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Dec 2020

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Muscle Cryoinjury and Quantification of Regenerating Myofibers in Mice
小鼠肌肉低温损伤及再生肌纤维的定量研究   

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Abstract

Cryoinjury, or injury due to freezing, is a method of creating reproducible, local injuries in skeletal muscle. This method allows studying the regenerative response following muscle injuries in vivo, thus enabling the evaluation of local and systemic factors that influence the processes of myofiber regeneration. Cryoinjuries are applicable to the study of various modalities of muscle injury, particularly non-traumatic and traumatic injuries, without a loss of substantial volume of muscle mass. Cryoinjury requires only simple instruments and has the advantage over other methods that the extent of the lesion can be easily adjusted and standardized according to the duration of contact with the freezing instrument. The regenerative response can be evaluated histologically by the average maturity of regenerating myofibers as indicated by the cross-sectional areas of myofibers with centrally located nuclei. Accordingly, cryoinjury is regarded as one of the most reliable and easily accessible methods for simulating muscle injuries in studies of muscle regeneration.

Keywords: Muscle injuries (肌肉损伤), Cryoinjury (低温损伤), Muscle regeneration (肌肉再生), Myofiber regeneration (肌纤维再生)

Background

Muscle injuries are a common type of injury that can result from various causes, from exertion to blast trauma. These injuries lead to a complex cascade of inflammatory and regenerative responses that result in the repair of the injured tissues. To characterize muscle injuries and the following responses and to evaluate the efficacy of potential treatments, various experimental models have been developed to replicate the process of muscle injury. In vitro approaches offer some practical advantages, and the advancement in 3D culture technique has enabled bioengineering of human cell-derived muscle tissue surrogates that may be used in muscle injury studies (Mills et al., 2017). In vivo models, however, remain imperative to understanding muscular injuries, as the complex processes involving the local and systemic responses that govern the overall regenerative process and the accompanying functional impairments/recovery can only be observed in a whole system of organisms. Rodent models of muscle injury are well-established and the most used systems for studying muscle regeneration today.


There are various modalities of injuries and their models, including laceration, crushing, blast injuries, volumetric muscle loss, myotoxins, and freezing. While all the modalities of injury go through common phases of healing (Huard et al., 2002; Jarvinen et al., 2005), some heterogeneity exists in the extent and quality of repair that follows regarding susceptibility/responsiveness of muscle stem cells to injury, tissue remodeling, inflammatory response, re-vascularization, and re-innervation (Caldwell et al., 1990; Lefaucheur and Sebille, 1995). Cryoinjuries, or traumatic injuries due to freezing, create localized injuries accompanied by substantial inflammatory and vascular responses (Warren et al., 2007), the extent of which can be adjusted and standardized according to the duration of the contact with the freezing instrument (Oh et al., 2016; Sinha et al., 2017; Endo et al., 2020). This makes it an easy and reliable method for generating reproducible outcomes, in contrast with the use of various myotoxins that lack comparative information needed for standardization. A step of skin incision is, however, required for the cryoinjury model, which could be considered a disadvantage over the use of injectable myotoxins. The cryoinjury model is applicable to a wide range of muscle injuries and is generally an appropriate choice for studying injuries without a substantial loss of muscle volume. In particular, cryoinjuries can simulate non-traumatic and mild traumatic injuries in striated muscles, including crushing injuries in skeletal muscle and myocardial infarction in cardiac muscles. Regenerative responses following cryoinjury can be identified histologically with clearly demarcated borders between injured and uninjured areas. Here, we describe a method of generating cryoinjuries in mouse skeletal muscle using dry ice and quantifying the regenerative response in myofibers.


