这项工作是支持的研究补助金（NIH / NIDCD 009269，012831和ARRA行政补充剂NIH补助）维宏林。我们要特别感谢UMBC蒂姆·福特先生在他的偷拍和处理的技术援助。我们还要感谢达芙妮布隆伯格博士UMBC的的CHERE小资女士和尼古拉斯·麦科勒姆先生从奥林巴斯美国公司，他们的设备援助偷拍。

1。鼠标鼻子准备在本研究中，我们使用成人的C57BL / 6背景的小鼠。所有动物护理和程序批准的动物护理和使用委员会（IACUC）美国马里兰大学巴尔的摩县。 1.1获取鼻子从paraformaldahyde固定小鼠<img src="/files/ftp_upload/50538/50538fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50538/50538fig1highres.jpg"/> 图1。鼠标头骨骨骼的头骨背视图。B：除去下颌骨的头骨与腹面观。从40日龄小鼠头骨。个别骨头的颜色更好的可视化。 <a href="https://www.jove.com/files/ftp_upload/50538/50538fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <ol><li>灌注transcardially解决个别的小鼠日É林， 等。协议，（2008）16。简单地说，小鼠深度麻醉，三溴乙醇（圣阿韦坦250微克/克体重），心脏灌注，用0.1M磷酸缓冲液（PB，30〜50毫升）中，然后由磷酸盐缓冲固定液中含有3％多聚甲醛，19 mM的L-赖氨酸单盐酸盐，0.23％的钠米，高碘酸（约35-50毫升）。也可以按照动物灌注35的朱庇特的文章中的步骤。 </li><li>使用一把剪刀切断的下颌骨（或下颚）和去除皮肤的头。 </li><li>从身体的其余部分分开，整个头部。 </li><li>取出的口感。此外，清洗，除去残留的结缔组织和肌肉的头骨取得试样，在图1A和1B所示的表面上。 </li><li>在解剖显微镜下，取出脑和嗅球颅骨覆盖。剪掉多余的组织和骨骼。没有特扩展的组织制剂，其中大脑和鼻子保持连接，只有头骨都被删除。对于免疫组化实验中，将组织后固定1.5小时，然后转移到0.1M磷酸盐缓冲盐水（PBS）与25％蔗糖过夜。鼻组织应保持加湿整个剥离，通过浸渍在缓冲​​的蔗糖溶液数次。 </li></ol> 1.2获取鼻子从新鲜安乐死小鼠<ol><li>个别小鼠转移到一个干净的笼中，并暴露在CO 2气体，随后用颈脱位5分钟后，最后的呼吸。为了减少血液中的鼻子组织，用一把剪刀开胸切心脏，使血液漏。 </li><li>重复步骤1.1.2至1.1.5，除了25％的蔗糖后的固定和冷冻保护。应保持的检体加湿保持与台氏盐含有（以mM计）：140钠氯化钾，氯，5 1的MgCl 2，1的 CaCl 2，10的丙酮酸钠，10个D-葡萄糖，10 N-2-羟乙基哌嗪-N&#39;-2-乙磺酸缓冲液（HEPES，在pH 7.4）调整。或者，折片的Kimwipes，浸泡，用台氏溶液，和这里下方的检体解剖时，偶尔浸入试样的台氏溶液或一些解决办法拖放到组织上，以保持它加湿以维持细胞和组织的可行性。 </li></ol> 2。门齿，前犁骨和上颌骨骨去除<ol><li>开始腹观点。找到犁骨和牙齿咬骨钳或镊子打破最腹侧部分骨，沿其长度打破犁骨。 </li><li>使用锯齿钳轻轻行走碎骨离犁鼻器（VNO），取下破裂的犁骨段。请注意，这些老鼠不到一个月以前，或者如果只有兴趣在教育部，两个STEps的，可以跳过。 </li><li>牢牢握住头钳。使用咬骨钳打破两个门牙的前部的两个门牙和犁骨之间的接合区域的前上颌骨。 </li><li>额的右侧上颌骨腹侧部上面的区域只是前颧弓颧板的背侧的水平。 </li><li>在鼻子翻转背的观点。用咬骨钳打破背颧板的右侧上颌骨前壁。整个右上颌前颧板要宽松。用细镊子轻轻任何相关颌骨鼻组织分开，然后轻轻抬起了骨头碎片。 </li><li>重复步骤2.4和2.5，，松开左门牙和左上颌前牙区，，鼻骨后已被删除，并删除它们。 </li></ol> 3。鼻骨和背颧板拆除<ol><li>使用锯齿钳或咬骨钳重新将剩余的额骨鼻甲骨只是尾椎。取出这块骨头，后尾部分的鼻骨可以使用镊子夹住。 </li><li>使用咬骨钳打破颧弓的前部，这是连接到颧板。 </li><li>颧板的背端外侧缘和细镊子轻轻翻转骨，并删除它。如果骨头不松，用咬骨钳轻轻松开去除。 </li><li>用细镊子或刀片松动之间的左，右鼻甲骨内侧缝合。 </li><li>使用锯齿镊子夹住尾端右鼻骨。轻轻地移动，从一侧到另一侧的骨到相关组织分开。移动骨一侧到另一侧移动钳沿尾第三鼻骨它是有用的。由于鼻骨分离，慢慢抬起骨，从尾端。 </li><li>而T他鼻骨轻微抬起，横向倾斜骨的骨内衬薄呼吸道上皮组织揭示了横向露头。用细镊子轻轻松开这个组织从鼻腔骨。继续解除鼻骨。当骨完全分离皮下组织，用剪刀切断鼻骨喙端。 </li><li>左鼻骨去除重复步骤3.1，3.5和3.6。 </li><li>鼻子的左侧，重复步骤3.2和3.3。 </li></ol> 4。颧骨外侧板拆除<ol><li>删除颧板一侧的时间。无论是颧板可以拆下第一。 </li><li>从腹侧的观点，打破休息，直到达到颧弓颧弓上颌骨逊色。 </li><li>从背部的角度看，轻轻地抓住颧弓和正向和侧向抬起。如果颧板仍连接到任何组织，采取罚款钳轻轻地切断的组织和骨之间的连接。 </li><li>鼻颧板的另一侧重复上述步骤。 </li></ol> 5。轨道骨去除<ol><li>从腹侧的观点，打破巴伦丁的磨牙骨之间的鼻子。 </li><li>使用咬骨钳的每一侧的鼻子打破3臼齿和上颌骨。 </li><li>打破和消除任何剩余的腹厚的骨片和后每一侧鼻孔的鼻甲。 </li></ol> 6。筛骨去除<ol><li>中断任何部分筛骨突出尾鳍鼻甲。这是必要的，以避免损失的鼻甲组织，除去薄件时，筛骨覆盖鼻甲。 </li><li>对于鼻子的右侧，将筛骨前缘细镊子轻轻取出。如果骨的部分仍然存在，重复这个过程，直到所有薄骨覆盖鼻甲已被删除。鼻甲骨不需要除去大部分的制剂。在老年小鼠，筛板变得脆弱。如果需要鼻腔组织cryosectioning是，用细镊子取出小块板，以减少潜在的损害，引起骨。 </li><li>重复步骤6.2），鼻子的左侧。 </li><li>切片之前删除任何剩余的碎骨。注：在动物超过一岁，隔骨posterodorsal地区是有点厚和硬。人们可以用细镊子取出这部分。镊子的尖端插入到骨背侧部的隔垫骨和衬里上皮组织分离的两侧。使用一对镊子，抢骨头和保持标本。使用另一双钳打破上层部分的中隔软骨下骨部分，轻轻取出。 </li></ol><ol><li>设置抽真空泵。 </li><li>将鼻子嵌入模具。淹没鼻子媒体华侨城。 </li><li>使用真空以除去空气气泡内鼻组织。这个过程最多需要5分钟。 </li><li>除去气泡后，将组织在所需的方向。 </li><li>冻结在模具使用干冰华侨城和组织。然后包埋组织立即cryosectioned，或储存于-80℃以备将来使用。 </li></ol>

