Die Autoren danken Joris Van Asselberghs und Ann Van Santvoort für ihre hervorragende technische Hilfe. Forschung wurde von der IWT-Vlaanderen (IWT SBO / 80020), der FWO Vlaanderen (G.0768.10), von der EG-FP6 Programm &quot;DiMI &#39;(LSHB-CT-2005-512146), das RP7 FTE-Projekt MEFOPA (Health finanziert -2.009-241.791), das RP7-Programm &quot;inMind&quot; (HEALTH-F2-2011-278850), die KU Leuven (IOF-KP / 07/001, OT / 08 / 052A, IMIR PF / 10/017), und die MJFox Foundation (Targetvalidierung 2010). A. Van der Perren und C. Casteels sind ein Postdoc-Stipendiaten des Flämischen Fonds für wissenschaftliche Forschung. K. Van Laere ist ein leitender klinischer Fellow des Flämischen Fonds für wissenschaftliche Forschung.

Alle Tierversuche sind in Übereinstimmung mit der Europäischen Gemeinschaften Richtlinie des Rates vom 24. November 1986 (86/609 / EWG) und genehmigt durch das Bioethical Komitee der Universität Leuven (Belgien) durchgeführt. 1. Rekombinante AAV Herstellung und Reinigung Hinweis: rAAV - Vektor Produktion und Reinigung wurde durchgeführt von der Leuven viraler Vektor Core (LVVC) , wie zuvor 17 beschrieben. <ol><li> Kurz gesagt, transfizieren subkonfluenten niedrig (&lt;50) Passage anhaftenden HEK 293T-Zellen ein 25 kD Lineares Polyethylenimin 150 nM NaCl Transfektionslösung und drei verschiedene Plasmide in einem Verhältnis von 1: 1: 1 in DMEM-Medium, 2% fötales Rinderserum. Nach 24 Stunden Inkubation bei 37 ° C in einem 5% CO 2, ersetzen Sie das Medium durch frisches DMEM - Medium 2% fötales Rinderserum. Hinweis: Die Plasmide, die die Konstrukte für die AAV7 Serotyp enthalten, verbesserte das AAV-Transfer-Plasmid die menschliche A53T Mutante α-SYN unter der Kontrolle des CMVie kodieren synapsin1 - Promotor und der pAdvDeltaF6 adenoviralen Helferplasmid 17. </li><li> Ernten Sie das Medium 5 Tage nach der transienten Transfektion und konzentrieren sich mit Tangentialstromfiltration 17. </li><li> Reinige die rAAV - Vektorpartikel aus dem konzentrierten Medium eine Iodixanol Stufengradienten mit 17. </li><li> Verwenden von Standardtechniken der Echtzeit-PCR für die genomische Kopie (GC) Bestimmung. In diesem Protokoll wurde ein Vektor - Titer von 3,0 E11 GC / ml verwendet , um ein α-SYN basierend Rattenmodell für 17 PD zu entwickeln. </li></ol> 2. Stereotactic Injektion von rAAV α-SYN Vektor in der SN der Ratte (Figur 2) <ol><li> Haus acht Wochen alt weiblichen Wistar-Ratten, die etwa 200 bis 250 g unter einem normalen 12 Stunden Licht / Dunkel-Zyklus mit freiem Zugang zu pelle Nahrung und Leitungswasser mit einem Gewicht. </li><li> Werfen die Ratte intraperitoneal (ip) Narkose eine Mischung von Ketamin enthält (60 mg / kg) und Medetomidin (0,4 mg / kg). Sobald die Ratte betäubt und nicht reagiertwenn die verschiedenen Pfoten quetschen, zu verwalten für die weitere Anerkennung einen Mikrotransponder subkutan der Ratte auf der Rückseite einen Mikrotransponder implanter verwenden. Prüfen, ob der Mikrotransponder richtig positioniert ist und durch das Lesegerät ausgelesen werden. </li><li> Schneiden Sie die Haare auf der Oberseite der Kopfhaut. Tragen Sie eine lokale Betäubung