Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture
PC12 细胞培养中建立的爆炸冲击诱导的初级脑损伤模型   

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Journal of Neuroscience Research
Sep 2015



While it is understood that structural damage occurs at the cellular level from the traumatic brain injury event, the effect on functional activity remains largely unknown. Simplified models such as in vitro models of primary explosive blast are critically needed to deconvolute mechanisms of cellular damage. This protocol details an in vitro indoor experimental system setup (Zander et al., 2015) using real military explosive charges to more accurately represent battlefield blast exposure, and probe the effects of primary explosive blast on dissociated neurons.

Keywords: Primary explosive blast model (初级炸药爆炸模型), Blast induced traumatic brain injury model (创伤性脑损伤诱导的爆炸模型), Cell culture (细胞培养)

Materials and Reagents

  1. 12 mm circular glass coverslips (Fisher Scientific, catalog number: 12-545-80 )
  2. 24-well plates (Corning, FalconTM, catalog number: 351147 )
  3. Sterile SealPlate® covers (Excel Scientific)
  4. PC12 cells (ATCC, catalog number: CRL-1721 )
  5. Mouse laminin (Corning, catalog number: 354239 )
  6. 100% ethanol
  7. Polylysine (Sigma-Aldrich, catalog number: P5899 )
  8. RPMI 1640 (Corning, catalog number: 10-041-CV )
  9. Horse serum (Corning, catalog number: 35-030-CV )
  10. Fetal bovine serum (FBS) (Fisher Scientific, catalog number: SH3007003IH )
  11. Dulbecco Modified Eagle’s Medium (DMEM) (Corning, catalog number: 10-013-CV )
  12. Calf serum (GE Healthcare Life Sciences, HycloneTM, catalog number: SH30072.03 )
  13. Nerve growth factor (NGF) (Corning, catalog number: 356004 )
  14. Spherical 1.77 grams/cm3 cyclotrimethylene trinitramine Class 5 (RDX Class V) charges
  15. HEPES (Fisher Scientific, catalog number: BP299500 )
  16. 10-gallon poly (methyl methacrylate) (PMMA)
  17. Calcein-AM (Thermo Fisher Scientific, catalog number: C3099 )
  18. Ethidium homodimer-1 (Thermo Fisher Scientific, catalog number: E1169 )
  19. Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: BP399500 )
  20. Lactate dehydrogenase cytotoxicity assay kit (LDH) (Abnova, catalog number: KA0785 )
  21. RIPA buffer (Alfa Aesar, catalog number: J62189-AP )
  22. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  23. The micro BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23235 )
  24. Hank’s Balanced Salt Solution (HBSS) (Corning, catalog number: 21-022-CV )
  25. Calcein (MP Biomedicals, catalog number: 190167 )
  26. Penicillin/Streptomycin, 50x (Corning, catalog number: 30-001-Cl )
  27. Antibiotic/antimycotic (see Recipes)
  28. Growth/complete medium (see Recipes)
  29. Differentiation medium (see Recipes)


  1. Incubator
  2. Aquarium
  3. Three piezoelectric high frequency dynamic pressure sensors (PCB Piezotronics, model: 102A )
  4. Sterile hood
  5. Oven
  6. Zeiss LSM5 Pascal equipped with Epiplan-Neofluar lenses
  7. Confocal laser scanning microscopy (CLSM)


  1. ImageJ v 1.34
  2. Zeiss LSM software (v


  1. Experimental setup and preparation
    1. Cell sample preparation
      1. Clean 12 mm circular glass coverslips using piranha etch [H2SO4:H2O2 = 70:30 (v/v)] for 30 min and then rinse thoroughly with deionized (DI) water.
      2. Sonicate slides 10 min/time, 3 times in 100% ethanol to sterilize.
      3. Place sterile coverslips in 24-well plates and coat in 100 µg/ml sterile polylysine solution for 30 min.
      4. Rinse the slides three times in sterile DI water and allow to air dry.
      5. Incubate the slides in 10 µg/ml sterile laminin at 4 °C overnight.
      6. After protein attachment, rinse the slides three times with sterile PBS and use immediately for cell culture.
      7. Culture cells in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum and 1% antibiotic/antimycotic “complete medium” at 37 °C and 5% CO2. Culture cells according to the supplier’s instructions.
      8. Seed cells at a density of 5,000 cells/well on the 12 mm coverslips in the 24-well plates in high-glucose DMEM with 1% horse serum, 0.5% calf serum and 1% antibiotic/antimycotic “differentiation medium”.
      9. After 24 h, add 100 ng/ml NGF to the differentiation medium. Subject the cells to blast 5 days after adding NGF.
    2. Blast induced injury in vitro setup
      1. Use a spherical 1.7 g/cm3 cyclotrimethylene trinitramine Class 5 charge to generate the blast (Figure 1).

