Die Autoren bedanken sich bei Dr. Jay Pepose zu helfen lehrt das Modell bei uns zu bedanken. Diese Arbeit wurde von den National Institutes of Health Grants EY11885 (PMS), EY21247 (PMS) und einer uneingeschränkten Finanzhilfe aus Forschung zur Blindheit zu Department of Ophthalmology Prevent unterstützt.

Animal Care Hinweis: Analgetika werden in diesem Modell nicht verwendet, da sie entzündungshemmend und sind somit würde das Modell ungültig. Ein. Herstellung von Virus <ol><li> Wachsen HSV-1 in Vero-Zellen (80% konfluent) mit einem ausgehend von 0,01 Verdünnung Infektionsmultiplizität (in T-150-Kolben, etwa 5 x 10 5 Plaque-bildende Einheiten) für 3-4 Tage, bis alle Zellen abgerundet-up und sind leicht durch das Drücken der Flasche verdrängt. </li><li> Entfernen Sie das Medium in sterile Zentrifugenröhrchen und Zentrifuge Sorvall Legend RT bei 4 ° C und 3.500 rpm für 15 min. Nach dem Entfernen Überstand (siehe 1.3), resuspendieren großen Pellets aus je 50 ml Röhrchen mit 5 ml Medium total. Beschallen für 1 min in zwei 30 sec platzt. </li><li> Überstand abnehmen und in 50 ml steriler Oak Ridge Rohren und Zentrifuge bei 9000 rpm, 1 Stunde lang bei 4 ° C in einer Beckman J2-21 Zentrifuge. Dies ist, um eine kleine virale Pellet herzustellen. Überstand verwerfen. </li><li> Kombinieren Sie beschallen von 1,2 with Pellet aus 1,3 und beschallen wieder für 45 Sekunden. Zentrifuge Sorvall RT Legende bei 1.500 UpM für 5 min bei 4 ° C. Speichern Sie den Überstand. Dies stellt das Virus Lager, die aliquotiert und bei -70 ° C gelagert </li><li> Titer Virus auf Vero-Zellen. Ich in der Regel Platte 10 0 bis 10 -8 Verdünnung (jeweils eine 10-fache Verdünnung) 2x aus 2 separate Verdünnung Sets. Titration des Virus auf 12-Well-sterile Platten durchgeführt. </li></ol> 2. Maus Infektion <ol><li> Injizieren jede Maus mit 0,1 ml des Narkosemittels cocktail/20 g Maus Körpergewicht (Ketamin 60 mg / kg Xylazin + 5 mg / kg, die in HBSS verdünnt wird). Dieser Cocktail verwendet wird, weil es eines der sichersten Mittel der Narkose, wenn eine ordnungsgemäße Dosierungen verwendet werden, und die Auswirkungen dieser Droge sind sehr kurzlebig ist. </li><li> Wenn Mäuse anästhesiert sind Kratzer auf der Hornhaut des rechten Auges mit einer 30-Gauge-Nadel mit einem Präpariermikroskop um sicherzustellen, dass die Hornhaut nicht perforiert ist. </li><li> Jede Maus receIved eine intraperitoneale Injektion von 1 ml gepooltes humanes Serum (Sigma, Temecula, CA; Anti-HSV Reaktivität mit einer wirksamen Dosis für 50% Neutralisation der viralen 1:800), um vor einer Beschädigung während Hornhäute primären Infektion zu schützen. </li><li> Anzahl der Mäuse entweder mit Ohrbügel oder nach Gehör Punsch. </li><li> Nach all der Mäuse wurden nummeriert; mit einem 20 ul Pipette, infizieren wir jedes Auge mit 10 6 PFU (5 ul). Wir infizieren nur ein Auge und in der Regel das rechte Auge. Wir legen auch 5 ul HBSS auf das andere Auge zu feucht halten, während sie betäubt sind. 5 ul ausreicht, um die Hydratisierung der Augen zu halten, da die Verfahren in weniger als 10 min durchgeführt, und die Tiere werden mit Anästhetikum dosiert entsprechend. Infektion wird durch Abtupfen Mäuse 3 Tage nach der Infektion mit einer sterilen hydratisierten Baumwolle Applikator, in 1 ml Vero Medien wurde bestätigt. Dann wurden 100 ul wird Vero-Zellen in 48-well Platten kultiviert zugegeben. Diese Platten werden täglich überwacht für zytopathischen Effekte<html> (Cpe). </li></ol> 3. Reaktivierung der Mäuse aus Latency <ol><li> Mäuse sind betäubt mindestens 5 Wochen nach der primären Infektion. Es sei angemerkt, dass wir Mäuse reaktiviert bis zu einem Jahr nach Primärinfektion und haben keinen signifikanten Unterschiede in Reaktion beobachtet werden. </li><li> Sobald sie anästhesiert TM20 Chromato-Vu Transilluminator, der UV-B emittiert bei einer Spitzenwellenlänge von 302 nm, so dass nur ein einzelnes Auge auf die UV-B-Licht ausgesetzt ist, platziert. Jede Maus auf 250 mJ von UV-B-Licht cm 2 belichtet. </li><li> Reaktivierung durch Abtupfen Augen mit einer sterilen Watteträger, die zuerst in 1 ml Vero Medien am Tag 0 gesetzt (vor UV-B-Exposition für spontane Ausscheider kontrollieren) und dann an den Tagen 1 bis 7 post-UV-B-Bestrahlung bestimmt. Der Tupfer Material wird bewertet, wie in 2.5 beschrieben). Für