This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).

Alle Tier Verfahren wurden in Übereinstimmung mit Empfehlungen im Leitfaden für die Pflege und Verwendung von Labortieren durch die National Institutes of Health und im Rahmen einer genehmigten Protokoll, das von der Institutional Animal Care und Use Committee am Scripps Research Institute durchgeführt. 1. Objektiv Dissection <ol><li> Euthanize Mäuse gemäß den Empfehlungen in den National Institutes of Health &quot;Leitfaden für die Pflege und Verwendung von Labortieren&quot; und zugelassene Institution Tier Protokolle verwenden. </li><li> Enucleate das Auge von Mäusen mit einer gebogenen Pinzette. Drücken Sie das Gewebe um das Auge mit der Zange aus der Steckdose, das Auge zu bringen, und dann reiß das Auge aus der Steckdose mit der Zange. Bringen Sie die Augen an die frische 1x phosphatgepufferter Salzlösung (PBS) in Dissektion Gericht. </li><li> Schneiden Sie den Sehnerv so nah an der Augapfel wie möglich. Sanft und vorsichtig einsetzen feinen Pinzette gerade in den Augapfel durch das Loch where der Sehnerv tritt aus dem hinteren. </li><li> machen sorgfältig einen Einschnitt mit einer Schere in den Augapfel vom hinteren zum Rand der Hornhaut. Nagetier Linsen besetzen ~ 30% des Auges. Nehmen Sie diese Einschnitte sorgfältig und nicht einlegen Pinzette oder Schere zu tief in das Auge eine Beschädigung der Linse zu vermeiden. </li><li> Geschnitten entlang der Verbindung zwischen der Hornhaut und Sklera mindestens um die halbe Augapfel. </li><li> Drücken Sie vorsichtig auf der Hornhaut, die Linse aus dem Auge durch die Öffnung in den Schritten 1.4 und 1.5 gemacht zu entfernen. </li><li> Verwenden Sie feine Spitze gerade Pinzette vorsichtig jede große Trümmer zu entfernen, die noch am Objektiv angebracht ist. Eine Sichtprüfung des Objektivs für Schäden vor den Steifigkeitsmessungen fortfahren. </li></ol> 2. Steifigkeitsmessungen <ol><li> Wiegen Sie mindestens 10 Deckgläser aus dem gleichen Feld einer Analysenwaage verwenden. Finden Sie das durchschnittliche Gewicht der Deck. Daher sollten Sie das gleiche Feld von Deckgläsern für alle Experimente. VornässenDie Deckgläser und rechten Winkelspiegel bei Raumtemperatur für mindestens 2 h in 1x PBS vor Experimente beginnen. </li><li> Füllen Sie die Messkammer (siehe Abbildung 1) mit 65 bis 75 ml 1x PBS. Die Messkammer wurde aus Plexiglas mit einer hauseigenen Maschinenhalle und Einschläge in der Kammer wurden durch eine Bohrmaschine auf die gewünschte Tiefe mit einem geeigneten Bohrer gemacht. Linsen verbleiben in 1x PBS bei Raumtemperatur für die Dauer der mechanischen Prüfung transparent. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53986/53986fig1.jpg" /> Abb . 1: Steifigkeitsmesskammer ein Foto , um die Abmessungen der maßgeschneiderte Steifigkeitsmesskammer mit einer Vielzahl von Einschläge von verschiedenen Tiefen und Formen zeigt. Die runden Einschläge, die 200 &amp; mgr; m oder 300 &amp; mgr; m tief (gelbe Pfeile) werden für die Messungen an der Maus Linsen verwendet werden. Divots sind 2 mm im Durchmesser und ~ 13-. 14 mm vom Rand der Kammer <a href="https://www.jove.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <ol start="3"><li> Platzieren Sie rechts Winkelspiegel in die Kammer mit einem konstanten Abstand von der Divot, die verwendet werden, wird die Linse zu halten. Stellen Sie sicher, dass der Spiegel nicht während des Experiments nicht bewegt. </li><li> Transfer seziert Linsen Messkammer sorgfältig mit Greifzange oder einer gebogenen Pinzette. </li><li> Nehmen Sie eine Draufsicht Bild des unbelasteten Linse direkt von oben. Nehmen Maus Objektiv Fotos bei 30-facher Vergrößerung mit Beleuchtung von Binokular (unten) und eine Faseroptik-Lichtquelle auf der linken und rechten Seite. Stellen Sie die faseroptische Stromversorgung zu 80% der maximalen Lichtintensität. Stellen Sie die Stromversorgung Ausgabe basierend auf Umgebungsbeleuchtung, Benutzereinstellung und Bildqualität nach Bedarf. </li><li> Nehmen Sie eine Seitenansicht Bild des unbelasteten Linse, die durch die ri zu sehen istght Winkelspiegel. Wenn die Kamera nicht kalibriert ist, nehmen Sie ein Bild von der Spiegelrand im Fokus. Der Spiegelrand ist 5 mm lang, und diese Messung später verwendet werden, können die Pixel / mm und dienen als Maßstab in den Bildern zu bestimmen. </li><li> Platz Linse in das Divot, und bestätigen, dass die Linse fest sitzt und gerade in der Divot. Nehmen Sie ein Bild von der Linse vor dem Laden. Die Linse sollte in der Divot an der vorderen oder hinteren Pol ruhen. </li><li> Platz 1 Deckglas vorsichtig auf die