1Department of Histology and Embryology,
2Department of Pathogeny Biology, Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang Province, China
Liu Zhi-xin, Teacher assistant, Department of Histology and Embryology, Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang Province, China
Correspondence to:
Liang Jun, Associate professor, Department of Histology and Embryology, Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang Province, China mdjzplj@126.com
Supported by: the Scientific Research Program of Mudanjiang Medical University, No.
B200812*
Received: 2009-07-21 Accepted: 2009-10-23 (20090721023/WL)
Liu ZX, Song BH, Zhao FS, Li YZ, Liang J. Extraction and purification of neonatal versus adult rat Schwann cells.
Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu.
2010;14(6):
1115-1119(China).
[http://www.crter.cn http://en.zglckf.com]
Extraction and purification of neonatal versus adult rat Schwann cells *
Liu Zhi-xin
1, Song Bao-hui
2, Zhao Fu-sheng
1, Li Yue-zhen
1, Liang Jun
1Abstract
BACKGROUND: Schwann cells are the seed cells of neural repair, and it is a key to harvest a large number of Schwann cells with high purity and activity.
OBJECTIVE: To compare the in vitro culture, purification, and morphology of Schwann cells between neonatal and adult rats, and investigate a simple and feasible culture method to harvest high-purity Schwann cells.
METHODS: Totally 30 Sprague-Dawley rats, comprising 20 neonatal (1-3 days after birth, neonatal group) and 10 adult (weighing 150-200 g, adult group) rats, were included. Following double-enzyme digestion and two incubations, Schwann cells were isolated and purified by differential attachment. Cell morphology and attaching speed were determined through the use of inverted microscope.
Cells were counted and cell purity was calculated. Cell proliferative ability was detected by MTT microcolorimetry. Curves of cell proliferation in each group were depicted to determine proliferative speed. Schwann cells were identified by S-100 immunochemistry.
RESULTS AND CONCLUSION: Compared with fibroblasts, neonatal rat Schwann cells exhibited faster, while adult rat Schwann cells showed slower, attaching speed. Both neonatal and adult groups yielded over 96% cell purity. MTT microcolorimetry results revealed that Schwann cells proliferated actively in neonatal and adult groups. Cell proliferative curves show that neonatal rat Schwann cells proliferated faster than adult rat Schwann cells (P < 0.05). S-100 immunochemistry results showed positive results in both groups. All these findings suggest that double-enzyme digestion and two incubations followed by differential attachment is a satisfactory method to harvest considerable Schwann cells with high purity and activity. Neonatal rat Schwann cells show stronger proliferative, attaching capacities than adult rat Schwann cells.
INTRODUCTION
Schwann cells have been shown to be closely related to the development and regeneration of peripheral nerves and play an extremely important role in maintaining peripheral nervous system function and repairing peripheral nerve injury[1-3]. For this reason, Schwann cells have been widely used in peripheral nerve tissue engineering[4]. A basis for research is to harvest a large number of purified Schwann cells.
Many methods have been presently developed to culture Schwann cells, such as explant culture[5], enzyme digestion[6], anti-mitosis[7], specific adhesion[8], immunoselection[9-10], differential attachment[11], and inhibition of fibroblasts[12]. Different methods have different advantages and drawbacks[13-15]. The explant culture was first reported by Askanas et al [16] in 1980.
Its advantage is to be able to harvest considerable high-purity Schwann cells, but it wastes long time, and cell proliferative capacity is influenced. The enzyme digestion was first reported by Brockes et al [17] in 1979.
Its advantage is to harvest considerable cells within short time, but it can not ensure cell purity. The immunoselection method can acquire high-purity cells but costs high. Based on the characteristics of nerve structure, the present study adopted double enzyme digestion and two incubations, followed by differential attachment to discard fibroblasts to enhance the purity of Schwann cells. A set of feasible methods were finally determined to harvest considerable Schwann cells with high purity and activity.
MATERIALS AND METHODS
Design
A cytological, in vitro experiment based on two
sample observation.
Time and setting
This study was performed at the Department of Histology and Embryology, Mudanjiang Medical University between December 2008 and June 2009.
Materials
The primary reagents and instruments used in this study are listed as follows.