        Figure 1. Explosive charge

      2. The charge standoff distance to the neuron cell chamber is measured as a clear spacing between the charge and the outer wall of the aquarium.
      3. Adjust the explosive charge standoff distance to generate different pressure exposure inside the cell culture wells. For multiple blast exposure, keep the samples inside the tank, and separate each exposure by 5-7 min intervals.
        Note: The time interval does not necessarily reflect the actual time between blasts on the battlefield. The interval is the minimum amount of time needed to clear the blast chamber for re-entry and set-up the next explosive.
      4. Three piezoelectric high frequency dynamic pressure sensors were used to measure the shockwave overpressure duration above the cultured samples (Figure 2).

        Figure 2. Pressure Sensor Holder. A. Piezoelectric pressure sensors in horizontal position inside testing aquarium. B. Piezoelectric pressure sensors in vertical position outside testing aquarium.

      5. All pressure gauges were mounted on top of the cell culture plate with a custom-designed lid, submerged approximately 4 inches underwater, and positioned side-on to the blast wave direction.
      6. The caps were designed such that the pressures in different rows or columns of the well plate could be measured by moving the pressure sensors to the desired locations.
      7. Two pencil gauges were positioned in front of the aquarium to measure the pressure in air before the shock wave moves into the water interface (Figures 3 and 4).

        Figure 3. Blast induced cell injury overview

        Figure 4. Schematic diagram showing positions of RDX explosive charge, aquarium tank, and sample

      8. Pressure-time history and peak pressure were recorded for each blast.
    3. Blast-induced injury of cells
      1. Just prior to the blast experiment in a sterile hood, remove the plastic well plate lid in a sterile hood and add sterile HEPES buffer (10 mM) in the well plate. Seal the well plates with sterile SealPlate® covers. Placed in a plastic bag to maintain sterility of the cultures. Transport the samples to the blast site and put the samples in an oven at 37 °C until the experiments.
      2. Submerge the samples horizontally on a sample holding stage in a 10-gallon poly (methyl methacrylate) (PMMA) aquarium containing water heated to 37 °C, as displayed in Figure 2.
      3. Mount, secure, and immerse under water horizontally the neuronal cell line culture well plates in the middle of the aquarium with the cells in the first row (1) facing the shock front.
      4. All blast experiments are performed without the well plate lid.
      5. Put the control samples in the incubator for the duration of the experiment.
      6. Put sham cells in the aquarium for 20 min (typical length of a triple blast experiment).
      Note: Control samples and sham samples were not identical. Control samples remained in the incubator in the lab while sham samples traveled to the blast site, were submerged in the aquarium tank and held in an oven (without CO2) for the duration of the blast experiment.
    4. Pressure measurements during the blast cell exposure (Figure 5)
      Subject the cells to single or multiple blast insults five days after seeding cells in differentiation medium supplemented with 100 ng/ml NGF. To minimize the movement, ensure good attachment of the cells to the coverslip. In addition, the dimensions of the well plate and coverslips need to be chosen carefully so that the coverslip fits tightly within the well.

      Figure 5. Blast-induced injury of cells. A. Aquarium tank showing pencil gauge. B. Sample well plate submerged in aquarium tank showing location of pressure sensors on lid.

