankel神经功能评分：D级6例,E级30例。结论：脊柱微创手术能够有效改善骨质疏松性椎体骨折患者骨骼力学变化,可有效保证其术后恢复情况,疗效较好,且不会引发严重并发症,值得临床广泛推广。     

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.     

Septin 6 localizes to microtubules in neuronal dendrites

In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs.     

A Proprioception Based Regulation Model to Estimate the Trunk Muscle Forces

Evaluation of loads acting on the spine requires the knowledge of the muscular forces acting on it, but muscles redundancy necessitates developing a muscle forces attribution strategy. Optimisation, EMG, or hybrid models allow evaluating muscle force patterns, yielding a unique muscular arrangement or/and requiring EMG data collection. This paper presents a regulation model of the trunk muscles based on a proprioception hypothesis, which searches to avoid the spinal joint overloading. The model is also compared to other existing models for evaluation. Compared to an optimisation model, the proposed alternative muscle pattern yielded a significant spine postero-anterior shear decrease. Compared to a model based on combination of optimisation criteria, present model better fits muscle activation observed using EMG (38% improvement). Such results suggest that the proposed model, based on regulation of all spinal components, may be more relevant from a physiologic point of view.     

Rac GTPase-activating protein (Rac GAP) α1-Chimaerin undergoes proteasomal degradation and is stabilized by diacylglycerol signaling in neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.     

Modulating micro-opioid receptor phosphorylation switches agonist-dependent signaling as reflected in PKCepsilon activation and dendritic spine stability

A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCε pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCε pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCε pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCε, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCε. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.     

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light''s intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy''s theoretical resolution limit of 200 nm.Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.     

生物活性玻璃提高骨质疏松绵羊椎弓根螺钉稳定性的体内实验研究

目的：研究生物活性玻璃（Bioactive Glass,BG）在骨质疏松绵羊体内强化椎弓根螺钉固定的力学效果,并观察钉道界面及材料吸收等情况。方法：4只成年雌性小尾寒羊,采用去势联合激素注射方法建立骨质疏松绵羊模型。选择绵羊L2至L5双侧椎弓根,随机化选取一侧直接拧入椎弓根螺钉（空白组）,对侧采用BG强化钉道后拧入椎弓根螺钉（实验组）。术后3月随机选取6个椎体（12枚椎弓根螺钉）,对螺钉骨质界面行显微CT分析和组织学观察。对剩余10个椎体（20枚椎弓根螺钉）行轴向拔出实验,分析螺钉固定强度。结果：术前绵羊腰椎BMD为0.818±0.0310 g/cm2,建模完成后为1.000±0.0316 g/cm2,BMD平均值下降22.38%,差异有统计学意义（P〈0.05）。实验组螺钉骨质界面的Tb.Th、Tb.N组较对照组分别增加143.60%和33.56%,差异有统计学意义（P〈0.05）。实验组钉道周围材料绝大部分已经降解吸收,大量新生骨组织紧密包裹螺钉;对照组钉道周围骨量较少,钉骨结合不紧密,实验组螺钉骨质界面结合优于对照组。实验组的最大轴向拔出力为1083.04±86.37N,空白组为871.76±79.03N,前者较后者提高25.26%,差异有统计学意义（P〈0.05）。结论：生物活性玻璃能显著改善骨质疏松情况下螺钉骨质界面的骨微观结构,进而提高椎弓根螺钉的把持力。     

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.     

N-methyl-D-aspartate (NMDA) receptor composition modulates dendritic spine morphology in striatal medium spiny neurons

Dendritic spines of medium spiny neurons represent an essential site of information processing between NMDA and dopamine receptors in striatum. Even if activation of NMDA receptors in the striatum has important implications for synaptic plasticity and disease states, the contribution of specific NMDA receptor subunits still remains to be elucidated. Here, we show that treatment of corticostriatal slices with NR2A antagonist NVP-AAM077 or with NR2A blocking peptide induces a significant increase of spine head width. Sustained treatment with D1 receptor agonist (SKF38393) leads to a significant decrease of NR2A-containing NMDA receptors and to a concomitant increase of spine head width. Interestingly, co-treatment of corticostriatal slices with NR2A antagonist (NVP-AAM077) and D1 receptor agonist augmented the increase of dendritic spine head width as obtained with SKF38393. Conversely, NR2B antagonist (ifenprodil) blocked any morphological effect induced by D1 activation. These results indicate that alteration of NMDA receptor composition at the corticostriatal synapse contributes not only to the clinical features of disease states such as experimental parkinsonism but leads also to a functional and morphological outcome in dendritic spines of medium spiny neurons.