Die Autoren möchten Dr. David Rowe danken, dass freundlicherweise die transgene Mäuse und Li Chen für die histologische Unterstützung.
Die Forschung berichtet in dieser Publikation wurde vom nationalen Institut der Dental & Craniofacial Forschung von den National Institutes of Health unter Preis Anzahl K08DE025914 und der amerikanischen Vereinigung der kieferorthopädischen Foundation zur Sumit Yadav unterstützt.

<ol>
	<li>LeResche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Temporomandibular+Disorders:+Implications+for+the+Investigation+of+Etiologic+Factors.">Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors.</a> Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).</li><li>Chen, 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=Altered+functional+loading+causes+differential+effects+in+the+subchondral+bone+and+condylar+cartilage+in+the+temporomandibular+joint+from+young+mice.">Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice.</a> Osteoarthr Cartil. 17 (3), 354-361 (2009).</li><li>Pirttiniemi, P., Kantomaa, T., Sorsa, T. <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+decreased+loading+on+the+metabolic+activity+of+the+mandibular+condylar+cartilage+in+the+rat.">Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat.</a> Eur J Orthod. 26 (1), 1-5 (2004).</li><li>Chavan, S. J., Bhad, W. A., Doshi, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+temporomandibular+joint+changes+in+Twin+Block+and+Bionator+appliance+therapy:+a+magnetic+resonance+imaging+study.">Comparison of temporomandibular joint changes in Twin Block and Bionator appliance therapy: a magnetic resonance imaging study.</a> Prog Orthod. 15 (57), (2014).</li><li>Dutra, E. H., 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=Cellular+and+Matrix+Response+of+the+Mandibular+Condylar+Cartilage+to+Botulinum+Toxin.">Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.</a> PLoS ONE. 11 (10), 0164599 (2016).</li><li>Benjamin, M., Ralphs, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fibrocartilage+cells.">Biology of fibrocartilage cells.</a> Int Rev Cytol. 233, 1-45 (2004).</li><li>Shen, G., Darendeliler, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Adaptive+Remodeling+of+Condylar+Cartilage-+A+Transition+from+Chondrogenesis+to+Osteogenesis.">The Adaptive Remodeling of Condylar Cartilage- A Transition from Chondrogenesis to Osteogenesis.</a> J Dent Res. 84 (8), 691-699 (2005).</li><li>Utreja, 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=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Kaul, 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=The+Effect+of+Altered+Loading+on+Mandibular+Condylar+Cartilage.">The Effect of Altered Loading on Mandibular Condylar Cartilage.</a> PLoS ONE. 11 (7), 0160121 (2016).</li><li>Dyment, N. 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=High-Throughput,+Multi-Image+Cryohistology+of+Mineralized+Tissues.">High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.</a> J Vis Exp. , e54468 (2016).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch Histol Cytol. 66 (2), 123-143 (2003).</li><li>Hayman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+(TRAP)+and+the+osteoclast/immune+cell+dichotomy.">Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.</a> Autoimmunity. 41 (3), 218-223 (2008).</li></ol>

Die Autoren haben keinen finanziellen Interessenkonflikt.