sowohl auf der Kopfhaut und den Ohren. Führen Sie den Rest des chirurgischen Eingriffs unter einer laminaren Strömung unter aseptischen Bedingungen. </li><li> Legen Sie die Ratten in einem stereotaktischen Kopfrahmen mit zwei Ohr Bars, einen Mund und eine Nase bar. Bedecken Sie den Körper der Ratte mit einem Papiertuch einen Abfall der Körpertemperatur zu vermeiden. Anwenden eines okularen Gleitmittel, die Augen vor dem Austrocknen zu verhindern. </li><li> Desinfizieren Sie die Kopfhaut mit jodium 1% in Isopropanol 70% und machen einen kleinen Schnitt in der Mittellinie der Kopfhaut. Kratzen Sie vorsichtig die Membranen auf dem Schädel entfernt und Spülen mit Kochsalzlösung. Lassen Sie den Schädel trocken für einige Minuten. Beachten Sie die Schädelnähte und die beiden Bezugspunkte: Bregma und Lambda. </li&gt; <li> Um den rAAV-Vektor in den SN injizieren, definieren die Koordinaten in Richtung Bregma (von vorne nach hinten: 5,3 mm; mediolateral: 2,0 mm und dorsoventrale: 7,2 mm von der Dura berechnet). Hinweis: Die dreidimensionalen Koordinaten für jede Region von Interesse berechnet werden kann, eine stereotaktischen Atlas des Rattengehirns mit, Bregma als anatomischen Bezugspunkt der Anwendung. </li><li> Füllen Sie eine 10 &amp; mgr; l Mikroinjektionsspritze (30 Gauge 20 mm) mit rAAV-Vektor und legen Sie sie in der stereotaktischen Instrument mit einem motorisierten Mikroinjektionspumpe angeschlossen. Kontrollieren Sie die Lautstärke , indem Sie einen Tropfen Vektor Freigabe und in einem polyvalenten Reinigungsmittel pH 9 (zB RBS) zu beseitigen. </li><li> Optisch prüfen, ob der Kopf gerade in der Kopfrahmen befestigt ist und die Links-Rechts-Achse zu bewerten. Sorgfältig visuell die von vorne nach hinten und mediolateral Koordinaten für Bregma und Lambda definieren und deren Höhe eine 30-Gauge-20 mm-Nadel in dorsoventraler Arm des stereotaktischen Rahmen mit messen. <ol><li> Erlauben Uhraximale von 0,3 mm Höhenunterschied zwischen Bregma und Lambda. Legen Sie die Nadel wieder auf Bregma und wenden die von vorne nach hinten und mediolateral Koordinaten durch die von vorne nach hinten und die mediolateral Arm des stereotaktischen Rahmen bewegen. </li></ol></li><li> Am Ort der Injektion, messen die Höhe des Schädels und sicherzustellen, dass er nicht mehr als 0,3 mm von der Höhe des Bregma unterscheidet. Bohren Sie ein Loch in den Schädel mit einem Durchmesser von etwa 2 mm. Messen Sie die Höhe der Dura, dies dient als Referenz der dorsoventrale anzuwenden koordinieren. Alternativ subtrahieren für den Schädel einer festen Dicke (0,9 mm). </li><li> Dringen die Dura eine 26-Gauge-Nadel und das Blut mit einem sterilen Gewebe absorbieren. Warten Sie, bis alle Blutungen, bevor Sie fortfahren gestoppt hat. </li><li> einfügen langsam die 10 &amp; mgr; l Mikroinjektionsspritze mit Vektor-Lösung in das Gehirn auf die vorgegebene Tiefe vorbelastet (dorsoventrale Koordinate). Warten Sie 1 min mit der Nadel an Ort und Stelle. Injizieren 3 ulder Vektorlösung (3,0 E11 Genomkopien / ml (mittlerer Vektor Dosis) oder 1,0 E12 GC / ml (high Vektor Dosis) von rAAV2 / 7 α-SYN oder eGFP