Name: intravital imaging of fluorescent protein expression in mice with a closed-skull traumatic brain injury and cranial window using a two-photon microscope
Uploaded: 2023-04-21T14:05:00
Description: Watch this Scientific Journal Video about Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope at Jo

我们感谢马萨诸塞大学陈医学院的 Miguel Sena-Esteves 博士赠送了 AAV（PHP.eB）-Syn1-EGFP 病毒，并感谢马萨诸塞大学陈医学院的 Debra Cameron 绘制了小鼠头骨草图。我们还要感谢 Bosco、Schafer 和 Henninger 实验室的现任和前任成员的建议和支持。这项工作由国防部 （W81XWH202071/PRARP） 资助给 DAB、DS 和 NH。

所有与动物相关的方案均按照美国国家研究委员会（US）委员会发布的《实验动物护理和使用指南》进行。该协议已获得马萨诸塞大学陈医学院（UMMS）机构动物护理和使用委员会的批准（许可证编号202100057）。简而言之，如研究示意图（图1）所示，动物接受病毒注射、TBI、窗口植入，然后按时间顺序进行活体成像。
注意：商业条款已被删除。有关使用?...

<ol>
	<li>Bowman, K., Matney, C., Berwick, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+traumatic+brain+injury+care+and+research:+a+report+from+the+National+Academies+of+Sciences,+Engineering,+and+Medicine.">Improving traumatic brain injury care and research: a report from the National Academies of Sciences, Engineering, and Medicine.</a> JAMA. 327 (5), 419-420 (2022).</li><li>National Academies of Sciences, Engineering, and Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traumatic Brain Injury: A Roadmap for Accelerating Progress. , (2022).</li><li>Xu, X., 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=Repetitive+mild+traumatic+brain+injury+in+mice+triggers+a+slowly+developing+cascade+of+long-term+and+persistent+behavioral+deficits+and+pathological+changes.">Repetitive mild traumatic brain injury in mice triggers a slowly developing cascade of long-term and persistent behavioral deficits and pathological changes.</a> Acta Neuropathologica Communications. 9 (1), 60 (2021).</li><li>Chen-Plotkin, A. S., Lee, V. M. Y., Trojanowski, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAR+DNA-binding+protein+43+in+neurodegenerative+disease.">TAR DNA-binding protein 43 in neurodegenerative disease.</a> Nature Reviews Neurology. 6 (4), 211-220 (2010).</li><li>Mackenzie, I. R., Rademakers, R., Neumann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+and+FUS+in+amyotrophic+lateral+sclerosis+and+frontotemporal+dementia.">TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia.</a> The Lancet. Neurology. 9 (10), 995-1007 (2010).</li><li>McKee, A. C., 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=The+first+NINDS/NIBIB+consensus+meeting+to+define+neuropathological+criteria+for+the+diagnosis+of+chronic+traumatic+encephalopathy.">The first NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy.</a> Acta Neuropathologica. 131 (1), 75-86 (2016).</li><li>Henninger, N., 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=Attenuated+traumatic+axonal+injury+and+improved+functional+outcome+after+traumatic+brain+injury+in+mice+lacking+Sarm1.">Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1.</a> Brain. 139, 1094-1105 (2016).</li><li>Bouley, J., Chung, D. Y., Ayata, C., Brown, R. H., Henninger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+spreading+depression+denotes+concussion+injury.">Cortical spreading depression denotes concussion injury.</a> Journal of Neurotrauma. 36 (7), 1008-1017 (2019).</li><li>Goldey, G. 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=Removable+cranial+windows+for+long-term+imaging+in+awake+mice.">Removable cranial windows for long-term imaging in awake mice.</a> Nature Protocols. 9 (11), 2515-2538 (2014).</li><li>Kugler, S., 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=Neuron-specific+expression+of+therapeutic+proteins:+evaluation+of+different+cellular+promoters+in+recombinant+adenoviral+vectors.">Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors.</a> Molecular and Cellular Neurosciences. 17 (1), 78-96 (2001).</li><li>von Jonquieres, G., 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=Glial+promoter+selectivity+following+AAV-delivery+to+the+immature+brain.">Glial promoter selectivity following AAV-delivery to the immature brain.</a> PLoS One. 8 (6), 65646 (2013).</li><li>Trachtenberg, J. 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=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420 (6917), 788-794 (2002).</li><li>Mostany, R., 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=Altered+synaptic+dynamics+during+normal+brain+aging.">Altered synaptic dynamics during normal brain aging.</a> The Journal of Neuroscience. 33 (9), 4094-4104 (2013).</li><li>Yang, Q., Vazquez, A. L., Cui, X. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+two-photon+imaging+of+the+neuroinflammatory+response+to+intracortical+implants+and+micro-vessel+disruptions+in+awake+mice.">Long-term in vivo two-photon imaging of the neuroinflammatory response to intracortical implants and micro-vessel disruptions in awake mice.</a> Biomaterials. 276, 121060 (2021).