die Abstriche, die infektiöse Virus haben die Virusmenge wird durch ein Standard-Plaque-Assay mit 12-Well-Platten kicherten. </li></ol>ove_title &quot;&gt; 4. Clinical Evaluation <ol><li> Mäuse werden zur klinischen Augenerkrankung durch einen maskierten Beobachter unter Verwendung einer binokularen Präpariermikroskop ausgewertet. </li><li> Hornhauttrübung auf einer Skala von 0 bis 4, wobei 0 bedeutet, klare Stroma bewertet, ein mildes Stromazellen Trübung zeigt, 2 mäßig Opazität zeigt mit erkennbaren Merkmalen Iris, zeigt 3 dichte Opazität unter Verlust von Iris definiert Detail außer Pupille Ränder, und 4 zeigt Insgesamt Deckkraft ohne Ansicht von dorsal. </li><li> Neovaskularisation wird auf einer Skala von 1 bis 8 durch Teilen der Hornhaut in 4 gleiche Quadranten und Bestimmen des Ausmaßes der Vaskularisierung in jedem dieser Quadranten bewertet. </li><li> Blepharitis wird wie folgt gemessen: 0, keine Läsionen, 1, minimal Augenlid Schwellung; 2, mäßige Schwellung und knusprigem Augenausfluss, 3, starke Schwellung, mäßige periokuläre Haarausfall und Hautveränderungen und 4, starke Schwellung mit den Augen verkrusteten geschlossen, schwere periokuläre Haarverlust und Hautläsionen. </li><li> Der Nachweis der Enzephalitis Inclusionded zurück, gebeugt zerzausten Haaren und offensichtliche neurologische Anomalien. Mäuse sind eingeschläfert, wenn sie deutliche Anzeichen von Encephalitis anzuzeigen. </li></ol> 5. Repräsentative Ergebnisse Das Modell, das wiederkehrende oben beschrieben wurde zuerst von Shimeld, et al. 1-3. Sie getestet und unserem Labor wurde mit einer modifizierten Version dieses Modells auf viele Manuskripte über die Jahre 4-15 veröffentlichen. Das Modell wurde verwendet, um die Rolle zu definieren, dass das Zytokin Interferon-γ spielt bei rezidivierenden HSV-1 Krankheit 10. Wie in Abbildung 3 gezeigt, hat Mäusen IFNgamma angezeigt schlimmer Hornhauterkrankung als Wildtyp-Mäusen 14. Es wurde auch erfolgreich auf den therapeutischen Wert von mehreren unterschiedlichen Vakzin-Konstrukte 6,10,11 definieren. Beachten Sie, dass in einem Fall der Impfstoff getestet gut funktioniert aber nicht prophylaktisch erweisen therapeutisch wirksame 6. Jedochwenn ein Replikations-inkompetent viraler Impfstoff getestet wurde, war es nicht nur wirksam, prophylaktisch, war aber auch sehr effektiv therapeutisch 10,11, siehe Abbildung 5 in Referenz 10. Vor kurzem haben wir dieses Modell verwendet werden, um festzustellen, ob Unterschiede zwischen den Geschlechtern bei Mäusen unterzogen wiederkehrende HSK nachweisbar waren. Wie Abbildung 1 zeigt, zeigen männliche C57BL / 6 Mäuse deutlich weniger Hornhauttrübung als weibliche C57BL / 6 Mäusen. Ebenso, wenn cornealer Neovaskularisierung wurde ausgewertet, nachgewiesen männlichen Mäusen signifikant weniger neue Gefäßwachstum in der Hornhaut als tat weiblichen Mäusen (Abbildung 2). Wir testen derzeit, ob dieser Phänotyp stammspezifische indem ähnliche Analyse in anderen Stämmen von Mäusen ist. Wir werden auch bestimmen, ob der Unterschied aufgrund des Fehlens von Testosteron oder der Gegenwart von Östrogen ist. <img alt="Abbildung 1" src="/files/ftp_upload/4276/4276fig1.jpg" /> Abbildung 1. Corneal Krankheit in männliche und weibliche C57BL / 6 Maus-Stämme nach der UV-B induzierte Reaktivierung. Latent infizierten Mäusen induziert wurden mit UV-B-Bestrahlung und Mäuse wurden für Hornhauttrübung für 35 Tage überwacht reaktivieren. Die Zahl der Mäuse für diese Studien verwendet wurden, waren wie folgt: Männchen, n = 15; Weibchen n = 15. Die Ergebnisse zeigen den Mittelwert ± SEM. * Es war signifikant größer Virus-induzierte Erkrankung weibliche C57BL / 6 Mäuse für Tage 14-35 (P = 0,001 bis 0,01). <img alt="Abbildung 2" src="/files/ftp_upload/4276/4276fig2.jpg" /> Abbildung 2. Corneal Krankheit in männliche und weibliche C57BL / 6 Maus-Stämme nach der UV-B induzierte Reaktivierung. Latent infizierten Mäusen induziert wurden mit UV-B-Bestrahlung und Mäuse wurden für korneale Neovaskularisierung für 35 Tage überwacht reaktivieren. Die Zahl der Mäuse für diese Studien verwendet wurden, waren wie folgt: Männchen, n= 15; Frauen, n = 15. Die Ergebnisse zeigen den Mittelwert ± SEM. * Es war signifikant größer Virus-induzierte Erkrankung weibliche C57BL / 6 Mäuse für Tage 14-28 (P = 0,001 bis 0,01).