Materials and Reagents

  1. 3.15% Chlorhexidine Gluconate and 70% Isopropyl Alcohol swabs (PDI, catalog number: S40750)

  2. 8-week-old C57BL/B male mice

  3. Isoflurane, USP (Patterson Veterinary, catalog number: 14043070406)

  4. Dry ice (or liquid nitrogen)

  5. Buprenorphine (Patterson Veterinary, catalog number: 07-892-5235)

  6. 3-0 PROLENE® polyprorylene suture (Ethicon, catalog number: 8665G)

  7. 10% formalin solution (Sigma, catalog number: HT501128)

  8. HistoPrepTM 70% Ethyl Alcohol (Fisher Scientific, catalog number: HC1500)

Equipment

  1. Stainless-steel metal rods, 5 mm diameter (Uxcell, catalog number: UX657206)

    Note: The metal rod should be chosen so its diameter to matches the muscle size. Inspect and measure the approximate width of the muscle and ensure that the diameter of the rod is smaller than the width of the muscle of all the animals undergoing cryoinjury.

  2. Surgical scissors and forceps (Roboz Surgical Instrument Co, catalog number: RS-5910; Symmetry Surgical, catalog number: 30-1185)

  3. Stainless steel scalpel, #15 (Healthcare Supply Pros, catalog number: MDS15215)

  4. Animal Clipper (Kent Scientific Corporation, catalog number: CL7300)

  5. Glass shell vials (Fisher Scientific, catalog number: 03-339-26A)

  6. Light microscope (Olympus, catalog number: UCMAD3)

  7. Shandom Microtome (Thermo Scientific, catalog number: A78100001K)

Software

  1. ImageJ (National Institute of Health, http://imagej.nih.gov/ij)

  2. GraphPad Prism version 9 (GraphPad Software, https://www.graphpad.com)

Procedure

  1. Cryoinjury

    1. Sterilize the surgical instruments and metal rod prior to the surgeries.

    2. Anaesthetize mice using isoflurane.

    3. Remove hair from the whole leg using a clipper and sterilize the skin anterior to the muscle of interest with chlorhexidine and alcohol swabs (Figure 1 Step 3).

    4. Using aseptic techniques, make an incision in the skin and muscle fascia and expose the underlying muscle using surgical scissors or a scalpel (Figure 1 Step 4a and 4b).

      Note: The size of the skin incision should be adjusted according to the size of the muscle and metal rod used (see Figure 1 Step 4a); it must allow the skin to be retracted wide enough so that the edges of the incised skin do not touch the metal rod. This usually requires an incision that spans over 80% of the muscle in the longitudinal direction, with the center of the incision aligning with that of the underlying muscle.

    5. Cool a metal rod in dry ice for 5 min, wipe the rod with the chlorhexidine and alcohol swabs, and apply the flat end of the metal rod to the center of the muscle for a given amount of time (see Figure 1 Step 5).

      Note: Standardize the size of the injury by maintaining the duration of the contact with the rod constant. Adjust the duration to achieve the desired extent of injury. A duration of 5 s is recommended for histological evaluation of injuries up to 10 d post-injury; 10 s are recommended for evaluation 14-21d postinjury. A pilot study may be required to establish the optimal duration of the cold injury for your animal model and study design.

    6. Suture the skin and muscle fascia to close the incision using 3-0 PROLENE® polyprorylene suture (Figure 1 Steps 6a and 6b).

    7. Provide the appropriate post-operative analgesia to the animal, according to the relevant guideline, and carefully and regularly observe them for at least 48 h after surgery.

      Example: 0.05 mg/kg buprenorphine subcutaneous injection every 12 h for 48 h.



      Figure 1. Induction of cryoinjury using a cooled stainless-steel metal rod. After the induction with sufficient anesthesia in the animal, cryoinjury can be inflicted in the following steps: removal of the hair and sterilization of the skin over the whole leg (3), exposure of the muscle (4a and 4b), application of the cooled metal rod over the muscle (5), and closure of the skin incision (6a and 6b).


  2. Muscle harvesting

    1. Euthanize mice according to institutional guidelines.

      Note: Cervical dislocation is a preferred method of euthanizing adult mice to minimize the time between the completion of euthanasia and muscle harvesting.

    2. Open the previous incision on the skin and remove the whole length of the injured muscle from the tendon attachments.

    3. Place the muscle in a 10% formalin solution in a glass shell vial.

    4. Replace formalin with 70% ethyl alcohol after 48 h for future sectioning and store the sample at room temperature (RT), avoiding direct sun and heat exposure.

