脑源性神经营养因子纳米微球对马尾束缢犬的神经保护

doi:10.3969/j.issn.1673-5374.2013.03.005

中国神经再生研究(英文版) ›› 2013, Vol. 8 ›› Issue (3): 233-243.doi: 10.3969/j.issn.1673-5374.2013.03.005

• 原著:周围神经损伤修复保护与再生 • 上一篇下一篇

脑源性神经营养因子纳米微球对马尾束缢犬的神经保护

收稿日期:2012-07-28 修回日期:2012-10-10 出版日期:2013-01-25 发布日期:2013-01-25

Changes in compressed neurons from dogs with acute and severe cauda equina constrictions following intrathecal injection of brain-derived neurotrophic factor-conjugated polymer nanoparticles

Junming Tan¹, Jiangang Shi², Guodong Shi², Yanling Liu³, Xiaohong Liu³, Chaoyang Wang¹, Dechun Chen¹, Shunming Xing¹, Lianbing Shen¹, Lianshun Jia², Xiaojian Ye², Hailong He², Jiashun Li²

1 Center of Trauma Repair and Reconstruction of Chinese PLA and Department of Orthopedics of the 98th Hospital of Chinese PLA, Huzhou 313000, Zhejiang Province, China
2 Department of Orthopedics, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
3 Department of Pathologic Laboratory of Chest Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

Received:2012-07-28 Revised:2012-10-10 Online:2013-01-25 Published:2013-01-25
Contact: Jiangang Shi, M.D., Associate professor, Chief physician, Department of Orthopedics, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China, tanjunm@sina.com.
About author:Junming Tan☆, M.D., Associate chief physician, Associate professor, Master’s supervisor.
Supported by:
This study was financially supported by grants from the Medical Scientific Fund and Intensive Research of Nanjing Military Area Command of Chinese PLA, No. Nan 2007-13 and Nan 08Z003; and the Medical Scientific Fund and Research of Chinese PLA during the 12th Five-Year Plan Period, No. CWS11J260.

摘要/Abstract

摘要：

实验以下腰骶部背根神经节的中央突受压缢48h制备犬急性多重马尾压缢模型，观察鞘内注射15mg可吸收缓释聚(乳酸-羟基乙酸)-脑源性神经营养因子纳米微球对犬背根神经节内神经元损伤的修复作用。分别于造模后1，2，4周取L7节段背根神经节行苏木精-伊红及免疫组化染色，发现鞘内给予脑源性神经营养因子可减轻受压迫的背根神经节感觉神经元变性和炎性反应，提高受压迫的背根神经节感觉神经元内脑源性神经营养因子的表达。同时鞘内给予脑源性神经营养因子可显著改善急性多重马尾压缢模型犬的神经功能。结果证实鞘内注射持续缓慢释放的脑源性神经营养因子纳米粒，可促进急性重度马尾综合征犬神经元组织形态及功能的修复。

关键词: 神经再生, 纳米微球, 缓释, 马尾综合征, 多重马尾压缢, 脑源性神经营养因子, 背根神经节, 神经保护, 基金资助文章, 图片文章

Abstract:

This study established a dog model of acute multiple cauda equina constriction by experimental constriction injury (48 hours) of the lumbosacral central processes in dorsal root ganglia neurons. The repair effect of intrathecal injection of brain-derived neurotrophic factor with 15 mg encapsulated biodegradable poly(lactide-co-glycolide) nanoparticles on this injury was then analyzed. Dorsal root ganglion cells (L₇) of all experimental dogs were analyzed using hematoxylin-eosin staining and immunohistochemistry at 1, 2 and 4 weeks following model induction. Intrathecal injection of brain-derived neurotrophic factor can relieve degeneration and inflammation, and elevate the expression of brain-derived neurotrophic factor in sensory neurons of compressed dorsal root ganglion. Simultaneously, intrathecal injection of brain-derived neurotrophic factor obviously improved neurological function in the dog model of acute multiple cauda equina constriction. Results verified that sustained intraspinal delivery of brain-derived neurotrophic factor encapsulated in biodegradable nanoparticles promoted the repair of histomorphology and function of neurons within the dorsal root ganglia in dogs with acute and severe cauda equina syndrome.

