Design
A randomized, controlled animal study.

Time and setting
Experiments were performed at the Fujian Medical University, Fujian Neurosurgical Institute from 2008 to 2011.

Materials
A total of 42 clean adult male Sprague-Dawley rats weighing 250–300 g were purchased from Shanghai Laboratory Animal Center (SCXK (Hu) 2003-0003).

Methods
Ischemic preconditioning and permanent middle cerebral artery occlusion establishment
Rats were intraperitoneally anesthetized with chloral hydrate (300 mg/kg). Ischemic preconditioning was induced by occluding the bilateral common carotid artery temporarily (15 minutes), followed by a period of reperfusion (48 hours). Subsequently, permanent middle cerebral artery occlusion was induced in the middle cerebral artery occlusion group and ischemic preconditioning + middle cerebral artery occlusion group using the intraluminal filament occlusion method as described in our previous report^[5].

Assessment of neurological deficits
Neurological deficit scores were assessed at 72 hours after middle cerebral artery occlusion as follows: 0, no observable deficits; 1, contralateral forelimb flexion; 2, decreased resistance to lateral push without circling; 3, circling to the contralateral side; 4, death.

Sample preparation
After 72 hours of middle cerebral artery occlusion, rats were sacrificed, perfused and the ischemic brain cortex was removed, fixed, and embedded in paraffin. Coronal sections (4 μm thick) were obtained and deparaffinized for Nissl staining, TUNEL staining and immunohistochemical analysis.

Nissl staining for nerve cell damage
To confirm the effect of ischemic preconditioning on focal cerebral ischemia-induced neuronal injury, Nissl staining (0.1% (w/v) toluidine blue; Amresco, Solon, OH, USA) was performed to observe the morphological characteristics of neurons in the ischemic brain. The sections were visualized with a light microscope (Olympus). The survival cell ratio was calculated.

TUNEL analysis for cell apoptosis
TUNEL staining was performed according to the manufacturer’s instructions. The paraffin-embedded sections were deparaffinized and incubated in the TdT enzyme at 37°C for 60 minutes, followed by two washes in standard saline citrate to stop the reaction. Brain sections were incubated in streptavidin horseradish- peroxidase solution for 30 minutes at room temperature. Diaminobenzidine was used as a color substrate, and the sections were counterstained with hematoxylin. The TUNEL-positive cell ratio was calculated under light microscopy.

Immunohistochemical analysis for nuclear factor kappa B expression
The paraffin-embedded sections were deparaffinized, then incubated with a mouse anti-rat monoclonal antibody against nuclear factor kappa B p65 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Biotinylated goat anti-mouse IgG (1:500; Zhongshan Biotechnology, Beijing, China) was used as a secondary antibody at 37°C for 2 hours. Diaminobenzidine was used as the chromogen, and the sections were counterstained with hematoxylin. The number of nuclear factor kappa B p65 immunopositive cells was calculated under light microscopy.

Western blot analysis for nuclear factor kappa B and cleaved caspase-3 expression
Total protein was extracted and separated on a 10% (w/v) sodium dodecyl sulphate polyacrylamide gel, transferred to nitrocellulose membrane, then incubated overnight with a mouse anti-rat monoclonal antibody against nuclear factor kappa B p65 (1:200; Santa Cruz Biotechnology), or a rabbit polyclonal antibody against cleaved caspase-3 (1:1 000; Cell Signaling, Boston, MA, USA) at 4°C overnight, followed by incubation with horseradish-peroxidase conjugated goat anti-rabbit/ mouse IgG at 4°C for 2 hours (1:2 000; KPL Inc., Gaithersburg, MD, USA). Protein expression was detected with an enhanced chemiluminescence detection system (KPL Inc.). Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as a loading control. The absorbance of nuclear factor kappa B p65, cleaved caspase-3 and GAPDH bands on the X-ray film were quantitatively analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA). The results were expressed as an absorbance ratio of nuclear factor kappa B p65/GAPDH and cleaved caspase-3/GAPDH.

Biochemical analysis for myeloperoxidase activity and malondialdehyde content
Myeloperoxidase activity in the rat brain was measured according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China). The results were expressed as unit per gram of tissue (U/g tissue). Malondialdehyde content in the rat brain was detected according to the manufacturer’s instructions (Jiancheng Bioengineering Institute). The results were expressed as μmol/g.

Statistical analysis
Experimental data were presented as mean ± SD. Statistical significance was assessed by one-way analysis of variance followed by Least Significant Difference test with SPSS 13.0 software (SPSS, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.

Funding: This study was supported by the National Natural Science Foundation of China, No. 81100987; the Natural Science Foundation of Fujian Province of China, No. 2011J05066; the Clinical Key Subject (Neurosurgery) Funding of Fujian Medical University.
Author contributions: Songsheng Shi was in charge of animal model establishment. Weizhong Yang was responsible for experimental design. Xiankun Tu obtained the funding and wrote the manuscript. Chunmei Chen and Chunhua Wang performed the experiments. All authors have read and agree to the manuscript as written.
Conflicts of interest: None declared.
Ethical approval: The surgical procedures were approved by Animal Ethics Committee of Fujian Medical University in China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.

