אנו אסירי תודה לד&quot;ר פרנק קירכהוף למתן GFAP-EGFP וזני עכבר CX3CR1-EGFP. העבודה נתמכה על ידי מענקים ממרכז ניידות הבינלאומית של פינלנד, טקס, בית הספר פיני בוגר מדעי המוח (FGSN) והאקדמיה של פינלנד.

<ol>
	<li>Lighthall, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cortical+impact:+A+new+experimental+brain+injury+model.">Controlled cortical impact: A new experimental brain injury model.</a> J. Neurotrauma. 5 (1), 1-15 (1988).</li><li>Lindgren, S., Rinder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+in+head+injury.">Experimental studies in head injury.</a> Biophysik. 2 (5), 320-329 (1965).</li><li>Shreiber, D. I., 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=Experimental+investigation+of+cerebral+contusion:+histopathological+and+immunohistochemical+evaluation+of+dynamic+cortical+deformation.">Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation.</a> J. Neuropathol. Exp. Neurol. 58 (2), 153-164 (1999).</li><li>Feeney, D. M., Boyeson, M. G., Linn, R. T., Murray, H. M., Dail, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+to+cortical+injury:+I.+Methodology+and+local+effects+of+contusions+in+the+rat.">Responses to cortical injury: I. Methodology and local effects of contusions in the rat.</a> Brain Res. 211 (1), 67-77 (1981).</li><li>Bardehle, 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=Live+imaging+of+astrocyte+responses+to+acute+injury+reveals+selective+juxtavascular+proliferation.">Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation.</a> Nat. Neurosci. 16 (5), 580-586 (2013).</li><li>Sword, J., Masuda, T., Croom, D., Kirov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+neuronal+and+astroglial+disruption+in+the+peri-contusional+cortex+of+mice+revealed+by+in+vivo+two-photon+imaging.">Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging.</a> Brain. 136 (5), 1446-1461 (2013).</li><li>Feng, 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Nolte, 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=GFAP+promoter-controlled+EGFP-expressing+transgenic+mice:+a+tool+to+visualize+astrocytes+and+astrogliosis+in+living+brain+tissue.">GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue.</a> Glia. 33 (1), 72-86 (2001).</li><li>Jung, 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20 (11), 4106-4114 (2000).</li><li>Nimmerjahn, A., Kirchhoff, F., Kerr, J. N. D., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulforhodamine+101+as+a+specific+marker+of+astroglia+in+the+neocortex+in+vivo.">Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.</a> Nat. Methods. 1 (1), 31-37 (2004).</li><li>Carr&#233;, 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=Technical+aspects+of+an+impact+acceleration+traumatic+brain+injury+rat+model+with+potential+suitability+for+both+microdialysis+and+PtiO2+monitoring.">Technical aspects of an impact acceleration traumatic brain injury rat model with potential suitability for both microdialysis and PtiO2 monitoring.</a> J. Neurosci. Methods. 140, 23-28 (2004).</li><li>Holtmaat, 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=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat. Protoc. 4 (8), 1128-1144 (2009).</li><li>Yang, G., Pan, F., Parkhurst, C. N., 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=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat. Protoc. 5, 201-208 (2010).</li><li>Cianchetti, F. A., Kim, D. H., Dimiduk, S., Nishimura, N., Schaffer, 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=Stimulus-evoked+calcium+transients+in+somatosensory+cortex+are+temporarily+inhibited+by+a+nearby+microhemorrhage.">Stimulus-evoked calcium transients in somatosensory cortex are temporarily inhibited by a nearby microhemorrhage.</a> PloS one. 8 (5), (2013).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J. Vis. Exp. 61, (2012).</li></ol>

יש המחברים אין לחשוף.

טראומה מוחית היא אירוע פתאומי, בלתי צפוי. כאן, אנו מתארים את מודל החיה שמתרבה ספקטרום של שינויים פתולוגיים שנצפו בחולים אנושיים לאחר פגיעה מוחית כגון ניוון מוחיים, חיסול של דנדריטים, בצקת במוח, צלקת גליה, דימומים בקליפת המוח בשילוב עם דימום תת עכבישי מוקד וחדירות מוגב...