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+calcium+imaging+of+neuronal+networks.">In vivo two-photon calcium imaging of neuronal networks.</a> Proceedings of the National Academy of Sciences. 100 (12), 7319-7324 (2003).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3 (4), 489-496 (2006).</li><li>Isshiki, 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=Enhanced+synapse+remodelling+as+a+common+phenotype+in+mouse+models+of+autism.">Enhanced synapse remodelling as a common phenotype in mouse models of autism.</a> Nature Communications. 5, 4742 (2014).</li><li>Mondo, 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=A+developmental+analysis+of+juxtavascular+microglia+dynamics+and+interactions+with+the+vasculature.">A developmental analysis of juxtavascular microglia dynamics and interactions with the vasculature.</a> The Journal of Neuroscience. 40 (34), 6503-6521 (2020).</li><li>White, M. A., 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=TDP-43+gains+function+due+to+perturbed+autoregulation+in+a+Tardbp+knock-in+mouse+model+of+ALS-FTD.">TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD.</a> Nature Neuroscience. 21 (4), 552-563 (2018).</li><li>Chou, A., 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=Inhibition+of+the+integrated+stress+response+reverses+cognitive+deficits+after+traumatic+brain+injury.">Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury.</a> Proceedings of the National Academy of Sciences. 114 (31), 6420-6426 (2017).</li><li>Padmashri, R., Tyner, K., Dunaevsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+a+cranial+window+for+repeated+in+vivo+imaging+in+awake+mice.">Implantation of a cranial window for repeated in vivo imaging in awake mice.</a> Journal of Visualized Experiments. (172), e62633 (2021).</li><li>Foda, M. A., Marmarou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.+Part+II:+Morphological+characterization.">A new model of diffuse brain injury in rats. Part II: Morphological characterization.</a> Journal of Neurosurgery. 80 (2), 301-313 (1994).</li><li>Flierl, M. A., 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=Mouse+closed+head+injury+model+induced+by+a+weight-drop+device.">Mouse closed head injury model induced by a weight-drop device.</a> Nature Protocols. 4 (9), 1328-1337 (2009).</li><li>Sun, W., 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=In+vivo+two-photon+imaging+of+anesthesia-specific+alterations+in+microglial+surveillance+and+photodamage-directed+motility+in+mouse+cortex.">In vivo two-photon imaging of anesthesia-specific alterations in microglial surveillance and photodamage-directed motility in mouse cortex.</a> Frontiers in Neuroscience. 13, 421 (2019).</li><li>Li, D., 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+Through-Intact-Skull+(TIS)+chronic+window+technique+for+cortical+structure+and+function+observation+in+mice.">A Through-Intact-Skull (TIS) chronic window technique for cortical structure and function observation in mice.</a> eLight. 2 (1), 1-18 (2022).</li><li>Paveliev, 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=Acute+brain+trauma+in+mice+followed+by+longitudinal+two-photon+imaging.">Acute brain trauma in mice followed by longitudinal two-photon imaging.</a> Journal of Visualized Experiments. (86), e51559 (2014).</li><li>Han, X., 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=In+vivo+two-photon+imaging+reveals+acute+cerebral+vascular+spasm+and+microthrombosis+after+mild+traumatic+brain+injury+in+mice.">In vivo two-photon imaging reveals acute cerebral vascular spasm and microthrombosis after mild traumatic brain injury in mice.</a> Frontiers in Neuroscience. 14, 210 (2020).</li><li>Jang, S. H., Kwon, Y. H., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrecoup+injury+of+the+prefronto-thalamic+tract+in+a+patient+with+mild+traumatic+brain+injury:+A+case+report.">Contrecoup injury of the prefronto-thalamic tract in a patient with mild traumatic brain injury: A case report.</a> Medicine. 99 (32), 21601 (2020).</li><li>Courville, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+coup-contrecoup+injuries+of+the+brain;+a+critical+review+of+recent+experimental+studies+in+the+light+of+clinical+observations.">The mechanism of coup-contrecoup injuries of the brain; a critical review of recent experimental studies in the light of clinical observations.</a> Bulletin of the Los Angeles Neurological Society. 15 (2), 72-86 (1950).</li><li>Drew, L. B., Drew, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contrecoup-coup+phenomenon:+a+new+understanding+of+the+mechanism+of+closed+head+injury.">The contrecoup-coup phenomenon: a new understanding of the mechanism of closed head injury.</a> Neurocritical Care. 1 (3), 385-390 (2004).</li></ol>