Linse. Warten Sie 2 min Kriechen zu ermöglichen, und nehmen Sie eine andere Seitenansicht Bild der geladenen Linse. </li><li> Weiterhin Zugabe von Deckgläsern, wie in Schritt 2.8 und unter Seitenansicht Bilder nach der Zugabe von jedem Deckglas, wie in Schritt 2,8 bis insgesamt 10 Deckgläser aufgebracht. </li><li> Entfernen Sie alle Deckgläser. Warten Sie 2 Minuten, und nehmen Sie eine Seitenansicht Bild der Linse (innerhalb und außerhalb des Divot), nachdem Sie alle Deckgläser zu entfernen. </li></ol> 3. Objektiv Nucleus Mess <ol><li> Um bestimine Linsenkerngröße, bewegen Sie die Linse auf eine saubere Petrischale mit 1x PBS gefüllt. </li><li> Sanft decapsulate die Linse fein gerade Pinzette. </li><li> Slough die kortikale Faserzellen aus, indem die Linse zwischen behandschuhten Fingern rollen. Der verbleibende Linsenkern wird wie eine harte Marmor fühlen. Verwenden Sie dieses Verfahren, um den Kern auf die Erwachsenen Linsen zu isolieren, um Alter von 1 Monat beginnen. Da der isolierte Kern ein starrer Körper ist, weitere mechanische Prüfung des Linsenkerns ist, kann nicht unter Verwendung dieses beschriebenen Verfahrens durchgeführt werden. </li><li> Spülen Sie vorsichtig den Linsenkern in 1x PBS in der Petrischale. </li><li> Setzen Sie den Linsenkern zurück in die Messkammer (nicht in der Divot), und nehmen Sie ein Bild des Linsenkerns durch den rechten Winkelspiegel. </li></ol><img alt="Figur 2" src="/files/ftp_upload/53986/53986fig2.jpg" /> Abbildung 2: a . Durch Deckgläser Druck Maus Objektive (A) Schematische und (B) Fotografie des experimental Setup einen 2 Monate alten Maus-Objektiv in einem 200 &amp; mgr; m tief Divot in der Messkammer gefüllt mit 1x PBS zeigt. Ein rechter Winkelspiegel und eine Digitalkamera auf einem Präpariermikroskop angebracht wurden verwendet, um Bilder der Linse während der Kompression durch Deckgläser zu sammeln. (C) Fotos von sagittalen Ansichten eines 2 Monate alten Wildtyp-Linse komprimiert, indem nacheinander Anzahl von Deck Erhöhung vorgesehen, um die Rohdaten zur Messung von axialen und äquatorialen Durchmesser und Berechnung axialen und äquatorialen Sorten während Deck-basierten Druckprüfung. Eine Reflexion der Linse kann manchmal in den Deckgläsern zu sehen (am deutlichsten sichtbar in der 1 Deckbild). Bei Messungen, ignorieren zu dem Scheitelpunkt der Linse, um die Reflexion und zu messen. (D) Fotos von sagittalen Blick auf die 2-Monate alten Wildtyp-Objektiv Nachverdichtung und der isolierten Linsenkern. Die Nachverdichtung Linse und isolierte Kern außerhalb des Divot sitzen. Maßstabsbalken, 1 mm. Diese Zahl wird geändert von Gokhin, et al. PLoS One, 2012 19. <a href="https://www.jove.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> 4. Bildanalyse <ol><li> Messen Sie äquatorialen und axialen Durchmesser der Linsen vor dem Laden und nach jedem Ladeschritt mit ImageJ oder ähnliche Software. Messen der Durchmesser jedes Linsenkern. Der Linsenkern ist nahezu kugelförmige so eine Messung bei jeder Ausrichtung 19,21 ausreichen. </li><li> Korrigieren Sie die Linse axialen Durchmesser von verwendet, um die Tiefe des Divot Zugabe. In der Messkammer, verdeckt das Divot 200 um (2 Monate alten Maus-Linsen) oder 300 um (4 Monate alte und 8 Monate alten Maus-Linsen) der axialen Dicke der Linse. </li><li> Berechnen der axialen und äquatorialen Stämme aus der Linsendurchmesser - Messungen unter Verwendung der Gleichung, ε = (d - d 0) / d 0 ist , wobei ε - Stamm ist, d der axiale oder eQuatorial Durchmesser bei einer gegebenen Last ist , und d 0 der entsprechenden axialen oder äquatorialen Durchmesser bei Nullast. </li><li> Zeichnen Sie die axialen und äquatorialen Sorten als Funktionen der auferlegten Last (in mg). </li><li> Zeichnen Sie die axial, äquatorialen und Kerndurchmesser. Berechnen und zeichnen Sie das Objektiv Seitenverhältnis durch die axiale Durchmesser durch den äquatorialen Durchmesser geteilt wird. </li><li> Berechnen und zeichnen Sie das Objektiv Volumen unter Verwendung der Gleichung, Volumen = 4/3 × π × r E 2 × r A, wobei r E ist der äquatoriale Radius und r A die axiale Radius aus dem Bild in Schritt 2.6 genommen , gemessen. Diese Gleichung nimmt die Linse ist eine Oblate Sphäroid (Ellipsoid) 1,22. </li><li> Berechnen und zeichnen Sie die Kernvolumen unter Verwendung der Gleichung, Volumen = 4/3 × π × r N 3, wobei r N der Radius des Linsenkerns ist , wie aus dem Bild in Schritt 3.5 genommen gemessen. Diese Gleichung nimmt den Linsenkern isa Kugel 19,21. </li><li> Berechnen und Plotten der nukleären Fraktion als das Verhältnis des Kernvolumens zum Objektiv Volumen. </li></ol>