Key words: neural regeneration, peripheral nerve injury, cauda equina syndrome, dorsal root ganglion, brain-derived neurotrophic factor, multiple cauda equina constrictions, neurotrophic factors, neural protection, grants-supported paper, photographs-containing paper, neuroregeneration

. 脑源性神经营养因子纳米微球对马尾束缢犬的神经保护[J]. 中国神经再生研究(英文版), 2013, 8(3): 233-243.

Junming Tan, Jiangang Shi, Guodong Shi, Yanling Liu, Xiaohong Liu, Chaoyang Wang, Dechun Chen, Shunming Xing, Lianbing Shen, Lianshun Jia, Xiaojian Ye, Hailong He, Jiashun Li. Changes in compressed neurons from dogs with acute and severe cauda equina constrictions following intrathecal injection of brain-derived neurotrophic factor-conjugated polymer nanoparticles[J]. Neural Regeneration Research, 2013, 8(3): 233-243.

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引言

材料方法

Design

A randomized, controlled animal experiment.

Time and setting

The experiment was performed at the Animal Care and Use Department of the Navy Institute, China, from September 2005 to June 2006.

Materials

A total of 18 adult mongrel male dogs, aged 18–48 months and weighing 10–15 kg, were purchased from the Institutional Animal Care and Use Committee of the Institute of Navy (license No. SYXK (Hu) 2007-0003). Animals were housed in individual runs, given free access to water and fed a dry certified canine diet. Animal room temperature and light cycle were controlled (targeted conditions: temperature range 18.3–25.5°C, 12-hour light/dark cycle). Humidity was not controlled but recorded regularly. Dogs were allowed to acclimate for a minimum of 7 days after receipt and conditioned to vests for 3 days prior to surgery. All protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[49].

Methods

Model establishment and intervention

Each animal received intramuscular injection (i.m.) of penicillin G procaine (20 000 U/kg) and atropine (0.04 mg/kg) before surgery. Dogs were anesthetized with a parenteral solution of Su-mian-xin II (0.08– 0.10 mL/kg, i.m., made by the Military Veterinarian Institute of the Academy of Military Medical Sciences, Changchun, Jilin Province, China), endotracheally intubated and artificially ventilated on a respirator with halothane (1–2%) in a mixture of oxygen and nitrous oxide (1:1). Lumbar laminectomy was performed and the dural sac of the exposed cauda equina comprising dorsal and ventral roots of L₇, S_1-3, Co_1-5 segments was tied by four loose 4-0 ligatures (Shanghai Jade Weaver Co., Ltd., Shanghai, China) with about 2 mm spacing. The entire cauda equina was constricted by 50–75% by the first tightened constriction, and the lower cauda equina was constricted by 25–50% by the other three tightened constrictions (Figure 3).

The central processes of the L₇–Co₅ DRG neurons were permanently constricted. The sham surgery group underwent cauda equina exposure, without ligation. Forty-eight hours after surgery, dogs from both control and experimental groups were again deeply anesthetized with the Su-mian-xinII (0.08–0.10 mL/kg, i.m.). The four constrictions on the cauda equina were removed through the original operative incision, and dogs in the experimental group were infused through intrathecal injection with 15 mg of encapsulated biodegradable poly(lactic-co-glycolic acid) nanoparticles carrying 2.5 mg of active BDNF (Pharmacy College of the Second Military Medical University, Shanghai, China).

DRG (L₇) preparation

Two animals, one from the control and experimental groups separately, were deeply anesthetized with the Su-mian-xin II (0.08–0.10 mL/kg, i.m.) after 1, 2 and 4 weeks following the second operation. Animals were transcardially perfused through the heart with 2 L PBS followed by 2 L of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The corresponding DRG (L₇) was removed and sliced into 5-µm transverse paraffin sections for histological and immunohistochemical study.

Neuronal changes and damage in compressed DRG, observed by hematoxylin-eosin staining

Samples were taken from DRG at L₇ and embedded in paraffin, before preparing 5-µm transverse sections. The sections were stained with hematoxylin-eosin and examined for the density of neurons in the DRG, and then observed using a light microscope (Olympus, Tokyo, Japan). To count the neurons, we chose a slice thickness of 5 µm and gap interval of more than 8 µm. Cell nuclei stained blue-black and cytoplasm stained salmon pink^[50].

