Yazarlar mükemmel teknik yardım için Joris Van Asselberghs ve Ann Van Santvoort teşekkür ederim. Araştırma AT-FP6 programı &#39;Dimi&#39; (LSHB-CT-2005-512146), 7.ÇP RTD proje MEFOPA (SAĞLIK tarafından, IWT-Vlaanderen (/ 80020 IWT SBO), FWO Vlaanderen (G.0768.10) tarafından finanse edildi -2.009-241791), 7.ÇP programı &#39;Inmind&#39; (SAĞLIK-F2-2011-278850), KU Leuven (IOF-KP / 07/001, OT / 08 / 052A, İmir PF / 10/017), ve MJFox Vakfı (Hedef doğrulama 2010). A. Van der Perren ve C Casteels Bilimsel Araştırma Flaman Fonu doktora sonrası adamlar vardır. K. Van Laere Bilimsel Araştırma Flaman Fonu üst düzey bir klinik üyesidir.

Tüm hayvan deneyleri Leuven Üniversitesi Biyoetik Komitesi (Belçika) tarafından 24 Kasım 1986 (86/609 / EEC) Avrupa Toplulukları Konseyi Direktifi uyarınca yürütülen ve onaylanır. 1. Rekombinant AAV Üretimi ve Saflaştırılması Not: Daha önce tarif edildiği gibi 17 rAAV vektör üretimi ve saflaştırılması Leuven Viral vektör çekirdek (LVVC) ile gerçekleştirilmiştir. <ol><li> Kısaca, transfeksiyonu konflüan düşük (&lt;50) geçiş, 25kD, doğrusal polietilenimin 1 bir oranda 150 nM NaCI transfeksiyon solüsyonu ve üç farklı plazmitler ile yapışık HEK 293T hücreleri: DMEM ortamı,% 2 fetal sığır serumu içinde 1: 1 arasındadır. % 5 CO2 içinde 37 ° C&#39;de inkübe edildikten 24 saat sonra, taze DMEM ortamı,% 2 fetal sığır serumu ile orta yerine. Not: plazmidler AAV7 serotipi için konstruktlar arasında, CMVie kontrolünde-sin α insan A53T mutantını kodlayan AAV aktarım plazmid sinaps gelişmişin1 promotör ve pAdvDeltaF6 adenoviral yardımcı plazmid 17. </li><li> Geçici transfeksiyondan sonra orta 5 gün hasat ve teğetsel akış filtrasyonu 17 kullanılarak konsantre. </li><li> Bir iyodiksanol basamaklı bir derece 17 kullanılarak konsantre ortamdan rAAV vektörü tanecikleri arındırın. </li><li> genomik kopya (GC) tayini için real-time PCR standart teknikleri kullanın. Bu protokol, 3.0 E11 GC bir vektör titresi / ml PD 17 için bir α-SYN tabanlı sıçan modeli geliştirmek için kullanıldı. </li></ol> rAAV a-SYN 2. Stereotaktik enjeksiyon Sıçan SN Vektör (Şekil 2) <ol><li> pelet haline yiyecek ve musluk suyu ücretsiz erişim ile normal 12 saat ışık / karanlık döngüsü altında yaklaşık 200-250 g ağırlığında Ev sekiz haftalık dişi Wistar rat. </li><li> intraperitoneal sıçan sun (ip), bir ketamin (60 mg / kg) ve medetomidini (/ kg, 0.4 mg) ihtiva eden anestezi. sıçan anestezi ve tepki vermiyor kezFarklı pençeleri sıkma yaparken, bir mikro-Transponder IMPLANTER kullanarak daha fazla tanınması için sıçan arkasında bir mikro-Transponder subkutan yönetmek. Mikro-Transponder doğru konumlandırılmış ve okuma cihazı tarafından okunabilir olup olmadığını kontrol edin. </li><li> Kafa derisinin üstünde saçlarını kesti. derisi ve