Tüm sıçanlar, Başkent Tıp Üniversitesi, Pekin Dostluk Hastanesi Hayvan Deney Komitesi tarafından onaylanan protokollere göre kullanıldı.
1. Cerrahi hazırlık, anestezi ve cerrahi teknikler
NOT: Bu çalışma için 200-220 g ağırlığında toplam 40 dişi Wistar sıçanı ticari olarak elde edilmiştir. 40 sıçandan 10'u rastgele kontrol grubu olarak seçildi ve geri kalanı test grubu olarak kabul edildi. Tüm hayvanlar, Başkent Tıp Üniversitesi, Pekin Dostluk Hastanesi'nin hayvan tesislerinde steril bir ortamda barındırıldı.
<ol><li>Sodyum pentobarbital intraperitoneal (40 mg/kg) uygulayarak genel anestezi uygulayın. Alternatif olarak,% 3-4% izofluran kullanarak anesteziyi indükleyin ve% 1 -% 3'te (solunan) tutun. Kuruluğu önlemek için gözlere oftalmik merhem uygulayın. Ardından, sıçanı cerrahi platforma yerleştirin ve termal destek sağlayın. NOT: İşlemin başında buprenorfin, 0.05 mg/kg, SC, 0.1-0.2 mL gibi analjezikler uygulayın.</li>
	<li>Ayak parmağı sıkışmasına yanıt vermemek suretiyle anestezi derinliğini kontrol edin. Kürkü tüm arka bölgeden tıraş bıçağı ile tıraş edin.</li>
	<li>Cerrahi bölgeyi, Klorheksidin veya Povidon iyot ve ardından İzopropil alkol gibi iki aşamalı bir ovmanın en az 3 döngüsü ile sterilize edin. Uzuvları cerrahi bantla sabitleyin ve cerrahi makasla sırtta yaklaşık 3 cm'lik bir medyan kesi yapın.</li>
	<li>Cerrahi makas kullanarak kesiyi deri altı dokulardan derinleştirin ve omurgaya bağlı kasları kesin.</li>
	<li>13. kaburgayı görsel olarak tanımlayın ve ortaya çıkarın (bu kaburgaya bağlı intervertebral boşluk T13\u2012L1 aralığıdır). 13. kaburgayı bir dikiş kullanarak işaretleyin.</li>
	<li>Tanımlamadan sonra, omurgaya bağlı kasları dikkatlice rezeke edin ve vertebral kolonu açığa çıkarın. Vertebral kolonun doğru tanımlanması için supraspinöz ligament ve interspinöz ligamenti rezeke edin. L4\u2012L5 seviyesini cerrahi makas ve forseps ile ortaya çıkarın. NOT: Supraspinöz ligament, ince deri altı dokusunun varlığı nedeniyle kolayca tanımlanabilir. Supraspinöz ligamentin rezeksiyonundan sonra, spinöz süreç arasındaki ligament interspinöz ligamenttir.</li>
	<li>L4\u2012L5 vertebral dikenli süreci ve omuriliği açığa çıkarmak için Kelly forseps kullanarak enine sürecin parçalarını dikkatlice inceleyin (Şekil 1).</li>
	<li>Konus medullarisin L4\u2012L5 seviyesinde tamamen açığa çıkarılması ve iridektomi makası ile konus medullarisin tamamen transekte edilmesi. Omuriliğin iyileşmesini engellemek için bazı doku ambalajları yerleştirin.</li>
	<li>Dış deri tabakasındaki üst kas ve cildi 4-0 emilemeyen dikiş kullanarak kapatın.</li>
	<li>Kontrol grubu için 1.1\u20121.7 adımlarını uygulayın ve conus medullaris'i olduğu gibi bırakın. İnsizyonu adım 1.9'a göre kapatın.</li>