פגיעה מוחית חריפה היא בעיה משמעותית בבריאות הציבור עם שכיחות גבוהה של פגיעה בתאונות דרכים, נפילות או תקיפות, ושכיחות גבוהה של מוגבלות כרוניות שלאחר מכן. גישות טיפוליות לטיפול בפגיעה מוחית יישארו סימפטומטי לחלוטין, ובכך להגביל את האפקטיביות של טיפול prehospital, כירורגית וקריטי. זה הופך את ההשפעה החברתית והכלכלית של פגיעה מוחית חמורה במיוחד. עבור מגוון רחב של סיבות, רוב הניסויים הקליניים לא הצליח להוכיח שיפור בהתאוששות לאחר פגיעה מוחית תוך שימוש בגישות טיפוליות חדשניות. מודלים של בעלי החיים הם קריטיים לפיתוח אסטרטגיות טיפוליות חדשות לקראת שלב שבו יעילות תרופה ניתן לחזות בחולים עם ניזק מוחי. נכון לעכשיו, כמה מודלים של בעלי חיים מבוססים היטב של חבלת הראש קיימים, כוללים השפעה מבוקרת קליפת המוח 1, פגיעת הקשה נוזל 2, עיוות בקליפת המוח דינמית 3, ירידה במשקל4, ופציעת תמונה 5. מספר המודלים הניסיוניים שנוצלו כדי ללמוד היבטים מורפולוגיים, מולקולריים והתנהגותיות מסוימים של פתולוגיה הקשורים חבלת הראש. עם זאת, אין מודל חיה אחת הוא מוצלח כולו באימות אסטרטגיות טיפוליות חדשות. פיתוח של מודלים של בעלי חיים אמינים, לשחזור ובשליטה של ​​פגיעה מוחית יש צורך להעריך את התהליכים פתולוגיים המורכבים. שילוב החדשני של הטכנולוגיות החדישות ביותר מיקרוסקופי ההדמיה וכתבי ניאון גנטי מקודדים מציע הזדמנות חסרת תקדים לחקור את כל השלבים של פגיעה מוחית, הכוללים פגיעה העיקרית, הפצה של הפגיעה העיקרית, פגיעה משנית, והתחדשות. בפרט, in vivo שני הפוטונים במיקרוסקופ הוא טכנולוגיה אופטית קוי ייחודית המאפשרת הדמיה של מבנים תאיים ואפילו subcellular בשכבות בקליפת המוח עמוקה של מוח מכרסם בזמן אמת. מספר סוגים של תאים וorgaניתן הדמיה Nelles בו זמנית על ידי שילוב סמני ניאון שונים. שימוש בכלי רב עוצמה זה, אנו יכולים לחזות שינויים מורפולוגיים ופונקציונליים דינמיים בחיים מוח בתנאי פוסט טראומתיים. היתרונות של במיקרוסקופ שני פוטונים vivo בלימוד פגיעה מוחית הודגמו לאחרונה על ידי קירוב ועמיתיו 6. שימוש במודל חבלה קל המוקדים בקליפת המוח, מחברים אלה הראו כי פציעה הדנדריטים חריפה בקליפת pericontusional היא מגודרת על ידי הירידה בזרימת הדם המקומית. יתר על כן, הם הראו כי קליפת המוח נפגעת מטבולית סביב אתר חבלת ניזוק עוד יותר על ידי שלילת קוטביות מתפשטת. נזק המשני זה משפיע על מעגלים סינפטיים, מה שהופך את ההשלכות של פגיעה מוחית טראומטית חמורות יותר. כאן, אנו מציעים את השיטה של ​​זין stereotaxic עם מחט מזרק, אשר יכול להיות בשילוב עם יישום תרופה אקטואלי בו זמנית, כמודל מתקדם למוח המקומיפציעה וככלי ללמוד השלכות pathophysiological של טראומה חריפה במוח של היונקים in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2A-sa dumb Tweezers, 115mm</td>
			<td style="width:71px;">XYtronic</td>
			<td style="width:119px;">XY-2A-SA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G &#189;&#8217;&#8217; needle</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">REF 304000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Animal trimmer, shaving machine</td>
			<td style="width:71px;">Aesculap</td>
			<td style="width:119px;">Isis GT420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binocular Microscope</td>
			<td style="width:71px;">Zeiss&#160;</td>
			<td style="width:119px;">Stemi 2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Biological Temperature Controller with stainless steel heating pad</td>
			<td style="width:71px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blunt microsurgical blade</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">REF 374769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Borosilicate tube with filament</td>
			<td style="width:71px;">Sutter Instruments</td>
			<td style="width:119px;">BF120-69-10</td>
			<td style="width:167px;">For glass pipette production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofene</td>
			<td style="width:71px;">Pfizer</td>
			<td style="width:119px;">Rimadyl vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chlorhexidine digluconate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C9394</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Dental cement</td>
			<td style="width:71px;">DrguDent, Dentsply</td>
			<td style="width:119px;">REF 640 200 271</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Dexamethasone</td>
			<td style="width:71px;">FaunaPharma</td>
			<td style="width:119px;">Rapidexon vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Disposable drills</td>
			<td style="width:71px;">Meisinger</td>