    5. Create five 6-8 μm thick paraffinized transverse sections of the injury site on glass slides using a microtome.

    6. Perform hematoxylin-eosin (HE) staining according to standard procedures.


  3. Histological evaluation

    1. Under a light microscope, identify regenerating myofibers recognizable by their centrally located nuclei. Avoid counting multi-nucleated myofibers (green arrows) and inflammatory cells (red arrow) (Figure 2).

    2. Take high-power field images (the use of 20× or 40× objectives is recommended) spanning the entire injury site.

    3. Using ImageJ or an equivalent software, measure the cross-sectional area (CSA) of a total of at least 1,000 distinct regenerating fibers per sample.



      Figure 2. Identification of regenerating myofibers in HE-stained transverse muscle sections. A. Regenerating myofibers (yellow circles) are distinguishable from the surrounding mature myofibers by their centrally located nuclei (black arrows). On day 5 post-injury (DPI 5), regenerating myofibers with centrally located nuclei are in the inflammatory foci (red arrows) (A). B. By day 10 post-injury (DPI 10), regenerating myofibers have formed an organized structure with substantially less inflammatory infiltration. Both images are of tibialis anterior muscle of 8-week-old C57BL/B male mice, with cryoinjury inflicted for 5 s. Scale bars = 100 μm.

Data analysis

Calculate the CSAs of at least 1,000 distinct regenerating fibers per sample. Create a contingency table (Figure 3A) for frequency distribution of various fiber sizes (steps of 100 μm2 are recommended for adequate statistical resolution) of all samples and draw a frequency histogram of the mean frequency or percentage frequency of the group (Figure 2B). Calculate the mean CSAs per sample of a given group (Figure 2C). Perform statistical analysis using unpaired the Student’s t-test or one-way ANOVA to evaluate the presence of statistically significant variation between groups. Data can be expressed as mean ± standard deviation (SD) or standard error of means (SEM), and significance may be set at P-value < 0.05.



Figure 3. Data analysis and presentation. A. Contingency table containing the number of myofibers per 1,000 distinct regenerating fibers with a cross-sectional area (CSA) within a given range. The range was separated by 100 μm2 steps in this example. B. Histogram showing the mean percentage frequency of different myofiber CSA ranges of seven samples. C. Graph of the mean and standard error of the average myofiber CSAs of seven samples.

Acknowledgments

This protocol is derived from “Loss of ARNT in skeletal muscle limits muscle regeneration in aging” (Endo et al., 2020). This work was funded by the National Institute of Aging (K76AG059996 to IS), the National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK036836 to AJW), the Glenn Foundation for Medical Research (AJW), and a Research and Education Committee Grant (IS) from the Boston Claude D. Pepper Center, supported by the National Institute of Aging (P30AG031679 to SB).

Competing interests

The authors declare no competing interests.

Ethics

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital and were conducted within the guidelines of the U.S. National Institutes of Health (NIH). IACUC approval ID: 2016N000375, valid between 2020-2023.

References

  1. Caldwell, C. J., Mattey, D. L. and Weller, R. O. (1990). Role of the basement membrane in the regeneration of skeletal muscle. Neuropathol Appl Neurobiol 16(3): 225-238.
  2. Endo, Y., Baldino, K., Li, B., Zhang, Y., Sakthivel, D., MacArthur, M., Panayi, A. C., Kip, P., Spencer, D. J., Jasuja, R., Bagchi, D., Bhasin, S., Nuutila, K., Neppl, R. L., Wagers, A. J. and Sinha, I. (2020). Loss of ARNT in skeletal muscle limits muscle regeneration in aging. FASEB J 34(12): 16086-16104.
  3. Huard, J., Li, Y. and Fu, F. H. (2002). Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 84(5): 822-832.
  4. Jarvinen, T. A., Jarvinen, T. L., Kaariainen, M., Kalimo, H. and Jarvinen, M. (2005). Muscle injuries: biology and treatment. Am J Sports Med 33(5): 745-764.
  5. Lefaucheur, J. P. and Sebille, A. (1995). Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast growth factor, transforming growth factor beta 1 or insulin-like growth factor I. J Neuroimmunol 57(1-2): 85-91.
  6. Mills, R. J., Voges, H. K., Porrello, E. R. and Hudson, J. E. (2017). Cryoinjury Model for Tissue Injury and Repair in Bioengineered Human Striated Muscle. Methods Mol Biol 1668: 209-224.
  7. Oh, J., Sinha, I., Tan, K. Y., Rosner, B., Dreyfuss, J. M., Gjata, O., Tran, P., Shoelson, S. E. and Wagers, A. J. (2016). Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging (Albany NY) 8(11): 2871-2896.
  8. Sinha, I., Sakthivel, D., Olenchock, B. A., Kruse, C. R., Williams, J., Varon, D. E., Smith, J. D., Madenci, A. L., Nuutila, K. and Wagers, A. J. (2017). Prolyl Hydroxylase Domain-2 Inhibition Improves Skeletal Muscle Regeneration in a Male Murine Model of Obesity. Front Endocrinol (Lausanne) 8: 153.
  9. Warren, G. L., Summan, M., Gao, X., Chapman, R., Hulderman, T. and Simeonova, P. P. (2007). Mechanisms of skeletal muscle injury and repair revealed by gene expression studies in mouse models. J Physiol 582(Pt 2): 825-841.

