Design

A randomized controlled animal experiment.

Time and setting

Experiments were performed between July 2010 and January 2012 in the Central Laboratory of Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, China.

Materials

Healthy adult male Sprague-Dawley rats of clean grade, aged 9 weeks, and weighing 250–280 g, were provided by the Experimental Animal Center of Soochow University, China, under license No. SYXK (Su) 2007- 0035. The animals were housed at 23 ± 2°C with 40–70% relative humidity under a natural light-dark cycle, and allowed free food and water. Prior to experiments, all rats were adapted to the environment for 1 week. Experimental rats were preoperatively fasted for      12 hours. Experimental animals were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[26].

Methods

Establishment of the focal cerebral ischemia model

A middle cerebral artery occlusion model was established in rats according to the suture method of Longa et al ^[27]. The model rats were anesthetized with 10% chloral hydrate and disinfected with iodophor, a median incision was made in the median skin of the cervical part, and then the right common carotid artery and external carotid artery were ligated. The distal end of the common carotid artery was clamped and another incision was made at 2 mm lateral to the common carotid artery bifurcation, following which a nylon line of 0.2 mm in diameter and 25 mm in length was inserted until resistance was encountered. The suture was removed 90 minutes after ischemia and reperfusion was then performed. The sham surgery group underwent the separation and exposure of blood vessels only, without a suture being inserted.

Criteria for a successful model

At 24 hours after reperfusion, the model animals that were scored 1–3 according to the neurological function scale of Longa et al ^[27] were included in the model group. Of the animals in the model group, one rat was killed by decapitation at 24 hours after reperfusion and its brain was frozen at –20°C. Coronal serial slices were then cut from the frozen tissue and stained in PBS containing 1% TTC for 20 minutes at 37°C in the dark. Normal brain tissue was stained dark red while ischemic brain tissue was stained white, thus confirming that the cerebral ischemia model was successfully established.

Frozen section preparation

Rats were fixed with 4% paraformaldehyde and killed by pulling the skull. Brain tissues were isolated and fixed in 4% paraformaldehyde for 6 hours and successively dehydrated in 20% and 30% sucrose solutions overnight. Continuous coronal frozen sections were cut at a thickness of 8 μm, and one out of every six sections was mounted on a poly-L-lysine (Sigma, St. Louis, MO, USA)- coated glass slide and stored at –20°C.

Immunofluorescence analysis of the expression of netrin-1 and its receptors, DCC and UNC5B, in brain tissues from ischemic and reperfusion rats

Brain slices from the same layers were used for immunofluorescence staining^[28]. Frozen sections were equilibrated at room temperature for 10 minutes, hydrated in 0.01 M PBS for 10 minutes, and blocked with 10% donkey serum (Jackson Immunoresearch, West Grove, PA, USA) for 1 hour at room temperature. After discarding the blocking solution, the sections were incubated with rabbit anti-netrin-1 polyclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-DCC polyclonal antibody (1:100; Santa Cruz Biotechnology), goat anti-UNC5B polyclonal antibody (1:100; Santa Cruz Biotechnology), mouse anti-rat neuronal nuclear antigen monoclonal antibody (1:100; Chemicon, Billerica, MA, USA), mouse anti-rat glial fibrillary acidic protein monoclonal antibody (1:200; Sigma) or mouse anti-rat CD11b monoclonal antibody (1:200; Sigma) diluted in 5% donkey serum at 4°C overnight. After washing three times with PBS for 10 minutes each, the slices were incubated with donkey anti-rabbit IgG-Alexa Fluor 488, or donkey anti-goat IgG-Alexa Fluor 488 and donkey anti-mouse IgG-Alexa Fluor 594 secondary antibodies (1:200; Invitrogen, Eugene, OR, USA) for 1 hour in the dark. The slices were then washed a further three times with PBS for 10 minutes each, mounted in DAPI-containing quenching sealing agent (Santa Cruz Biotechnology), and stored at 4°C. Negative control slices were incubated with PBS instead of antibodies. Sections were analyzed by confocal laser scanning microscopy (Nikon A1, Tokyo, Japan) to determine the localization of immuno-positive cells at the infarct site and in the cerebral cortex, and also the co-localization of neurons, astrocytes and microglial cells.

(1) Netrin-1 induces the directional migration of nerve cells and plays an important role in the repair of adult central nervous system injuries.
(2) We confirmed the upregulated expression of netrin-1 and its receptor, deleted in colorectal cancer, in the cortex of rats after focal cerebral ischemia and reperfusion injury, both of which reached their peak expression level at 14 days and began to decrease at 21 days.
(3) Netrin-1 expression was observed in neuronal cell bodies by laser confocal microscopy, particularly in neurons at the edges of small vessels, while it was scarcely expressed in astrocytes and microglial cells.