kulakların hem bir lokal anestezik uygulayın. aseptik teknikler kullanılarak bir laminer akış altında cerrahi geri kalanını gerçekleştirin. </li><li> İki kulak çubukları, bir ağız ve burun çubuğunu kullanarak bir stereotaktik kafa çerçevesi içinde sıçan yerleştirin. vücut ısısında bir düşüş önlemek için bir kağıt battaniye ile sıçan vücudu kaplar. kurutma gözleri önlemek için bir göz yağ sürün. </li><li> izopropanol% 70 jodium% 1 ile saçlı deri dezenfekte ve kafa derisi orta hatta küçük bir kesi yapmak. Yavaşça kafatası membranlar kazınması ve tuzlu su ile durulayın. birkaç dakika kafatası kurumasını bekleyin. Bregma ile lambda: kranial sütürler ve iki referans noktaları dikkate alın. </li&gt; <li> SN içine rAAV vektör enjekte, Bregma doğru koordinatları define (ön-arka: 5,3 mm; mediyolateral: 2.0 mm ve dorsoventral: dura hesaplanan 7.2 mm). Not: her bir ilgi bölgesi için üç boyutlu koordinatlar, anatomik bir referans noktası olarak bregmadan uygulanması, sıçan beyninin bir stereotaksik Atlası kullanarak hesaplanabilir. </li><li> rAAV vektörü ile bir 10 ul mikroenjeksiyon şırınga (30 ölçer 20 mm) doldurun ve motorlu bir mikroenjeksiyon pompasına bağlı stereotaksik alet içine koyun. Vektör bir damla bırakarak ses düzeyini kontrol ve bir çok değerlikli temizlik deterjanı pH 9 (örneğin RBS) olarak ortadan kaldırır. </li><li> Görme baş düz kafa çerçeveye sabit olup olmadığını kontrol edin ve sol-sağ eksen değerlendirir. Dikkatle görsel bregma ile lambda için ön-arka ve mediolateral koordinatlarını tanımlamak ve stereotaktik çerçevenin dorsoventral kolunda bir 30 ölçü 20 mm iğne kullanılarak onların yüksekliğini ölçmek. <ol><li> am izinBregma ile lambda arasındaki yükseklik 0.3 mm farkı edilen maksimum eğim. geri Bregma üzerinde iğne yerleştirin ve ön-arka ve stereotaktik çerçevenin mediyolateral kolunu hareket ettirerek ön-arka ve mediyolateral koordinatlarını geçerlidir. </li></ol></li><li> Enjeksiyon yerinde, kafatasının yüksekliğini ölçmek ve bregma yükseklikten fazla 0,3 mm farklılık olmamasını sağlamak. Yaklaşık 2 mm çaplı kafatasında bir delik delin. Dura yüksekliğini ölçmek, bu dorsoventral koordinat uygulamak için bir referans olarak hizmet verecek. Seçenek olarak kafatası (0.9 mm) sabit bir kalınlığı çıkarılır. </li><li> 26 gauge iğne kullanılarak dura nüfuz ve steril bir doku ile kan emer. Tüm kanama devam etmeden önce durmasını bekleyin. </li><li> Yavaş yavaş (koordinat dorsoventral) 10 ul mikroenjeksiyon şırınga önceden belirlenmiş bir derinlikte beyin içine vektörü çözeltisi ile önceden yüklenmiş yerleştirin. yerinde iğne ile 1 dakika bekleyin. 