Dieses Manuskript beschriebenen Verfahren zur Messung der morphometrische und zelluläre Analyse der murinen mandibulares Kondylen und Mandibeln. Die radiologischen morphometrische Maße können auch verwendet werden, andere Knochen aus kleinen Versuchstieren zu analysieren. Darüber hinaus die zelluläre Analyse (Zelle Quantifizierung und Knorpel Entfernung Zuordnung) beschränken sich nicht auf das Nagetier mandibulares Kondylus, sondern kann zur histologischen Abschnitte von zahlreichen Geweben zu quantifizieren.
Transgenen Mausmodellen auszudrücken fluoreszierende Reporter sind hervorragende Werkzeuge, um Veränderungen in der Genexpression visuell zu verstehen. Die Doppel-Kollagen fluoreszierende Reporter Maus-Modell (Col2a1XCol10a1) in diesem Bericht verwendeten war besonders geeignet für die Untersuchung des MCC, da transgene in der Region prehypertrophic und hypertrophe des MCC so ausgedrückt werden, dass Kollagen-Verteilung konnten ausgewertet werden. Wenn die Forscher keinen Zugang zu einem transgenen Mausmodell fluoreszierende Reporter zum Ausdruck zu bringen, können andere Methoden der histologischen Ausdruck Proteinanalytik, z. B. Immunfluoreszenz, verwendet werden.
Für die morphometrische Messungen ist der entscheidende Schritt vor der Einnahme der Schrank Röntgenbild Bilder, alle Weichteile des Unterkiefers oder der Knochen zu analysierenden entfernen. Übermäßige Muskel- oder Weichgewebe an den Knochen befestigt könnte die realen Messungen der Strukturen maskieren. Es ist wichtig, die Grenzen des Kabinetts Röntgenbild Systems beachten. Wie jedes x-ray es ist eine 2-dimensionale Darstellung einer 3-dimensionalen Struktur und Überschneidungen von Strukturen und Artefakte sollten vorsichtig interpretiert werden. Darüber hinaus sollte die Positionierung der Knochen in der radiologischen Gehäuse übereinstimmen.
Die zelluläre Quantifizierung durch zählen der Anzahl von positiven Pixel ist eine effiziente Methode zur histologischen Quantifizierung in einer Zelle-reiche Region wie der MCC, aber die Einschränkung dieses Ansatzes ist, dass es nicht die genaue Anzahl der Zellen liefert. Darüber hinaus, da es nur die ausgewählten Pixel quantifiziert, empfiehlt es sich, alle Bilder mit der gleichen "Stichprobe" Pixel, um die Quantifizierung der Pixel von unterschiedlicher Intensität zu vermeiden, bei der Analyse der verschiedenen Bilder zu quantifizieren.
Eine allgemeine Ratschlag bei der Quantifizierung der histologischen Abschnitte ist, mehrere Schnittserien jeder Probe zu analysieren (in dieser Handschrift wurden drei Schnittserien für jedes Kondylus analysiert), da Variationen innerhalb jeder Probe in der Regel eingehalten werden.
Die Methodik, die in unseren Experimenten erwähnt ist einfach, einfach zu bedienen und kann in jeder mineralisierten Gewebe-Studie in die Reporter Mäuse verwendet werden verwendet werden. Mit der vorhandenen Methodik ist es möglich, die Zellen zu visualisieren, die für TRAP (Col2a1 oder Col10a1) oder EdU (Col1a1 oder Col2a1) gefärbt sind, durch die Visualisierung der Co Lokalisierung von Zellen.