1.实验证实局灶性脑缺血再灌注损伤大鼠轴突导向因子1及其受体在缺血周围皮质中表达上调，两者随时间的变化规律具有一致性：在脑缺血再灌注后14d达高峰，21d后表达减弱。
2.激光共聚焦显微镜发现轴突导向因子1在神经元胞体内表达，并表达于小血管边缘，但在星形胶质细胞及小胶质细胞中无表达。

1实验设计构思介绍：

以netrin-I为代表的netrin家族是目前最受关注的轴突导向因子之一。在胚胎发育期，轴突导向因子1通过吸引、排斥的双重机制对轴突生长和细胞迁移发挥导向作用。近期的研究发现，轴突导向因子1参与成年神经系统损伤后的结构和功能重建。有实验表明，轴突导向因子1能够抑制神经细胞凋亡，促进成年小鼠中枢神经系统的新生血管形成，这为很多神经系统疾病的临床治疗提供了新的希望。

目前关于轴突导向因子1信号如何发挥脑缺血后的神经保护作用，其受体在局灶性脑缺血后表达变化的报道却结果各异。为了解决这一争论，我们通过线栓法建立大鼠大脑中动脉缺血再灌注模型。在脑缺血再灌注不同时间点（1，7，14，21d），应用免疫荧光标记和激光共聚焦显微镜，检测轴突导向因子1及受体结直肠癌缺失基因抗原、UNC5B在缺血灶和缺血周围区域的表达变化，以及它们在神经元、星形胶质细胞、小胶质细胞中的定位情况。实验清楚地阐明了轴突导向因子1及其受体结直肠癌缺失基因抗原在缺血周围大脑皮质的表达规律，并明确其在缺血后神经细胞中的定位，这些结果有助于深入探讨轴突导向因子1在缺血再灌注损伤中的作用。

2国内外同类研究水平的介绍：

以往的研究集中在中枢神经发育过程中轴突导向因子1对轴突生长、细胞迁移的引导机制上，近来轴突导向因子1及受体在中枢神经系统损伤修复中的神经保护、血管生成作用成为研究热点。在坐骨神经自体免疫性炎症中，轴突导向因子1和结直肠癌缺失基因抗原在病变后11-24 d表达显著增高，且主要在轴突以及巨噬细胞和雪旺氏细胞的细胞膜和细胞质中表达，被认为可对抗自体免疫性损伤，有助于细胞生存和轴突再生。而在成年小脑和脊髓损伤后，去神经支配的小脑颗粒细胞UNC5H3表达增高，在损伤后8 d，UNC5H(1-3)和netrin-I在病灶部位的巨噬细胞、活化的小胶质细胞或脑膜成纤维细胞等处显著表达，并可持续数月之久，被认为能通过参与胶质瘢痕形成而抑制损伤后轴突再生。在小鼠大脑中动脉闭塞模型中，外源性轴突导向因子1经侧脑室注射后能减小梗死范围、抑制p53介导的神经细胞凋亡；通过腺病毒载体使轴突导向因子1过表达，可改善大脑中动脉缺血再灌注大鼠的运动功能评分。研究发现轴突导向因子1过表达，可以促进成年大脑局部类动脉样的新生血管形成，抑制血管内皮细胞凋亡，提示其具有血管内皮细胞生长因子样作用，促进血管再生。

3与其他同类研究的比较：

关于轴突导向因子1及其受体在局灶性脑缺血后表达变化的报道有以下3个研究。Wu等在小鼠中动脉栓塞模型中发现，缺血后UNC5B表达上调，结直肠癌缺失基因抗原表达无明显改变；而Tsuchiya发现在大鼠大脑中动脉缺血再灌注后轴突导向因子1及其受体结直肠癌缺失基因抗原在梗死周围表达明显升高，Stanley等则报道大鼠大脑中动脉缺血再灌注后梗死周围UNC5B表达无改变。三者之间的结论有所差异，考虑选取的动物、观察时间点以及取材部位不同所致。文章制备大鼠大脑中动脉缺血再灌注模型，对轴突导向因子1及其受体结直肠癌缺失基因抗原、UNC5B的表达规律进行系统的研究。结果发现，再灌注1d后，缺血周围大脑皮质轴突导向因子1和结直肠癌缺失基因抗原的表达开始增加，14d时达到高峰，21d后表达下降；而UNC5B的表达无明显变化，这一结果支持Tsuchiya与Stanley的研究。实验进一步采用神经细胞特征性标记物，进行免疫组化双标染色，证实轴突导向因子1在神经元胞体内表达，星形胶质细胞及小胶质细胞不表达轴突导向因子1。结直肠癌缺失基因抗原则神经元和星形胶质细胞的胞膜及突起。文章结果既阐明了轴突导向因子1信号在缺血后的表达规律，也进一步明确了轴突导向因子1和结直肠癌缺失基因抗原在神经细胞中的定位。