<ol>
	<li>Shimeld, C., Hill, T. J., Blyth, B., Easty, D. <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+model+of+recurrent+herpetic+eye+disease+in+mice.">An improved model of recurrent herpetic eye disease in mice.</a> Curr. Eye Res. 8, 1193-1205 (1989).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+latent+infection+and+induction+of+recurrent+herpetic+eye+disease+in+mice.">Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.</a> J. Gen. Virol. 71, 397-404 (1990).</li><li>Shimeld, C., Hill, T. J., Blyth, W. A., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+protects+the+mouse+eye+from+damage+after+herpes+simplex+virus+infection+by+limiting+spread+of+virus+in+the+nervous+system.">Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system.</a> J. Gen. Virol. 71, 681-687 (1990).</li><li>Laycock, K. A., Lee, S. F., Brady, R. H., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+model+of+recurrent+herpes+simplex+viral+keratitis+induced+by+ultraviolet+B+radiation.">Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.</a> Invest. Ophthalmol. Vis. Sci. 32, 2741-2746 (1991).</li><li>Keadle, T. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+ameliorates+corneal+disease+in+a+mouse+model+of+recurrent+herpetic+keratitis.">IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis.</a> Microbial Path. 38, 13-21 (2005).</li><li>Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+recombinant+glycoprotein+D+subunit+vaccine+on+the+development+of+primary+and+recurrent+ocular+infection+with+herpes+simplex+virus+type+1+in+mice.">Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.</a> J. Inf. Dis. 176, 331-338 (1997).</li><li>Keadle, T. L., Laycock, K. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinflammatory+cytokines+IL-1+and+TNF-a+are+required+for+recurrent+herpetic+keratitis+in+NIH+mice.">Proinflammatory cytokines IL-1 and TNF-a are required for recurrent herpetic keratitis in NIH mice.</a> Invest. Ophth. Vis. Sci. 41, 96-102 (2000).</li><li>Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+cytokine+expression+in+a+murine+model+recurrent+herpetic+stromal+keratitis.">Corneal cytokine expression in a murine model recurrent herpetic stromal keratitis.</a> Ocular Immunol. Inflam. 9, 193-205 (2001).</li><li>Keadle, T. L., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++and+CD8++cells+are+key+participants+in+the+development+of+recurrent+herpetic+stromal+keratitis+in+mice.">CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice.</a> Microbial Path. 32, 255-262 (2002).</li><li>Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+immunization+with+a+virion+host+shutoff-defective,+replication-incompetent+herpes+simplex+virus+type+1+strain+limits+recurrent+herpetic+ocular+infection.">Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection.</a> J. Virol. 76, 3615-3625 (2002).</li><li>Keadle, T. L., Laycock, K. A., Morris, J. L., Leib, D. A., Morrison, L. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+vaccination+with+vhs(-)+herpes+simplex+virus+reduces+the+severity+of+recurrent+herpetic+stromal+keratitis+in+mice.">Therapeutic vaccination with vhs(-) herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice.</a> J. Gen. Virol. 83, 2361-2365 (2002).</li><li>Keadle, T. L., Morris, J. L., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aminoguanidine+on+primary+and+recurrent+ocular+herpes+simplex+virus+infection.">The effects of aminoguanidine on primary and recurrent ocular herpes simplex virus infection.</a> Nitric Oxide. 13, 247-253 (2005).</li><li>Stuart, P. M., Morris, J. E., Sidhu, M., Keadle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL3+protects+mice+from+corneal+pathology+during+recurrent+HSV-1+infection.">CCL3 protects mice from corneal pathology during recurrent HSV-1 infection.</a> Front. Biosci. 13, 4407-4415 (2008).</li><li>Keadle, T. L., Alexander, D. E., Leib, D. A., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+gamma+is+not+required+for+recurrent+herpetic+stromal+keratitis.">Interferon gamma is not required for recurrent herpetic stromal keratitis.</a> Virology. 380, 46-51 (2008).</li><li>Carr, D. J., Austin, B. A., Halford, W. P., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Interferon-gamma+by+an+adenovirus+vector+blocks+herpes+simplex+virus+Type+1+reactivation+in+vitro+and+in+vivo+independent+of+RNase+L+and+double-stranded+RNA-dependent+protein+kinase+pathways.">Delivery of Interferon-gamma by an adenovirus vector blocks herpes simplex virus Type 1 reactivation in vitro and in vivo independent of RNase L and double-stranded RNA-dependent protein kinase pathways.</a> J. Neuroimmunol. 206, 39-43 (2009).</li></ol>