<ol>
	<li>Naclerio, R. M., Pinto, J., Assanasen, P., Baroody, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+ability+of+the+nose+to+warm+and+humidify+inspired+air.">Observations on the ability of the nose to warm and humidify inspired air.</a> Rhinology. 45, 102-111 (2007).</li><li>Bjermer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nose+as+an+air+conditioner+for+the+lower+airways.">The nose as an air conditioner for the lower airways.</a> Allergy. 54, 26-30 (1999).</li><li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li><li>Bryant, B., Silver, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemisthesis: The common chemical sense. , (2000).</li><li>Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+the+nasal+cavity+in+rats+and+mice.">Comparative morphometry of the nasal cavity in rats and mice.</a> J. Anat. 135, 83-88 (1982).</li><li>Halpern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+and+function+of+the+vomeronasal+system.">The organization and function of the vomeronasal system.</a> Annu. Rev. Neurosci. 10, 325-362 (1987).</li><li>Rodolfo-Masera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Su+l'esquoestizenza+di+un+particulare+organo+olfacttivo+nel+setto+nasale+della+cavia+e+di+altri+roditori.">Su l'esquoestizenza di un particulare organo olfacttivo nel setto nasale della cavia e di altri roditori.</a> Arch. Ital. Anat. Embryol. 48, 157-212 (1943).</li><li>Levai, O., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+pattern+of+nerve+fibers+from+the+septal+organ:+DiI-tracing+studies+with+transgenic+OMP+mice.">Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice.</a> Histochemistry and Cell biology. 120, 483-492 (2003).</li><li>Storan, M. J., Key, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septal+organ+of+Gruneberg+is+part+of+the+olfactory+system.">Septal organ of Gruneberg is part of the olfactory system.</a> J. Comp. Neurol. 494, 834-844 (2006).</li><li>Restrepo, D., Arellano, J., Oliva, A. M., Schaefer, M. L., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+views+on+the+distinct+but+related+roles+of+the+main+and+accessory+olfactory+systems+in+responsiveness+to+chemosensory+signals+in+mice.">Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice.</a> Horm. Behav. 46, 247-256 (2004).</li><li>Breer, H., Fleischer, J., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sense+of+smell:+multiple+olfactory+subsystems.">The sense of smell: multiple olfactory subsystems.</a> Cell Mol. Life Sci. 63, 1465-1475 (2006).</li><li>Munger, S. D., Leinders-Zufall, T., Zufall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsystem+organization+of+the+mammalian+sense+of+smell.">Subsystem organization of the mammalian sense of smell.</a> Annu. Rev. Physiol. 71, 115-140 (2009).</li><li>Finger, T. E., St Jeor, V. L., Kinnamon, J. C., Silver, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+substance+P-+and+CGRP-immunoreactive+nerve+fibers+in+the+nasal+epithelium+of+rodents.">Ultrastructure of substance P- and CGRP-immunoreactive nerve fibers in the nasal epithelium of rodents.</a> J. Comp. Neurol. 294, 293-305 (1990).</li><li>Papka, R. E., Matulionis, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+substance-P-immunoreactive+nerves+with+the+murine+olfactory+mucosa.">Association of substance-P-immunoreactive nerves with the murine olfactory mucosa.</a> Cell Tissue Res. 230, 517-525 (1983).</li><li>Baraniuk, J. N., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasonasal+reflexes,+the+nasal+cycle,+and+sneeze.">Nasonasal reflexes, the nasal cycle, and sneeze.</a> Curr. Allergy Asthma Rep. 7, 105-111 (2007).</li><li>Lin, W., Ogura, T., Margolskee, R. F., Finger, T. E., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM5-expressing+solitary+chemosensory+cells+respond+to+odorous+irritants.">TRPM5-expressing solitary chemosensory cells respond to odorous irritants.</a> J. Neurophysiol. 99, 1451-1460 (2008).</li><li>Ogura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+microvillous+cells+in+the+mouse+main+olfactory+epithelium+and+effect+of+acetylcholine+on+olfactory+sensory+neurons+and+supporting+cells.">Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells.</a> J. Neurophysiol. 106, 1274-1287 (2011).</li><li>Finger, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solitary+chemoreceptor+cells+in+the+nasal+cavity+serve+as+sentinels+of+respiration.">Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8981-8986 (2003).</li><li>Gulbransen, B. D., Clapp, T. R., Finger, T. E., Kinnamon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+solitary+chemoreceptor+cell+responses+to+bitter+and+trigeminal+stimulants+in+vitro.">Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro.</a> J. Neurophysiol. 99, 2929-2937 (2008).</li><li>Zhao, K., Dalton, P., Yang, G. C., Scherer, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+modeling+of+turbulent+and+laminar+airflow+and+odorant+transport+during+sniffing+in+the+human+and+rat+nose.">Numerical modeling of turbulent and laminar airflow and odorant transport during sniffing in the human and rat nose.</a> Chemical Senses. 31, 107-118 (2006).