４文章创新性的分析：

以netrin 1和cerebral ischemia为关键词检索Pubmed数据库，截止到2012-03-01，共检索到8篇文献，其中最近1篇为2012年，2011年4篇，2008年2篇，最早为2007年（1篇），表明实验具有一定的创新性和先进性。具体检索结果如下： 

(1) Lu H, Wang Y, He X, et al. Netrin-1 hyperexpression in mouse brain promotes angiogenesis and long-term neurological recovery after transient focal ischemia. Stroke. 2012;43(3):838-843.

(2) Liu N, Huang H, Lin F, et al. Effects of treadmillexercise on the expression of netrin-1 and its receptors in rat brain after cerebral ischemia. Neuroscience. 2011;194:349-358.

(3) Sun H, Le T, Chang TT, et al. AAV-mediated netrin-1 overexpression increases peri-infarct blood vessel density and improves motor function recovery after experimental stroke. Neurobiol Dis. 2011;44(1):73-83.

(4) Lu H, Wang Y, Yuan F, et al. Overexpression of netrin-1 improves neurological outcomes in mice following transient middle cerebral artery occlusion. Front Med. 2011;5(1):86-93.

(5) Grenz A, Dalton JH, Bauerle JD, et al. Partial netrin-1 deficiency aggravates acute kidney injury. PLoS One. 2011;6(5):e14812.

(6) Wu TW, Li WW, Li H. Netrin-1 attenuates ischemic stroke-induced apoptosis.Neuroscience. 2008;156(3):475-482.

(7) Hou ST, Jiang SX, Smith RA. Permissive and repulsive cues and signalling pathways of axonal outgrowth and regeneration. Int Rev Cell Mol Biol.2008;267:125-181.

(8) Tsuchiya A, Hayashi T, Deguchi K, et al. Expression of netrin-1 and its receptors DCC and neogenin in rat brain after ischemia. Brain Res. 2007;1159:1-7.

5所投文章国内外研究状况的微观分析：

以往的研究集中在中枢神经系统发育过程中轴突导向因子1对轴突生长、细胞迁移的引导机制上，近来轴突导向因子1及受体在中枢神经系统损伤修复中的神经保护、血管生成作用成为研究热点。在坐骨神经自体免疫性炎症中，轴突导向因子1和结直肠癌缺失基因抗原在病变后11-24 d表达显著增高，且主要在轴突以及巨噬细胞和雪旺氏细胞的细胞膜和细胞质中表达，被认为可对抗自体免疫性损伤，有助于细胞生存和轴突再生。而在成年小脑和脊髓损伤后，去神经支配的小脑颗粒细胞UNC5H3表达增高，在损伤后8 d，UNC5H(1-3)和轴突导向因子1在病灶部位的巨噬细胞、活化的小胶质细胞或脑膜成纤维细胞等处显著表达，并可持续数月之久，被认为能通过参与胶质瘢痕形成而抑制损伤后轴突再生。在小鼠大脑中动脉闭塞模型中，外源性轴突导向因子1经侧脑室注射后能减小梗死范围、抑制p53介导的神经细胞凋亡；通过腺病毒载体使轴突导向因子1过表达，可改善大脑中动脉缺血再灌注大鼠的运动功能评分。研究发现轴突导向因子1过表达，可以促进成年大脑局部类动脉样的新生血管形成，抑制血管内皮细胞凋亡，提示其具有血管内皮细胞生长因子样作用，促进血管再生。

6外审专家意见及作者回复：

问题1：作者在文中提到“假手术组及手术对侧大脑皮质中轴突导向因子1免疫反应阴性”，但大量国内外文献报道哺乳动物在发育过程及发育完善后中枢神经系统均有轴突导向因子1表达，如前脑及脊髓灰质等，以及缺血后对侧大脑皮质也有轴突导向因子1表达上调4，出现这样的矛盾，如何解释?