Kiefergelenk ist ein einzigartiges tragende Gelenk in der kraniofazialen Region gelegen und besteht aus Faserknorpel gebildet. Die MCC des Kiefergelenks ist unerlässlich für die Funktion der Gelenke, einschließlich ungehindert Kieferbewegung beim sprechen und arbeiten, aber es ist häufig von degenerativen Erkrankungen wie Arthrose1betroffen. Kiefergelenk hat die Fähigkeit zur Anpassung an äußere Reize und laden Umbauten, führt zu strukturellen und zellulären Veränderungen auf die Komponenten des MCC2,3,4,5. Die tragenden Eigenschaften des MCC lässt sich durch die Wechselwirkungen zwischen seinen Bestandteilen, einschließlich Wasser, das Kollagennetz und dicht gepackte Proteoglykane. Der MCC hat vier verschiedene zelluläre Zonen, die verschiedene Arten von Kollagen und nicht-Kollagen Proteine ausdrücken: 1) der oberflächlichen oder artikuläre Zone; (2) die proliferative Zone, bestehend aus undifferenzierten mesenchymalen Zellen und das reagiert auf Anforderungen laden; (3) die prehypertrophic Zone, bestehend aus Reife Chondrozyten Kollagen Typ 2 zum Ausdruck zu bringen; und 4) die hypertrophen zone, die Region, wo die hypertrophen Chondrozyten auszudrücken Kollagen Typ 10 sterben, und Verkalkung zu unterziehen. Die nicht mineralisierten Region ist reich an Proteoglykanen, die Resistenz gegen Druckkräfte6zur Verfügung zu stellen.
Gibt es kontinuierliche Mineralisierung in der hypertrophen Zone des MCC, wo der Übergang von werden zur Osteogenesis auftritt, garantiert die robuste mineralische Struktur des subchondralen Knochens des Unterkiefers Kondylus7. Zellveränderungen in den Regionen Latein und mineralisierten führen letztlich zu morphologischen und strukturelle Veränderungen in den Unterkiefer Kondylus und Unterkiefer. Aufrechterhaltung der Homöostase der alle zellulären Regionen des MCC und die Mineralisierung des subchondralen Teils sind unerlässlich für die Gesundheit, die Tragfähigkeit und die Integrität des Kiefergelenks.
Die mehrere Kollagen transgenen Mausmodell (wie von Utreja Et Al.beschrieben) 8 ist ein großartiges Werkzeug verwenden, um Veränderungen im Kollagen Ausdruck zu verstehen, weil alle transgene im MCC ausgedrückt werden. Für eine eingehende histologische Bewertung werden histologische Flecken zur Matrix Deposition, Mineralisierung, Zellproliferation, Apoptose, sowie Protein-Expression in den verschiedenen Zellschichten des MCC zu studieren.
In diesem werden Manuskript, histologische und morphometrische Analysen zur zelluläre und strukturelle Veränderungen in der MCC und subchondralen Knochen des Unterkiefers Kondylus von Mäusen zu bewerten. Darüber hinaus wird eine Zelle Quantifizierungsmethode, für die Analyse von fluoreszierenden histologische Bilder und für die Zuordnung von leichten Objektträger, beschrieben. Zusammenpressende statische Kiefergelenk laden-Methode, die zelluläre und morphologische Veränderungen an der MCC und subchondralen Knochen9verursacht, wird auch veranschaulicht, um unsere Methoden zu überprüfen.
Die hier beschriebenen Methoden einsetzbar, morphometrische und histologische Veränderungen im Unterkiefer Kondylus und Unterkiefer von Nagetieren zu bestimmen oder zu anderen Regionen der Endochondral Verknöcherung und die Morphologie der zusätzlichen mineralisierten Gewebe zu analysieren.

<table width="896">
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			<td height="21" style="width:392px;height:21px;">MX20 Radiography System</td>
			<td style="width:219px;">Faxitron X-Ray LLC&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">Digimizer Image software&#160;</td>
			<td style="width:219px;">MedCalc Software</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:392px;height:21px;">Shandon Cryomatrix embedding resin</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:392px;height:21px;">Manual microscope Axio Imager Z1</td>
			<td style="width:219px;">Carl Zeiss</td>
			<td style="width:119px;">208562</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">yellow fluorescent protein filter&#160; - EYFP</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49003</td>
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			<td height="21" style="width:392px;height:21px;">cyan fluorescent protein filter - ECFP</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49001</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">red fluoresecent protein filter - Cy5</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49009</td>
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			<td height="21" style="width:392px;height:21px;">sodium acetate anhydrous</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
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			<td height="23" style="width:392px;height:23px;">sodium L-tartrate dibasic dihydrate&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">228729</td>
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			<td height="21" style="width:392px;height:21px;">sodium nitrite&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
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			<td height="21" style="width:392px;height:21px;">ELF97 substrate</td>
			<td style="width:219px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">E6600</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ClickiT EdU Alexa Fluor 594 HCS kit</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">C10339&#160;</td>
			<td>includes EdU (5-ethynyl-2&#39;-deoxyuridine)&#160;</td>
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			<td height="21" style="width:392px;height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="width:392px;height:21px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>S3264</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">Sodium phosphate monobasic&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">71505</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">Toluidine Blue O&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3260</td>
			<td></td>
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			<td height="25" style="width:392px;height:25px;">Adobe Photoshop&#160;</td>
			<td style="width:219px;">Adobe Systems Incorporated</td>
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			<td height="24" style="width:392px;height:24px;">Phosphate buffered saline tablets (PBS)</td>
			<td style="width:219px;">Research Products International</td>
			<td style="width:119px;">P32080-100T</td>
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			<td height="21" style="height:21px;">CNA Beta III Nickel-Free Archwire</td>
			<td>Ortho Organizers, Inc.</td>
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			<td>GraphPad Software, Inc.</td>
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a morphometric and cellular analysis method for the murine mandibular condyle

