人工神经导管原材料选择与功能设计的研究进展

人工神经导管（nerve guidance conduits,NGCs）作为一种合成的神经移植物,为神经再生提供结构与营养支持。理想的神经导管对生物相容性、机械强度、拓扑结构和导电性等均有较高要求,因此需对神经导管的设计不断改进并建立更完善的周围神经再生策略,以期满足临床需求。虽然NGCs在周围神经损伤的治疗中已经取得一定进展,但其对长距离神经离断伤的结构与功能修复仍不理想。本文分别从原材料选择、结构设计、治疗因子搭载及自供电元件集成4个方面对神经导管的设计进行综述,归纳总结NGCs在周围神经损伤治疗中的研究进展,以期推动NGCs的迭代更新与临床转化。     

神经导管研究与进展

随着机械化程度的提高和交通事业的发展,周围神经损伤发生率大幅度的上升。虽然通过手术的方法可以进行端对端的缝合,但是神经恢复的效果仍然不理想,特别是存在神经短距离缺损时,神经导管可为神经的修复提供一个合适的微环境,使得神经纤维能够再生并顺利到达远端。本文介绍了受损神经恢复效果的评价标准,神经导管材料的研究进展,神经生长因子对神经再生的影响因素及作用机理,和神经导管的制备方法,且在总结前人研究的基础上,给出适合于神经导管支架所需的材料和结构要求。     

PLGA支架降解对血管化功能影响的体外研究

目的:研究聚乳酸-羟基乙酸(PLGA)支架材料降解产物对血管内皮细胞增殖、迁移和小管样结构形成的影响。方法:将PLGA支架材料放入磷酸盐缓冲液(PBS)中体外无菌降解1、2、4周。用降解液处理人脐静脉内皮细胞株HUVEC,采用Brdu ELISA法、Transwell小室法和小管形成实验检测PLGA支架材料降解液对血管内皮细胞增殖、迁移和小管样结构形成的影响。结果:PLGA支架材料1周降解液对内皮细胞的迁移和小管形成无明显影响,对内皮细胞增殖有一定的促进作用。随着降解时间的延长,2周降解液抑制内皮细胞的迁移和小管形成,4周的降解液对内皮细胞增殖、迁移和小管形成均有抑制作用。结论:PLGA支架材料降解初期有对血管内皮细胞的增殖有促进作用,降解后期可能由于降解过程中产生的酸性物质累积增多,影响了血管内皮细胞的生长和功能,从而抑制新生血管的形成。     

血管与导管选择对PICC置管引发并发症的影响

目的:通过比较PICC置管的血管与导管选择,探讨其对并发症发生率的影响。方法:2005年10月至2006年7月共336例恶性肿瘤病人应用B/BRAUN单腔导管,"可分裂"穿刺针355型173例,257型163例分别选择头静脉、贵要静脉、颈外静脉进行观察。结果:头静脉病人>50%出现并发症,其中30%出现中途拔管;贵要静脉<10%出现并发症,90%完成治疗计划;颈外静脉2例因固定不妥导致导管脱出。结论:在非高速度滴注的情况下,尽量选用小管径的导管;对血管的选择应当首选责要静脉,优选右侧,穿刺点最好过肘关节,其次选择颈外静脉优选右侧;选择PICC置管操作应慎重,操作之前做好详细的评估。     

聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞促进大鼠坐骨神经损伤修复的研究

目的:观察聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞修复大鼠坐骨神经缺损的效果。方法:将24只SD大鼠随机分为4组,制备右侧坐骨神经5mm缺损模型,A组聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞移植组;B组聚己内酯神经导管复合骨髓间充质干细胞移植组;C组壳聚糖神经导管复合骨髓间充质干细胞移植组;D组自体神经移植组。术后每2周进行坐骨神经功能指数检测,12周时行电生理、腓肠肌湿重恢复率、组织学观察和免疫组织化学检测。结果:坐骨神经功能指数显示,A组运动功能恢复速度较B、C组快,但比D组慢。A组电生理和腓肠肌湿重恢复率的检测结果与C、D组相比无统计学意义(P0.05),但优于B组(P0.05)。组织学观察,A组再生神经纤维排列密集。S-100免疫组织化学结果表明A组有大量雪旺细胞增生。结论:聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞能够促进周围神经损伤修复,效果与壳聚糖神经导管、自体神经相同,优于聚己内酯神经导管。     