3 ul enjekte0.25 ul hacmi motorlu mikroenjeksiyon pompa kullanılarak (rAAV2 / 7 α-sin ya eGFP kontrol vektörü 3.0 E11 genom kopya / ml (orta vektör doz) ya da 1.0 E12 GC / ml (yüksek vektör doz)) vektörü çözeltisi / min. </li><li> Enjeksiyondan sonra, yavaş yavaş çıkarmadan önce başka bir 5 dakika boyunca yerde iğne tutmak. kaplanmış örgülü polyester 3.0 kullanarak kafa derisi Dikiş,% 70 izopropanol içinde% 1 jodium ile dezenfekte ve yavaşça stereotaktik enstrüman hayvan kaldırmak. Öncelikle, daha sonra iki kulak çubukları burun ve ağız çubuğu gevşetin. </li><li> Anestezi tersine çevirmek için, 0.5 mg / kg atipamezol ile intraperitoneal sıçan enjekte ve uyanır kadar 38 ° C&#39;lik bir ısıtma plakası üzerinde temiz bir kafeste sıçan yerleştirin. vücut ısısında bir düşüş önlemek için bir kağıt battaniye ile sıçan örtün. </li><li> İlk saat için yiyecek ve su kolay erişim sağlar. İlk birkaç gün sıçan izleyin. Gerekirse analjezi uygulanır. Not: t dikiş kaldırmak için gerek yokturO kafatası. 1-2 hafta sonra kafatası tamamen tamir edilir ve stiches gevşemiş. </li></ol> Non-invaziv PET Görüntüleme, Davranışsal Testler ve immünohistokimyasal Analizi 3. rAAV2 Değerlendirilmesi / 7 α-SYN Enjekte Sıçanlar <ol><li> (DAT) dopamin taşıyıcısı ölçmek küçük hayvan pozitron emisyon tomografisi (PET) ve DA Transporter, örneğin bir izleyici kullanarak bağlama, non-invaziv bireysel hayvanlarda zamanla nigrostriatal dopaminerjik nörodejenerasyon kinetiği takip etmek [18 F] -FECT 16 . </li><li> dopaminerjik nörodejenerasyon seviyesi kendiliğinden ön ayakları kullanımını değerlendirmek, sıçanlarda motor bozukluk neden silindir testi fareler tabi yeterli olup olmadığını incelemek için. <ol><li> Bir arka sonrasında duvar ve iniş boyunca dikey hareketleri sırasında cam silindir ve video kaset davranışı geniş açık 20 cm sıçan yerleştirin. 20 toplam her forepaw tarafından yapılan kişilerin sayısını skorrehber. Puanlama kriterlerinin ayrıntılı bilgi için bkz Schallert ve ark., 18 toplam ön ayakları temas yüzdesi (sol artı sağ forepaw) olarak bozulmuş ön ayakları temas (örneğin sol forepaw) sayısını ifade eder. Not: eşit iki pençeleri kullanarak Olmayan lezyonlu kontrol sıçanların bu teste yaklaşık% 50 puan olmalıdır. </li></ol></li><li> transgen ifade ve dopaminerjik hücre kaybı seviyesini değerlendirmek için immünohistokimyasal (İHK) analizi yapın. <ol><li> Farklı son aşamalarında, sodyum pentobarbital (60 mg / kg, ip) bir fazla dozu ile kurban fareleri ve PBS 19 içinde% 4 paraformaldehid, ardından, soğuk tuz ile intrakardiyak perfüzyon gerçekleştirin. gece boyunca 4 ° C &#39;de beyin sabitlemek ve titreşimli mikrotom kullanılarak 50 um kalınlığında koronal beyin kesitler halinde kesilmiştir. </li><li> α-SYN ifade seviyelerini ve le analiz α-SYN ve tirozin hidroksilaz karşı antikorlar kullanılarak serbest yüzen bölümlerde İHK boyama gerçekleştirinnörodejenerasyon 16 vel. </li></ol></li></ol>