Das Kiefergelenk (TMJ) hat die Fähigkeit zur Anpassung an äußere Reize und laden Änderungen beeinflussen die Position der Kondylen sowie die strukturelle und zellulären Komponenten des Unterkiefers kondylären Knorpels (MCC). Dieses Manuskript beschreibt Methoden für die Analyse dieser Veränderungen und eine Methode für das Laden des Kiefergelenks in Mäusen (d. h. zusammenpressende statische TMJ laden) zu verändern. Die strukturellen Bewertung hier abgebildete ist eine einfach morphometrische Ansatz, der nutzt die Digimizer-Software und wird in Röntgenaufnahmen kleiner Knochen durchgeführt. Darüber hinaus die Analyse der zellulären ändert führt zu Veränderungen im Kollagen Ausdruck, Knochenaufbau, Zellteilung und Proteoglykan Verteilung im MCC beschrieben. Die Quantifizierung dieser Änderungen in histologischen Abschnitten - durch zählen der positiven fluoreszierenden Pixeln mit Software und messen die Entfernung Zuordnung von Bildern und gefärbten Bereich mit Digimizer - wird auch demonstriert. Die hier gezeigten Methoden beschränken sich nicht auf das murine TMJ, aber auf zusätzliche Knochen der kleinen Versuchstieren und in anderen Regionen der Endochondral Verknöcherung verwendet werden konnte.

Deskriptive Statistik wurden durchgeführt, um die Verteilung der morphometrische Maße (mandibularis Länge, kondylären Länge, kondylären Breite) und histologische Analysen untersuchen. Ergebnisse wurden im Vergleich zwischen den geladen (z.B. Mäuse ausgesetzt zusammenpressende Belastung mit der Beta-Titan-Feder) und Kontrollgruppe (z.B. passende Kontroll-Mäusen, die keinem Eingriff erhalten haben). Statistisch signifikante Unterschiede zwischen den waren bestimmt durch ungepaarten t-Test und ein p-Wert von < 0,05 wurde als um statistisch signifikant zu sein.
Morphometrische Messungen, wie in Schritt 4 und Abbildung 3, wurden in den Mandibeln von der geladenen und Kontrollgruppen durchgeführt. Die geladenen Gruppe präsentiert mit deutlich erhöhten Unterkiefer Länge (geladen: 16.62 ± 0,23 mm versus Kontrolle: 16.21 ± 0,2 mm; p < 0,05) und Kondylus Kopf Länge (geladen: 4,6 mm, SD 0,08 mm versus Kontrolle: 4,4 mm; SD 0,06; p < 0,05) im Vergleich zur Kontrollgruppe. Allerdings gab es keinen signifikanten Unterschied in der Kondylus Breite zwischen Gruppen (geladen: 3,06 ± 0,12 mm versus Kontrolle: 2,9 ± 0,11; p = 0,09).
Quantifizierung der Kollagen-Verteilung mit der in Schritt 8 deutlich offenbart beschriebenen Methode erhöht Col10a1 Ausdruck in der MCC die Kondylen des Arbeitskreises geladen im Vergleich zur Kontrolle (geladen: 21 % ± 4,46 % versus Kontrolle: 7,50 % ± 2,03 %; p < 0,005; Abbildung 8 " A und D). Auf der anderen Seite gab es keine statistisch Unterschied in Col2a1 Ausdruck zwischen der geladenen und Steuern Gruppen (geladen: 4,85 % ± 1.95 % versus Kontrolle: 2,92 % ± 1,89 %; p = 0,13; Abbildung 8 B und E). Darüber hinaus zeigte Analyse der TRAP Tätigkeit erhöhte TRAP-positiven Regionen subchondralen und Umgebung die Kondylen des geladenen Mäuse (geladen: 5.28 % ± 1,45 % versus Kontrolle: 2,41 % ± 1,39 %; p < 0,05; Abbildung 8 " C und F). In ähnlicher Weise, wir beobachteten erhöhten Zellproliferation, wie durch erhöhte EdU-positiven Zellen in der MCC der geladenen Gruppe im Vergleich zu Kontroll-Mäusen (geladen: 6,23 % ± 1,89 % versus Kontrolle: 1,90 % ± 1,03 %; p < 0,05; Abbildung 9 " A und C).
Proteoglykan Verteilung im MCC von geladen und Kontroll-Mäusen wurde durch die Auswertung Toluidin blau gefärbten Bereich und der Entfernung Karte quantifiziert, wie in Schritt 9 und Abbildung 7beschrieben. Wir fanden signifikant erhöhte Entfernung Zuordnung in der MCC Kondylen der geladenen Gruppe im Vergleich zur Kontrolle (geladen: 210.22 µm ± 4.11 µm im Vergleich zu Kontrolle: 187.36 µm ± 8.64 µm; p < 0,005; Abbildung 9 ( B und D). Proteoglykan-gefärbten Bereich war jedoch nicht statistisch anders zwischen den geladenen und Steuern Gruppen (geladen: 203,897.93 µm2 ± 10,171.00 µm2 versus Kontrolle: 202,875.09 µm2 ± 33,419.09 µm2; p = 0,94; Abbildung 9 B und E)
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55998/55998fig8.jpg"> Abbildung 8 : Repräsentative Ergebnisse: (A) Col10a1-positiven Zellen in der sagittalen Abschnitten des MCC der Kondylen von Kontrolle und geladenen Mäuse. Es gibt vermehrt Col10a1-positiven Zellen in der geladenen Gruppe (D). (B) Col2a1-positiven Zellen in Kontrolle und geladenen Mäuse. Es gibt keinen Unterschied in Col2a1-positiven Zellen zwischen den Gruppen (E). FANGEN Sie (C) Färbung in Kondylen Kontroll-und geladenen Mäuse. TRAP-Überaktivität in der geladenen Gruppe im Vergleich zur Kontrolle (F). Die Histogramme (D-F) repräsentieren die Mittel ± SD für n = 4 pro Gruppe. ** Signifikanter Unterschied zwischen Steuerung und geladenen Gruppen (p < 0,005). Maßstabsleiste = 200µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig8large.jpg" target="_blank">Klicken Sie bitte hier, um eine größere Version dieser Figur.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55998/55998fig9.jpg"> Abbildung 9:repräsentative Ergebnisse: (A) EdU Färbung in Kondylen Kontroll-und geladenen Mäuse. Erhöhten Zellproliferation in der geladenen Gruppe, Zellen vertreten durch erhöhte EdU-positiven (C). (B) Toluidin blauer Färbung in die Kondylen des Kontroll- und geladenen Mäuse. Erhöhte Dicke Knorpel in die geladenen Mäuse dargestellt durch erhöhte Entfernung Zuordnung in der experimentellen Gruppe (D). Keinen Unterschied in der Proteoglykan-gefärbten Bereich zwischen der Steuerung und geladenen Gruppen (E). Histogramme (C-E) repräsentieren die Mittel ± SD für n = 4 pro Gruppe. ** Signifikanter Unterschied zwischen Steuerung und geladenen Gruppen (p < 0,005). Maßstabsleiste = 200 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig9large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>