Kontrollvektor) des motorisierten Mikroinjektionspumpe mit einem Durchsatz von 0,25 &amp; mgr; l unter Verwendung von / Minute </li><li> Nach der Injektion, halten Sie die Nadel an Ort und Stelle für eine weitere min 5, bevor es langsam zu entfernen. Nähen Sie die Kopfhaut mit beschichteten geflochtenen Polyester 3.0, desinfizieren mit 1% jodium in 70% Isopropanol und sanft das Tier aus dem Gerät zu entfernen Stereotaxie. Lösen Sie zuerst die Nase und den Mund Bar, dann die beiden Ohr Bars. </li><li> Um die Anästhesie umkehren, injizieren die Ratte intraperitoneal mit 0,5 mg / kg Atipamezol und legen Sie die Ratte in einen sauberen Käfig auf einer Heizplatte von 38 ° C, bis er aufwacht. Decken Sie die Ratte mit einem Papiertuch einen Abfall der Körpertemperatur zu verhindern. </li><li> Bieten Sie einfachen Zugang zu Nahrung und Wasser für die ersten Stunden. Überwachen Sie die Ratte für die ersten paar Tage. Bei Bedarf anwenden Analgesie. Hinweis: Es gibt keine Notwendigkeit, die Maschen von t zu entfernen,er Schädel. Nach 1-2 Wochen wird der Schädel vollständig repariert und die Stiche lösen. </li></ol> 3. Bewertung der rAAV2 / 7 α-SYN injizierten Ratten mit nicht-invasiven PET-Bildgebung, Verhaltenstests und immunhistochemischen Analyse <ol><li> Um die Kinetik der nigrostriatale dopaminergen Neurodegeneration nicht-invasiv im Laufe der Zeit in den einzelnen Tieren verfolgen, zu quantifizieren Dopamin - Transporter (DAT) Bindung unter Verwendung von Kleintier - Positronen - Emissions - Tomographie (PET) und einen Tracer des DA - Transporter zB [18 F] -Fect 16 . </li><li> Um zu untersuchen, ob die Höhe der dopaminergen Neurodegeneration ausreicht motorischen Beeinträchtigungen bei den Ratten zu induzieren, die Ratten zu dem Zylinder Test unterziehen, um spontane forelimb Gebrauch zu bewerten. <ol><li> Legen Sie die Ratte in einer Breite von 20 cm Klarglaszylinder und Video das Verhalten bei vertikalen Bewegungen entlang der Wand und der Landung nach einem hinten. Ergebnis die Anzahl der Kontakte von jedem forepaw für insgesamt 20 gemachtKontakte. Ausführliche Beschreibung der Bewertungskriterien siehe Schallert et al. 18 Express die Anzahl der beeinträchtigten forelimb Kontakte (zB linke forepaw) als Prozentsatz der gesamten forelimb Kontakte (links und rechts forepaw). Hinweis: Nicht-lädierten Kontrollratten beide Pfoten mit ebenso sollte etwa 50% in diesem Test punkten. </li></ol></li><li> Führen Sie immunhistochemischen (IHC) Analyse der Ebene der Transgen-Expression und dopaminergen Zellverlust zu bewerten. <ol><li> Zu verschiedenen Endstadien opfern die Ratten mit einer Überdosis von Natriumpentobarbital (60 mg / kg, ip) und eine intrakardiale Perfusion mit kalter Kochsalzlösung mit 4% Paraformaldehyd in PBS 19 gefolgt zuführen. Fixieren Sie die Gehirne über Nacht bei 4 ° C und schnitt 50 um dicke koronale Hirnschnitten einen vibrierenden Mikrotom. </li><li> Führen Sie IHC-Färbung auf frei schwebenden Abschnitte Antikörper gegen α-SYN und Tyrosin-Hydroxylase unter Verwendung des Ausdrucks α-SYN Ebenen zu analysieren und die level der Neurodegeneration 16. </li></ol></li></ol>