<ol>
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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=Dynamic+mechanical+properties+of+human+lenses.">Dynamic mechanical properties of human lenses.</a> Exp Eye Res. 80 (3), 425-434 (2005).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+the+human+lens.">Elastic properties of the human lens.</a> Exp Eye Res. 11 (1), 143 (1971).</li><li>Krueger, R. R., Sun, X. K., Stroh, J., Myers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+increase+in+accommodative+potential+after+neodymium:+yttrium-aluminum-garnet+laser+photodisruption+of+paired+cadaver+lenses.">Experimental increase in accommodative potential after neodymium: yttrium-aluminum-garnet laser photodisruption of paired cadaver lenses.</a> Ophthalmology. 108 (11), 2122-2129 (2001).</li><li>Burd, H. J., Wilde, G. S., Judge, 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=An+improved+spinning+lens+test+to+determine+the+stiffness+of+the+human+lens.">An improved spinning lens test to determine the stiffness of the human lens.</a> Exp Eye Res. 92 (1), 28-39 (2011).</li><li>Glasser, A., Campbell, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Pau, H., Kranz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+sclerosis+of+the+human+lens+with+age+and+its+relevance+to+accommodation+and+presbyopia.">The increasing sclerosis of the human lens with age and its relevance to accommodation and presbyopia.</a> Graefes Arch Clin Exp Ophthalmol. 229 (3), 294-296 (1991).</li><li>Hollman, K. W., O'Donnell, M., Erpelding, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+elasticity+in+human+lenses+using+bubble-based+acoustic+radiation+force.">Mapping elasticity in human lenses using bubble-based acoustic radiation force.</a> Exp Eye Res. 85 (6), 890-893 (2007).</li><li>Baradia, H., Nikahd, N., Glasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lens+stiffness+measurements.">Mouse lens stiffness measurements.</a> Exp Eye Res. 91 (2), 300-307 (2010).</li><li>Gokhin, D. 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=Tmod1+and+CP49+synergize+to+control+the+fiber+cell+geometry,+transparency,+and+mechanical+stiffness+of+the+mouse+lens.">Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.</a> PLoS One. 7 (11), e48734 (2012).</li><li>Sindhu Kumari, 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=Role+of+Aquaporin+0+in+lens+biomechanics.">Role of Aquaporin 0 in lens biomechanics.</a> Biochem Biophys Res Commun. , (2015).</li><li>Fudge, D. 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=Intermediate+filaments+regulate+tissue+size+and+stiffness+in+the+murine+lens.">Intermediate filaments regulate tissue size and stiffness in the murine lens.</a> Invest Ophthalmol Vis Sci. 52 (6), 3860-3867 (2011).</li><li>Kuszak, J. R., Mazurkiewicz, M., Zoltoski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+secondary+fiber+development+and+growth:+I.+Nonprimate+lenses.">Computer modeling of secondary fiber development and growth: I. Nonprimate lenses.</a> Mol Vis. 12, 251-270 (2006).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <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+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li></ol>

Die Autoren haben nichts zu offenbaren.