简介

[摘要]冰冻损伤或冷冻损伤是一种在骨骼肌中产生可复制的局部损伤的方法。此方法允许研究的再生反应以下肌肉损伤体内,从而使所述的本地评估和影响肌纤维再生的处理全身性因素。ç ryoinjuries适用于该研究的肌肉injur的各种形式Ÿ ,特别是非创伤性和外伤,而不会损失大量的肌肉。冷冻损伤仅需要简单的仪器,并且与其他方法相比具有优势,即可以根据与冷冻仪器接触的持续时间轻松调整病变程度并使其标准化。第r egenerative响应可以在组织学上由平均来评估到期通过用位于中央的细胞核的肌纤维的横截面区域所指示的再生肌纤维。因此,冷冻损伤被认为是一个最可靠和方便的方法小号在螺柱模拟肌肉拉伤IES的肌肉再生。

[背景]肌肉损伤是injur的常见类型ý ,可能导致从各种原因,从消耗稻瘟病创伤。牛逼HESE伤害导致的炎症和再生反应的复杂的级联反应,导致了修复的损伤组织。为了表征肌肉受伤和下面的反应和评价efficac Ÿ潜在的治疗方法,各种实验模型已发展到复制肌肉injur的过程ÿ 。体外方法提供了一些实用的优势,并且3D培养技术的进步已使生物工程化人类细胞衍生的肌肉组织替代物成为可能,可用于肌肉损伤研究(Mills等人,2017)。体内模型中,然而,仍然迫切需要理解肌肉损伤,如涉及复杂过程的局部和全身反应小号支配整个再生过程和伴随的功能障碍/只能在生物体的整个系统中观察的恢复。肌肉的啮齿动物模型injur Ÿ是完善和今天研究肌肉再生最常用的系统。

有伤的各种方式和他们的模型,其中包括裂伤,破碎,爆炸伤,容积肌肉损失,myotoxins ,并冻结。虽然所有的方式伤害经过治疗的常见阶段(华德等,2002; Jarvinen等人。,2005),一些heterogeneit ÿ存在小号的程度和维修的质量遵循有关的肌肉干细胞的敏感性/响应损伤,组织重塑,炎症反应,血管重建和神经支配(Caldwell等,1990;Lefaucheur和Sebille ,1995)。Cryoinjur IES ,或创伤性损伤,由于冻结,产生局部损伤伴有实质炎症和血管反应(沃伦等人,2007),其程度可被调节和标准化根据持续时间与所述接触的冷冻仪( Oh等人,2016; Sinha等人,2017; Endo等人,2020)。与缺乏标准化所需的比较信息的各种肌毒素相比,这使它成为一种可产生可靠结果的简便而可靠的方法。的步骤皮肤切口的,但是,需要的冷冻损伤模型,可以考虑在使用注射的缺点myotoxins 。与c ryoinjury模型适用于宽范围的肌肉损伤的,通常是用于研究伤害无肌体积的大量损失的合适的选择。尤其是,冷冻伤可以模拟横纹肌的非创伤性和轻度创伤,包括骨骼肌的挤压伤和心肌的心肌梗塞。以下cryoinjur再生反应Ÿ可以用组织学和受伤未受伤的区域之间明确划分边界进行识别。在这里,我们描述了一种使用干冰在小鼠骨骼肌中产生冷冻损伤并量化肌纤维再生反应的方法。