<ol>
	<li>Magen, I., Chesselet, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+Parkinson's+disease+The+state+of+the+art.">Genetic mouse models of Parkinson's disease The state of the art.</a> Prog Brain Res. 183, 53-87 (2010).</li><li>Masliah, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+loss+and+inclusion+body+formation+in+alpha-synuclein+mice:+implications+for+neurodegenerative+disorders.">Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.</a> Science. 287, 1265-1269 (2000).</li><li>Freichel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+cognitive+decline+and+amygdala+pathology+in+alpha-synuclein+transgenic+mice.">Age-dependent cognitive decline and amygdala pathology in alpha-synuclein transgenic mice.</a> Neurobiol Aging. 28, 1421-1435 (2007).</li><li>Fleming, S. M., Fernagut, P. O., Chesselet, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+of+parkinsonism:+strengths+and+limitations.">Genetic mouse models of parkinsonism: strengths and limitations.</a> NeuroRx. 2, 495-503 (2005).</li><li>Kahle, P. 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=Selective+insolubility+of+alpha-synuclein+in+human+Lewy+body+diseases+is+recapitulated+in+a+transgenic+mouse+model.">Selective insolubility of alpha-synuclein in human Lewy body diseases is recapitulated in a transgenic mouse model.</a> Am J Pathol. 159, 2215-2225 (2001).</li><li>Chesselet, M. F., Richter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+Parkinson's+disease+in+mice.">Modelling of Parkinson's disease in mice.</a> Lancet Neurol. 10, 1108-1118 (2011).</li><li>Deroose, C. M., Reumers, V., Debyser, Z., Baekelandt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+genes+at+work+in+the+living+brain+with+non-invasive+molecular+imaging.">Seeing genes at work in the living brain with non-invasive molecular imaging.</a> Curr Gene Ther. 9, 212-238 (2009).</li><li>Manfredsson, F. P., 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=rAAV-mediated+nigral+human+parkin+over-expression+partially+ameliorates+motor+deficits+via+enhanced+dopamine+neurotransmission+in+a+rat+model+of+Parkinson's+disease.">rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson's disease.</a> Exp Neurol. 207, 289-301 (2007).</li><li>Vercammen, L., 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=Parkin+protects+against+neurotoxicity+in+the+6-hydroxydopamine+rat+model+for+Parkinson's+disease.">Parkin protects against neurotoxicity in the 6-hydroxydopamine rat model for Parkinson's disease.</a> Mol Ther. 14, 716-723 (2006).</li><li>Winklhofer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parkin+protein+as+a+therapeutic+target+in+Parkinson's+disease.">The parkin protein as a therapeutic target in Parkinson's disease.</a> Expert opinion on therapeutic targets. 11, 1543-1552 (2007).</li><li>Kirik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson-like+neurodegeneration+induced+by+targeted+overexpression+of+alpha-synuclein+in+the+nigrostriatal+system.">Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.</a> J Neurosci. 22, 2780-2791 (2002).</li><li>Kirik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nigrostriatal+alpha-synucleinopathy+induced+by+viral+vector-mediated+overexpression+of+human+alpha-synuclein:+a+new+primate+model+of+Parkinson's+disease.">Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease.</a> Proc Natl Acad Sci U S A. 100, 2884-2889 (2003).</li><li>Lauwers, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology+and+neurodegeneration+in+rodent+brain+induced+by+lentiviral+vector-mediated+overexpression+of+alpha-synuclein.">Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of alpha-synuclein.</a> Brain pathology. 13, 364-372 (2003).</li><li>Klein, R. L., King, M. A., Hamby, M. E., Meyer, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+cell+loss+induced+by+human+A30P+alpha-synuclein+gene+transfer+to+the+rat+substantia+nigra.">Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra.</a> Hum Gene Ther. 13, 605-612 (2002).</li><li>Vander Perren, A., Van den Haute, C., Baekelandt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Vector-Based+Models+of+Parkinson's+Disease.">Viral Vector-Based Models of Parkinson's Disease.</a> Curr Top Beh Neurosci. , (2014).</li><li>Van der Perren, 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=Longitudinal+follow-up+and+characterization+of+a+robust+rat+model+for+Parkinson's+disease+based+on+overexpression+of+alpha-synuclein+with+adeno-associated+viral+vectors.">Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors.</a> Neurobiol Aging. , (2014).</li><li>Van der Perren, 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=Efficient+and+stable+transduction+of+dopaminergic+neurons+in+rat+substantia+nigra+by+rAAV++2/1,+2/2,+2/5,+2/6.2,+2/7,+2/8+and+2/9.">Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9.</a> Gene Ther. , (2011).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Soueid, J., Nokkari, A., Makoukji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+Methods+of+Animal+Brain+Surgery:+Perfusion,+Brain+Removal,+and+Histological+Techniques.+Brain+Neurotrauma:+Molecular,+Neuropsychological,+and+Rehabilitation+Aspects.">Techniques and Methods of Animal Brain Surgery: Perfusion, Brain Removal, and Histological Techniques. Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects.</a> Frontiers in Neuroengineering. , (2015).</li><li>Dale, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+Lewy+bodies+and+pale+bodies+in+Parkinson's+disease.">Relationships between Lewy bodies and pale bodies in Parkinson's disease.</a> Acta Neuropathol. 83, 525-529 (1992).</li><li>Dawson, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+flies+and+mice.">Neurobiology of flies and mice.</a> Science. 288, 631-632 (2000).</li><li>Dawson, T., Mandir, A., Lee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+PD:+pieces+of+the+same+puzzle?.">Animal models of PD: pieces of the same puzzle?.</a> Neuron. 35, 219-222 (2002).</li><li>LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+beta-sheet+amyloid+fibril+structures+with+thioflavin+T.">Quantification of beta-sheet amyloid fibril structures with thioflavin T.</a> Methods Enzymol. 309, 274-284 (1999).</li><li>Oliveras-Salva, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV2/7+vector-mediated+overexpression+of+alpha-synuclein+in+mouse+substantia+nigra+induces+protein+aggregation+and+progressive+dose-dependent+neurodegeneration.">rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration.</a> Mol Neurodegener. 8, (2013).</li></ol>