木质部导管空穴化研究中的几个热点问题

 导管的空穴化和栓塞化现象是目前国际上水分生理生态研究的一个热点。该文对导管空穴化和栓塞化研究中出现的几个热点问题进行了概括和总结。1）在研究导管空穴化的实验手段上,超声波传感器探测法具有一定的局限性;目前至少存在4种可能的原因来解释木质部压力探针法（XPP）和压力室法所测得的导管水柱张力不一致的现象;近来出现的核磁共振成像法可以进行导管空穴化的无损伤检测。2）导管解剖学特征与形成空穴的可能性之间的关系可能与树种相关。3）导管空穴化引起气孔关闭的作用机制目前还不太清楚。4）植物防止空穴化产生的能力与适应干旱能力之间的关系还没有定论。5）单独用根压来解释空穴化导管的重新注水机制是不全面的,还存在其它重新注水机制。     

人造血管移植物自然内皮化的研究

人造血管移植作为自体血管代用品已被广泛使用了解内皮细胞的功能,来源,血管内皮化的过程,形成机理及影响内皮化的因素,对防治血栓形成,内膜增生,维持移植血管通畅率有重要意义。     

血管内皮生长因子的神经保护作用

血管内皮生长因子（vascular endothelial growth factor,VEGF）是内皮细胞特异性的生长因子,大多数关于VEGF的研究都是致力于其在血管生长方面的作用,而近年来有大量文献报道VEGF具有神经营养和促神经发生作用,它能够直接作用于神经元细胞和神经胶质细胞甚至是神经干细胞,促进其生长及存活。VEGF的多种功能使其和多种神经退行性疾病相关,如阿茨海默病,肌萎缩侧索硬化症,帕金森病等。导入VEGF基因能够改善肌萎缩侧索硬化症、帕金森病动物模型的病情。     

骨组织工程血管化的研究进展

血管化是组织工程骨成活的关键,组织工程骨的血管化过程与生理情况下的血管发生相似,但又有其独特性,并受多种因素影响。基于对组织工程骨血管化过程的认识,研究者通过联合细胞培养、促血管化生长因子、显微外科等方法重建血运。本文综述了当前骨组织工程的血管化研究进展。     

Laminin-incorporated nerve conduits made by plasma treatment for repairing spinal cord injury

To better direct the repair of damaged axons following spinal cord injury (SCI), we designed a nerve conduit (NC) modeled after the intact spinal cord, which would enable the axons to cross the lesioned area to rejoin on the other side. The NC consisted of a porous chitosan scaffold and was incorporated with laminin (LN) on the inner surface through oxygen plasma treatment. According to the BBB, CBS, and treadmill analyses, we found that following the implantation of the laminin-coated NC (LN-NC) the rats showed a tendency towards behavior improvement and functional recovery. Histology and immunocytochemical analyses indicated that the NC groups were capable of leading the damaged axons through the lesioned area without triggering inflammation or apoptosis. Together with the significantly enhanced expression of local GAP-43 in the LN-NC groups, as evidenced by western blot analysis, axon re-growth mediated by LN-NC was found to compare better than that by NC group. These results suggest a new possible approach to repairing SCI and, in general, a model which will be useful for other multidisciplinary procedures for complex neurological situations.     