</ol>2. Hayvan kurtarma
<ol><li>Sıçanları ameliyattan sonraki ilk saat boyunca sıcaklık kontrollü bir inkübatörde (37 ° C) tutun ve sternal veya aktif olarak hareket edene kadar izleyin. NOT: Tam kurtarma işlemi yaklaşık yarım saat sürer.</li>
	<li>Hayvanı yeterli yiyecek ve su içeren temiz bir kafese aktarın. Sıçanları ayrı kafeslerde tutun. NOT: Transeksiyonun başarısı, test grubundaki sıçanlar sadece ön ayakların yardımıyla hareket ederken, kontrol grubundaki sıçanlar normal şekilde yürüyebildiklerinde belirtilir.</li>
</ol>3. Operasyon sonrası yönetim
<ol><li>İntraperitoneal olarak bir antibiyotik olan Penisilin G'yi (hayvan başına 50.000 U / mL) enjekte edin. Buprenorfin, 0.05 mg / kg, SC, 0.1-0.2 mL gibi analjezikleri ameliyat sonrası 48 saat boyunca her 6-12 saatte bir uygulayın.</li>
	<li>İşemeye yardımcı olmak için idrar kesesini hipogastriyumda sıkıştırın. Bunu altı hafta boyunca günde iki kez aynı anda (sabah 8 ve akşam 8) gerçekleştirin. NOT: Detrusor'un normal daralmasının kaybı, DU'nun sembolüdür.</li>
	<li>Tüm sıçanları metabolik kafeslerde barındırın, her biri miksiyon ve inkontinansı izlemek için daha önce tartılmış emici bir kağıdın üzerine yerleştirilmiş bir idrar toplama hunisi içerir.</li>
	<li>İşemiş hacmi (VV) ve artık idrar hacmini ayrı ayrı gösteren emici kağıdın ağırlık değişimini toplayın ve not edin.</li>
</ol>4. Ürodinamik test
<ol><li>Ameliyattan altı hafta sonra, aşağıdaki gibi ürodinamik ölçüm ekipmanı kullanarak bir sistometrogram uygulayın.<ol><li>Sıçanları periton boşluğuna (3 mL/kg) %10 kloral hidrat enjekte ederek anestezi altına alın.</li>
		<li>İşeme için mesaneyi sıkıştırın, ardından sıçanı bir bant kullanarak cerrahi platforma sabitleyin.</li>
		<li>Epidural kateteri (3F) mesaneye yerleştirin ve ürodinamik ölçüm ekipmanını, epidural kateteri ve infüzyon pompasını üç uzuvlu tüp ile bağlayın.</li>
		<li>Ürodinamik ölçüm için fizyolojik salini 0,2 mL/dak hızında pompalayın (bakınız Malzeme Tablosu). MCC ve DOP'u ve mesanenin uygunluğunu kaydedin (δ mesane hacminin detrusörün δ basıncına bölünmesiyle hesaplanır).</li>
	</ol></li>
</ol>5. İstatistiksel analiz
<ol><li>Ticari olarak temin edilebilen yazılımları kullanarak istatistiksel analiz gerçekleştirin.</li>
	<li>Verilerin normalliğini test etmek için Kolmogorov-Smirnov testini kullanın.</li>
	<li>Normal olarak dağılmış değişkenleri standart sapmalarla ortalama değerler olarak ifade edin. Her iki gruptaki sistometrogram parametrelerini karşılaştırmak için iki kuyruklu eşleştirilmiş Öğrenci t-testlerini kullanın. NOT: p < 0,05, aradaki farkın istatistiksel anlamlılığa sahip olduğunu gösterir.</li>
</ol>