Die Autoren erklären, dass es keine tatsächlichen oder potenziellen Interessenkonflikt.

Es gibt mehrere wichtige Schritte innerhalb des Protokolls. Der Vektor-Titer sowie der Vektor Reinheit beeinflusst direkt die phänotypische Ergebnis des Modells. Übermäßige Vektor-Titer oder unzureichend gereinigte Vektor Chargen können in nicht-spezifische Toxizität führen. Daher ist die Verwendung von hochwertigen Vektor Chargen und geeignete Kontrollvektoren unverzichtbar. Ferner wird die genaue Positionierung des Kopfes der Ratte in den stereotaktischen Rahmen und die genaue Bestimmung der Koordinaten essenti...

Um die Pathophysiologie von PD studieren und neue therapeutische Strategien zu entwickeln, besteht ein dringender Bedarf an Tiermodellen, die eng an die Neuropathologie ähneln, Physiologie und motorischen Symptome der menschlichen PD. Je höher der prädiktive Wert ist, desto besser können wir neue Therapien von Tiermodellen für die Patienten zu übersetzen. Die Entdeckung von alpha-Synuclein (α-SYN) als erstes PARK-Gen 1997 führte zu der Entwicklung der ersten genetischen PD-Modellen. Viele transgene Mäuse, die menschliche Wildtyp (WT) oder eine Mutante (A30P, A53T) α-SYN wurden in den letzten zehn Jahren erzeugt überexprimieren. Die Gehalte an α-SYN-Überexpression haben sich bei der Entwicklung der Pathologie von entscheidender Bedeutung zu sein. Auch die Mausstamm, das Vorhandensein oder Fehlen von endogenem α-SYN und ob die in voller Länge oder eine verkürzte Form exprimiert wird , spielt eine Rolle (detaillierte Überprüfung durch Magen und Chesselet 1). Die Überexpression von sowohl WT und mehreren klinischen Mutanten des menschlichen &amp;# 945; -syn in transgenen Mäusen induziert pathologischen Akkumulation von α-SYN und neuronale Dysfunktion 2-6. Doch bis jetzt sind die meisten transgenen α-SYN Maus-Modellen keine klaren dopaminergen Zellverlust und Dopamin-abhängige Verhaltensdefiziten anzuzeigen. Diese Hürde wurde durch direkte Ausrichtung der Substantia nigra (SN) mit viralen Vektoren überexprimiert, α-SYN überwinden. Virale Vektoren sind aus Viren stammen, die Zellen leicht infizieren können, stellen genetischem Material in ihren Wirtsgenom und zwingen die Wirtszelle das virale Genom, um zu replizieren, um neue Viruspartikel produzieren. Viren können zu nicht-replizierenden viralen Vektoren konstruiert werden, die ihre Fähigkeit behalten, in Zellen einzudringen und Gene einzuführen. Durch Teile des viralen Genoms zu löschen und sie durch die Gene von Interesse ersetzt, die Anwendung des Vektors in einer einzigen Runde Infektion ohne Replikation in der Wirtszelle führen, die allgemein als &quot;Transduktion&quot; bezeichnet. Virale Vektoren cein sowohl für die Überexpression und gene silencing verwendet werden. Das exprimierte Transgen ein Reporterprotein (zB grün fluoreszierende Protein oder Glühwürmchen - Luciferase) 7, ein therapeutisches Protein für Gentherapie - Anwendungen sein können 8-10 oder, wie wir in diesem Papier konzentrieren wird, um ein krankheitsbedingtes Protein für Krankheitsmodelle verwendet 11 -14. Virusvektor-vermittelte Genabgabe bietet eine alternative Möglichkeit Transgenen im ZNS mit mehreren Vorteilen auszudrücken. Mit lokalen Transgen Lieferung, können bestimmte Hirnregionen ausgerichtet sein. Ferner kann die Transgenexpression im Erwachsenenalter die Gefahr der Kompensationsmechanismen während der Entwicklung abnimmt induziert werden. Außerdem können Modelle in unterschiedlichen Arten und Stämme erstellt werden. Und schließlich können verschiedene Transgene leicht kombiniert werden. Verwendung von viralen Vektoren können hohe Transgen-Expressionsniveaus erreicht werden, was wichtig sein könnte, da der Beginn der Erkrankung und der Schwere auf der Ebene der ÜBERBEL häufig abhängigression. Mehrere Vektorsysteme basieren auf verschiedenen Viren entwickelt worden. Die Wahl des Vektorsystems hängt von der Größe des Gens von Interesse, die erforderliche Dauer der Genexpression, die Zielzelle und die biologische Sicherheit Probleme. Zur stabilen Gentransfer in das Gehirn, lentivirale (LV) und rekombinantem adeno-assoziierter viraler (rAAV) -Vektoren sind nun die Vektorsysteme der Wahl angesehen, da sie auf effiziente und langfristige Genexpression im Nagetier Gehirn führen. Für spezifische Targeting der dopaminergen Neuronen (DN) des SN, rAAV Vektoren sind Vektoren LV allmählich outcompeted wegen ihrer höheren Titern und Transduktionseffizienz von DN. Die besten α-SYN Basis Nagetiermodellen derzeit haben aus einem kombinierten Einsatz mit neueren AAV - Serotypen (rAAV 1, 5, 6, 7, 8) und optimierte Vektorkonstrukte, Titer und Reinheit 15,16 entwickelt. Der Vektor-Titer sowie der Vektor Reinheit direkt beeinflusstdas phänotypische Ergebnis des Modells. Übermäßige Vektor-Titer oder unzureichend gereinigte Vektor Chargen können in nicht-spezifische Toxizität führen. Daher sind geeignete Kontrollvektoren unverzichtbar. Erhebliche Zeitinvestitionen in den viralen Vektor Produktion, Upscaling und Reinigungsverfahren wurden ebenfalls als wesentlich erwiesen reproduzierbare und qualitativ hochwertige Vektor-Chargen zu erhalten.