在这项研究中，将 AAV 注射、TBI 给药和颅窗植入头柱相结合，对小鼠大脑皮层（IV 层和 V 层）内 EGFP 标记的神经元进行纵向成像分析，以观察 TBI 对皮层神经元的影响。这项研究指出，这里选择的 TBI 部位，在海马体上方，为颅窗植入提供了一个相对平坦和宽阔的表面。相反，颅骨在该部位的前方相对较窄，因此很难确保头柱能够有效地接触颅骨表面。虽然本研究中仅使用了TDP-43Q331K / Q331K...

创伤性脑损伤 （TBI） 可由运动损伤、车辆碰撞和军事战斗引起，是一个全球性的健康问题。TBI 可导致生理、认知和行为缺陷，以及终身残疾或死亡 1,2。TBI 的严重程度可分为轻度、中度和重度，绝大多数为轻度 TBI （75%-90%）3。人们越来越认识到，TBI，特别是TBI的重复发生，可以促进神经元变性，并成为多种神经退行性疾病的危险因素，包括阿尔茨海默病（AD），肌萎缩侧索硬化症（ALS），额颞叶痴呆（FTD）和慢性创伤性脑病（CTE）4,5,6.然而，TBI诱导的神经退行性变的分子机制仍不清楚，因此是一个活跃的研究领域。为了深入了解神经元如何对 TBI 做出反应并从中恢复，本文描述了一种在 TBI 后通过纵向活体成像在小鼠中监测荧光标记的目标蛋白的方法，特别是在神经元内。
为此，本研究展示了如何将用于闭合颅骨 TBI 的外科手术（类似于之前报道的7,8）与植入颅内窗口的外科手术相结合，用于下游活体成像，如 Goldey 等人所述9.值得注意的是，先植入颅窗，然后在同一区域进行TBI是不可行的，因为诱发TBI的体重下降的影响可能会损坏颅窗并对小鼠造成无法弥补的伤害。因此，该方案旨在在同一手术过程中进行 TBI，然后将颅窗直接植入撞击部位。在一次手术中结合 TBI 和颅窗植入的一个优点是减少了小鼠接受手术的次数。此外，它允许人们监测对 TBI 的即时反应（即，在小时的时间尺度上），而不是在以后的手术过程中植入窗口（即，在 TBI 后几天的时间尺度上开始初始成像）。与通过固定组织免疫染色等传统方法监测神经元蛋白相比，颅窗和活体成像平台也具有优势。例如，活体成像需要较少的小鼠，因为同一只小鼠可以在多个时间点进行研究，而不是在离散时间点需要单独的小鼠队列。此外，可以随着时间的推移监测相同的神经元，从而允许跟踪同一细胞内的特定生物学或病理事件。
作为概念证明，这里证明了突触蛋白启动子下增强的绿色荧光蛋白 （EGFP） 的神经元特异性表达10。这种方法可以扩展到 1） 通过利用其他细胞类型特异性启动子来扩展不同的脑细胞类型，例如少突胶质细胞的髓鞘碱性蛋白 （MBP） 启动子和星形胶质细胞的神经胶质纤维酸性蛋白 （GFAP） 启动子11 ，2） 通过将其基因与 EGFP 基因融合不同的目标蛋白，以及 3） 共表达融合到不同荧光团的多种蛋白质。在这里，EGFP 通过颅内注射通过腺相关病毒 （AAV） 递送进行包装和表达。使用减重装置进行闭合性颅骨 TBI，然后植入颅窗。神经元EGFP的可视化是通过颅窗实现的，使用双光子显微镜检测体内的EGFP荧光。使用双光子激光，可以以最小的光损伤更深入地穿透皮质组织，从而可以对单个小鼠内的相同皮质区域进行数天至数月的重复纵向成像12,13,14,15。总之，这种将 TBI 手术与活体成像相结合的方法旨在促进对导致 TBI 诱发疾病病理学的分子事件的理解16,17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adjustable Precision Applicator Brushes</td>
			<td>Parkell</td>
			<td>S379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD insulin syringe</td>
			<td>BD</td>
			<td>NDC/HRI#08290-3284-38</td>
			<td>5/16&#34; x 31G</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue</td>
			<td>NDC67618-151-17</td>
			<td>including 7.5% povidone iodine</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>PAR Pharmaceutical</td>
			<td>NDC 42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>HIKMA Pharmaceutical</td>
			<td>NDC 0143-9924-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Mixing Dish</td>
			<td>Parkell</td>
			<td>SKU: S387</td>
			<td>For dental cement preparation</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>ZORO</td>
			<td>catlog #: G9531702</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalyst</td>
			<td>Parkell</td>
			<td>S371</td>
			<td>full name: &#34;C&#34; Universal TBB Catalyst</td>
		</tr>
		<tr>
			<td>Dental cement powder</td>
			<td>Parkell</td>
			<td>S396</td>
			<td>Radiopaque L-Powder for C&#38;B Metabond</td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>Foredom</td>
			<td>H.MH-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill controller</td>
			<td>Foredom</td>
			<td>HP4-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Phoenix</td>
			<td>NDC 57319-519-05</td>
			<td></td>
		</tr>
		<tr>
			<td>EF4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150113</td>
