黄岑苷干预慢性应激大鼠海马新生神经元树突的形态

doi:10.3969/j.issn.1673-5374.2013.06.002

摘要/Abstract

摘要：

连续14 d皮下注射40 mg/kg糖皮质激素建立成年SD大鼠慢性应激模型，同时灌胃黄岑苷50 mg/kg，观察其对慢性应激大鼠神经发生的影响。结果显示皮下注射糖皮质激素可显著降低海马中doublecortin阳性神经元数目，且糖皮质激素导致的海马神经元减少以II型doublecortin阳性神经元为主，而I型doublecortin阳性神经元不受影响。黄岑苷可显著提高糖皮质激素诱导的慢性应激模型大鼠海马中I型和II型doublecortin阳性神经元数目。此外，黄岑苷可逆转糖皮质激素注射引起的doublecortin阳性神经元树突形态萎缩。提示黄岑苷可促进成体动物海马的神经发生。

关键词: 神经再生, 中医药, 神经退行性疾病, 神经发生, 黄岑苷, 应激, 海马, 神经元, doublecortin, 树突, 认知, 情绪调节, 基金资助文章, 图片文章

Abstract:

Chronic stress models, established in adult Sprague-Dawley rats through a 14-day subcutaneous injection of 40 mg/kg corticosterone, once per day, were given a daily oral feeding of 50 mg/kg baicalin. The study was an attempt to observe the effect of baicalin on neurogenesis in chronically stressed rats. Results showed that subcutaneous injection of corticosterone significantly decreased the total number of doublecortin-positive neurons in the hippocampus. The reduced cell number caused by corticosterone was mainly due to the decrease of class II doublecortin-positive neurons, but the class I doublecortin-positive neurons were unaffected. Baicalin treatment increased the number of both class I and class II doublecortin-positive neurons. In addition, doublecortin-positive neurons showed less complexity in dendritic morphology after corticosterone injection, and this change was totally reversed by baicalin treatment. These findings suggest that baicalin exhibits a beneficial effect on adult neurogenesis.

Key words: neural regeneration, traditional Chinese medicine, neurogenesis, neurodegenerative disease, baicalin, stress, hippocampus, neurons, doublecortin, dendrites, cognition, mood regulation, grants-supported paper, photographs-containing paper, neuroregeneration

. 黄岑苷干预慢性应激大鼠海马新生神经元树突的形态[J]. 中国神经再生研究(英文版), 2013, 8(6): 496-505.

Xinghua Jiang, Junmei Xu, Dingquan Zou, Lin Yang, Yaping Wang. Baicalin influences the dendritic morphology of newborn neurons in the hippocampus of chronically stressed rats[J]. Neural Regeneration Research, 2013, 8(6): 496-505.

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引言

材料方法

Design

A randomized, controlled, animal experiment.

Time and setting

All the experiments were conducted from September 2011 to May 2012 in the Anesthesia Research Institute, Central South University, China.

Materials

Animals

Twelve 2-month-old male Sprague-Dawley rats, weighing 220–250 g, were obtained from the Department of Laboratory Animal at Central South University, China, with license number SCXK (Xiang) 2009-0004. The animals were maintained at a controlled temperature and 12-hour light/dark cycle with access to food and water ad libitum. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[75].

Drug

Baicalin, 95% purity, was purchased from Sigma-Aldrich (Cat No. 572667, St. Louis, MO, USA).

Methods

Establishment of corticosterone-induced stress models and drug treatment

Corticosterone (Sigma-Aldrich) was suspended and sonicated in sesame oil as previously described^[13]. The ready-to-use suspension was subcutaneously injected into PBS + corticosterone and baicalin + corticosterone groups at a dose of 40 mg/kg daily for 14 days to induce stress. At the same time, an oral feeding with 50 mg/kg baicalin dissolved in 0.01 M PBS was started, and was given to the baicalin + corticosterone group for 14 days. The PBS + corticosterone rats were orally given 0.01 M PBS at the same volume. The PBS + vehicle rats were orally given 0.01 M PBS plus vehicle injection (sesame oil) for 14 days; the baicalin + vehicle rats were orally dosed with 50 mg/kg baicalin dissolved in 0.01 M PBS plus vehicle injection for 14 days.