Reagent/instrument Source
Dulbecco's modified eagle's medium (DMEM), bovine serum albumin(BSA)
Hyclone, USA
Fetal bovine serum(FBS), trypsin-ethylenediamine tetraacetic acid(EDTA), type I collagenase, laminin
Gibco, USA
Polylysine, basic fibroblast growth factor (bFGF), methyl thiazolyl tetrazolium (MTT), S-100 antibody (FITC-labeled)
Sigma, USA
D-Hank’s solution Solarbio, China 25 mL culture flask, 60 mm culture
dish
Orange, Belgium 10 mL centrifuge tube, 6-well plate,
96-well plate
Costar, USA
CO2 incubator(MCO-18AC) Sanyo, Japan Refrigerated centrifuge (5804R) Eppdorff,
Germany Inverted phase contrast microscope
(IX71-22PH)
Olympus, Japan Anatomical microscope
(SMZ-800), Fluorescent biological microscope (80I)
Nikon, Japan
Superclean bench (SW-CJ-2F) Shanghai Boxun Industry &
Commerce Co., Ltd., China
Totally 30 Sprague-Dawley (SD) rats, comprising 20 neonatal (1-3 days after birth) and 10 adult (weighing 150-200 g) rats, of either gender and of SPF grade, were provided by Laboratory Animal Center of Harbin Medical University in China. 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[18].
Methods Grouping
According to cell source, a neonatal group and an adult group were used. Rats from each group were randomly selected.
Pre-degeneration
For neonatal rats: the proximal end of dissociated sciatic nerve was ligated at 4 mm inferior to the piriformis muscle, and incision was sutured, and artificial raising was performed. For adult rats:
the dissociated sciatic nerve was cut off at 1 mm inferior to the piriformis muscle, and incision was sutured layer by layer.
Isolation, culture, and purification of Schwann cells In the neonatal group, ten neonatal rats pre-degenerated for 3 days were included. Precisely, under the anatomical microscope, following stripping of the epineurium of bilateral sciatic nerves, nerve tissue was chipped into 0.5 mm3 small blocks, digested with 0.16% trypsin-EDTA for 10 minutes, followed by 0.1% type Ⅰ collagenase for 20 minutes (vibrating once every 5 minutes), digestion-terminated, centrifuged, re-suspended with 5 mL DMEM containing 20 μg/L bFGF and 10% FBS, inoculated into a 25 mL culture flask, and incubated in a 5% CO2 incubator at 37 ℃.
Twenty-four hours later, nerve tissue was collected, centrifuged, and thoroughly, gently re-suspended. Twenty minutes after new inoculation, culture medium was renewed to culture the adherent cells. Eight visual fields were randomly selected for cell counting and photographing.
In the adult group, five adult rats pre-degenerated for 7 days were used. Precisely, under the anatomical microscope, following stripping of the epineurium and perineurium, nerve tissue was chipped into 0.5 mm3 small blocks, digested with 0.16% trypsin-EDTA for 15 minutes, followed by 0.1% type I collagenase for 30 minutes, and digestion-terminated. Cells were collected and incubated in the incubator. Twenty-four hours later, tissue was collected and cultured twice by differential attachment, each 30 minutes. The non-adhering cells were collected and cultured in laminin- and polylysine- precoated culture flask using DMEM containing 20 μg/L bFGF and 10% FBS. Eight visual fields were randomly selected for cell counting and photographing.
Determination of cell proliferation by MTT microcolorimetry and depicting of Schwann cell growth curves
Cell proliferation was detected by MTT microcolorimetry[19]. Primary and passage Schwann cell suspension from each group was inoculated in a 96-well plate at 108/L. Eight wells were allocated for each passage of cells in each group, 200 μL suspension for each well. Five days later, 5 g/L MTT reagent was added, 20 μL/well, and there was a 4-hour incubation procedure in a 5% CO2 incubator at 37 ℃. Thereafter, the supernatant was discarded, and dimethyl sulfoxide, 150 μL/well, was added, followed by 10-minute vibration. The absorbance value at 490 nm
was measured for depicting cell proliferation curves.
Identification of cells by S-100 immunofluorescence staining
The purified passage cells were inoculated onto
polylysine-coated slides at 108/L, incubated for 48 hours, fixed by paraformaldehyde, blocked with ABV solution, stained with S-100 protein dyes, and finally photographed under
fluorescence microscope.