			<td style="width:119px;">HP 310&#160;104&#160;001&#160;001&#160;008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dulbeco&#8217;s PBS 10X</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D1408</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont #5 forceps, 110 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">91150-20</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Ealing microelectrode puller</td>
			<td style="width:71px;">Ealing</td>
			<td style="width:119px;">50-2013</td>
			<td style="width:167px;">Vertical puller for glass pipette production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eyes-ointment</td>
			<td style="width:71px;">Novartis</td>
			<td style="width:119px;">Viscotears</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foredom drill control</td>
			<td style="width:71px;">Foredom&#160;</td>
			<td style="width:119px;">FM3545</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foredom micro motor handpiece</td>
			<td style="width:71px;">Foredom</td>
			<td style="width:119px;">MH-145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Gas anesthesia platform for mice</td>
			<td style="width:71px;">Stoelting</td>
			<td align="right" style="width:119px;">50264</td>
			<td style="width:167px;">Assembled on stereotaxic instrument</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Hemostasis Collagen Sponge</td>
			<td style="width:71px;">Avitene, Ultrafoam BARD</td>
			<td style="width:119px;">Ref 1050050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:71px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:71px;">Intervet</td>
			<td style="width:119px;">Ketaminol vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Mai Tai DeepSee laser</td>
			<td style="width:71px;">Spectra-Physics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal holder</td>
			<td style="width:71px;">Neurotar</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro dressing forceps, 105 mm</td>
			<td style="width:71px;">Aesculap</td>
			<td style="width:119px;">BD302R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Microfil</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">MF34G-5</td>
			<td style="width:167px;">Micro syringe filling capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Multiphoton Laser Scanning Microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">FV1000MPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NanoFil Syringe 10 microliter</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">NANOFIL</td>
			<td style="width:167px;">Hamilton syringe</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Nonwoven swabs 5&#215;5</td>
			<td style="width:71px;">Molnlycke Health Care</td>
			<td style="width:119px;">Mesoft</td>
			<td style="width:167px;">Surgical tampons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylic glue</td>
			<td style="width:71px;">Henkel</td>
			<td style="width:119px;">Loctite 401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Round glass coverslip&#160;</td>
			<td rowspan="2" style="width:71px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 thickness&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Small animal stereotaxic instrument</td>
			<td style="width:71px;">David Kopf Instruments</td>
			<td align="right" style="width:119px;">900</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Stoelting mouse and neonatal rat adaptor</td>
			<td style="width:71px;">Stoelting</td>
			<td align="right" style="width:119px;">51625</td>
			<td style="width:167px;">Assembled on stereotaxic instrument.</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Student iris scissors, straight 11.5 cm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">91460-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Sulforhodamine 101</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">S-359</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">UMP3-1</td>
			<td style="width:167px;">Microinjector and controller</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Xylazine</td>
			<td style="width:71px;">Bayer Health Care</td>
			<td style="width:119px;">Rompun vet</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