Keine Interessenskonflikte erklärt.

Die wiederkehrende Modell Herpes Stromakeratitis (HSK) hier beschrieben ermöglicht die Ermittler auf die menschliche HSK in einem Modell, das mehr im Einklang mit dem, was während der Krankheit beim Menschen beobachtet wird, zu studieren. Somit sind das Modell der Stärken, die Krankheit im Zusammenhang mit einer Immunsystem, das bereits durch stimulierte während primäre Erkrankung auftritt. Da diese Mäuse bereits eine Immunantwort gegen HSV-1 hat, sind die Faktoren, die diese Immunantwort zu reaktivieren wahrschei...

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5796</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-C1</td>
		</tr>
		<tr>
			<td>Pen/ Strep</td>
			<td>Sigma</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>52-21</td>
		</tr>
		<tr>
			<td>Transilluminatior</td>
			<td>UVP</td>
			<td>95-0452-02</td>
		</tr>
		<tr>
			<td>Sonicatior</td>
			<td>Branson</td>
			<td>Sonifier 450</td>
		</tr>
		<tr>
			<td>Oak Ridge Tubes</td>
			<td>Nalgene</td>
			<td>Z 720410</td>
		</tr>
		<tr>
			<td>Flasks- 7150</td>
			<td>Corning</td>
			<td>430823</td>
		</tr>
		<tr>
			<td>Plates- 12 well</td>
			<td>Corning</td>
			<td>CLS 3513</td>
		</tr>
		<tr>
			<td>Plates- 48 well</td>
			<td>Corning</td>
			<td>CLS 3548</td>
		</tr>
		<tr>
			<td>Sterile Conical Tubes- 30 ml</td>
			<td>Corning</td>
			<td>CLS 430828</td>
		</tr>
		<tr>
			<td>Sterile Cotton-tipped Applicators</td>
			<td>Fisher</td>
			<td>23-400-125</td>
		</tr>
		<tr>
			<td>30G Needles</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
		</tr>
		<tr>
			<td>25G Needles</td>
			<td>BD</td>
			<td>305122</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>BD</td>
			<td>309604</td>
		</tr>
	</tbody>
</table>

