Yazarlar, hem Kuzey Amerika Pediatrik Ortopedi Derneği Biomet Omurga Hibesi hem de Ulusal Sağlık Enstitüleri Klinik ve Translasyonel Bilim Enstitüsü KL2 Hibesi ve HH Lee Cerrahi Araştırma Hibesi'nin bu deneyler için ana finansman kaynakları olarak alındığını kabul etmek istemektedir.

Tüm hayvanlar, Hayvan Refahı Yasası (AWA), 1996 Laboratuvar Hayvanlarının Bakımı ve Kullanımı Kılavuzu, Laboratuvar Hayvanlarının İnsani Bakımı ve Kullanımı için PHS Politikası ve ayrıca Hayvan Bakımı ve Kullanımı Eğitim Kılavuzunda belirtilen kurumun politika ve prosedürlerinde belirtilen federal düzenlemelerde tanımlanan iyi hayvan uygulamalarına sıkı sıkıya bağlı olarak ele alınmıştır. ve tüm hayvan çalışmaları, California Üniversitesi Los Angeles Şansölyesi'nin Hayvan Araştırma Komitesi (ARC) tarafından onaylandı.
1. S. aureus biyolüminesan gerinim seçimi
<ol><li>İlgilenilen aşı olarak biyolüminesan S. aureus suşu Xen36'yı kullanın. NOT: Bu suş, septik bir hastadan alınan klinik bir izolat olan ebeveyn suşu S. aureus ATCC-49525'ten türetilmiştir. S. aureus ( S. aureus) Xen36, optimize edilmiş ve ana bilgisayarın doğal plazmidine entegre edilmiş bir luxABCDE operonunu benzersiz bir şekilde kullanır. 19 Sonuç olarak, Xen36 suşu, 490 nm'lik bir tepe dalga boyu emisyonuna sahip mavi-yeşil bir biyolüminesan ışık üretebilir. Bu emisyon sinyali sadece metabolik olarak aktif olan canlı bakteriyel organizmalar tarafından üretilir.</li>
</ol>2. S'nin Hazırlanması. Aşılama için Aureus
<ol><li>S. aureus Xen36'yı potansiyel kirleticilerden izole etmek için Luria Broth'a 200 μg/mL kanamisin artı %1.5 agar ekleyin, lux operon19'a bağlı kanamisin direnç genini kullanın.</li>
	<li>S. aureus Xen36 bakterilerini triptik soya agar plakalarına (triptik soya suyu [TSB] artı %1.5 agar) yerleştirin ve 37 °C'de 12-16 saat inkübe edin.</li>
	<li>S. aureus Xen36'nın tek kolonilerini izole edin ve TSB'de 12-16 saat boyunca 37 ° C'de çalkalayan bir inkübatörde (200 rpm) ayrı ayrı kültürleyin.</li>
	<li>Elde edilen kültürü 1:50 oranında seyreltin.</li>
	<li>Midlogaritmik faz bakterilerini izole etmek için 37 ° C'de 2 saat daha kültür.</li>
	<li>Bakterileri fosfat tamponlu salin (PBS) içinde üç kez peletleyin, yeniden süspanse edin ve yıkayın.</li>
	<li>Absorbansı 600 nm'de ölçün. İdeal OD600, 0.700 ile 0.750 arasındadır ve bu da 4x105 CFU/mL'ye karşılık gelir. İstenilen bakteriyel aşılamayı (1 x 103 CFU / 2 μL) elde etmek için seri seyreltmeler gerçekleştirin. NOT: Kronik bir enfeksiyonun kurulması için optimal Xen36 konsantrasyonunun 1 x 103 CFU olduğu bulunmuştur. Daha düşük bakteri dozu, konakçı bağışıklık sistemi tarafından temizlendi ve daha yüksek dozlama, yara parçalanmasına neden oldu. Yara parçalanması, derin implant enfeksiyonu ile yüzeysel yara enfeksiyonu arasında ayrım yapmaz ve bu nedenle bu modelde önlenir (Şekil 1)20.</li>
</ol>3. Fareler
<ol><li>12 haftalık erkek C57BL / 6J vahşi tip fareler kullanın.</li>
	<li>Bir seferde en fazla 4 tane olan kafeslerdeki ev fareleri.</li>
	<li>Suyu her zaman hazır bulundurun. 12 saatlik bir aydınlık/karanlık döngüsü sürdürün ve döngünün karanlık evresinde deney yapmayın.</li>
	<li>Floresan sinyalizasyonu ile potansiyel parazit nedeniyle besleme için yonca içermeyen yem kullanın.</li>