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			<td height="20" style="height:20px;width:391px;">Female 8 weeks old Wistar rats</td>
			<td style="width:227px;">Janvier</td>
			<td style="width:187px;">/</td>
			<td style="width:681px;">200-250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Nimatek)</td>
			<td>Eurovet animal health</td>
			<td>804132</td>
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			<td></td>
		</tr>
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			<td height="20" style="height:20px;">&#160;Local anesthetic for scalp and ears: Xyloca&#239;ne 2% gel</td>
			<td>Astrazeneca</td>
			<td>0137-547</td>
			<td></td>
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			<td height="20" style="height:20px;">Terramycine</td>
			<td>Pfizer</td>
			<td>0132-472</td>
			<td></td>
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			<td height="20" style="height:20px;">Bupr&#233;norphine (Vetergesic)</td>
			<td>Ecuphar</td>
			<td>2623-627</td>
			<td></td>
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			<td height="20" style="height:20px;">Jodium 1% isopropanol</td>
			<td>VWR</td>
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			<td height="20" style="height:20px;">stereotactic head frame</td>
			<td>Stoeling</td>
			<td>/</td>
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			<td height="20" style="height:20px;">Hamilton Syringe (30 gauge -20mm -pst 2)</td>
			<td>Hamilton/ Filter Service</td>
			<td>7803-07</td>
			<td></td>
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			<td height="20" style="height:20px;">atipamezole (Antisedan)</td>
			<td>Orion-Pharma/Elanco</td>
			<td>1300-185</td>
			<td></td>
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			<td height="20" style="height:20px;">rAAV A53T &#945;-SYN vector</td>
			<td>LVVC, KU Leuven</td>
			<td>/</td>
			<td>https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular-virology-and-gene-therapy/lvvc/</td>
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			<td height="20" style="height:20px;">sodium pentobarbital (Nembutal)</td>
			<td>Ceva Sant&#233;</td>
			<td>0059-444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microtome</td>
			<td>Microm</td>
			<td>HM650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal synuclein Ab</td>
			<td>Chemicon</td>
			<td>5038</td>
			<td>1:5000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal TH Ab</td>
			<td>Chemicon</td>
			<td>152</td>
			<td>1:1000</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph</td>
			<td>Siemens/ Concorde Microsystems</td>
			<td>/</td>
			<td></td>
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			<td height="20" style="height:20px;">radioligand: 18F-FECT</td>
			<td>In house</td>
			<td>/</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-dopa: Prolopa 125</td>
			<td>Roche</td>
			<td></td>
			<td>6mg/kg i.p.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, Glutamax</td>
			<td>Life Technologies</td>
			<td>N&#176; 31331-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Foetal bovine serum</td>
			<td>Life Technologies</td>
			<td>N&#176; 10270-106</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">25 kD linear polyethylenimine (PEI)</td>
			<td>Polysciences</td>
			<td>/</td>
			<td></td>
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			<td height="20" style="height:20px;">OptiPrep Density Gradient Medium: Iodixanol</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optimen</td>
			<td>Life Technologies</td>
			<td>N&#176; 51985-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paxinos 1 watston steretactic atlas, fourth Edition</td>
			<td>Elsevier</td>
			<td>/</td>
			<td></td>
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development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector

Um die molekularen Mechanismen der Parkinson-Krankheit (PD) und die Entwicklung neuer therapeutischer Strategien zu untersuchen, setzen wissenschaftliche Forscher an Tiermodellen. Die Identifizierung von PD-assoziierten Genen hat zu der Entwicklung von genetischen PD Modelle geführt. Die meisten transgenen α-SYN Mausmodelle entwickeln schrittweise α-SYN Pathologie aber nicht klar dopaminergen Zellverlust und Dopamin-abhängige Verhaltensdefiziten anzuzeigen. Diese Hürde wurde durch direkte Ausrichtung der Substantia nigra mit viralen Vektoren überexprimiert, PD-assoziierten Gene zu überwinden. Lokale Genübertragung mittels viraler Vektoren bietet eine attraktive Möglichkeit, Transgene in das zentrale Nervensystem zu exprimieren. Spezifischen Hirnregionen ausgerichtet werden können (zB der Substantia nigra), kann die Expression im adulten Einstellung und hohe Expressionsniveaus erreicht werden kann induziert werden. Ferner können verschiedene Vektor-Systeme auf der Basis verschiedener Viren verwendet werden. Das Protokoll legt alle entscheidenden Schritte, um einen viralen Vektor auszuführenInjektion in der Substantia nigra der Ratte einen viralen Vektor-basierte alpha-Synuclein-Tiermodell für die Parkinson-Krankheit zu entwickeln.

Das Gesamtkonzept des Experiments ist in Abbildung 1 dargestellt 2 rAAV / 7-vermittelte Überexpression von A53T α-SYN induziert Dopamin-abhängige motorische Defizite. Um zu untersuchen , ob die Höhe der α-SYN - Überexpression ausreichend ist , motorischen Beeinträchtigungen bei Ratten zu induzieren, wir die Ratten zur Zylindertest unterzogen , um spontane forelimb Verwendung <str...

Watch this Scientific Journal Video about Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector at JoVE.co

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

Entwicklung eines Alpha-Synuklein Based Rattenmodell für Parkinson-Krankheit durch stereotaktische Injektion eines rekombinanten Adeno-assoziierten viralen Vektor,

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

development-an-alpha-synuclein-based-rat-model-for-parkinson-s

Research

JoVE Journal

Medicine

13.2K Aufrufe.  KU Leuven. This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease. 

Serie: Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

High-Efficiency Transduktion von Liver Cancer Cells von rekombinanten Adeno-assoziierten Virus Serotyp 3 Vektoren

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Forschung

Medizin

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Entwicklung eines Einseitig-läsionierten 6-OHDA Mausmodell der Parkinson-Krankheit

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI-Imaging-Massenspektrometrie von Neuropeptiden bei Parkinsonpatienten

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Eine mögliche Zebrafisch-Modell der Kidney Disease: Knockdown von<em&gt; Wnt5a</em&gt; Ursachen Zysten in Zebrafisch-Nieren

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Verwendung Adeno-assoziierte Virus als Werkzeug Retinal Barrieren in Disease Study

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Intrarenale Injektion von<em&gt; Escherichia coli</em&gt; In einem Rattenmodell der Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Entwicklung eines Direkt Pulp-Kappungsmodell für die Beurteilung von Pulpal Wundheilung und Reparative Dentinbildung bei Mäusen

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Entwicklung von rekombinanten Proteinen zur Behandlung von chronischen Schmerzen

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Entwicklung eines uterosakralen Ligament-Suspensionsrattenmodells

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...