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	<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>

Yazarlar herhangi bir çıkar fiili ya da potansiyel bir çatışma olduğunu beyan ederim.

protokolü içinde birkaç kritik adımlar vardır. vektör titresi yanı sıra vektör saflık doğrudan modelin fenotipik sonuçlarını etkiler. Aşırı vektör titreleri ya da yeterli saflaştırılmış vektör toplu spesifik olmayan toksisite ile sonuçlanabilir. Bu nedenle, yüksek kaliteli vektör gruplar ve uygun kontrol vektörlerin kullanılması kaçınılmazdır. Bundan başka, stereotaksik çerçeveye sıçan kafasının tam olarak konumlandırılması ve koordinatları doğru belirlenmesi substantia nigr...

PD patofizyolojisini incelemek ve yeni tedavi stratejilerinin geliştirilmesi amacıyla, yakından insan PH nöropatoloji, fizyoloji ve motor semptomları benzer hayvan modellerinde için acil bir ihtiyaç vardır. yüksek öngörü değeri, daha iyi biz hastalara hayvan modellerinde yeni tedaviler çevirebilir. 1997 yılında ilk PARK geninde gibi alfa-sinüklein keşfi (α-sin) ilk genetik PD modelini gelişmesine yol açmıştır. İnsan doğal tip (WT) veya mutant (A30P, A53T) aşırı ifade eden transjenik farelerin, bir çok α-EŞZ son on yıl içinde oluşturulmuştur. α-EŞZ aşırı ifadesinin düzeyleri patoloji gelişiminde çok önemli olduğu kanıtlanmıştır. Ayrıca fare suşu, endojen α-sin ve tam uzunlukta veya kesik bir şekilde ifade edilip varlığı veya yokluğu, (Magen ve Chesselet 1 ile ayrıntılı bir inceleme) rol oynar. WT ve insan çeşitli klinik mutantlar hem aşırı ekspresyonu &amp;# 945; transgenik farelerin -SYN α-SYN patolojik birikimi ve nöronal disfonksiyon 2-6 uyarır. Ancak, şimdi en transgenik α-SYN fare modelleri berrak dopaminerjik hücre kaybı ve dopamin-bağımlı davranış açıkları görüntülemek için başarısız kadar. Bu engel α-SYN fazla sentezleyen viral vektörlerle substantia nigra (SN) doğrudan hedefleme ile üstesinden oldu. Viral vektörler kolayca hücreleri enfekte ev sahibi genoma genetik malzemeyi vermek ve yeni virüs parçacıklarının üretilmesi amacıyla viral genomu çoğaltmak için konakçı hücrenin zorlayabilir virüslerden türetilir. Virüsler hücrelerine girip genlerin katılmasıdır yeteneklerini muhafaza replike olmayan viral vektörler için tasarlanmış olabilir. Viral genomun parçalarını silme ve ilgi genleri tarafından değiştirmek suretiyle, vektör uygulanması genellikle &quot;transdüksiyon&quot; olarak adlandırılan bir konakçı hücre, replikasyon olmaksızın tek bir tur enfeksiyona neden olur. Viral vektörler CıBir aşırı ekspresyonu, gen susturma hem de kullanılabilir. Ifade transgen bir raportör protein (örneğin, yeşil fluoresan protein ya da ateşböceği lusiferaz) 7, bu yazıda üzerinde durulacak bir terapötik gen terapi uygulamaları 8-10 protein ya da olabilir, bir hastalıkla ilişkili proteinin hastalıkta modelleme 11 için kullanılan -14. Viral vektör aracılığıyla gen teslimi birçok avantaj CNS&#39;de transgenleri ifade için alternatif bir yöntem sağlar. lokal transgen uygulama kullanıldığında spesifik beyin bölgelerinde hedeflenebilir. Dahası, transgen ifade gelişimi sırasında telafi mekanizmalarının riskinin azaltılması yetişkinlik döneminde teşvik edilebilir. Ayrıca, modeller farklı türler ve suşları oluşturulabilir. Ve son olarak, farklı transgenler kolaylıkla birleştirilebilir. viral vektörler kullanılarak yüksek transgen sentezleme seviyeleri hastalığın başlama ve şiddeti genellikle overexp düzeyine bağlı olduğundan çok önemli olabilir, elde edilebilirdışarı yansıması. farklı virüsler dayalı çeşitli vektör sistemleri geliştirilmiştir. bir vektör sisteminin seçimi, ilgi konusu genin, gen ekspresyonu, hedef hücre ve biyo-güvenlik sorunları gerekli süre büyüklüğüne bağlıdır. bu kemirgen beyin etkili ve uzun vadeli gen ifadesini yol açacağından beyin, lentiviral (IV) &#39;de sabit gen transferinin ve rekombinant adeno-ilişkili viral, (rAAV), vektörler artık tercih edilen vektör sistemleri kabul edilmektedir. SN dopaminerjik nöronların (DN) özel hedeflemesi için rAAV vektörleri yavaş yavaş nedeniyle yüksek titreleri ve DN transdüksiyon verimliliği LV vektörleri outcompeted var. Şu anda mevcut en iyi α-EŞZ göre kemirgen modelleri yeni AAV serotiplerinin kullanan bir birleşik yaklaşım adlı geliştirilmiştir (rAAV 1, 5, 6, 7, 8) ve vektör yapıları, titerleri, ve saflık 15,16 optimize edilmiştir. vektör titresi yanı sıra vektör saflık doğrudan etkiliyorModelin fenotipik sonucu. Aşırı vektör titreleri ya da yeterli saflaştırılmış vektör toplu spesifik olmayan toksisite ile sonuçlanabilir. Bu nedenle, uygun kontrol vektörleri vazgeçilmezdir. Viral vektör üretimi, yükseltme ve saflaştırma işlemlerinde önemli zaman yatırımı da tekrarlanabilir ve yüksek kaliteli vektör toplu elde etmek için gerekli kanıtlanmıştır.