	<li>Deneyin tamamı boyunca hayvanların refahını sağlamak için araştırma veya veteriner personelinin fareleri günlük olarak değerlendirmesini sağlayın.</li>
</ol>4. Fare cerrahi prosedürleri
<ol><li>Fareleri yaklaşık 5 dakika boyunca bir izofluran (% 2) odasına yerleştirerek anesteziyi indükleyin. Solunumun uyanık olduğundan daha ritmik ve daha yavaş kalmasını ve zararlı uyaranlara yanıt olarak değişmemesini izleyerek uygun anestezi derinliğini onaylayın (ör., cerrahi manipülasyon, ayak parmağı sıkışması).</li>
	<li>Anestezi uygulanmış fareleri bir hazırlık istasyonuna aktarın ve kemirgen makası ile sakrumdan üst torasik omurgaya kadar tüyleri alın.</li>
	<li>Cildi alternatif betadin çözeltisi ve izopropil alkolün üçlü yıkamasıyla temizleyin ve sterilize edin.</li>
	<li>Yüzüstü pozisyonda anestezi uygulanmış ve sterilize edilmiş fareleri steril bir cerrahi yatağa aktarın, burun konisi yoluyla inhale izofluran (% 2) uygulanarak anesteziyi koruyun.</li>
	<li>Kalçaları maksimum düzeyde esnetin ve lomber 4 vertebral gövdeye yaklaşmak için dizin omurga seviyesindeki konumunu belirleyin.</li>
	<li>15 bıçaklı cerrahi neşter ile deriden uzunlamasına 2 cm'lik bir kesi yapın.</li>
	<li>Orta hattı doğrulamak için dikenli süreçleri palpe edin ve insizyonu kemiğe kadar devam ettirin.</li>
	<li>L4 spinöz işleminin sağ tarafında subperiostal olarak diseksiyon yapın ve enine sürece lateral olarak uzanın.</li>
	<li>5-0 sefalad ve kaudad boyutunda, emilebilir örgülü bir sütürü fasyadan L4 gövdesine geçirin ve gelecekteki kapanmaya hazırlık olarak açık bırakın.</li>
	<li>25 G'lik bir spinal iğne kullanarak, 25 G'lik bir spinal iğne kullanarak L4'ün dikenli işlemini raybalayın ve uzun kol döşeme sefaladı ile lamina boyunca 0,1 mm çapında, 1 cm uzunluğunda "L-şekilli" cerrahi sınıf paslanmaz çelik bir implant yerleştirin.</li>
	<li>İmplantı 1 x 103 CFU/2 μL biyolüminesan S. aureus Xen36 ile aşılayın ve tüm solüsyonun implanta temas etmesine dikkat edin.</li>
	<li>İmplant üzerinde aşı tutulmasını sağlamak için aşılamadan sonra daha önce geçirilen emilebilir sütürü bağlamadan önce yaklaşık 10 saniye bekleyin.</li>
	<li>Emilebilir sütür ile cildi koşu tarzında kapatın.</li>
	<li>Ağrı kesici ilaçları deri altı buprenorfin (0.1 mg / kg) enjeksiyonu yoluyla hemen postop ve daha sonra 3 gün boyunca her 12 saatte bir uygulayın.</li>
	<li>Bir ısıtma yastığındaki fareleri kurtarın ve normal aktiviteye dönüş için izleyin.</li>
	<li>İmplantın uygun yerleşimini doğrulamak için postoperatif radyografiler alın.</li>
</ol>5. Bakteri yükünü ölçmek için uzunlamasına in vivo biyolüminesans görüntüleme
<ol><li>Fareleri inhale izofluran (% 2) ile uyuşturun. Solunumun uyanık olduğundan daha ritmik ve daha yavaş kalmasını ve zararlı uyaranlara yanıt olarak değişmemesini izleyerek uygun anestezi derinliğini onaylayın (ör., cerrahi manipülasyon, ayak parmağı sıkışması).</li>
	<li>Kemirgen makası ile sakrumdan üst torasik omurgaya kadar kılları alın.</li>
	<li>İn vivo biyolüminesans görüntüleme (BLI)19 gerçekleştirmek için fareleri biyolüminesans görüntüleme platformunun görüş alanına yükleyin.</li>
	<li>Biyolüminesan sinyali 5 dakikalık bir çekim süresi boyunca yakalayın. 15 cm'lik görüş alanına (B) sahip büyük gruplama ayarlarını kullanın.</li>