Main outcome measures
Schwann cell morphology, attaching speed and cell purity, Schwann cell growth curves, and cell identification by S-100 immunofluorescence
Design, enforcement, and evaluation
The first and fifth authors designed this study. All authors performed experimental procedure. The fourth and fifth authors evaluated experimental data. All authors received professional trainings. A blind method evaluation was not employed.
Statistical analysis
All data were statistically processed by the first author using SPSS 17.0 software and expressed as Mean ± SD. t test and one-way analysis of variance were employed. A level of P <
0.05 was considered statistically significant.
RESULTS
Observation of Schwann cell morphology and purity by inverted phase microscope
In the neonatal group, after 20 minutes of culture, neonatal rat Schwann cells began to adhere to the flask wall and exhibited a round body surrounded by a halation circle with strong refraction.
Four hours later, individual cells exhibited an oval shape, and most of cells took a round or oval appearance. Forty-eight hours later, most of cells exhibited a spindle-shaped appearance, with small cell body, strong refraction. Schwann cells stretched out thin, long processes with intumescent, irregular, thin and flat ends. Large processes had small spinous processes, which interweaved with others. Granule-free cytoplasm contained small, round nucleus, in which nucleolus was visible. Schwann cell purity was over 97%
(Figure 1).
Figure 1 After 48-hour primary culture, neonatal rat Schwann cells stretched out processes and exhibited strong refraction (Inverted phase microscope, × 200)
In the adult group, after 45 minutes of culture, adult rat
Schwann cells began to adhere to the flask wall. Six hours later, cell morphology slightly changed. Forty-eight hours later, Schwann cell morphology was similar to neonatal rat Schwann cells, but cell body was bigger, fraction was weaker, processes were shorter and thicker, and exhibited a comma-shaped intumescence. There were few processes, which were hardly connected with adjacent cells. The round or oval nucleus was surrounded by fine granular cytoplasm and contained a visible nucleolus. Schwann cell purity was over 96% (Figure 2).
Proliferative capacity of Schwann cells
The proliferative capacity of Schwann cells from each group was relatively weak, but following passage, the proliferative capacity was gradually strengthened (P < 0.01), and peaked at the third generation. There was no significant difference in cell proliferative capacity between the 3rd and 4th generations in the same group (P > 0.05). There was significant difference in cell proliferative capacity between the neonatal and adult groups at the 2nd generation (P < 0.05). Significant difference also existed between the two groups at the 3rd, 4th generations (P <
0.01, Figure 3).
Cell identification by S-100 immunofluorescence staining Most of purified Schwann cells exhibited positive green fluorescence in the cytoplasm (Figure 4).
DISCUSSION
The in vitro proliferation and purity of Schwann cells are relatively difficult owing to poor proliferative capacity and fibroblast contamination. With further research, pre-injury has been used to solve the problem of poor proliferative capacity of Schwann cells[20]. However, there has been no optimal treatment of fibroblast contamination. Based on the histological structure characteristics of the nerve and different attaching speeds of cells, the present study developed the extraction and purification methods of Schwann cells.
Removal of epineurium and perineurium
The nerve contains epineurium, perineurium, and endoneurium.
Each layer has considerable fibroblasts. Neonatal rat sciatic nerve is very tiny, so it is very difficult to excise the epineurium, in particular the perineurium, under the microscope. Adult rat sciatic nerve is very thick, so it is easy to excise the epineurium, but it remains difficult, however feasible, to excise the
perineurium. The present study excised rat epineurium and perineurium to reduce the source of fibroblasts as far as possible, which is different from previous findings[21]. Because perineurium and endoneurium reside in the lateral myelin sheath, so fibroblast abscission is first primarily performed. In the present study, in the first inoculation after digestion, nerve tissue should be preserved as far as possible, and the Figure 2 After 48-hour primary culture, adult rat Schwann
cells exhibited similar morphology to neonatal rat Schwann cells, but cell body was larger, fraction was weaker, and processes were shorter and thicker (Inverted phase microscope, × 200)
Figure 3 Schwann cell proliferation curves from the neonatal and adult groups, as detected by methyl thiazolyl tetrazolium microcolorimetry (P0: primary cells, P1-4: passage cells)
Neonatal group Adult group 0.8
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Proliferative capacity
P0 P1 P2 P3 P4
Schwann cell generations
Figure 4 Schwann cells positive for S-100 protein exhibited green fluorescence in the cytoplasm (×100)
a: Neonatal group
b: Adult group
suspended fibroblasts were removed by attachment (Figure 5a);
in the second inoculation, nerve tissue blocks were rapidly re-suspended to separate the fibroblasts from tissue for later differential attachment, which reduces the source of fibroblasts and avoids decreased Schwann cell activity caused by long-term retention in in vitro tissue (Figure 5b). These are different from previous findings[22-24].