Sample collection and tissue processing

After treatment, all animals were anesthetized by over-dose of 2% sodium phenobarbital and transcardially prefixed with 4% paraformaldehyde. All the brains were post-fixed for 48 hours in 4% paraformaldehyde and immersed in 30% sucrose solution until each brain sank to the bottom of the solution. Serial coronary frozen sections (40 µm thickness) were cut with a freezing microtome and prepared for immunohistochemistry.

Immunohistochemistry for doublecortin expression

The brain sections were brought to room temperature and were blocked with a solution containing 0.01 M PBS, 0.1% Triton X-100 and 10% normal goat serum solution for 30 minutes, and subsequently incubated at 4°C overnight with polyclonal rabbit anti-doublecortin (1:500; Cell Signaling Technology, Boston, MA, USA). After thorough washes, sections were then incubated with goat anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA, USA) and followed by avidin-biotin complex (1:200; Vector Laboratories) at room temperature for 60 minutes, respectively. Chromogen reactions were performed with diaminobenzidine (Sigma-Aldrich) and 0.1% H₂O₂ for 10 minutes. The sections were dehydrated in graded alcohol and cleared in toluene, and then coverslipped. The stained sections were observed using a CHBS light microscope (Olympus, New York Microscope Company Inc., NY, USA).

Analysis of the number of doublecortin-positive cells

To determine the number of doublecortin-positive cells in the hippocampus, six sections (at 440 µm intervals) were selected in each animal and processed for immunohistochemical staining. The doublecortin-positive cells in the dentate gyrus in the dorsal part of hippocampus were counted using Stereo Investigator software (Micro-Bright Field Biotechnology, New York, NY, USA) by a skilled and blinded experimenter. The software randomly offered 30–50 visual fields under 40 × lens and the qualified cells were counted. The selected doublecortin-positive cells were further divided into class I and class II doublecortin-positive cells, as previously described^{[35, 53]}. Briefly, the most mature class II doublecortin-positive cells displayed a primary dendrite that was orientated perpendicular to the subgranular zone. The class I doublecortin-positive cells were located in the subgranular zone, without a dendrite, or only a short dendrite.

Dendritic complexity of doublecortin-positive neurons

Dendritic outline of doublecortin-positive cells including dendritic length and number of intersections (branch points) in the granule cells layer of the hippocampal dentate gyrus were traced using Filament tracer software (Bitplane Inc., South Windsor, CT, USA) and then analyzed by Imaris track software (Bitplane Inc.). A qualified neuron for analysis displayed a comparatively independent dendritic tree with at least tertiary branches. Tracings were analyzed by Sholl analysis in concentric circles (diameter 10–200 µm) with their center in the cell body^[39]. In brief, the dendritic length counted as the length of the tracing between every two concentric circles. The intersection number was the number of tracings crossing every circle. All tracings were done by a skilled experimenter blind to the groups and treatments. A higher value in dendritic length or the number of intersections represented a neuron with more complex dendritic branches.

Statistical analysis

All data were presented as mean ± SEM. Comparisons of multiple groups were done by two-way analysis of variance-dependent experimental designs and followed by Student-Newman-Keuls test for intergroup comparisons with SPSS 16.0 statistical software (SPSS, Chicago, IL, USA). Significance was set at a probability level of P < 0.05.