Investigating the mechanical shear-plane between core and sheath elements of peripheral nerves

The mechanical architecture of rat sciatic nerve has been described as a central core surrounded by a sheath, although the way in which these structures contribute to the overall mechanical properties of the nerve is unknown. We have studied the retraction responses of the core and sheath following transection, together with their tensile properties and the interface between them. Nerves were harvested and maintained at their in situ tension and then either transected entirely, through the sheath only, or through an exposed section of the core. The retraction of each component was measured within 5 min and again after 45 min. Post mortem loss of retraction was tested 0 min or 60 min after excision. For fresh nerves, immediate retraction was 12.68% (whole nerve), 5.35% (sheath) and 4% (core), with a total retraction of 15%, 7.21% and 5.26% respectively. For stored nerves, immediate retraction was 5.33% (whole nerve) and 5.87% (sheath), with an extension of 0.78% for core, and a total retraction of 6.71% and 7.87% and an extension of 1.74%, respectively. Tensile extension and pullout force profiles were obtained for the sheath, the core and the interface between them. These showed a consistent hierarchy of break strengths that would, under increasing load, result in failure of the interface, then the core and finally the sheath. These data reflect the contributions of material tension and fluid swelling pressure to total retraction, and the involvement of an energy-dependent process that runs down rapidly post mortem. This study increases our understanding of the composite nature of peripheral nerve tissue architecture and quantifies the material properties of the distinct elements that contribute to overall mechanical function.Preliminary forms of this study were presented at: (1) British Matrix Biology Society Meeting, Manchester, March 2001; (2) Tissue and Cell Engineering Society Meeting, Keele, United Kingdom, September 2001.Financial support was provided by the EU framework 5 programme in neural tissue engineering (QLK3-CT-1999-00625).     

蛇毒神经生长因子促进周围神经再生的诱发电位观察

目的：研究蛇毒神经生长因子（sNGF)对大鼠坐骨神经损伤后诱发电位的影响,评价蛇毒神经生长因子在促进周围神经再生中的作用。方法：建立大鼠坐骨神经钳夹模型,局部滴加药物和术后肌注sNGF,通过脊髓诱发电位（SEP）,运动诱发电位（MEP）评定,观察坐骨神经修复情况。结果：sNGF治疗后可使伤后SEP,MEP提早出现,结论：蛇毒提取的NGF对大鼠坐骨神经损伤修复具有促进作用。     

Effects of Axotomy on Distribution and Concentration of Elements in Rat Sciatic Nerve

X-ray microprobe analysis was used to determine the effects of axotomy on distribution and concentration (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in frozen, unfixed sections of rat sciatic nerve. Elemental concentrations were measured in axoplasm, mitochondria, and myelin at 8, 16, and 48 h after transection in small-, medium-, and large-diameter fibers. In addition, elemental composition was determined in extraaxonal space (EAS) and Schwann cell cytoplasm. During the initial 16 h following transection, axoplasm of small fibers exhibited a decrease in dry weight concentrations of K and Cl, whereas Na and P increased compared to control values. Similar changes were observed in mitochondria of small axons, except for an early, large increase in Ca content. In contrast, intraaxonal compartments of larger fibers showed increased dry weight levels of K and P, with no changes in Na or Ca concentrations. Both Schwann cell cytoplasm and EAS at 8 and 16 h after injury had significant increases in Na, K, and Cl dry weight concentrations, whereas no changes, other than an increase in Ca, were observed in myelin. Regardless of fiber size, 48 h after transection, axoplasm and mitochondria displayed marked increases in Na, Cl, and Ca concentrations associated with decreased K. Also at 48 h, both Schwann cell cytoplasm and EAS had increased dry weight concentrations of Na, Cl, and K. The results of this study indicate that, in response to nerve transection, elemental content and distribution are altered according to a specific temporal pattern. This sequence of change, which occurs first in small axons, precedes the onset of Wallerian degeneration in transected nerves.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

神经生长因子药效学研究

目的考察神经生长因子对视神经损害的疗效。方法腹腔注射二硫化碳致视神经损害的大鼠模型,给药组球后注射或肌内注射神经生长因子,每天一次,每周6d,共计3周,空白对照组球后注射等量生理盐水,于治疗前后测定大鼠模式翻转（FREP）和闪光视觉诱发电位。结果球后注射高剂量组大鼠在治疗10d和20d时各波潜伏时比对照组有显著缩短,而球后注射低剂量组和肌内注射高剂量组大鼠于治疗10d时FVEP各波潜伏时也显著缩短,肌内注射低剂量组大鼠于治疗20d时FEP的P1和P2波潜伏时也显著或非常显著缩短。结论神经生长因子对大鼠视神经损害有明显的治疗作用。     