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Yazarların açıklayacak hiçbir şeyleri yoktur.

DÜ, hem erkeklerde hem de kadınlarda alt üriner sistem semptomlarının yaygın bir nedenidir. Etkilenenlerin yaşam kalitesini (Qol) önemli ölçüde azaltabilen az sayıda tedavi seçeneğine sahip karmaşık bir semptom takımyıldızıdır18. DÜ'nün multifaktöriyel olduğuna inanılmasına rağmen, patogenezinin anlaşılması ilkeldir. Çalışmalar, DÜ'nün patogenezinin miyojenik ve nörojenik faktörlerle ilişkili olabileceğini göstermiştir.
Miyojeni...

Defrüsör yetersizliği (DU), halen çalışılan tipik bir alt üriner sistem disfonksiyonudur. DU Uluslararası Kontinans Derneği (ICS)1 tarafından tanımlanmış olmasına rağmen, bu hastalığa atıfta bulunmak için çok sayıda farklı terminoloji kullanılmaktadır, örneğin, "detrusor yetmezliği", "akonttil mesane", "detrusor arefleksia"2. DU, 2002 yılında Uluslararası Kontinans Derneği (ICS) tarafından tanımlandığı gibi, mesane boşaltma için zamanın uzun süreli artmasına neden olan ve böylece normal bir süre içinde tam mesane boşalmasının sağlanamamasına neden olan, azaltılmış güç ve sürenin daralmasıdır.
DU, alt üriner sistem semptomları olan erkeklerin %48'ini ve kadınların (>70 yaş arası)3 %12'sini etkileyebilir. Multifaktöriyel gibi görünüyor ve etkili bir tedavi mevcut değil. Multipl skleroz4, diabetes mellitus5, Parkinson hastalığı6 veya serebral inme7 gibi nörojenik mesane disfonksiyonu olan hastalarda DU'nun her yerde olduğu bildirilmektedir. DU ayrıca laparoskopik histerektomi, prostatektomi veya küçük pelvis8'deki diğer cerrahi müdahaleler gibi iyatrojenik sinir hasarından da kaynaklanabilir. Patofizyoloji değişiklikleri ve DÜ'nün mevcut tedavileri, çalışma için uygun bir hayvan modelinin bulunmaması nedeniyle hala kafa karıştırıcıdır.
Miktüritasyon refleksi, pontin miktüritasyon merkezini, sakral parasempatik çekirdeği ve daha kıdemli korteks merkezlerini birleştiren spino-bulbospinal yollar tarafından kontrol edilir9. Miktürition refleksinin aktivasyonu ve sürdürülmesi esas olarak duyusal sinyallerin mesaneden daha kıdemli korteks merkezlerine düzenli olarak taşınmasına bağlıdır. Duyusal disfonksiyonun DU'ya katkıda bulunduğu varsayılabilir.
Alt üriner sistem disfonksiyonlarıyla ilgili deneysel hayvan çalışmalarının çoğu, aşırı aktif mesane (OAB) modellerine odaklanmıştır10. Bu modeller OAB patofizyolojisi ve prognozu hakkında makul bir anlayış sağlar. Bununla birlikte, sadece birkaç DU modeli bildirilmiştir, örneğin, supraspinal yaralanma (lokal lezyonlar, deserebrasyon ve orta serebral arter tıkanıklığı), omurilik transeksiyonu veya kontüzyon yaralanması, sistemik (örneğin, siklofosfamid) veya tahriş edici veya enflamatuar ajanların intravezikal uygulaması (örneğin, asit, akrolein ve lipopolisakkarit)11,12,13,14 . Bu yöntemler arasında DU13'ün hayvan modelinin oluşturulmasında sadece omurilik transeksiyonu veya kontüzyon yaralanması yöntemi kullanılabilir. Pontin miktüritasyon merkezinin ve yüksek korteks merkezlerinin yaralanmasını içeren girişimler ciddi travma nedeniyle terk edildi. Bu nedenle, DU'yu minimum yan etkilerle indüklemek için miksiyon refleks merkezinde doğru bir yer bulmak için daha fazla dikkat edilmektedir.
Daha önce de belirtildiği gibi, DU'yu indükleme mekanizmalarından biri, miksiyon refleksinin sinyal yoluna zarar vermek için omuriliğe zarar vermektir. Allen'ın ağırlık bırakma yöntemi, yaralı omurilikleri olan laboratuvar hayvanları oluşturmak için geliştirilmiştir15. Bununla birlikte, bu yöntem hakkında daha fazla deneysel veri bulunmamaktadır. Dahası, hayvanların bir kısmı DU olmadan inmeden sonra omurga fonksiyonunu geri kazandığından, bir DU hayvan modeli16 oluşturmak için mükemmel bir yöntem olarak düşünülemez.
1987'de Bregman, DU hayvan modelini üretmek için omuriliğin transeksiyonu sürecini çıkardı ve deneysel veriler elde etti17. Bununla birlikte, bu yöntem DU hayvan modelini oluşturmak için uygulanmamıştır. O zamanlar, araştırmacılar hala DU'nun patogenezi konusunda kafaları karışıktı. OAB veya DU indüksiyonu ile ilişkili omurilikteki yerler birbirine bitişik olduğundan, DU17'yi indüklemek için omuriliğe doğru hasar bölgesini bulamadılar. OAB ve DU bu yöntemle birlikte veya ayrı ayrı tanıtıldı. Bu nedenle, bu yöntem DU'yu tanıtmasına rağmen, kesin değildi ve DU'nun oluşumunun ve işlenmesinin anlaşılması için kullanılamazdı.
Yukarıda belirtildiği gibi, DU'nun uygun bir hayvan modelinin olmaması, DU'nun çalışmasının önündeki ana engellerden biridir. Araştırmacılar sürekli olarak DU'nun patolojisini simüle edebilecek doğru ve yönetilebilir bir model aramaktadır. DU için tedavi seçenekleri bile son 20 yılda önemli ölçüde iyileşmemiştir. Toplu olarak, DU'nun bir hayvan modelini oluşturmak için standart bir protokol tanımlamaya büyük ihtiyaç vardır.
Bu nedenle, bu yazıda, konus medullaris transeksiyonu ile DU'nun sıçan modelini başarılı bir şekilde oluşturmak için bir yöntem açıklanmaktadır. Konüs medullaris'i ayırmak için L4\u2012L5 seviyesinde transeksiyon yapıldı. Maksimum sistometrik kapasite (MCC), detrusor açma basıncı (DOP) ve mesanenin uyumu kaydedildi ve protokolü doğrulamak için analiz edildi. Aşağıda belirtilen protokol, DU hayvan modelini oluşturmak için hem fizibiliteyi hem de güvenilirliği standartlaştırılmış bir şekilde birleştirir ve DU'nun oluşumunu ve işlenmesini simüle eder. Protokol, DU'nun daha ileri çalışmaları için bir teknik olarak kullanılabilir.