讨论

文章快阅

延伸阅读

1 中文内容介绍

在成年的中枢神经系统中，成年的神经发生是一种长期存在的生物学活动，被大量的研究认为与成体的认知功能和情绪调节密切相关。在本次实验研究中，我们首先建立了慢性应激大鼠模型，计划给予黄岑苷，一种从黄芩中提取的黄酮类化合物，据前期动物研究推测黄岑苷可能同样对神经发生过程可以产生一定的影响。研究中，通过连续14d给予40 mg/kg糖皮质激素证实可以导致SD大鼠大脑海马区域神经发生的减少，而在开始糖皮质激素注射的同时给予每日口服50mg/kg的黄岑苷作为干预措施。我们的研究结果显示皮下注射糖皮质激素显著降低海马中doublecortin (DCX) 阳性神经元数目。糖皮质激素导致的减少的神经元以II型DCX阳性神经元为主，而I型DCX阳性神经元基本上不受糖皮质激素的影响。而黄岑苷的治疗在海马区明显通过增加I型和II型DCX阳性神经元数目，从而提高糖皮质激素处理大鼠的神经发生的增加。此外，糖皮质激素注射可引起DCX阳性神经元细胞的树突形态学结构发生萎缩改变，而这种形态学改变可以又可以被黄岑苷干预所逆转。总结：本研究通过在体实验研究证实，应用黄岑苷对成体的神经发生有增强的作用，提示黄芩苷可能在改善认知功能和情绪调节中有潜在的治疗作用和临床应用前景。

2 实验设计思路

在成年的中枢神经系统中，存在着持续的新生神经元的区域：室管膜下区和海马的齿状回区。这些成体存在的新生神经元不仅参与调控认知功能和情绪，而且了解成体神经发生的机制对中枢神经系统损伤后的修复有重大的指导意义。所以开发和研究促进成体神经发生的药物，成为中枢神经系统损伤和修复的主要研究方向之一。黄岑苷作为黄芪苷的提取物，被广泛各种急性感染疾病的治疗。近年来，黄岑苷在促进各类干细胞向神经元分化的作用慢慢受到关注，但均限于体外的实验数据。本研究拟用黄岑苷干预应激的成年大鼠，以了解黄岑苷处理对于应激大鼠海马神经发生的保护作用。此外，成体的神经发生可分为几个阶段：前体细胞的增殖，向神经元分化，新生神经元的成熟与迁移，以及新生神经元向现有通路的整合。研究采用DCX染色标记新生的未成熟的神经元，通过对DCX阳性细胞的数目和树突形态学的分析，针对性的了解黄岑苷对于在体神经前体细胞的神经分化和成熟两个阶段的作用。

3 与国内外研究的比较

近年来，国内外研究促进和抑制成体神经发生的药物的报道很多。但运用现代生物技术，研究中药和中药提取物对neurogenesis和神经保护作用才刚刚起步。本研究的创新点在于两点：（1）研究了药物黄岑苷在在体动物上对于神经前体细胞的神经分化和成熟的影响，首次为黄岑苷的促进神经发生作用提供了在体实验证据；（2）在对于新生神经元的数目和形态研究上，采取了分类的研究方法。分别探讨了黄岑苷对DCX阳性的I类和II类新生神经元数目和形态的影响。在大多数关于海马在体的神经发生都忽略了这个问题。

4 专家意见与答疑

专家意见：dendritic length and the number不能代表 Dendritic morphology，特别是没有分析class I and class II DCX positive cells的差异。

作者答疑：dendritic length and the number of intersections 不能完全代表 Dendritic morphology。但是，如果想对树突形态（dendritic analysis）做出定量的分析，就需要借助图像分析软件，如本文中用到的sholl analysis, sholl analysis 是最常规用到的树突形态分析的软件之一, sholl analysis采用间距为10 µm的同心圆体系去分析由实验者trace 出来的神经元树突轮廓，这里的dendritic length显示的就是sholl analysis 每两个同心圆之间所有的突起长度，the number of intersections 是突起的轮廓与每一个同心圆的交点的数目。所以突起轮廓越长，分支越多，就会有越长的dendritic length 和越大的 the number of theintersection。所以，这两个指标能够一定程度的反映dendritic morphology，或者说反映出 the complexity of the dendrites（突起形态的复杂性）。在做sholl analysis 或者其他的突起分析时，对于神经元的选取是有严格的要求的，常规对于DCX+的细胞来说，需要细胞具有三级或者以上的树突分支才可以用来做，而且分支要和周围神经元的分支尽量不会有相互影响，这样的神经元才会被选择，所以根据这个要求，所有的class I的DCX+ cells 在形态上是不符合树突形态分析的（讨论中也有提到）。因此，没有做class I和class II DCX+ cells 的差异分析。