  2. Cell analysis
    1. Cell morphology and viability analysis
      For single blast exposure, assess the cell viability at 2 and 24 h post-blast by staining the cells with calcein-AM and ethidium homodimer-1, following the protocol outlined by Life TechnologiesTM.
      1. Briefly, remove the medium and rinse the cells with PBS three times.
      2. Incubate the cells in a PBS solution containing 2 µM calcein-AM and 4 µM ethidium homodimer-1 for 30 min at 37 °C.
      3. Using the 543 nm and 488 nm lasers for the viability assay: Image samples by confocal laser scanning microscopy (CLSM) on a Zeiss LSM5 Pascal equipped with Epiplan-Neofluar lenses. Image the cells in the multi-track mode with a 543-nm laser with a minimum of n = 5 random areas for each of a minimum of 3 replicate samples using the 10x and 20x objectives.
      4. CLSM are processed using image area analysis tools in ImageJ v 1.34, National Institutes of Health. Convert images to binary and thresholds set for each channel (live cells and dead cells).
      5. Calculate the percent area using the analyze particles tool.
      6. Correct the smaller cell size for the dead cells compared to live cells by multiplying the percentage of area of dead cells by the average live cell size/average dead cell size.
      7. Calculate percentage of live cells by taking the live cell percent area over the total live and corrected dead cell percent area.
      8. Assess neurite morphology via the calcein AM stain (Figure 6).
      9. Measure the length and bead diameter using the Zeiss LSM software.

      Figure 6. Viability of PC12 neurons 24 h after exposure to single and multiple explosive blasts of ca. 32 psi. I: CSLM images with live/dead stain. (A) One blast, (B) Two blasts, (C) Three blasts. Arrows indicate selected dead cells. Insets: magnified images of dead cells. II. Viability quantification. *P < 0.05 compared with control, sham, and one blast. Scale bars = 100 µm in A-C; 50 µm for insets

    2. Membrane permeability assays
      1. Sample media from the extracellular bath at 1, 2, 4 and 24 h post-blast.
      2. Perform the LDH assay according to the manufacturer’s instructions.
      3. Remove the media and rinse the samples with PBS and dry the coverslips for few minutes.
      4. Add approximately 20 µl of RIPA buffer with 1% (vol) protease inhibitor to each coverslip.
      5. Scrap the cells immediately and analyze using the Micro BCA protocol or put on ice for 30 min and then stored at -80 °C until analysis.
      6. Normalize all values to the total protein determined by a micro BCA protein assay.
      7. Probe calcein uptake by rinsing the samples with HBSS (without calcium and magnesium) and then incubating in 0.3 mM calcien in HBSS for 10 min.
      8. Rinse the samples thoroughly with HBSS and image using the 488 nm laser on the CLSM system.
      9. Fix imaging gains and offsets to allow semi-quantitative comparison between samples. 
      10. Collect phase-contrast images to ensure cells are in focus.
      11. Measure fluorescence intensities by selecting individual cells using the region of interest (ROI) feature and calculating the mean intensity using the histogram feature using the software (Figure 7).

      Figure 7. Membrane permeability changes as a function of calcein dye uptake of PC12 neurons exposed to explosive blast of ca. 32 psi. *P < 0.05 compared with control and sham, n = 3 


  1. Antibiotic/antimycotic
    10,000 IU penicillin
    10,000 µg/ml streptomycin
    25 g/ml amphotericin 
  2. Growth/complete medium
    High-glucose DMEM
    10% heat-inactivated horse serum
    5% fetal bovine serum
    1% antibiotic/antimycotic
  3. Differentiation medium
    High-glucose DMEM
    1% horse serum
    0.5% calf serum
    1% antibiotic/antimycotic
    100 ng/ml NGF


This research was supported by the U.S. Army Research Laboratory’s Director’s Science Initiative, DSI-13-WMR-2D-005.


  1. Zander, N. E., Piehler, T., Boggs, M. E., Banton, R. and Benjamin, R. (2015). In vitro studies of primary explosive blast loading on neurons. J Neurosci Res 93(9): 1353-1363.