<table>
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Wuhan Prosai Company</td>
			<td>EY-C1178</td>
			<td>pump for urodynamic measurement</td>
		</tr>
		<tr>
			<td>10% chloral hydrate</td>
			<td>Shandong Yulong Co., Ltd</td>
			<td>H37022673</td>
			<td>3mL/kg&#65292;administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td>
			<td>H12020275</td>
			<td>0.05mg/kg subcutaneously 24h and 48h postoperation</td>
		</tr>
		<tr>
			<td>Epidural Catheter</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VABR3L</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Alta Technology Co., Ltd</td>
			<td>1ST5637</td>
			<td>50,000 unit/ml per animal</td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Beijing solabo Technology Co., Ltd</td>
			<td>NK-WF0001</td>
			<td>40 mg/kg, administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Suture line(4-0)</td>
			<td>ETHICON</td>
			<td>VCP422H</td>
			<td>suture the injury</td>
		</tr>
		<tr>
			<td>Three-limb tube</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VAB3T</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Trace infusion pump</td>
			<td>Zhejiang Smith Medical Instrument Co., Ltd</td>
			<td>20162540335</td>
			<td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Urodynamic measurement equipment</td>
			<td>Medical Measurement SystemsB.V.</td>
			<td>08-0467</td>
			<td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>HFK Biotechnology Co.Ltd,Beijing ,China</td>
			<td>SCXK2012-0023</td>
			<td>200-220g</td>
		</tr>
	</tbody>
</table>