</li><li>Ressler, K. J., Sullivan, S. L., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zonal+organization+of+odorant+receptor+gene+expression+in+the+olfactory+epithelium.">A zonal organization of odorant receptor gene expression in the olfactory epithelium.</a> Cell. 73, 597-609 (1993).</li><li>Vassar, R., Ngai, J., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+segregation+of+odorant+receptor+expression+in+the+mammalian+olfactory+epithelium.">Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium.</a> Cell. 74, 309-318 (1993).</li><li>Fulle, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor+guanylyl+cyclase+expressed+specifically+in+olfactory+sensory+neurons.">A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 3571-3575 (1995).</li><li>Juilfs, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+olfactory+neurons+that+selectively+express+cGMP-stimulated+phosphodiesterase+(PDE2)+and+guanylyl+cyclase-D+define+a+unique+olfactory+signal+transduction+pathway.">A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 3388-3395 (1997).</li><li>Lin, W., Arellano, J., Slotnick, B., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odors+detected+by+mice+deficient+in+cyclic+nucleotide-gated+channel+subunit+A2+stimulate+the+main+olfactory+system.">Odors detected by mice deficient in cyclic nucleotide-gated channel subunit A2 stimulate the main olfactory system.</a> The Journal of Neuroscience: The Official journal of the Society for Neuroscience. 24, 3703-3710 (2004).</li><li>Ishii, T., Omura, M., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+two-+and+three-color+fluorescent+RNA+in+situ+hybridization+of+the+main+and+accessory+olfactory+epithelia+in+mouse.">Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse.</a> J. Neurocyt. 33, 657-669 (2004).</li><li>Lee, A. C., Tian, H., Grosmaitre, X., Ma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice.">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chemical Senses. 34, 695-703 (2009).</li><li>Packard, A., Schnittke, N., Romano, R. A., Sinha, S., Schwob, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeltaNp63+regulates+stem+cell+dynamics+in+the+mammalian+olfactory+epithelium.">DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 31, 8748-8759 (2011).</li><li>Matthews, J. B., Mason, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+decalcifying+agents+on+immunoreactivity+of+formalin-fixed,+paraffin-embedded+tissue.">Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue.</a> Histochem J. 16, 771-787 (1984).</li><li>Athanasou, N. A., Quinn, J., Heryet, A., Woods, C. G., McGee, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decalcification+agents+on+immunoreactivity+of+cellular+antigens.">Effect of decalcification agents on immunoreactivity of cellular antigens.</a> J. Clin. Pathol. 40, 874-878 (1987).</li><li>Hegg, C. C., Irwin, M., Lucero, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+store-mediated+signaling+in+sustentacular+cells+of+the+mouse+olfactory+epithelium.">Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium.</a> Glia. 57, 634-644 (2009).</li><li>Spehr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+the+main+olfactory+system+in+social+recognition+of+major+histocompatibility+complex+peptide+ligands.">Essential role of the main olfactory system in social recognition of major histocompatibility complex peptide ligands.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 26, 1961-1970 (2006).</li><li>Ma, M., Chen, W. R., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation.">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J. Neurosci. Methods. 92, 31-40 (1999).</li><li>Cygnar, K. D., Stephan, A. B., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+responses+of+mouse+olfactory+sensory+neurons+using+the+air-phase+electroolfactogram+recording.">Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording.</a> J. Vis. Exp. (37), e1850 (2010).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Lin, W., Margolskee, R., Donnert, G., Hell, S. W., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+neurons+expressing+transient+receptor+potential+channel+M5+(TRPM5)+are+involved+in+sensing+semiochemicals.">Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 2471-2476 (2007).</li><li>Lin, W., Ezekwe, E. A., Zhao, Z., Liman, E. R., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM5-expressing+microvillous+cells+in+the+main+olfactory+epithelium.">TRPM5-expressing microvillous cells in the main olfactory epithelium.</a> BMC Neurosci. 9, 114 (2008).</li><li>Ogura, T., Krosnowski, K., Zhang, L., Bekkerman, M., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoreception+regulates+chemical+access+to+mouse+vomeronasal+organ:+role+of+solitary+chemosensory+cells.">Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells.</a> PLoS One. 5, e11924 (2010).</li><li>Sathyanesan, A., Feijoo, A. A., Mehta, S. T., Nimarko, A. F., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profile+of+G-protein+&#946;&#947;+subunit+gene+transcripts+in+the+mouse+olfactory+sensory+epithelia.">Expression profile of G-protein &#946;&#947; subunit gene transcripts in the mouse olfactory sensory epithelia.</a> Frontiers in Cellular Neuroscience. 7, 84 (2013).</li></ol>