细胞毒性CD8 + T细胞能够通过其T细胞受体(TCR)与主要组织相容性复合物(MHC)分子呈递的小免疫原性肽(抗原)之间的特异性相互作用特异性识别和杀死靶细胞。抗原特异性细胞毒性T细胞的抗原识别能力和体外裂解活性可以在所谓的铬51(<51> Cr)释放测定中功能性评估,其是几乎50年前在我们的机构中​​发展起来的(Brunner等人,1968年)。放射性标记的内源性抗原呈递缺陷的细胞[例如,用于抗原呈递(TAP)缺陷型T2细胞的转运蛋白],并用感兴趣的MHC稳定转染(例如 ,HLA-A2 sup + +)通常在这个4小时测定期间用作靶标。或者,内源性呈递免疫原性抗原的Cr标记的病毒感染或肿瘤细胞系可以作为靶细胞(例如,用于评估肿瘤识别)。
  在肽滴定测定(部分A)中,用抗原性肽的系列稀释物对放射性标记的靶细胞进行脉冲,并在效应物(例如,CD8 + T细胞克隆)与靶细胞( Cr-T2细胞)比(E:T)为10:1的混合物在96孔V型底板中37℃温育4小时。在肿瘤杀伤试验(B部分)中,将细胞毒性CD8 +效应细胞以不同的比率与支原体51-标记的靶细胞系(通常以E:T比例在特异性抗原肽(1μM)的存在或不存在的情况下为30:1,10:1,3:1和1:1),并在37℃下孵育4小时。在测试结束时,使用液体闪烁计数器在上清液中测定从裂解的靶细胞释放的放射性的量。然后计算特异性裂解的百分比以及EC 50(即最大杀死的50%)和EMax值,提供关于抗原特异性功能亲合力的定量信息(即,基于通过限定的TCR的抗原识别和分析的T细胞的最大杀伤能力的T细胞功能的相对效率)。...

关键字:初级炸药爆炸模型, 创伤性脑损伤诱导的爆炸模型, 细胞培养


  1. 12mm圆形玻璃盖玻片(Fisher Scientific,目录号:12-545-80)
  2. 24孔板(Corning,Falcon ,目录号:351147)
  3. 无菌SealPlate ®盖(Excel Scientific)
  4. PC12细胞(ATCC,目录号:CRL-1721)
  5. 小鼠层粘连蛋白(Corning,目录号:354239)
  6. 100%乙醇
  7. 聚赖氨酸(Sigma-Aldrich,目录号:P5899)
  8. RPMI 1640(Corning,目录号:10-041-CV)
  9. 马血清(Corning,目录号:35-030-CV)
  10. 胎牛血清(FBS)(Fisher Scientific,目录号:SH3007003IH)
  11. Dulbecco改良的Eagle培养基(DMEM)(Corning,目录号:10-013-CV)
  12. 小牛血清(GE Healthcare Life Sciences,Hyclone TM ,目录号:SH30072.03)
  13. 神经生长因子(NGF)(Corning,目录号:356004)
  14. 球形1.77克/厘米3 环三亚甲基三硝胺5类(RDX Class V)费用
  15. HEPES(Fisher Scientific,目录号:BP299500)
  16. 10加仑聚(甲基丙烯酸甲酯)(PMMA)
  17. Calcein-AM(Thermo Fisher Scientific,目录号:C3099)
  18. 乙锭同型二聚体-1(Thermo Fisher Scientific,目录号:E1169)
  19. 磷酸盐缓冲盐水(PBS)(Fisher Scientific,目录号:BP399500)
  20. 乳酸脱氢酶细胞毒性测定试剂盒(LDH)(Abnova,目录号:KA0785)
  21. RIPA缓冲液(Alfa Aesar,目录号:J62189-AP)
  22. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  23. 微BCA蛋白测定试剂盒(Thermo Fisher Scientific,目录号:23235)
  24. Hank's平衡盐溶液(HBSS)(Corning,目录号:21-022-CV)
  25. Calcein(MP Biomedicals,目录号:190167)
  26. 青霉素/链霉素,50x(Corning,目录号:30-001-Cl)
  27. 抗生素/抗真菌剂(见配方)
  28. 生长/完全培养基(见配方)
  29. 分化介质(参见配方)


  1. 孵化器
  2. 水族馆
  3. 三个压电高频动态压力传感器(PCB Piezotronics,型号:102A)
  4. 无菌罩
  5. 烤箱
  6. Zeiss LSM5 Pascal配备了Epiplan-Neofluar镜头
  7. 共聚焦激光扫描显微镜(CLSM)