Watch this Scientific Journal Video about A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle at JoVE.com

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Dieses Manuskript stellt Methoden für die Analyse morphometrische und zelluläre Veränderungen innerhalb des Unterkiefers Kondylus von Nagetieren.

Eine morphometrische und zelluläre Analysemethode für das Murine mandibulares Kondylus

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

a-morphometric-cellular-analysis-method-for-murine-mandibular

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K Aufrufe.  University of Connecticut Health Center. Dieses Manuskript stellt Methoden für die Analyse morphometrische und zelluläre Veränderungen innerhalb des Unterkiefers Kondylus von Nagetieren.

Serie: A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Verwendung von Drehstabtest als Methode für die qualitative Analyse von Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Forschung

Biologie

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murine Renal Transplantation

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Ein High-Throughput-Verfahren zur global Studie der Organellen-Morphologie in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Ein High-Throughput-Methode für Zebrabärblinge Sperma Kryokonservierung und In Vitro Fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Eine schnelle High-Throughput-Verfahren zur Abbildung Ribonukleoproteine ​​(RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Blaue Native Polyacrylamid-Gelelektrophorese (BN-PAGE) zur Analyse von Multiproteinkomplexen aus Zelllysaten

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Eine Explantation Assay zur Beurteilung Cellular Verhalten des Cranial Mesenchym

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Einzellige Mikroinjektions für Zellkommunikationsanalyse

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolierung von murinen Ventil Endothelzellen

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...