作者宣称，他们有没有竞争经济利益。

在这里，我们展示了一个隔离完好鼠标鼻子的嗅觉和呼吸道组织的顺序取出骨头，同时保留周围组织的一步一步的过程。我们发现，小心骨去除的全部，甚至可以保留最微妙的组织。我们也分享洞察可能修改这项技术，我们在其中分离大脑和鼻子组织共同维护神经连接。这种新方法提供了一个隔离装置，用于在一个单一的免疫组化，RNA 原位杂交，以便进一步处理和生理实验标本的整个嗅觉...

哺乳动物鼻腔包含各种类型的组织和器官，服务于不同的功能。鼻腔，使上呼吸道的入口部分，它允许空气流入和流出肺部旅行。吸入的空气通过鼻腔的温度和湿度进行调节1，以及作为清洁或过滤除去刺激性和毒性的物质和感染性微生物的2。这两种治疗方法进行鼻上皮及上皮下的组织，包括腺体和血管，是至关重要的保护下呼吸道和肺部。除了它的作用在呼吸和上皮防御，鼻组织也包含外周感觉的嗅觉和三叉神经系统，使空气中的化学物质的检测范围广泛的设备。根据该系统被激活，在鼻子的化学品的感官检测既可以引起感的气味，刺激或疼痛3,4。 的外周的嗅觉系统是复杂的，由几个解剖分离鼻腔内的嗅觉器官。其中，主要的嗅觉上皮细胞（MOE）是最大的，它构成了约45-52％的鼻上皮细胞在啮齿类动物中5和位于后方区域。在腹侧区域中，有一对管状结构被称为犁鼻器6，坐在沿鼻中隔的每一侧。两个额外的嗅感觉神经元的小分组，称为室间隔器官马塞拉7,8和在Gruneberg神经节9，位于沿腹隔垫和背进入区域的鼻腔。这些外周器官中含有神经上皮细胞形态特色鲜明，细胞标记物的表达，和生理功能。他们共同检测成千上万的气味分子与精致的灵敏度10-12。 除了嗅觉器官，鼻腔内还设有其他感官系统。它是已知的，三 ​​叉神经肽能神经纤维出现在鼻腔上皮细胞，特别是呼吸道上皮13,14。这些纤维检测刺激性和有毒化学品，并负责启动保护性反射，如咳嗽和打喷嚏4,15。有刺激性的异味和苦味化合物也可以被检测到最近发现的孤化学感受细胞（SCCS），其中有许多是由三叉神经纤维16-19支配人口。这些的SCC位于鼻腔及犁鼻进入管道的入口区域中的高密度，暗示它们可能也有助于保护功能16-18。因此，鼻上皮细胞可显着不同的功能，形态，取决于它们的细胞的组合物解剖位置。 即使在一个单一的和专门的上皮细胞，存在地区差异。教育部就是这样一个例子。各种鼻甲教育部线，这是很复杂的卷曲结构。因为他们，不同地区的教育部体验不同的空气流速，因此，不同的扩散，空气中的气味分子的清除率20。此外，它被称为表达一种特定气味受体的嗅感觉神经元（OSNS）位于四规避教育部21,22区。这个位置如何差异会影响OSN气味的反应主要是不知道的。此外，一些的OSN种群具有区域偏好。鸟苷酸环化酶-D（GC-D）表达的OSN有带状分布，有利于地区的ectoturbinates 23,24的小路，小路。最近，我们发现了一个典型的嗅觉神经元亚群表达瞬时受体电位通道M5（TRPM5），优先地位于在横向和腹侧区域25。这些结果表明，本部不统一。然而，如何使这些区域的差异影响嗅编码的不理解。这部分是保存完好的解剖组织，用目前的方法获得完整的鼻上皮的难度，因为彻底的生理教育部的调查和鼻子已经有限。 鼻腔上皮细胞主要是四周前，包括鼻，上颌骨，腭骨，颧骨，筛骨颅骨。在成年老鼠和其它啮齿类动物模型，这些骨头是硬而难以去除，不破坏密切相关的鼻部组织，特别是细腻鼻甲。通常，使用的化学物质为基础的脱钙软化骨骼以允许cryosectioning鼻腔组织形态，免疫组织化学， 原位杂交研究，然而，依赖丁动物的年龄，脱钙过程可以持续7天过夜24,26-28。这种治疗方法也是有限的，因为它需要组织固定液保存。此外，化学脱钙可恶劣影响的一些敏感的抗体免疫标记29,30。对于生理研究，活组织是必需的，因此，这些实验往往是孤立的OSN或MOE切片得到的新生儿的头骨薄而软17,31,32进行。生理研究也可以利用整装准备分裂头25,33,34，但通常只有内侧表面的鼻子是方便，限制了对其他方面的生理记录。 在这里，我们描述了一个有效的，手动的的去骨方法来准备完整的鼻腔组织保存了原有的解剖组织形态。我们依次取出骨头的前在解剖显微镜下，几乎完全完整的鼻上皮暴露同时保持薄鼻甲骨骼完好除非老鼠很旧，cryosectioning的头骨是必要的。我们还可以延长保存鼻组织嗅球之间的连接，以及大脑的其余部分，从而促进外周和中枢的电路的同时检查方法。我们的方法可以被用于制备多聚甲醛固定，以及新鲜的，活的鼻组织。因此，我们的方法将有利于呼吸，嗅觉，鼻腔损伤和疾病的形态学，免疫组织化学和生理研究。