回复：已有的文献[1-3]表明，轴突导向因子1在胚胎期的底板、脊髓腹侧、侧脑室腹侧、小脑皮质、纹状体中大量表达，起着引导轴突生长和神经细胞定向迁移的作用。但是出生后轴突导向因子1的表达下降，新生期主要位于小脑、耳蜗、纹状体、中脑腹侧的被盖区内；在成年后发现脊髓、小脑和纹状体、黑质中有轴突导向因子1的表达。

实验发现轴突导向因子1、结直肠癌缺失基因抗原表达变化集中在梗死周围的大脑皮质，对侧皮质及对照组皮质则无改变，提示轴突导向因子1可能在梗死及修复中发挥作用。

文献[4]摘要中提到，在对照组及缺血组小鼠大脑皮质中轴突导向因子1表达升高。这一结果是由于注射了携带轴突导向因子1基因的腺病毒载体，其目的是证实病毒转染有效。在文献4的结果1中提到，缺血组脑内注射生理盐水或携带绿色荧光蛋白的腺病毒载体后，轴突导向因子1几乎不能检出，也说明对照侧皮质中无轴突导向因子1的表达。

此外轴突导向因子1在不同种属中的表达存在差异，在小鼠及大鼠缺血后的表达规律可能也有所差别。

[1] Manitt MA, Colicos KM, Thompson E, et al. Widespread expression of netrin - 1 by neurons and oligodendrocytes in the adult mammalian spinal cord. J Neurosci.2001(21):3911-3922.

[2] Livesey FJ, Hunt SP. Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral, and cerebellar development. Mol Cell Neurosci. 1997(8):417-429.

[3] Guijarro P, Simo S, Pascual M, et al. Netrin1 exerts a chemorepulsive effect on migrating cerebellar interneurons in a Dcc-independent way. Mol Cell Neurosci. 2006(33):389-400.

[4] Gillespie LN, Marzella PL, Clark GM, et al. Netrin-1 as a guidance molecule in the postnatal rat cochlea. Hear Res. 2005(199):117-123.

问题2：实验对缺血再灌注不同时间点梗死周围轴突导向因子1、结肠癌缺失蛋白阳性细胞进行了定量统计，是如何选择 “梗死周围”进行拍照及计数统计的?并请出示轴突导向因子1和结肠癌缺失蛋白阳性表达变化规律的统计数据。

回复：缺血周围区域的确定：对于大鼠大脑中动脉缺血再灌注模型，缺血侧大脑半球距离嗅球尖端7-11mm、矢状裂至外侧裂上1/3的皮质组织由大脑前动脉和中动脉双重供血，缺血较轻，表现为半暗区的变化。通过TTC染色可以清楚地区别梗死核心区以及缺血周围皮质组织。

在组织切片进行荧光标记后也可以通过形态学特征识别缺血周围区域。梗死核心区内细胞破坏、组织结构丧失，半暗带组织结构基本保持完整。实验同时进行神经元核抗原、胶质纤维酸性蛋白、CD11b和轴突导向因子1、结直肠癌缺失基因抗原、UNC5B的免疫荧光双标记，有助于定位。梗死核心区内神经元标记缺失，在缺血周围区域则出现大量反应性星形胶质细胞及活化的小胶质细胞（表现为胶质纤维酸性蛋白及CD11b荧光强度增加、细胞体积增大、阿米巴样等形态）^[1-2]。

[1] Shi J, Panickar KS, Yang SH, et al. Estrogen attenuates over-expression of ß-amyloid precursor protein messager RNA in an animalmodel of focal ischemia. Brain Res. 1998;1-2:87-92.

[2]Li Y, Powers C, Jiang N. Intact, injured, necrotic and apoptotic cells after focal cerebral ischemia in the rat. J Neurol Sci. 1998;156(2):119-132.

阳性细胞计数：对照组皮质无轴突导向因子1及结直肠癌缺失基因抗原阳性表达，故只对缺血组进行相对定量。每个脑组织选取5张切片进行分析，在相同的荧光强度、400倍放大的条件下，于梗死周围皮质选取5个不连续的视野拍照、计数阳性细胞，取平均值。采用SPSS 13.0统计软件，计量资料以平均值±标准差表示，单向方差分析，P<0.05为差异有统计学意义。具体统计结果如下：

表1大鼠缺血周围皮质轴突导向因子1和结直肠癌缺失基因抗原阳性表达细胞数

^aP<0.01，vs 1d；^bP<0.01，vs 7d；^cP<0.01，vs 14d。实验检测8只大鼠。

由于视野的选择、计数的标准上有一定的主观因素干扰，形态学观察仅作为半定量结果，所以在文章中未列入统计数据。作者正在后续的试验中通过western blot、实时定量PCR方法进一步证实上述表达规律。