Schwann cell morphology and attaching capacity After attachment, neonatal rat Schwann cells exhibited a well-stacked, splendent cell body. Other cell bodies or cell processes were seen passing through the passage between many Schwann cell bodies and bottom wall, which indicates a relatively weak attaching capacity. The large processes had intumescent, irregular, thin and flat ends, which attached to bottom wall. In addition, large processes stretched out small spinous processes, which also attached to bottom wall.
Adjacent processes were interwoven by small processes, demonstrating that Schwann cell attachment primarily depends on the ends of large processes and small processes. Adult rat Schwann cells exhibited similar morphology to neonatal rat Schwann cells, but cell body was larger, and fraction was weaker, demonstrating that neonatal and adult rat Schwann cells exhibited attaching capacity.
Attachment speed
A key to in vitro culture of Schwann cells is to discard the mixed fibroblasts[25]. Differential attachment has been a common method at home and abroad. Results from this study revealed that compared with the fibroblasts, neonatal Schwann cells exhibited a faster attaching speed, which was different from
previous findings[6, 23, 26], and the underlying mechanisms need to be investigated, while adult Schwann cells showed a slower attaching speed, which was consistent with previous results[13, 21, 23].
Generally, the proliferative capacity of cells would be decreased with aging. The present study detected the proliferative capacity of Schwann cells by MTT microcolorimetry, demonstrating that neonatal rat Schwann cells exhibited stronger proliferative capacity than adult rat Schwann cells and that the use of MTT microcolorimetry can acquire high-activity Schwann cells. In addition, during the process of culture, purification, the drugs inhibiting fibroblast growth was not used, which avoids the drug toxicity-caused injury to Schwann cells.
REFERENCES
[1] Reynolds ML, Woolf CJ. Reciprocal Schwann cell-axon interactions. Curr Opin Neurobiol. 1993;3(5):683-693.
[2] Bergey PD, Axel L. Focal hypertrophic cardiomyopathy simulating a mass: MR tagging for correct diagnosis. AJR Am J Roentgenol. 2000;174(1):242-244.
[3] Rhona M, Kristjan RJ, Angela B, et al. Schann cells as regulations of nerve development. Physio paris. 2002;96:17-24.
[4] Langer R. Tissue Engineering. Science. 1993;260(5110):920.
[5] Labrador RO, Butí M, Navarro X. Influence of collagen and laminin gels concentration on nerve regeneration after resection and tube repair. Exp Neurol. 1998;149(1):243-252.
[6] Mei YF, Wang J, Yan YH, et al. The method of cold enzyme digestion applied in isolating and culturing schwann cells from the newborn rat. Shenjing Jiepouxue Zazhi. 2007;23(2):205-208.
[7] Wood PW. Separation functional Schwann cells and nerons from normal peripheral nerve tissue. Brain Res. 1976;115:361.
[8] Pleasure D, Kreider B, Shuman S, et al. Tissue culture studies of Schwann cell proliferation and differentiation. Dev Neurosci.
1985;7(5-6):364-373.
[9] Huang M, Luo ZJ, Xiao W, et al. Experimental study of separation and purification of Schwann cells by immunomagnetic heads method. Zhongguo Jiaoxing Waike Zazhi. 2008;16(8):599-601.
[10] Assouline JG, Bosch EP, Lim R. Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies. Brain Res. 1983;277(2):389-392.
[11] Kreider BQ, Messing A, Doan H, et al. Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion. Brain Res. 1981;207(2):433-444.