	<li>Bakteri yükünü izlemek için postoperatif 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 ve 35. günlerde (veya belirli deneysel tasarıma dayalı diğer günlerde) 5.1-5.4 adımlarını tekrarlayın.</li>
	<li>BLI verilerini renk skalası aracılığıyla sunun ve gri tonlamalı bir fotoğrafın üzerine bindirin. BLI'yi toplam akı (saniyedeki fotonlar) veya ortalama maksimum akı (fotonlar/saniye/santimetre2/steradyan) cinsinden ölçmek için BLI yazılımını kullanarak standart bir oval ilgi bölgesini (ROI) izole edin.</li>
</ol>6. İmplantlara ve çevresindeki dokuya yapışan bakterileri ölçün
<ol><li>Fareleri POD 35'te ötenazi yapın veya AVMA Yönergelerine uygun olarak karbondioksite maruz kalma ile alternatif bir ameliyat sonrası tercih tarihi. İkincil servikal çıkık ile ötenaziyi onaylayın.</li>
	<li>Sırt derisini Adım 4.3'e göre sterilize edin ve fareyi steril bir cerrahi alana yüzüstü yerleştirin.</li>
	<li>15 bıçaklı bir cerrahi neşter kullanarak önceki kesiği keskin bir şekilde kesin.</li>
	<li>L4 spinöz sürecini kör bir şekilde incelemek ve cerrahi implantı tanımlamak için steril makas kullanın.</li>
	<li>L4 dikenli işlemde implantı nazikçe bükmek ve konumundan çıkarmak için bir iğne ucu kullanın.</li>
	<li>Steril forseps ve makas kullanarak, cerrahi implantı hemen çevreleyen yaklaşık 0.1 g dikenli işlem kemiği ve yumuşak dokuyu toplayın ve 4 keskin homojenleştirici boncuklu küçük konik gergedan tüpüne 1 mL PBS'ye yerleştirin.</li>
	<li>Hasattan önce ve sonra konik boruyu tartarak yumuşak dokunun ağırlığını kaydedin.</li>
	<li>İmplantı TSB'de 0.5 mL% 0.3 Tween-80'e yerleştirin ve 15 dakika boyunca sonikasyon yapın.</li>
	<li>Elde edilen implant süspansiyonunu 2 dakika vorteksleyin ve gece boyunca 12-16 saat boyunca kültürleyin.</li>
	<li>Daha önce implantı çevreleyen 1 ml PBS'ye yerleştirilmiş yumuşak doku ve spinöz işlemleri homojenizatör kullanarak homojenize edin.</li>
	<li>Elde edilen yumuşak doku süspansiyonunu 5 dakika boyunca vorteksleyin ve gece boyunca 12-16 saat boyunca kültürleyin.</li>
	<li>Gece kültüründen sonra, sırasıyla implanttan ve çevresindeki dokulardan CFU sayın. Yumuşak doku için hasat edilen toplam CFU / g ve sonikasyonlu implant için CFU / mL olarak ifade edilen değer.</li>
</ol>

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Yazarların ifşa edecek herhangi bir çıkar çatışması yoktur.

Omurgadaki implantla ilişkili enfeksiyonlar, 1,2,3,4,5 hastaları için kötü sonuçlara işaret eder. Vücuttaki diğer birçok bölgeden farklı olarak, omurgadaki enfekte donanım, instabilite ve nörolojik uzlaşma riski nedeniyle sıklıkla çıkarılamaz. Sistemik antibiyotik tedavisine dirençli biyofilm bakterileri ortamındaki bu benzersiz zorluk, t...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

Bu yöntemin amacı, spinal implant enfeksiyonunun (SII) yeni bir fare modelini tanımlamaktır. Bu model, konakçı, patojen ve/veya implant değişkenlerinin etkisini in vivo olarak esnek bir şekilde değerlendirmek için ucuz ve doğru bir araç sağlamak üzere tasarlanmıştır. Bu modelde spinal implant enfeksiyonları için potansiyel terapötiklerin ve tedavi stratejilerinin test edilmesi, daha büyük hayvan modellerinde ve klinik çalışmalarda uygulamadan önce araştırma geliştirmeye rehberlik etmeyi amaçlamaktadır.