recurrent herpetic stromal keratitis in mice, a model for studying human hsk

Herpetic Augenkrankheit, genannt Herpes Stromakeratitis (HSK), ist ein potentiell blendenden Infektion der Hornhaut, die zu über 300.000 klinische Besuche pro Jahr für die Behandlung. Zwischen 1 und 2 Prozent der Patienten mit klinischer Erkrankung erleben Verlust des Sehvermögens der infizierten Hornhaut. Die überwiegende Mehrheit dieser Fälle sind die Folge der Reaktivierung einer latenten Infektion durch Herpes simplex-Virus Typ I und nicht aufgrund einer akuten Erkrankung. Interessanterweise ist die akute Infektion das Modell häufig verwendet, um diese Krankheit zu studieren. Es wurde jedoch das Gefühl, dass ein immer wiederkehrendes Modell HSK wäre reflektiert, was geschieht, während der klinischen Erkrankung. Die wiederkehrenden Tiermodelle für HSK haben beide Kaninchen und Mäuse eingesetzt. Der Vorteil von Kaninchen ist, dass sie erleben Reaktivierung aus der Latenz abwesend alle bekannten Reiz. Das heißt, es ist schwierig, die Rolle, die viele immunologische Faktoren bei rezidivierendem HSK spielen zu erforschen, weil die Kaninchen-Modell nicht die immungische und genetische Ressourcen, die Maus hat. Wir wählten das Mausmodell für wiederkehrende HSK verwenden, weil es den Vorteil davon, dass es viele Ressourcen zur Verfügung hat und auch wir wissen, wann die Reaktivierung wird auftreten, da die Reaktivierung durch Bestrahlung mit UV-B-Licht induziert wird. Bisher hat dieses Modell der Laboratorien verwenden darf es zu mehreren immunologischen Faktoren, die wichtig für diese Erkrankung sind zu definieren. Es hat uns auch die Möglichkeit, sowohl therapeutische und Wirksamkeit des Impfstoffes zu testen.

Watch this Scientific Journal Video about Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK at JoVE.com

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Die meisten Studien über Herpes Hornhauterkrankung verwenden Primärinfektion Modell. Allerdings bedeutet primäre Infektion mit HSV-1 in der Regel nicht für die menschliche Erkrankung führen. Hier beschreiben wir ein wiederkehrendes Muster von herpetischer korneale Erkrankung, die näher Verhalten menschlicher Erkrankung.

Wiederkehrende Herpetic Stromakeratitis in Mäuse, ein Modell für das Studium der menschlichen HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

recurrent-herpetic-stromal-keratitis-mice-model-for-studying-human

Research

JoVE Journal

Immunology and Infection

11.9K Aufrufe.  Saint Louis University. Die meisten Studien über Herpes Hornhauterkrankung verwenden Primärinfektion Modell. Allerdings bedeutet primäre Infektion mit HSV-1 in der Regel nicht für die menschliche Erkrankung führen. Hier beschreiben wir ein wiederkehrendes Muster von herpetischer korneale Erkrankung, die näher Verhalten menschlicher Erkrankung.

Serie: Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Ex Vivo Organotypische Corneal Modell der akuten Epithelial Herpes Simplex Virus Typ I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Forschung

Neurowissenschaften

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Ein Laser-induzierten Maus-Modell der chronischen Ocular Hypertension zu Sehfehler Charakterisierung

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medizin

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex vivo Infektion von murinen Epidermis mit Herpes Simplex Virus Typ 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Immunologie und Infektion

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Etablierung des dualen humanisierten TK-NOG-Mausmodells für HIV-assoziierte Leberpathogenese

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Chronische, akute und reaktivierte HIV-Infektion in humanisierten immundefizienten Mausmodellen

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Maus Footpad Inokulation Modell zur Untersuchung viral-induzierte neuroentzündliche Reaktionen

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Porcine Hornhautgewebe Explant, um die Wirksamkeit von Herpes Simplex Virus-1 Antiviralen zu untersuchen

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Kontaktüberempfindlichkeit als murines Modell der allergischen Kontaktdermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Biotechnik

Protokolle zur Schädigung von sensorischen Fasern der Hornhaut für Studien zur Axonregeneration

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Autor im Rampenlicht: Förderung der Hornhautinnervationsforschung durch innovative Modelle 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

Drei Strategien zur Induktion von neurotropher Keratitis und Nervenregeneration in der murinen Hornhaut

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...