[12] Zhu J, Wang WJ, Ding WL. An efficient method for culturing schwann cell in vitro. Jiepou Kexue Jinzhan. 2007;13(2):144-147.
[13] Pannunzio ME, Jou IM, Long A, et al. A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods. 2005;149(1):74-81.
[14] Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods. 2003;122(2):195-200.
[15] Yang J, Wu L, Huang T, et al. The experimental study on the improved method of Schwann cells' passage culture. Zhongguo Linchuang Jiepouxue Zazhi. 2005;23(1):17-23.
[16] Askanas V, Engel WK, Dalakas MC, et al. Human Schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol. 1980;37(6):329-337.
[17] Brockes JP, Fields KL, Raff MC. Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res. 1979;165(1):105-118.
[18] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[19] Ma X, Yu GY, Zhang ZK, et al. Purification of cultured Schwann cells derived from wallerian degenerating facial nerves in rats.
Jiepou Xuebao. 2001;32(3):205.
[20] Keilhoff G, Fansa H, Schneider W, et al. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods.
1999;89(1):17-24.
[21] Qu W, Jiang HJ, Li DJ, et al. Experimental study of Schwann cell culture from adult SD rats. Shiyong Shouwaike Zazhi.
2008;22(1):30-32.
[22] Yang J, Zhang LS, Wu L, et al. Effects of conditioned mediums of fibroblast on the growth and proliferation of Schwann cells.
Zhongguo Linchuang Kangfu. 2004;35(8):7956-7957.
Figure 5 Twenty-four hours after two inoculations (×100) a: The first inoculation
b: The second inoculation, arrow indicates a tissue block
医学英文句型正误辨析：本刊英文部
[23] Pan LC, Yin ZS, Wang W, et al. Experimental study of methods of culturing and purifying newborn and adult rat Schwann cells.
Zhongguo Kangfu Yixue Zazhi. 2005;20(11):817-819.
[24] Linan XF, Hou TS, Fu Q, et al. Study of culturing primary Schwann cells of rats by explant and enzyme digestion technique. Zhongguo Jizhu Jisui Zazhi. 2008;18(9):703-706.
[25] Han Y, Tang CW, Wang JB, et al. Experimental study of purifying culture Schwann cells with Geneticin. Zhonghua Xianwei Waike Zazhi. 1997;20:277-280.
[26] Huang T, Qin JQ, Wang HL, et al. Culture and proliferation of Schwann cells obtained from lesioned peripheral nerves of newborn rats. Zhongguo Linchuang Kangfu.
2004;8(17):3294-3295.
SD 大鼠许旺细胞提取及纯化：乳鼠和成年鼠的对照*
刘志新1，宋宝辉2，赵富生1，李月珍1，梁 军1 (牡丹江医学院，1组织学与胚胎学教研室，2原生物学教研室，黑龙江省牡丹江市 157011)
刘志新，男，1981 年生，甘肃省会宁县人，
汉族，2006 年牡丹江医学院毕业，助教，主 要从事神经系统损伤修复的研究。
通讯作者：梁 军，副教授，牡丹江医学院 组织学与胚胎学教研室，黑龙江省牡丹江市 157011
牡丹江医学院科研项目(B200812)*
摘要
背景：许旺细胞是神经创伤修复的种子细胞，
获取大量高纯度、高活性许旺细胞是研究的 关键。
目的：比较乳鼠和成年鼠许旺细胞的体外培 养、纯化和形态学的差别，探讨简单可行的、
可以获得高纯度许旺细胞的培养方法。
方法：出生1~3 d SD 大鼠 20 只和成年大鼠 10 只(体质量 150~200 g)，实验按细胞的来 源分为新生组和成年组。双酶分步消化，二 次接种差速分离纯化细胞；倒置显微镜观察 细胞形态、贴壁速度；细胞计数，纯度计算；
MTT 法检测细胞增殖能力，绘制两组许旺细 胞的增殖曲线，判定增殖速度；S-100 免疫 化学法鉴定细胞。
结果与结论：乳鼠的许旺细胞贴壁速度快于 成纤维细胞，成鼠的许旺细胞贴壁速度慢于
成纤维细胞，两组许旺细胞纯度均达96%以
上；MTT 法检测两组许旺细胞增殖均活跃，
由增殖曲线显示乳鼠许旺细胞增殖更快(P <
0.05)；S-100 免疫化学反应均呈阳性。提示
双酶分步消化，二次接种差速分离纯化细胞，
可以获取纯度高、活性良好的许旺细胞；乳 鼠许旺细胞的增殖、贴壁能力更强。
关键词：许旺细胞；细胞培养；坐骨神经；
乳鼠；成年鼠
doi:10.3969/j.issn.1673-8225.2010.06.035 中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2010)06-01115-05 刘志新，宋宝辉，赵富生，李月珍，梁军.SD 大鼠许旺细胞提取及纯化：乳鼠和成年鼠的 对 照[J]. 中 国 组 织 工 程 研 究 与 临 床 康 复 , 2010,14(6):1115-1119.