Omurga cerrahisi sonrası implanta bağlı enfeksiyon yıkıcı bir komplikasyondur ve ne yazık ki elektif omurga cerrahisi geçiren hastaların yaklaşık %3-8'inde görülür 1,2,3,4,5 ve çok seviyeli veya revizyon cerrahisi geçiren hastaların %65'ine kadarı 6. Spinal implant enfeksiyonlarının tedavisi genellikle birden fazla hastaneye yatış, birden fazla ameliyat ve uzun süreli antibiyotik tedavisi gerektirir. SII'ler, nörolojik uzlaşma, sakatlık ve artmış mortalite riski dahil olmak üzere kötü hasta sonuçlarına işaret eder. SII'nin yönetimi son derece pahalıdır ve hasta başına 900.000 dolardan fazlaya mal olur7.
Staphylococcus aureus, SII 8,9,10,11'in en yaygın virülan patojenidir. Bakteriler donanımı doğrudan ameliyat sırasında, ameliyat sonrası dönemde yaradan veya daha sonra hematojen yayılma yoluyla tohumlayabilir. Metal implantların varlığında, S. aureus, bakterileri antibiyotik tedavisinden ve bağışıklık hücrelerinden koruyan biyofilm oluşturur. Enfekte olmuş donanımın çıkarılması bir enfeksiyonun etkili bir şekilde ortadan kaldırılmasına yardımcı olabilirken, bu genellikle omurgada istikrarsızlığa neden olmadan ve nörolojik uzlaşma riski taşımadanmümkün değildir 12.
Enfekte olmuş donanımın ekstrüktüre edilmemesi durumunda, SII'yi önlemek, tespit etmek ve tedavi etmek için yeni yaklaşımlara ihtiyaç vardır. Tarihsel olarak, yeni tedavilerin güvenliğini ve etkinliğini etkin bir şekilde değerlendirmek için SII'nin sınırlı hayvan modelleri olmuştur. SII'nin önceki hayvan modelleri, çok sayıda hayvan ve koloni sayımı, histoloji ve kültür dahil olmak üzere ötenazi gerektiren veri noktalarının toplanmasını gerektirir13,14,15. Uzunlamasına in vivo izlemeden yoksun olan bu modeller, hayvan başına yalnızca bir veri noktası sağlar ve bu nedenle pahalı ve verimsizdir.
Diz artroplastisi enfeksiyonunun bir fare modelini inceleyen önceki çalışma, enfeksiyon yükünü uzunlamasına izlemek için noninvaziv in vivo optik görüntülemenin değerini ve doğruluğunu ortaya koymuştur16. Biyolüminesansın tespiti, bakteri yükünün tek bir hayvanda uzunlamasına bir zaman boyunca insanca, doğru ve verimli bir şekilde ölçülmesini sağlar. Ayrıca, önceki çalışmalar, in vivo biyolüminesans ile implantlara bağlı CFU'lar arasında yüksek bir korelasyon olduğunu göstermiştir17. Zaman içinde enfeksiyonu takip etme kapasitesi, implantla ilişkili enfeksiyonun daha incelikli bir şekilde anlaşılmasına yol açmıştır. Ek olarak, boylamsal enfeksiyonun bu şekilde izlenmesi, antibiyotik tedavisinin ve yeni antimikrobiyallerin etkinliğinin doğru bir şekilde değerlendirilmesine olanak sağlamıştır16,17,18.
Bu araçlardan yararlanarak, postoperatif spinal implant enfeksiyonu modelini geliştirdik ve doğruladık. Sunulan yöntemde, bakteri yükünü uzunlamasına izlemek için SII'nin in vivo fare modelini oluşturmak için bir biyolüminesan S. aureus Xen36 aşısı kullanıyoruz16,17,18. Bu yeni model, daha büyük hayvan modellerinde ve klinik çalışmalarda uygulanmadan önce SII için potansiyel tespit, önleme ve tedavi stratejilerini verimli bir şekilde test etmek için değerli bir araç sağlar.