Effects of long term nitrite therapy on functional recovery in experimental ischemia model

Our data have shown that nitrite therapy can rescue the ischemic brain when injected <3 h after cerebral ischemic-reperfusion (I/R) injury and its effects can be prolonged to 4.5 h in combination with memantine. We investigated whether or not long-term nitrite therapy is beneficial in ischemic brains. Sodium nitrite (1-100 μg/kg ip) or saline were administered to rats subjected to focal I/R injury for 7 days beginning 24 h after I/R. Behavioral tests for 5 weeks revealed better functional recovery in the high-dose nitrite group than the control group. Other nitrite groups with relatively low doses showed no functional benefits. Hemispheric atrophy was attenuated by approximately 30% in the high-dose nitrite group. High-dose nitrite therapy also reduced inflammatory cytokine levels and caspase activity in the subacute period, and increased BrdU⁺MAP2⁺ and BrdU⁺laminin⁺ cells, and vascular density in the 5-week ischemic brain. Long-term nitrite therapy, when initiated 24 h after I/R, corrected the subacute hostile environment, induced tissue and vascular regeneration, and improved functional recovery. Early and subsequent long term nitrite therapy may be effective in the management for ischemic stroke patients.     

Acidic FGF enhances functional regeneration of adult dorsal roots

It has been well documented that the regeneration of sensory axons severed in the dorsal roots into the spinal cord is largely inhibited in adult mammals. We investigated whether peripheral nerve grafts combined with acidic fibroblast growth factor (aFGF) could induce the regeneration of transected dorsal roots in adult rats, as evaluated by cortical somatosensory evoked potentials (SEPs). Median nerve (forelimb) stimuli produced consistent responses in the primary somatosensory cortex of normal rats, but these were completely eliminated after the transection of cervical 6th - 8th roots. The dorsal root stumps were immediately anastomosed to the cord with intercostal nerve grafts. Subsequently, aFGF in fibrin glue was administered to the grafted area. Four to twenty weeks after rhizotomy, six of the seven rats receiving such reconstruction had recovery of SEPs. The reappearing SEPs typically showed similar waveforms and latencies as normal ones. They were eliminated by retransection of the repaired roots, thus verifying their source as the regenerated roots. We present here substantial evidence that aFGF enhances the functional restoration of cut dorsal roots. Cortical SEPs is considered a useful tool in evaluating such regeneration. These results may offer therapeutic potential in the treatment of dorsal root injuries.     

Proteomic identification of a novel isoform of collapsin response mediator protein-2 in spinal nerves peripheral to dorsal root ganglia

Primary afferent fibers are originated from pseudounipolar sensory cells in dorsal root ganglia (DRG) and transmit external stimuli received in the skin to the spinal cord. Here we undertook a proteomic approach to uncover the polarity of primary afferent fibers. Lumbar spinal nerve segments, peripheral and central to DRG, were dissected from 5-wk-old Wistar rats and the lysates were subjected to large-sized 2-DE at pH 5-6. Among approximately 800 protein spots detected in the central and peripheral fractions, one of the unique spots in the peripheral fraction with MW of 60 kDa and pI of 5.6 was identified as an isoform of collapsin response mediator protein-2 (CRMP-2) by MALDI-TOF MS and Western blots with anti-CRMP-2 antibodies that recognize 1-17 and 486-528 residues. Since this novel spot was detected only in the peripheral fraction, but not in the central fraction, DRG, and other regions of the brain, it was named periCRMP-2. The C-terminal fragment of CRMP-2 was not detected in periCRMP-2 by MS analyses. Expression of periCRMP-2 decreased following sciatic nerve injury. These results suggest that periCRMP-2 is a C-terminal truncated isoform polarized in the peripheral side of spinal nerves and may be involved in nerve degeneration and regeneration.