[http://www.crter.org http://cn.zglckf.com]
(Edited by Wang XD/Song LP/Wang L)
中文 修后 修前
首先在脑电图上用空间过滤法消除伪迹，然 后寻找出痫样放电，比较致痫源导联组合及 相关系数；参考自由导联组合以增强痫源电 位、抑制背景脑电的干扰，比较各导联痫样 放电时程，将找出的痫样波叠加以增加信噪 比，在头相镶嵌图上确认偶极子的部位，将
分析出的原发痫波偶极子与MRI图像融合成
为具有三维坐标的偶极子图像。典型病例脑 电联合偶极子定位。
The electroencephalogram was processed off-line to filter out scanner artifacts using the spatial filtering method. Epileptiform dis- charges were detected. Epileptogenic foci lead combinations and correlation coefficients were compared. Electric potential of the epileptogenic focus was enhanced, and the interference of background electroencephalogram was re- sisted. Duration of epileptiform discharges was compared between different lead combina- tions. Epileptiform waves were overlapped to increase the signal-to-noise ratio. Dipole loca- lization was defined. Primary epileptiform wave dipole and magnetic resonance imaging images were integrated into a dipole image with three-dimensional coordinates. An electroen- cephalogram combined with dipole localization in a typical case is shown.
The electroencephalogram was processed off-line to filter out scanner artifacts by using the spatial filtering method. Epileptiform dis- charge was detected. Epileptogenic foci lead combinations and correlation coefficients were compared. Electric potential of the epilepto- genic focus was enhanced, and the interfe- rence of background electroencephalogram was resisted. Duration of epileptiform dis- charges was compared between combina- tions of different leads. Epileptiform waves were overlapped to raise the signal-to-noise ratio. Dipole localization was defined. Primary epileptiform wave dipole and magnetic re- sonance imaging images were integrated into a dipole image with three-dimensional coor- dinates. Electroencephalogram combined with dipole localization in a typical case is shown.
细胞计数：根据Paxinos and Watson 图谱和 Alvarez-Bolado and Swanson 大鼠脑发育图 谱，选择所有切片的中脑黑质作为阳性细胞 计数区，利用光学显微镜，在低倍到高倍等 不同的倍数下，观察各组N-cadherin 阳性细 胞形态。然后，在高倍视野下，按从头侧端
至尾侧端的方向，隔2 张取 1 张切片进行细
胞计数，计数每张切片单位面积(实验中以 100×100 μm2为单位面积)内 N-cadherin 阳 性细胞数目。
Cell counting: According to coordinates from the Paxinos and Watson rat atlas, as well as the Alvarez-Bolado and Swanson rat atlas, subs- tantia nigra sections were identified to quantify positive cells using an optical microscope.
Morphology of N-cadherin-positive cells was observed under varied magnifications.
N-cadherin-positive cells were quantified in a 100 × 100-µm2 grid in 1:3 sections.
Cell count: According to coordinates from the Paxinos and Watson rat atlas, as well as the Alvarez-Bolado and Swanson rat atlas, subs- tantia nigra sections were identified to quantify positive cells using an optical microscope.
Morphology of N-cadherin-positive cells was observed under varied magnification.
N-cadherin-positive cells were quantified in 100 × 100-µm2 grid of 1:3 sections.
ISSN 1673-8225 CN 21-1539/R 2010年版权归《中国组织工程研究与临床康复》杂志社所有