Semiquantitative scoring of pathological sections was performed independently by two pathological investigators who were blinded to the animal groups. Thus, tissue sections from each block were described qualitatively with local reactions, local inflammatory cells, and pathological change, such as chromatolysis, neuronal loss, edema or necrosis being noted. For comparison purposes, material from each animal was rated on a semiquantitative scale of 0 to IV, where 0 represented a lack of pathological findings and IV represented a severe inflammatory reaction that involved corresponding tissues and structures. These values were reported and submitted for a rank order comparison^[51].

Immunohistochemical staining for BDNF expression in DRG neurons

Samples were immersed in PBS containing 30% sucrose for 24 hours at 4°C for cryoprotection. Free floating sections were immediately sliced into 5-µm paraffin sections, deparaffinized and dehydrated. Sections were subsequently washed in distilled water and then PBS, followed by microwave retrieval with citrate buffer solution (pH 6.0) for 3 minutes and 30 seconds. Sections were then rinsed in distilled water and PBS again after natural cooling. Specimens were exposed to 0.3% H₂O₂ in PBS for 10 minutes to inactivate endogenous peroxidase, and then rinsed in PBS. Sections were subsequently incubated with a rabbit monoclonal anti-BDNF antibody of low-density lipoprotein (1:50; Boster Biotech Corp, Wuhan, Hubei Province, China) for 1 hour at 37°C. BDNF was immunostained using the ENVISION system (DAKO, Carpinteria, CA, USA) according to the manufacturer’s instructions. Staining therefore continued by rinsing in the PBS, incubation with secondary sheep anti-rabbit IgG and labeled with horseradish peroxidase (1:250; Sigma, St. Louis, MO, USA). After washing in PBS, the sections were developed with 0.05% 3,3’ diaminobenzidine tetrahydrochloride (Sigma) and 0.006% H₂O₂ in PBS for 10 minutes. The slides were counterstained with Meyer’s hematoxylin for 10 minutes, differentiated with 75% hydrochloric acid-alcohol for 30 seconds, and then the sections were washed, dried, dehydrated and mounted on neutral resin. Positive signal was located in the cytoplasm of neurons and gliocytes and stained with buffy^[50] under light microscopy (Toshiba, Tokyo, Japan).

Neurofunctional evaluation

Neurological evaluation of motor function in the posterior limbs of each animal was performed independently by two investigators blinded to the animal groups. Each animal was graded three times for 40 minutes each time according to the Tarlov’s scoring system^[51]. The average value was obtained before deep anesthesia and transcardial perfusion.

The ratio of Tarlov’s motor scales was applied to assess neurological function. The calculation used is shown below^[52]:

Statistical analysis

Nonparametric Wilcoxon’s signed-ranks test was used to compare the semiquantitative scoring of the three groups using SPSS 10.0 software (SPSS, Chicago, IL, USA), and two-sample t-test was used for the analysis of Tarlov’s motor scale between control and experimental groups. Data are expressed as mean ± SD. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1实验设计构思介绍：
(1) 这个急性重度马尾束缢诱导马尾综合征的实验模型，类似于临床下腰椎暴裂性骨折伴马尾神经损伤及巨大椎间盘突出症导致严重马尾神经压迫的患者。同时这个实验性多重马尾压缢模型，还包括连同束缢腹侧根躯体和内脏运动的轴突一起，持续对中枢传入的多种束缢，可提供一个研究相应脊髓和背根神经节节段对应力、疼痛或炎症反应的可靠模型。
(2) 实验证实多节段压迫比单节段压迫更易导致马尾神经损害，多重马尾压缢造模后可及明显的马尾神经血液循环障碍、脑脊液循环障碍及其水肿、华勒氏变性、脱髓鞘等病理变化，从而提示至少机械性和血管性因素均参与了马尾综合征的发生发展过程。
(3) 研究证实背根神经节内初级感觉神经元中央支在束缢下，因轴突流、轴突反应障碍造成背根神经节内神经元的变化，包括神经元变性坏死、胞核进行性中央性Nissl小体溶解增加及显著的神经胶质、神经纤维变性。同时，这个累及下腰骶脊髓神经元的急性重度狗马尾综合征，可及下腰骶脊髓神经元的明显变性坏死、一定程度的脊髓溶解坏死及脊髓不同大小神经元内脑源性神经营养因子反应性的变化。这个脊髓内神经元形态学的变化，可能代表了对急性压迫的一种代偿性反应，且下腰骶脊髓内脑源性神经营养因子表达的增加有助于神经元的存活。
(4) 研究示多重马尾压缢后48h的背根神经节神经元胞浆内可及不同程度的脑源性神经营养因子阳性表达，且围绕背根神经节的卫星细胞亦呈脑源性神经营养因子阳性表达，而正常对照组呈阴性。由此认为实验性狗相应的背根神经节和脊髓内脑源性神经营养因子的增量调节，可能在阻止神经元变性上起到保护作用外，还可能有助于持续性炎症和神经痛的发生发展。
(5) 相比较对照组，实验组相应脊髓和背根神经节内神经元有明显的脑源性神经营养因子阳性表达，提示急性重度马尾综合征早期存在显著的BNDF增量调节，进一步提示脑源性神经营养因子是脊髓和背根神经节早期重度马尾综合征的一个生存因子，因为脑源性神经营养因子的增量调节仍可能是马尾综合征病理过程中阻止神经元变性的一个有效因素。
(6) 一定剂量的外缘性脑源性神经营养因子进入椎管可能起到了阻止感觉神经元的细胞凋亡，因为脑源性神经营养因子可通过调节转录因子等上游元件调节神经元内基因的表达，如c-fos、c-jun等而抑制细胞凋亡、坏死的发生，这种效应可能是通过诱导T受体的表达增高来实现的。
(7) 下腰骶髓内脑源性神经营养因子表达的变化，对马尾综合征的病理生理学机制有着重要的作用，因为急性重度马尾束缢很可能阻断了经正常背侧根顺向转运到背侧角的脑源性神经营养因子/TrkB受体介导的信号通路，以适应神经性疼痛行为的发生和发展。
(8) 实验表明，神经根内血流受损和神经纤维形态改变与神经根压迫相关的神经根性症状有关。看来由背根神经节内初级感觉神经元产生的各种神经递质及通过背侧根神经元中央支的轴突流转运到脊髓背侧角，与神经根压迫相关的根性痛和感觉障碍的发生发展有关。
(9) 这个急性重度多重马尾压缢模型，能导致下腰骶脊髓及相应背根神经节内神经元的不可逆性损伤。临床脊柱外科医师经常遇到因腰椎间盘突出症或腰椎管狭窄症等致马尾神经和神经根压迫的患者，经手术减压后仍存在较长时间的感觉障碍和麻木，尤其术前有下肢感觉障碍较长病史的患者。这个实验提示有神经根和/或马尾神经压迫的患者，其功能障碍并不局限于压迫部位的变性，而且病变可能累及背根神经节和脊髓背侧角的感觉神经元。故对有慢性感觉障碍和麻木的患者，术前医师必须告知其术后并不能立即缓解。

2 国内外同类研究水平的介绍：
目前国内外关于马尾神经综合征的研究本研究居于领先水平。
(1) 急性多重马尾压缢模型国外建立，但如此急性重度严重的压迫，系首创；且研究方向在脑源性神经营养因子，亦首创。
(2) 此急性重度马尾束缢诱导马尾综合征的实验模型，类似于临床急性下腰椎暴裂性骨折伴马尾神经损伤及急性巨大椎间盘突出症导致严重马尾神经压迫的患者，有利于临床研究，有利于揭示相关的临床意义。
(3) 采用可吸收缓释脑源性神经营养因子－聚(乳酸-羟基乙酸)纳米微球，研究脑源性神经营养因子对马尾神经综合征神经组织广泛损伤的防治作用，观察动物的神经功能恢复情况及脑源性神经营养因子置于椎管内对周围神经生长或再生的影响，系国内外首创。
(4) 国外近年研究和国内史建刚副教授的前期研究表明，马尾神经损伤后，其局部病理改变如不能得到及时改善，则形成恶性循环，可产生顺行性、逆行性变性及传递性神经元变性，由马尾神经的局部损害到广泛损害，以至于产生跨细胞的中枢神经元变性，而致使其结构和功能难以修复。其中腰骶段相应脊髓神经元的损害是广泛损害的中心环节，而神经营养因子的缺乏则是导致下腰骶髓神经元和背根神经节损害、限制轴突再生的主要因素之一。我们研究的目的就是在急性重度马尾神经损伤模型中通过蛛网膜下腔持续给予外源性脑源性神经营养因子治疗并观察其相应病理生理变化及其疗效。