			<td>Head Dia/Lgth/mm 1.0/4.2</td>
		</tr>
		<tr>
			<td>Ethonal</td>
			<td>Fisher Scientific</td>
			<td>04355223EA</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>FG1/4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150413</td>
			<td>Head Dia/Lgth/mm 0.5/0.4</td>
		</tr>
		<tr>
			<td>FG4 carbide bit</td>
			<td>Microcopy</td>
			<td>Lot# C150309</td>
			<td>Head Dia/Lgth/mm 1.4/1.1</td>
		</tr>
		<tr>
			<td>Headpost</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-manufactured</td>
		</tr>
		<tr>
			<td>Heating apparatus</td>
			<td>CWE</td>
			<td>TC-1000 Mouse</td>
			<td>equiped with the stereotaxic instrument and be used while operating surgery</td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>CVS pharmacy</td>
			<td>E12107</td>
			<td>extra heating device and be used after surgery</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal</td>
			<td>NDC 46066-755-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane induction chamber</td>
			<td>Vetequip</td>
			<td>89012-688</td>
			<td>induction chamber for short</td>
		</tr>
		<tr>
			<td>Isoflurane volatilizing machine</td>
			<td>Vetequip</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane volatilizing machine holder</td>
			<td>Vetequip</td>
			<td>901801</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica surgical microscope</td>
			<td>Leica</td>
			<td>LEICA 10450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant ophthalmic ointment</td>
			<td>Picetal</td>
			<td>NDC 46066-753-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Marker pen</td>
			<td>Delasco</td>
			<td>SMP-BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Norbrook</td>
			<td>NDC 55529-040-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection pump and its controller</td>
			<td>World Precision Instruments</td>
			<td>micro4 and UMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>Hamilton 80014</td>
			<td>1701 RN, 10 &#956;L gauge for syringe and 32 gauge for needle, 2 in, point style 3</td>
		</tr>
		<tr>
			<td>Mosquito forceps</td>
			<td>CAROLINA</td>
			<td>Item #:625314</td>
			<td>Stainless Steel, Curved, 5 in</td>
		</tr>
		<tr>
			<td>Depilatory agent</td>
			<td>McKesson Corporation</td>
			<td>N/A</td>
			<td>Nair Hair Aloe &#38; Lanolin Hair Removal Lotion</td>
		</tr>
		<tr>
			<td>Microscope 1</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>Nikon microscope for cranial window preparation</td>
		</tr>
		<tr>
			<td>Microscope 2</td>
			<td>Zeiss</td>
			<td>LSM 7 MP</td>
			<td>two-photon microscope</td>
		</tr>
		<tr>
			<td>Multiphoton laser</td>
			<td>Coherent</td>
			<td>Chameleon Ultra II, Model: MRU X1, VERDI 18W</td>
			<td>laser for two-photon microscopy</td>
		</tr>
		<tr>
			<td>Non-absorbable surgical suture</td>
			<td>Harvard Apparatus</td>
			<td>catlog# 59-6860</td>
			<td>6-0, with round needle</td>
		</tr>
		<tr>
			<td>Norland Optical Adhesive 81</td>