  1. ImageJ v 1.34
  2. 蔡司LSM软件(v


  1. 实验设置和准备
    1. 细胞样品制备
      1. 使用piranha蚀刻清洁12mm圆形玻璃盖玻片[H 2 SO 4:H 2 O 2 2 = 70: 30(v/v)] 30分钟,然后用去离子(DI)水彻底冲洗
      2. 超声处理幻灯片10分钟/次,在100%乙醇中消毒3次
      3. 将无菌盖玻片置于24孔板中,并在100μg/ml无菌聚赖氨酸溶液中包被30分钟。
      4. 在无菌去离子水中冲洗载玻片三次,并使其风干
      5. 将载玻片在10μg/ml无菌层粘连蛋白中在4℃下孵育过夜
      6. 蛋白质附着后,用无菌PBS冲洗载玻片三次,并立即用于细胞培养
      7. 在37℃和5%CO 2下,在补充有10%热灭活的马血清和5%胎牛血清和1%抗生素/抗真菌"完全培养基"的RPMI 1640培养基中培养细胞。 根据供应商的说明培养细胞。
      8. 在具有1%马血清,0.5%小牛血清和1%抗生素/抗真菌"分化培养基"的高葡萄糖DMEM中的24孔板中的12mm盖玻片上以5,000细胞/孔的密度种细胞。
      9. 24小时后,向分化培养基中加入100ng/ml NGF。 使细胞在加入NGF后5天进行blast。
    2. 爆炸诱导的损伤体外设置
      1. 使用球形1.7g/cm 3环三亚甲基三硝胺5级电荷产生爆炸(图1)。


      2. 到神经元细胞室的电荷远离距离被测量为电荷和水族箱的外壁之间的清晰间隔。
      3. 调整爆炸负荷间隔距离,以在细胞培养孔内产生不同的压力暴露。对于多次爆炸暴露,将样品保持在罐内,并将每次暴露分开5-7分钟间隔。
      4. 使用三个压电高频动态压力传感器来测量培养样品上方的冲击波超压持续时间(图2)。

        图2.压力传感器支架 A.压力传感器位于水族箱内的水平位置。 B.压力传感器位于水族箱外的垂直位置。

      5. 所有压力表都安装在细胞培养板的顶部,具有定制设计的盖子,浸没在水下约4英寸,并且定位在冲击波方向的侧面。
      6. 盖被设计成使得可以通过将压力传感器移动到期望位置来测量孔板的不同行或列中的压力。
      7. 两个铅笔量规定位在水族箱前面,以测量在冲击波进入水界面之前空气中的压力(图3和图4)。

        图3. blast诱导的细胞损伤概述


      8. 记录每次爆破的压力 - 时间历史和峰值压力。
    3. 爆炸引起的细胞损伤
      1. 恰好在无菌罩中的爆炸实验之前,在无菌罩中移除塑料孔板盖,并在孔板中加入无菌HEPES缓冲液(10mM)。用无菌SealPlate盖密封孔板。置于塑料袋中以保持培养物的无菌性。将样品运送到爆炸现场,并将样品放在37℃的烘箱中直到实验
      2. 将样品水平浸没在装有加热至37℃的水的10加仑聚甲基丙烯酸甲酯(PMMA)水族箱中的样品保持台上,如图2所示。
      3. 在水下水平安装,固定和浸没神经元细胞系培养孔板在水族箱的中间,其中第一排(1)的细胞面向休克前沿。
      4. 所有爆炸实验均在没有孔板盖的情况下进行
      5. 在实验期间将对照样品放入培养箱中。
      6. 将假细胞在水族箱中20分钟(三重爆炸实验的典型长度)。
      注意:对照样品和假样品不相同。对照样品保留在实验室的培养箱中,同时将运送到喷丸位点的假样品浸没在水族箱中并在喷射实验期间保持在烘箱(无CO 2)中。/em>
    4. 在胚细胞暴露期间的压力测量(图5)
      使细胞在补充有100ng/ml NGF的分化培养基中接种细胞5天后进行单次或多次blast攻击。为了最小化运动,确保细胞与盖玻片的良好附着。此外,需要仔细选择孔板和盖玻片的尺寸,使得盖玻片紧密地配合在孔内。