acute brain trauma in mice followed by longitudinal two-photon imaging

למרות טראומה מוחית חריפה לעתים קרובות תוצאות מנזק בראש בתאונות שונות ומשפיעה על חלק ניכר מהאוכלוסייה, אין טיפול יעיל לזה עדיין. מגבלות של מודלים של בעלי חיים המשמשים כיום לעכב הבנה של מנגנון פתולוגיה. מיקרוסקופיה multiphoton מאפשרת לימוד תאים ורקמות במוח של בעלי חיים ללא פגע אורכים בתנאים פיסיולוגיים ופתולוגיים. כאן, אנו מתארים שני מודלים של פגיעה מוחית חריפה למדו באמצעות הדמיה שני פוטונים של תאים במוח התנהגות בתנאי פוסט טראומתיים. אזור במוח שנבחר הוא נפגע עם מחט חדה כדי לייצר טראומה של רוחב ועומק שבשליטת parenchyma המוח. השיטה שלנו משתמשת בזין stereotaxic עם מחט מזרק, אשר יכול להיות בשילוב עם יישום תרופה בו זמנית. אנו מציעים כי בשיטה זו יכולה לשמש ככלי מתקדם ללמוד מנגנונים תאיים של השלכות pathophysiological של טראומה חריפה במוח של יונקים <em&gt; In vivo. בסרטון הזה, אנו משלבים פגיעה מוחית חריפה עם שתי הכנות: דילול חלון וגולגולת גולגולת. כמו כן נדונונו ביתרונות ומגבלות של שני ההכנות להדמית multisession של התחדשות המוח לאחר טראומה.

יש לנו אופטימיזציה שני הליכי פעולה: 1) חלון כרוני גולגולת ו2) דלילה גולגולת, להדמיה מוחית פוסט טראומטית בעכברים מהונדסים. בתצוגה סכמטית של ההכנות הניסיוניות מוצגת באיור 1. זין טראומטי על ידי מחט פלדה של OD 0.3 מ&quot;מ (30 G) מוחל (איור 1 א) גם נקדח. חלון הכנת גו?...

Watch this Scientific Journal Video about Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging at JoVE.com

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

טראומה מוחית חריפה היא פגיעה חמורה שאין לה טיפול הולם עד כה. מיקרוסקופיה multiphoton מאפשרת לימוד אורכי התהליך של התפתחות טראומה המוחית חריפה וחיטוט אסטרטגיות טיפוליות במכרסמים. שני דגמים של טראומה מוחית חריפה למדה אצל בשני פוטונים הדמיה vivo של המוח הם הפגינו בפרוטוקול זה.

טראומה מוחית חריפה בעכברים אחריו שני פוטונים הדמיה אורכית

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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12.4K Views.  University of Helsinki.  טראומה מוחית חריפה היא פגיעה חמורה שאין לה טיפול הולם עד כה. מיקרוסקופיה multiphoton מאפשרת לימוד אורכי התהליך של התפתחות טראומה המוחית חריפה וחיטוט אסטרטגיות טיפוליות במכרסמים. שני דגמים של טראומה מוחית חריפה למדה אצל בשני פוטונים הדמיה 

Video: Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Pseudofracture: טראומה חריפה היקפיים דגם רקמות

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

לחץ אוורור מבוקר כדי לגרום פגיעה ריאות חריפה על עכברים

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

השימוש fMRI אתגר תרופתי, מחקר קדם קליניים: בקשה מערכת 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

פגיעה קריעת צוואר הרחם מבוקרת בעכברים

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma עכבר דגם orthotopic שמירה על אילוצי המוח של רקמת ריאה, פיסיים ומתאים לשני פוטונים במיקרוסקופ Intravital

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

הזרקת intracardiac מונחה ההדמיה אולטראסאונד לפתח מודל עכבר של שד גרורות סרטן מוח ואחריו האורכי MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

קנדידה אלביקנס Biofilm פיתוח על גופי חוץ-רפואי רלוונטיים בתת עורי מודל עכבר ואחרי פליטת אור הדמיה

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

אֹרכִּי<em&gt; In vivo</em&gt; הדמיה של Cerebrovasculature: רלוונטיות למחלות של מערכת העצבים המרכזית

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

מודל פולשנית לניתוח שבר Endochondral ריפוי בעכברים בתנאים מתוקננים ביו-מכני

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

גרימת פציעה חריפה של הריאה בעכברים על ידי הישירה ליפופוליסכראסטיליאלגרלציה

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...