			<td>Norland Products</td>
			<td>NOA 81</td>
			<td></td>
		</tr>
		<tr>
			<td>No-Snag Needle Holder</td>
			<td>CAROLINA</td>
			<td>Item #: 567912</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick base liquid</td>
			<td>Parkell</td>
			<td>S398</td>
			<td>&#34;B&#34; Quick Base For C&#38;B Metabond</td>
		</tr>
		<tr>
			<td>Regular scissor 1</td>
			<td>Eurostat</td>
			<td>eurostat es5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Regular scissor 2</td>
			<td>World Precision Instruments</td>
			<td>No. 501759-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Round cover glass 1</td>
			<td>Warner instruments</td>
			<td>CS-5R Cat# 64-0700</td>
			<td>for 5 mm of diameter</td>
		</tr>
		<tr>
			<td>Round cover glass 2</td>
			<td>Warner instruments</td>
			<td>CS-3R Cat# 64-0720</td>
			<td>for 3 mm of diameter</td>
		</tr>
		<tr>
			<td>Rubber rings</td>
			<td>Orings-Online</td>
			<td>Item # OO-014-70-50</td>
			<td>O-Rings</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Bioworld</td>
			<td>L19102411PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissor 1</td>
			<td>World Precision Instruments</td>
			<td>No. 91500-09</td>
			<td>tip straight</td>
		</tr>
		<tr>
			<td>Spring scissor 2</td>
			<td>World Precision Instruments</td>
			<td>No. 91501-09</td>
			<td>tip curved</td>
		</tr>
		<tr>
			<td>Stereotaxic platform</td>
			<td>KOPF</td>
			<td>Model 900LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel</td>
			<td>Item #: 1647358</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical Caliper</td>
			<td>World Precision Instruments</td>
			<td>No. 501200</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps 1</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>Catlog# 0508-5/45-PO</td>
			<td>style 5/45, curved</td>
		</tr>
		<tr>
			<td>Surgical forceps 2</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>catlog# 0103-5-PO</td>
			<td>style 5, straight</td>
		</tr>
		<tr>
			<td>Surgical forceps 3</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>catlog# 72912</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps 4</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>Catlog# 0508-5/45-PO</td>
			<td>style 5/45, curved</td>
		</tr>
		<tr>
			<td>Surgical gauze</td>
			<td>ZORO</td>
			<td>catlog #: G0593801</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Leica</td>
			<td>Leica KL300 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>UV box</td>
			<td>Spectrolinker</td>
			<td>XL-1000</td>
			<td>also called UV crosslinker</td>
		</tr>
		<tr>
			<td>Vaporguard</td>
			<td>Vetequip</td>
			<td>931401</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M Animal Care</td>
			<td>Part Number:014006</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital imaging of fluorescent protein expression in mice with a closed-skull traumatic brain injury and cranial window using a two-photon microscope