(1)   Ischemic preconditioning reduced lipid peroxidation levels and neurological deficits following ischemic brain injury in the rat.

(2)   Ischemic preconditioning attenuated neuronal apoptosis following rat brain injury.

(3)   Ischemic preconditioning suppressed nuclear factor kappa B and cleaved caspase-3 expression in the rat brain.

实验从现象描述层次、组织学层次、分子生物学层次探讨缺血预处理对大鼠缺血性脑损伤的保护作用及其潜在的分子机制，结果显示：

①  缺血预处理可减轻缺血性脑损伤大鼠脑内脂质过氧化水平及神经功能损伤。

②  缺血预处理可减少缺血性脑损伤大鼠脑组织神经细胞凋亡。

③  缺血预处理可抑制凋亡相关因子核因子κB及cleaved caspase-3的表达。

1实验设计构思介绍：

实验在前期研究基础上，成功建立大鼠脑缺血动物模型，并通过设置对照组、模型组、缺血预处理干预组，在现象描述层次、组织学层次、分子生物学层次上，探讨：（1）缺血预处理对大鼠缺血性脑损伤的保护作用（现象描述层次），（2）缺血预处理对大鼠脑缺血损伤神经细胞损伤和神经细胞凋亡的抑制作用（组织学层次），（3）缺血预处理减轻脑缺血性损伤和神经细胞凋亡的潜在分子机制——对核因子κB和裂开的caspase-3的抑制作用（分子生物学层次）。实验设计较为严谨，层层递进，从不同角度、不同层次充分论证了缺血预处理对缺血性脑损伤的保护作用及其潜在的分子机制，为今后缺血性脑损伤的治疗策略提供理论依据。

2 国内外同类研究水平的介绍：

国内外相关研究表明缺血预处理可以减轻脑缺血诱导的脑损伤，多数研究仅停留于现象描述层次，仅仅从脑梗死体积、神经功能评分等方面验证缺血预处理对脑缺血损伤的保护作用，而较少探讨缺血预处理对脑缺血损伤保护作用的机制。近年来，一部分研究者开始致力于研究缺血预处理对缺血性脑损伤的神经保护的作用机制，他们发现缺血预处理可以通过抑制炎症反应和炎症基因的表达从而减轻缺血性脑损伤，还可以通过抑制血脑屏障的破坏减轻缺血性脑损伤。炎症反应、血脑屏障的破坏和神经细胞凋亡是缺血性脑损伤的3个重要的病理生理机制。缺血预处理是否通过抑制神经细胞凋亡减轻缺血性脑损伤呢，结果尚未明确，同时也缺少相关的分子生物学机制。这也正是文章的创新之处。

3与其他同类研究的比较：

如文中所述，国内外虽然已探讨过缺血预处理对缺血性脑损伤的保护作用，但是，缺血性脑损伤的神经保护机制尚未明确，关于缺血预处理对脑缺血神经细胞凋亡抑制作用的相关研究，为数很少。在分子生物学层次方面，我们第一次报道了缺血预处理对核因子κB和裂开的caspase-3的抑制作用，这是该文章的亮点，也是精华部分，国内外文献未见报道。在实验设计上，我们从不同层次（现象描述层次、组织学层次、分子生物学层次）探讨缺血预处理对大鼠缺血性脑损伤的保护作用及其潜在的分子机制，实验设计较为严谨，层层递进，这也是本文区别于国内外文献的特色之一。

4 文章先进性：

主要体现在以下3点：①研究内容有一定的创新性，在前人及本课题组前期研究的基础上，探讨缺血预处理的神经保护作用机制，围绕了当今国际的热点研究，但并不重复前人的研究，研究了缺血预处理对脑缺血神经细胞凋亡的抑制作用，并进一步探讨缺血预处理抑制神经细胞凋亡的分子机制——对核因子κB和裂开的caspase-3的抑制作用，从而说明预处理的神经保护作用，据我们所知，该研究内容国内外尚无明确的报道，有一定的创新性。②实验方法上有一定的特色，本文所采用的实验方法，如神经功能评分、尼氏染色、TUNEL染色、免疫组化分析、Western blot等方法，这些实验技术看似朴素无华，但实为缺血性脑中风基础研究的经典技术，能够充分的论证所要说明的问题，各种实验方法合理有效、相互结合，更有力地说明了缺血预处理的神经保护作用。③在实验设计上，从不同层次（现象描述层次、组织学层次、分子生物学层次）探讨缺血预处理对大鼠缺血性脑损伤的保护作用及其潜在的分子机制，实验设计较为严谨，层次分明，逐步深入，是本文的重要特色之一。

选题时在检索文献方面采用的数据库是Medline数据库，检索时间从1989~至今，范围大，检索关键词为缺血预处理、神经细胞凋亡、核因子κB、TUNEL、裂开caspase-3，未检出相关文献。