      图5.乳腺诱导的细胞损伤 A.显示铅笔量的水族箱。 B.样品孔板浸没在水族箱中,显示盖子上压力传感器的位置
  2. 细胞分析
    1. 细胞形态学和生存力分析
      对于单爆炸暴露,根据Life Technologies TM概述的方案,通过用钙黄绿素-AM和乙锭同型二聚体-1染色细胞来评估胚细胞后2和24小时的细胞活力。
      1. 简而言之,取出培养基并用PBS冲洗细胞三次。
      2. 将细胞在含有2μM钙黄绿素-AM和4μM乙锭同型二聚体-1的PBS溶液中在37℃下孵育30分钟。
      3. 使用543nm和488nm激光用于生存力测定:通过共聚焦激光扫描显微术(CLSM)在配备有Epiplan-Neofluar透镜的Zeiss LSM5 Pascal上的图像样品。使用543nm激光器以多道模式对单元格进行成像,使用10x和20x物镜,最少3个重复样本中的每个最小n = 5个随机区域。
      4. CLSM使用ImageJ v 1.34中的图像区域分析工具进行处理,National Institutes of Health。将图像转换为二进制和为每个通道设置的阈值(活细胞和死细胞)
      5. 使用分析粒子工具计算面积百分比。
      6. 通过将死细胞面积的百分比乘以平均活细胞大小/平均死细胞大小来校正死细胞与活细胞相比的更小的细胞大小。
      7. 通过取活细胞百分比面积占总活率和校正死细胞百分比面积计算活细胞百分比。
      8. 通过钙黄绿素AM染色评估神经突形态(图6)
      9. 使用Zeiss LSM软件测量长度和珠粒直径。

      图6.暴露于单个和多个爆炸性爆炸物后24小时的PC12神经元的活力。 32psi。 I:具有活/死染色的CSLM图像。 (A)一次爆炸,(B)两次爆炸,(C)三次爆炸。 箭头表示选择的死细胞。 插图:死细胞的放大图像。 II。 活力定量。 * P 0.05与对照,假手术和单爆炸相比。 在A-C中比例尺=100μm; 插图为

    2. 膜渗透性测定
      1. 在爆发后1,2,4和24小时从细胞外浴中取样培养基
      2. 根据制造商的说明进行LDH测定
      3. 取出介质,用PBS冲洗样品,并擦拭盖玻片几分钟
      4. 向每个盖玻片中加入约20μl含有1%(vol)蛋白酶抑制剂的RIPA缓冲液
      5. 立即刮取细胞,使用Micro BCA方案分析或放在冰上30分钟,然后储存在-80℃直到分析。
      6. 将所有值归一化为从微BCA蛋白测定确定的总蛋白
      7. 通过用HBSS(无钙和镁)冲洗样品,然后在0.3mM钙蛋白的HBSS中孵育10分钟来探测钙黄绿素摄取。
      8. 用HBSS彻底冲洗样品,使用CLSM系统上的488 nm激光进行成像。
      9. 修正成像增益和偏移量,以便在样本之间进行半定量比较。
      10. 收集相位对比图像,以确保单元格在焦点。
      11. 通过使用感兴趣区域(ROI)特征选择单个细胞并使用软件使用直方图特征计算平均强度来测量荧光强度(图7)。

      图7.作为钙黄绿素染料摄取的PC12神经元的暴露于爆炸性爆炸的膜渗透性变化。 32 psi。 * P 0.05与对照组和假手术组相比,n = 3 


  1. 抗生素/抗真菌剂
    10,000 IU青霉素
    10,000μg/ml链霉素 25μg/ml两性霉素
  2. 生长/完全培养基
    5%胎牛血清 1%抗生素/抗真菌剂
  3. 分化媒介
    100 ng/ml NGF




  1. Zander,NE,Piehler,T.,Boggs,ME,Banton,R.and Benjamin,R。(2015)。  体外对原发性爆炸负荷对神经元的研究。 J Neurosci Res 93(9):1353-1363。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Piehler, T., Zander, N. and Benjamin, R. (2016). Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture. Bio-protocol 6(16): e1907. DOI: 10.21769/BioProtoc.1907.