3与其他同类研究的比较：
目前关于神经再生的一个很重要挑战是脊髓损伤后逐渐建立的脊髓环境，非常不利于轴突生长，如何突破轴突再生的分子障碍及其抑制机制，已经取得了一定的进展。例如，移植细胞基质（如干细胞）进入损伤的脊髓，以提供轴突生长更有利的环境。挑战轴突再生的研究，不仅限于轴突生长的外部障碍，而且由于中枢神经系统神经元本身较低的内在再生反应能力，需要克服SCI后的神经元修复障碍。目前实验性轴突再生治疗的动物模型，对功能康复起主要作用的可能就是这种可塑性，实验研究的目的是尽可能观察到受伤部位已断裂的轴突能产生长距离的轴突再生。这也是本文的一个研究出发点，尽管研究重点在背根神经节及其神经元，但脑源性神经营养因子仍是研究神经再生的一个方向和出发点，仍有广阔的前景。目前改善中枢神经系统内轴突再生，主要集中在扩大神经元的内在再生能力或减弱中枢神经系统对轴突再生的抑制环境。国内外关于脑源性神经营养因子鞘内注射的相关研究：这些发现提示周围炎症和轴突切断术后，背根神经节内不同类型神经元的初级传入均可激活ERK，通过靶源性NGF的修饰并经脑源性神经营养因子表达的转录调节，从而有助于维持炎症痛和神经痛。而本文的结论是鞘内注射持续缓慢释放的脑源性神经营养因子纳米粒，在治疗急性重度马尾综合征的狗模型中疗效明显。

4 文章先进性：
文章构建的可吸收缓释脑源性神经营养因子－聚(乳酸-羟基乙酸)纳米微球，研究脑源性神经营养因子对马尾神经综合征神经组织广泛损伤的防治研究，观察动物的神经功能恢复情况及脑源性神经营养因子置于椎管内外对周围神经生长或再生的影响，在所检索的文献数据库中未有类似的研究报道（以关键词Cauda equina syndrome (CES);and/or Dorsal root ganglion (DRG);and/or Brain-derived neurotrophic factor (BDNF); and/or multiple cauda equina constrictions (MCEC)检索近5年（2007年－2012年）Pub-Med数据库，亦未及类似报道）。

5专家意见与答疑：
专家意见：引言对后根神经节及前后根的解剖介绍过多，关于作者为什么研究脑源性神经营养因子对后根神经节损伤的修复作用的原因说明的较少，没突出本研究的立体依据。
作者答疑：按专家意见，已删除部分对“后根神经节及前后根的解剖介绍”.
增加脑源性神经营养因子对后根神经节损伤的修复作用的原因：
“ In models of inflammatory and neuropathic pain, BDNF synthesis is greatly increased in different populations of dorsal root ganglion (DRG) neurons.This has been well demonstrated in the rat rubrospinal system (a motor control system) after acute and chronic spinal cord injury. Following a cervical spinal cord injury in which the rubrospinal tract is cut, the immediate administration of brain derived neurotrophic factor (BDNF) into the spinal cord injury site promotes significant rubrospinal axonal regeneration and prevents axotomy-induced atrophy and/or death of rubrospinal neurons. BDNF applied directly to the rubrospinal cell bodies within the brainstem either acutely or even 1 year after injury elicits a reversal of axotomy-induced neuronal atrophy, an upregulation of genes important for regeneration, and the promotion of rubrospinal axonal growth. This indicates that the acutely injured rubrospinal axons at the spinal cord injury site are responsive to BDNF and thereby conduct the appropriate intracellular signaling pathways to augment the intrinsic growth propensity of these CNS neurons. It illustrates the principle that the development of effective therapeutic strategies that use the administration of neurotrophic factors(such as Brain-derived neurotrophic factor) will require an understanding of the biologic responsiveness of the target tissue, responsiveness that may in fact change with time and thus differ between the acute and chronic injury settings.

脑源性神经营养因子纳米微球对马尾束缢犬的神经保护

Changes in compressed neurons from dogs with acute and severe cauda equina constrictions following intrathecal injection of brain-derived neurotrophic factor-conjugated polymer nanoparticles

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