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			<td style="width:227px;">Janvier</td>
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			<td style="width:681px;">200-250 g</td>
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			<td height="20" style="height:20px;">&#160;Local anesthetic for scalp and ears: Xyloca&#239;ne 2% gel</td>
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			<td height="20" style="height:20px;">stereotactic head frame</td>
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			<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>
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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>
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			<td height="20" style="height:20px;">microtome</td>
			<td>Microm</td>
			<td>HM650</td>
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			<td height="20" style="height:20px;">rabbit polyclonal synuclein Ab</td>
			<td>Chemicon</td>
			<td>5038</td>
			<td>1:5000</td>
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			<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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			<td height="20" style="height:20px;">Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph</td>
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			<td height="20" style="height:20px;">radioligand: 18F-FECT</td>
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			<td height="20" style="height:20px;">L-dopa: Prolopa 125</td>
			<td>Roche</td>
			<td></td>
			<td>6mg/kg i.p.</td>
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			<td height="20" style="height:20px;">DMEM, Glutamax</td>
			<td>Life Technologies</td>
			<td>N&#176; 31331-093</td>
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			<td height="20" style="height:20px;">Foetal bovine serum</td>
			<td>Life Technologies</td>
			<td>N&#176; 10270-106</td>
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			<td height="20" style="height:20px;">25 kD linear polyethylenimine (PEI)</td>
			<td>Polysciences</td>
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			<td height="20" style="height:20px;">OptiPrep Density Gradient Medium: Iodixanol</td>
			<td>Sigma</td>
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			<td height="20" style="height:20px;">Optimen</td>
			<td>Life Technologies</td>
			<td>N&#176; 51985-026</td>
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			<td height="20" style="height:20px;">Paxinos 1 watston steretactic atlas, fourth Edition</td>
			<td>Elsevier</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

Parkinson hastalığı (PH) moleküler yollar incelemek ve yeni tedavi stratejilerinin geliştirilmesi amacıyla, bilimsel araştırmacılar hayvan modellerinde güveniyor. PD-ilişkili genlerin belirlenmesi, genetik PD modelini geliştirilmesine yol açmıştır. En transgenik α-SYN fare modelleri kademeli α-SYN patolojiyi geliştirmek ama net dopaminerjik hücre kaybı ve dopamin-bağımlı davranış açıkları görüntülemek için başarısız. Bu engel PD-ilişkili genleri fazla sentezleyen viral vektörlerin ile substantia nigra doğrudan hedefleme ile üstesinden oldu. viral vektörler kullanılarak yerli gen dağıtım merkezi sinir sisteminde transgenleri ifade için çekici bir yol sağlar. Spesifik beyin bölgelerinde (örneğin, substantia nigra) hedeflenebilir, sentezleme yetişkin ortamda indüklenebilir ve yüksek ekspresyon seviyeleri elde edilebilir. Bundan başka, çeşitli virüsler ile ilgili olarak farklı vektör sistemleri kullanılabilir. protokol bir viral vektör gerçekleştirmek için bütün önemli adımlar özetlenirSıçan substantia nigra enjeksiyon Parkinson hastalığı için bir viral vektör bazlı alfa-sinüklein hayvan modeli geliştirmek için.

Deney genel şeması, Şekil 1 &#39;de gösterilmektedir A53T α-sin bölgesinin rAAV 2/7 aracılı aşırı dopamin bağımlı motor bozukluklar neden olmaktadır. Α-EŞZ aşırı ekspresyonu seviyesi sıçanlarda motor bozukluk uyarılması için yeterli olup olmadığını incelemek için, kendiliğinden ön ayakları kullanımı (Şekil 3A) değerlendirmek için si...

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

Bir rekombinant adeno-ilişkili viral vektörün stereotaktik enjeksiyon yoluyla Parkinson Hastalığı için bir alfa-sinüklein Bazlı Fare Modeli Geliştirme

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 Görüntüleme.  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. 

Video: 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.

Rekombinant Adeno Associated Virus serotip 3 Vektörler Karaciğer Kanseri Hücreleri Yüksek Verimlilik İletimi

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 ...

Tıp

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.

Parkinson Hastalığının tek taraflı lezyonlanmıştır 6-OHDA Mouse Model Geliştirme

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.

Parkinson Hastalığında nöropeptitler MALDI Görüntüleme Kütle Spektrometresi

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.

Polikistik Böbrek Hastalığı Olası Zebra balığı Modeli: demonte<em&gt; Wnt5a</em&gt; Zebra balığı Böbrekler Kistler Nedenler

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.

Aracı Olarak Adeno-ilişkili virüs kullanarak Hastalığında Retina Engelleri Eğitim için

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.

Intrarenal enjeksiyon<em&gt; Escherichia coli</em&gt; Piyelonefritli Sıçan Modelinde

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.

Pulpa Değerlendirilmesi için Doğrudan Selüloz-kapatma Modelinin Geliştirilmesi Yara İyileşmesi ve Farelerde Onarım dentin oluşumu

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.

Kronik ağrı tedavisinde rekombinant proteinler gelişimi

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.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

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.

Uterosakral Ligament Süspansiyon Sıçan Modelinin Geliştirilmesi

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