detrusor underactivity model in rats by conus medullaris transection

Sunulan protokolün amacı, konus medullaris transeksiyonu yoluyla sıçanda bir detrusor underactivity (DU) modeli oluşturmaktır. 200-220 g ağırlığındaki toplam 40 dişi Wistar sıçanına (kontrol grubu: 10 sıçan; test grubu: 30 sıçan) laminektomi uygulandı ve test grubunda konus medullaris L4\u2012L5 düzeyinde transekte edildi. Tüm sıçanlar altı hafta boyunca aynı çevre koşulları altında barındırıldı ve beslendi. Test grubunda, altı hafta boyunca günde iki kez idrar işemesi yapıldı ve ortalama rezidüel idrar hacmi kaydedildi. Her iki gruba da sistometrogram yapıldı. Mesanenin maksimum sistometrik kapasitesi (MCC), detrusor açma basıncı (DOP) ve uyumu kaydedildi ve hesaplandı. Test grubu, ameliyat sonrasında, spinal şok sırasında ve sonrasında önemli idrar retansiyonu gösterdi. Bununla birlikte, kontrol grubunda herhangi bir anormallik gözlenmedi. Kontrol grubuyla karşılaştırıldığında, MCC ve test grubundaki mesanenin uyumu test grubundan anlamlı derecede yüksekti (3.24 ± 2.261 mL'ye karşı 1.04 ± 0.571 mL; 0.43 ± 0.578 mL / cmH 2 O karşı 0.032 ± 0.016 mL / cmH 2 O), test grubundaki DOP kontrolden daha düşüktü (20.28 ± 14.022 cmH 2 O'ya karşı 35 ± 13.258 cmH2 Conus medullaris transeksiyonu ile DU'nun hayvan modelini oluşturmanın bu yöntemi, DU'nun patofizyolojisini daha iyi anlamak için mükemmel bir fırsat sunmaktadır.

Conus medullaris transeksiyonunun tüm prosedürü deneyimli cerrahlar tarafından 45 dakika içinde tamamlanabilir. Laboratuvarımızda 100'ün üzerinde konus medullaris transeksiyon ameliyatı gerçekleştirilmiştir. Başarı oranı, sıçanların sağkalımı ve DU'nun başarılı indüksiyonu ile tanımlandığı gibi% 95'in üzerindedir. Ürodinamik test DU indüksiyonunu doğruladı.
Deneyimlerimize dayanarak, DU indüksiyonu rezidüel idrar hacmi ile önceden değerlendirilebilir. İd...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Bu yazıda sıçanlarda conus medullaris transeksiyonu ile detrusor az aktivite modeli oluşturmak için bir yöntem sunulmaktadır. Detrusor az aktivitesi bu hayvanlarda başarıyla uyarıldı. Model, idrar yolu fonksiyonunu incelemek için kullanılabilir.

Conus Medullaris Transeksiyonu ile Sıçanlarda Detrusor Düşük Aktivite Modeli

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K Görüntüleme.  Capital Medical University. Bu yazıda sıçanlarda conus medullaris transeksiyonu ile detrusor az aktivite modeli oluşturmak için bir yöntem sunulmaktadır. Detrusor az aktivitesi bu hayvanlarda başarıyla uyarıldı. Model, idrar yolu fonksiyonunu incelemek için kullanılabilir.

Video: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Optik Sinir transeksiyonu: Santral Sinir Sistemi Yetişkin Nöron Apoptoz Bir Model

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Nörobilim

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Memeli MSS Optik Sinir transeksiyonu sonra Deneysel İşlemler Yöntemleri

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Topikal Transgenik Rats nesil tarafından<em&gt; Utero</em&gt; Elektroporasyon ve<em&gt; In vivo</em&gt; Biyoparlaklık Eleme

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Motor Sinir transeksiyonu ve Canlı Zebra balığı Glial Hücre Davranışlarının Time-lapse Görüntüleme

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Larva Zebra balığı Omurilik Kesisi

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Sinir Devresi Temelini Bellek Oluşumunda Diseksiyon için Yenidoğan Sıçanlarda bir Lateralize Koku Öğrenme Modeli

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Kısmi optik sinir transeksiyon Sıçanlarda: ikincil dejenerasyonu retina Ganglion hücrelerinin değerlendirmek için yeni bir operatif yaklaşım ile kurulan bir Model

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Alt akut serebral Microhemorrhages sıçanlarda Lipopolysaccharide enjeksiyonla indüklenen

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...