Es gibt mehrere wichtige Überlegungen bei der Verwendung dieser Methode Linse Steifigkeit zu messen. Zuerst werden die Deckgläser auf die Linse bei einem leicht schrägen Winkel aufgetragen (8-8,5 °) in Bezug auf den Boden der Kammer (θ). Dies wird eine sehr kleine Komponente der Last anwenden aquatorial anstatt axial. Allerdings ist diese äquatorialen Last als vernachlässigbar , da sin θ ≈ 0,1 19. Wenn dieses Verfahren für größere Linsen angepasst ist, müsste der Winkel der Deckgläser auf den B...

The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses. 
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness. 
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Fine tip straight forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11252-40</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdissection scissors, straight edge</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11272-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seizing forceps</td>
			<td style="width:139px;">Hammacher</td>
			<td style="width:119px;">HSC 702-93</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12565154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm petri dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">0875713A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18mm x 18mm glass coverslips</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Measurement chamber with divots to hold lenses</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Custom-made (see Figure 1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Right-angle mirror</td>
			<td style="width:139px;">Edmund Optics</td>
			<td style="width:119px;">45-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light source</td>
			<td style="width:139px;">Schott/Fostec</td>
			<td style="width:119px;">8375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illuminated dissecting microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">SZX-ILLD100</td>
			<td>With SZ-PT phototube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital camera</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">Coolpix 990</td>
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sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses

Die Augenlinse ist ein transparentes Organ, das Licht bricht und konzentriert sich auf die Netzhaut ein klares Bild zu bilden. Beim Menschen Vertrag Ziliarmuskeln die Linse zu verformen, zu einer Erhöhung führt in der Linse &quot;optische Leistung auf nahe Objekte scharf, ein Verfahren, wie Unterkunft bekannt. Altersbedingte Veränderungen in der Linsensteifigkeit haben Presbyopie in Verbindung gebracht worden, eine Verminderung der Linse die Fähigkeit aufzunehmen, und durch Erweiterung, die Notwendigkeit für eine Lesebrille. Auch wenn die Maus Linsen aufnehmen oder nicht entwickeln Presbyopie, Mausmodelle können einen unschätzbaren genetisches Werkzeug für das Verständnis Objektiv Pathologien bieten, und die beschleunigte Alterung bei Mäusen beobachtet, ermöglicht die Untersuchung von altersbedingten Veränderungen in der Linse. Dieses Protokoll zeigt, eine einfache, präzise und kostengünstiges Verfahren für Maus Linsensteifigkeit Bestimmung unter Verwendung von Glasdeckgläser sequentiell Druckbelastungen auf die Linse Erhöhung anzuwenden. Repräsentative Daten bestätigen, dass die Maus Linsen steifer mit dem Alter, wiemenschliche Linsen. Dieses Verfahren ist sehr gut reproduzierbar und kann potenziell Testlinsen von größeren Tieren zu mechanisch skaliert werden.

Die Steifigkeit und die Abmessungen der 2-, 4- und 8-Monate alten Maus Linsen wurden gemessen. Die Mäuse wurden alle Wildtyp-Tiere auf einem reinen Hintergrund C57BL6 Stamm aus der Zuchtanlage TSRI Tier erhalten, und jede Linse wurde mit 1 bis 10 Deckgläser geladen. Die axialen und äquatorialen Stämme wurden als Funktion der aufgebrachten Last berechnet, indem die axialen und äquatorialen Durchmesser der Linse nach der Zugabe von jedem Deckglas zu messen und dann jede Änderung des ...

Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Sequential Anwendung von Glasplättchen die Drucksteifigkeit der Maus Objektiv zu beurteilen: Stamm und morphometrische Analysen

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

sequential-application-glass-coverslips-to-assess-compressive

Research

JoVE Journal

Biology

7.2K Aufrufe.  The Scripps Research Institute. Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

Serie: Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Objektiv Transplantation in Zebrafisch und ihre Anwendung in der Analyse von Eye-Mutanten

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Forschung

Biologie

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Die Reinigung des M. magneticum Stamm AMB-1 Magnetosom assoziierten Protein MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

Ausweiden der Maus Glaskörper und Netzhaut für Proteomik-Analysen

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Biophysikalische Assays auf die mechanischen Eigenschaften der Interphase Zellkern Probe: Substrat Dehnungs-Anwendung und Mikronadel Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Interzelluläre Gap Junction-Kommunikation: Eine funktionelle Biomarker zur Beurteilung Unerwünschte Auswirkungen von Schadstoffen und Toxinen und gesundheitlichen Vorteile von Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

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 structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

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 ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Objektiv-kostenlose Videomikroskopie für die Dynamik und Quantitative Analyse der adhärente Zellkultur

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Anwendung der Elektrophysiologie Messung die Aktivität von Electro-neutralen Transporter zu studieren

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...