<table border="1">
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Dissection</td>
 </tr>
 <tr>
 <td>Rongeur, 1.0 mm Jaw width</td>
 <td>World Precision Instruments (WPI)</td>
 <td>501333</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont 3 </td>
 <td>WPI</td>
 <td>503235 </td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont 55</td>
 <td>WPI</td>
 <td>14099</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine forceps, Dumont AA</td>
 <td>Fine Science Tools (FST)</td>
 <td>11210-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Specimen forceps, Serrated</td>
 <td>VWR</td>
 <td>82027-440</td>
 <td></td>
 </tr>
 <tr>
 <td>Operating scissors</td>
 <td>WPI</td>
 <td>501753</td>
 <td></td>
 </tr>
 <tr>
 <td>Iris scissors, Straight</td>
 <td>Miltex</td>
 <td>V95-304</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>Olympus</td>
 <td>SZ40</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Tissue embedding</td>
 </tr>
 <tr>
 <td>Optimum cutting temperature (OCT) compound</td>
 <td>Sakura Finetek</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic embedding mold</td>
 <td>VWR</td>
 <td>15160-215 </td>
 <td></td>
 </tr>
 <tr>
 <td>Aspirator vacuum pump</td>
 <td>Fisher Scientific</td>
 <td>09-960-2</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 	 <td></td>
 <td></td>
 <td></td>
 <td>Section staining </td>
 </tr>
 <tr>
 <td>Neutral red</td>
 <td>ACROS Organic</td>
 <td>CAS 553-24-2</td>
 <td>Nuclei staining</td>
 </tr>
</table>

an effective manual deboning method to prepare intact mouse nasal tissue with preserved anatomical organization

哺乳动物的鼻子是一个多功能的器官，具有复杂的内部结构。鼻腔内衬各种上皮细胞，如嗅觉，呼吸，和鳞状上皮细胞明显不同的解剖位置，形态和功能。在成年小鼠中，与各种颅骨覆盖鼻子，限制了实验获得的内部结构，尤其是在后等为主体的嗅觉上皮细胞（MOE）。在这里，我们描述了一个有效的方法，获得几乎整个完整的鼻腔组织与腌制解剖组织。使用手术工具，在解剖显微镜下，我们依次取出头骨周围鼻腔组织。这个程序可以进行多聚甲醛固定，解剖新鲜，皮肤的鼠标头。去骨整个过程约需20-30分钟，这是明显短于传统化工为主的实验所需时间钙化。此外，我们提出了一个简单的方法来去除气泡被困鼻甲之间，这是获得完整的细水平线或者冠状或矢状切面​​鼻组织准备的关键。鼻组织用我们的方法制备可用于整个装载整个上皮细胞的观察，以及形态，免疫细胞化学，RNA 原位杂交，和生理的研究，尤其是在特定区域的检查和比较，还研究。

使用这种方法，可以可靠地获得几乎完全完整的鼻组织图2A示出的图像的成年人鼻试样从多聚甲醛固定磁头。在这个标本，所有四个子嗅觉感官器官，包括教育部，室间隔器官中，Gruneberg节，VNO，都完好无损。此外，呼吸道上皮细胞和上皮下的组织，如腺体和血管，将被保留。我们已经成功地使用此方法，在一些研究中，我们调查形态，分布，神经的神经支配，细胞标记的表达在各专...

Watch this Scientific Journal Video about An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization at JoVE.com

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

有效的人工剔骨方法编写完整的鼠标鼻组织与保存的解剖组织

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

an-effective-manual-deboning-method-to-prepare-intact-mouse-nasal

Microsoft Bing

Research

JoVE Journal

Biology

34.5K 次观看.  University of Maryland Baltimore County. 

视频: An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

<meta charset="utf8"></head><body>鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.