关键字:肌肉损伤, 低温损伤, 肌肉再生, 肌纤维再生



材料和试剂


1. 3.15%葡萄糖酸洗必泰和70%异丙醇棉签(PDI,目录号:S40750)      2. 8周大的C57BL / B雄性小鼠      3. USP异氟烷(Patterson兽医,目录号:14043070406)      4.干冰(或液氮)      5.丁丙诺啡(帕特森兽医,目录号:07-892-5235)      6. 3-0 PROLENE ® polyprorylene缝线(Ethicon的,目录号:8665G)      7. 10%福尔马林溶液(Sigma,目录号:HT501128)      8. HistoPrep TM 70%乙醇(Fisher Scientific,目录号:HC1500)      设备


直径5毫米的不锈钢金属棒(Uxcell ,目录号:UX657206)
注意:该米等人杆应当选择,以便其直径以匹配上课肌肉大小。检查和测量肌肉和的大致宽度Ë nsure该杆的直径比所有经历冷冻损伤动物的肌肉的宽度小。


手术剪刀和镊子(Roboz Surgical Instrument Co ,目录号:RS-5910; Symmetry Surgical,目录号:30-1185 )
不锈钢手术刀,#15(赞成医疗保健,目录号:MDS15215)
动物剪(肯特科学公司,目录号:CL7300)
玻璃瓶(Fisher Scientific,目录号:03-339-26A)
光学显微镜(奥林巴斯,目录号:UCMAD3)
Shandom切片机(Thermo Scientific ,目录号:A78100001K)
软件


ImageJ(国立卫生研究院,http://imagej.nih.gov/ij)
GraphPad Prism版本9(GraphPad软件,https : //www.graphpad.com )
程序


冷冻伤
手术前要对手术器械和金属棒进行消毒。
使用异氟醚麻醉小鼠。
使用限幅器的整个腿清除头发和皮肤消毒前向的与感兴趣的肌肉氯己定和酒精棉签(图URE 1步骤3) 。
使用无菌技术,使皮肤和肌肉筋膜的切口,并使用外科剪刀或暴露下面的肌肉一个解剖刀(图URE 1步骤4a和4b) 。
注意:皮肤切口的大小应根据所使用的肌肉和金属杆的尺寸来调节(参见图1小号TEP图4a ); 它必须允许皮肤缩回足够宽,以使切开的皮肤边缘不会碰到金属棒。这通常需要一个切口,在纵向方向上的肌肉的跨度超过80% ,与所述切口对齐的与下面的肌肉的中心。


冷却在干冰金属棒5分钟,擦拭用的氯己定和酒精棉签杆小号,并应用金属棒的平坦端至肌肉对的给定时间量的中心(参见图URE 1个步骤5 )。
注:规范大小的维持伤害持续时间的与杆的接触不断。调节持续时间,以实现所述injur的期望程度ÿ 。建议在伤后10 d内持续5 s进行组织学评估;10个小号建议用于评价14 - 21D伤后。可能需要进行初步研究,以为您的动物模型和研究设计确定最佳的冷伤害持续时间。


缝合皮肤和肌肉筋膜关闭使用切口3-0 PROLENE ® polyprorylene缝合(图URE 1步骤小号6a和6b)。             
提供了适当的术后镇痛动物,雅阁荷兰国际集团以相关指引,并仔细LY和定期LY OBSERV Ë他们手术后至少48小时。
例如:每12小时皮下注射0.05 mg / kg丁丙诺啡48小时。


图1.使用冷却的不锈钢金属棒诱导冷冻伤。诱导后用足够的麻醉的动物,冷冻损伤可通过以下步骤来造成:切除的皮肤在整个腿(3)的毛发和灭菌,肌肉的曝光(4a和4b) ,所述冷却的应用金属棒覆盖肌肉(5),并闭合皮肤切口(6a和6b)。