该协议的目标是演示在暴露于外源性刺激后，如何纵向可视化动物大脑特定细胞类型中目标蛋白的表达和定位。在这里，显示了闭合颅骨创伤性脑损伤（TBI）的施用和同时植入颅窗以进行随后的小鼠纵向活体成像。向小鼠颅内注射在神经元特异性启动子下表达增强绿色荧光蛋白（EGFP）的腺相关病毒（AAV）。2至4周后，在AAV注射位置使用体重下降装置对小鼠进行重复TBI。在同一手术过程中，将小鼠植入金属头柱，然后在TBI撞击部位植入玻璃颅窗。使用双光子显微镜在暴露于创伤的同一大脑区域检查EGFP的表达和细胞定位，历时数月。

作为该协议的概念证明，在3个月大时将表达AAV-Syn1-EGFP的病毒颗粒注射到雄性TDP-43Q331K / Q331K小鼠（C57BL / 6J背景）19的大脑皮层中。值得注意的是，也可以使用野生型 C57BL/6J 动物，但这项研究是在 TDP-43 Q331K/Q331K 小鼠中进行的，因为该实验室专注于神经退行性疾病研究。注射 AAV 后 4 周进行 TBI 手术。在相同的手术环境中，植入了头柱和颅窗。在TBI手术后第0天，1周...

Watch this Scientific Journal Video about Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope at Jo

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

这项研究表明，向小鼠提供重复性创伤性脑损伤，并同时植入颅窗，以便随后使用双光子显微镜对神经元表达的 EGFP 进行活体成像。

使用双光子显微镜对闭合颅骨创伤性脑损伤和颅窗小鼠荧光蛋白表达进行活体成像

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

我们的协议提供了一种可视化活小鼠对机械应力的神经元反应的方法，这可以提供创伤性脑损伤如何影响大脑的直接证据。该技术可以监测同一动物相同大脑位置的靶蛋白，用于创伤性脑损伤后的急性期和慢性期。可以相应地改变目的基因和病毒启动子，以研究大脑中特定细胞类型中的其他蛋白质。首先，将眼药膏涂抹在麻醉小鼠的眼睛上。用普通剪刀修剪，去除头顶的头发。在建立 12 至 15 毫米长的中线切口后，使用弯曲的尖端弹簧剪刀切除颅骨左右半球的皮肤。颅骨暴露后，用无菌棉签轻轻擦拭，并用无菌生理盐水冲洗，去除骨膜。颅骨区域干燥后，在前囟后方 2.5 毫米处和右侧矢状缝线外侧 2 毫米处标记创伤性脑损伤 （TBI） 撞击部位。快速将鼠标从立体定位框架中取出，并将头部放在 TBI 设备下方的缓冲垫上。将撞击器尖端与标记的撞击部位对齐。将系留的尼龙绳拉到鼠标头部上方 15 厘米处，抬起金属柱，然后松开，让重物自由落到换能杆上，换能杆与 TBI 部位的颅骨顶部接触。将麻醉小鼠的头部放在手术显微镜下的立体定位框架上。使用FG4硬质合金钻头以低转子速度轻轻缓慢地钻头颅骨表面，以去除剩余的骨膜，并形成粗糙的颅骨表面，使牙胶与颅骨牢固结合。使用生理盐水冲洗并清除颅骨表面的骨灰。为了将侧肌与颅骨分开，在眼睛后方约5毫米处，连接顶骨和颞颅骨的缝合线所在的位置，轻轻插入细闭合的尖端，并轻轻地向后移动闭合的尖端，直到羊羔形缝合线。分离将发生颅窗植入的一侧的侧肌。用生理盐水洗掉手术部位的碎屑，并用纱布擦干该区域。要植入头柱，请使用记号笔和手术卡尺在前囟后方 2.5 毫米处标记中心点，在右半球矢状缝合线处标记 1.5 毫米。然后在干净干燥的头骨上描摹开颅手术的周长。松开耳杆并旋转头部，使开颅平面完全水平，然后再次拧紧耳杆。使用棉签涂抹器的木棒在头柱的前后边缘添加两小滴强力胶。将钛头柱放在开颅手术的中心上方，并快速调整它以放置在将植入颅窗的同一平面内。轻轻按压直到强力胶干燥，通常需要大约 30 秒。通过充分混合 300 毫克水泥粉、六滴 QuickBase 液体和一滴催化剂，在预冷的陶瓷盘中制备牙科水泥。快速将大量牙科水泥混合物涂抹在描摹的圆周的外周，并覆盖任何暴露的骨表面。但是，不要覆盖开颅手术的部位。松开耳杆并将头柱固定在金属框架上，以确保头稳定，以便沿着标记的开颅手术周进行精确钻孔。要进行开颅手术，请使用手术卡尺验证标记圆圈的直径，如前所述，并根据需要进行调整，使颅窗紧贴开颅手术内。使用电动牙钻，首先使用 FG4 硬质合金钻头沿标记圆的外侧蚀刻和稀释颅骨，从而形成一条使颅骨变薄的轨道。用生理盐水冲洗该区域以洗去骨尘。继续使用 FG 1/4 硬质合金钻头稀释头骨，直到头骨薄且透明。定期停止钻孔，并再次用无菌盐水冲洗整个区域，以减少钻头的热量并洗去骨灰。使用EF4硬质合金钻头完成颅骨变薄，然后继续变薄并沿着轨道钻穿颅骨的其余部分。将 0.5 毫米细尖镊子插入破裂的地方，轻轻向上提起骨瓣，而不会压入下面的大脑。取出骨瓣后，用生理盐水冲洗开颅手术区域。使用弯曲的尖端手术镊子，轻轻去除可见的蛛网膜物质。使用直尖手术镊子拿起无菌玻璃窗，三毫米玻璃盖玻片朝下。将玻璃窗放置在开颅部位上方并调整，以确保窗户可以紧贴开颅手术边缘。五毫米的玻璃盖玻片在顶部。如前所述准备牙科水泥，并等待大约六分钟，直到水泥变得糊状和粘稠。在等待水泥变得糊状和厚实的同时，通过立体定位机械手对窗户施加足够的压力，以检查头骨是否可以牢固而紧密地接触玻璃窗。使用可调节的精密涂抹刷在窗边添加少量水泥，以将玻璃窗与头骨密封。等待大约 10 分钟，让水泥完全干燥，然后轻轻松开，然后取下窗户上方的机械手。如果多余的水泥覆盖了窗户，则使用带有 FG4 硬质合金钻头的牙钻修剪牙科水泥。术后约4小时，进行双光子成像，在浅表水平血管系统呈黑色，在约400微米的深水平，EGFP蛋白表达弥漫分布于整个细胞体。在 TBI 后 1 周零 4 个月，在第零天时间点观察到的脉管系统模式被用作定位相同成像区域的参考。脉管系统模式与第0天相似。同样，在整个细胞体中检测到EGFP表达。第0天和第4个月的荧光强度在浅表和深层水平上相似。然而，一周的荧光强度低于第0天和第4个月的荧光强度，这可能是由于其他TBI模型报道的翻译抑制。在操作开颅手术步骤时要格外小心，以免损伤脑组织。不要将钻头插入大脑。使用这种技术，研究人员可以在 TBI 后的不同阶段纵向可视化同一细胞中不同感兴趣的蛋白质，提供有关创伤后该蛋白质在哺乳动物大脑中的定位、表达和溶解度的信息。

Research

JoVE Journal

Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...