B.肌肉收获


根据机构指南对小鼠实施安乐死。
注意:颈椎脱位是euthan的优选方法中定义的成年小鼠以最小化e的结束之间的时间uthanasia和肌肉收获。


在皮肤上打开先前的切口,并在腱鞘处从肌腱上去除受伤肌肉的整个长度。
将肌肉放入玻璃瓶小瓶中的10%福尔马林溶液中。
48小时后,用70%的乙醇代替福尔马林,以备将来切片,并将样品保存在室温(RT)下,避免阳光直射和暴露在热源下。
创建五个6-8微米厚的石蜡包埋的受伤部位在载玻片上的横截面使用切片机。
按照标准程序进行苏木精-伊红(HE)染色。
C.组织学评估 在光学显微镜下,确定可通过位于中心的细胞核识别的再生肌纤维。避免计算多核肌纤维(绿色箭头)和炎性细胞(红色箭头)(图2)。
取高倍视野图像(在使用20 ×或40 ×目标的建议)跨越整个损伤坐即
使用I mageJ或等效软件,每个样品测量总共至少1,000条不同的再生纤维的横截面积(CSA)。
图2.鉴定HE- stain横肌切片中的再生肌纤维。A.再生的肌纤维(黄色圆圈)通过位于中心的核(黑色箭头)与周围的成熟肌纤维区分开。在损伤后第5天(DPI 5),具有位于中心的核的再生肌纤维在炎性病灶中(红色箭头)(A)。B.损伤后第10天(DPI 10),再生的肌纤维已经形成了组织结构,其炎症浸润明显减少。这两个图像都是8周大的C57BL / B雄性小鼠胫前肌的图像,造成了5 s的冷冻损伤。比例尺= 100μm 。


数据分析


计算的CSA至少1 ,每个样品000不同的再生纤维。创建用于各种纤维尺寸的频度分布的列联表(图3A)(100微米的步骤2的所有样品的被推荐用于足够的统计分辨率)和绘制平均频率或频率直方图百分比的组的频率(图2B) 。计算给定组的每个样本的平均CSA (图2C)。使用不成对的执行统计分析在S tudent的吨-test或单因素ANOVA来评价组间统计学显著变异的存在。数据可以表示为均值±标准差(SD)或均值标准误(SEM),并且显着性可以设置为P值<0.05。


图3.数据分析和表示。A. Ç含有每1000种的肌纤维的数量ontingency表不同再生纤维具有的横sectio纳尔面积(CSA)的给定范围内。的范围内通过100微米分离2个步骤在该实施例中。B. ħ istogram示出的平均百分比不同的肌纤维的频率CSA的范围7个样品。C. ģ拉夫的平均肌纤维的平均值和标准误差的CSA的7个样品。


致谢


该方案源自“骨骼肌中ARNT的丢失限制了衰老过程中的肌肉再生” (Endo等人,2020年)。这项工作由美国国立老龄研究所(IS-K76AG059996),美国糖尿病与消化及肾脏疾病研究所(AJW的P30 DK036836),格伦医学研究基金会(AJW)和研究与教育委员会拨款资助(IS)来自美国国家老龄研究所(Boston Claude D. Pepper Center)的支持(P30AG031679至SB)。


利益争夺


作者宣称没有利益冲突。


伦理


所有动物手术均已获得布里格姆妇女医院的机构动物护理和使用委员会(IACUC)的批准,并在美国国立卫生研究院(NIH)的指导下进行。IACUC批准ID:2016N000375,有效日期为2020-2023。


参考


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Mills,RJ,Voges,HK,Porrello ,ER和Hudson,JE(2017)。生物工程人类横纹肌组织损伤和修复的冷冻损伤模型。方法分子生物学1668:209-224。              
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引用:Endo, Y., Karvar, M. and Sinha, I. (2021). Muscle Cryoinjury and Quantification of Regenerating Myofibers in Mice. Bio-protocol 11(11): e4036. DOI: 10.21769/BioProtoc.4036.
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