<table>
	<tbody>
		<tr>
			<td>Analytical Balance ME104</td>
			<td>Mettler Toledo</td>
			<td>30029067</td>
			<td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td>
		</tr>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD 211825</td>
			<td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td>
		</tr>
		<tr>
			<td>Biomate 3S UV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208300</td>
			<td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td>
		</tr>
		<tr>
			<td>Bioshield 720+ swinging bucket rotor</td>
			<td>Thermo Scientific</td>
			<td>75003183</td>
			<td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td>
		</tr>
		<tr>
			<td>Branson Ultrasonics 2510R-MTH (Sonicator)</td>
			<td>Branson Ultrasonics</td>
			<td>CPX952217R</td>
			<td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td>
		</tr>
		<tr>
			<td>Bullet Blender Storm Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&#8482; minimizes sample heat up. Uses 1.5ml screw-cap RINO&#174; tubes or snap-cap Eppendorf&#174; Safe-lock&#8482; tubes.</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Electron Microscopy Sciences</td>
			<td>66118-10</td>
			<td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be 
			used exclusively for research purposes. The Germinator 500 should not be used as a substitute for 
			traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine 
			sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been 
			designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026282</td>
			<td>Single 150L</td>
		</tr>
		<tr>
			<td>IVIS Lumina X5 Imaging System</td>
			<td>Perkin Elmer</td>
			<td>CLS148590</td>
			<td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&#8212;and answers&#8212;on anatomical and molecular aspects of disease.</td>
		</tr>
		<tr>
			<td>MAXQ 4450 Digtial Incubating Bench Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE4450</td>
			<td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline</td>
			<td>Fisher Bioreagents</td>
			<td>BP24384</td>
			<td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td>
		</tr>
		<tr>
			<td>SORVALL LEGEND X1R 120V Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004261</td>
			<td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus - Xen36</td>
			<td>Perkin Elmer</td>
			<td>119243</td>
			<td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td>
		</tr>
		<tr>
			<td>TUTTNAUER AUTOCLAVE 2540E 120V</td>
			<td>Heidolph Tuttnauer</td>
			<td>23210401</td>
			<td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Fisher Bioreagents</td>
			<td>BP338-500</td>
			<td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td>
		</tr>
		<tr>
			<td>Vortex mixer VX-200</td>
			<td>Labnet Internation</td>
			<td>S0200</td>
			<td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Pfizer Injectables/Hospira</td>
			<td>00409-4888-10</td>
			<td>0.9% Sodium Chloride Injection, USP</td>
		</tr>
	</tbody>
</table>

in vivo mouse model of spinal implant infection

Omurga implantı enfeksiyonları, tanının zor olması ve cerrahi eradikasyonun mekanik spinal stabilite ile çelişmesi nedeniyle kötü sonuçlara işaret eder. Bu yöntemin amacı, spinal implant enfeksiyonları için potansiyel terapötikleri ve tedavi stratejilerini test etmek için ucuz, hızlı ve doğru bir in vivo araç sağlamak üzere oluşturulmuş yeni bir spinal implant enfeksiyonu (SII) fare modelini tanımlamaktır.
Bu yöntemde, paslanmaz çelik bir k-telinin 12 haftalık C57BL/6J vahşi tip farelerin L4 spinöz sürecine transfikse edildiği ve 1 x 103 CFU biyolüminesan bir Staphylococcus aureus Xen36 bakteri suşu ile aşılandığı bir posterior yaklaşım omurga cerrahisi modeli sunuyoruz. Fareler daha sonra ameliyat sonrası 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 ve 35. günlerde in vivo biyolüminesans için uzunlamasına görüntülenir. Standartlaştırılmış bir görüş alanından alınan biyolüminesans görüntüleme (BLI) sinyalleri, in vivo bakteri yükünü ölçmek için ölçülür.
İmplantlara ve implant çevresi dokuya yapışan bakterileri ölçmek için farelere ötenazi yapılır ve implant ve çevresindeki yumuşak doku hasat edilir. Bakteriler sonikasyon ile implanttan ayrılır, gece boyunca kültürlenir ve daha sonra koloni oluşturan birimler (CFU'lar) sayılır. Bu yöntemden elde edilen sonuçlar, in vivo S. aureus biyolüminesans (ortalama maksimum akı) ve ötenazi sonrası CFU sayıları ile ölçülen uzunlamasına bakteri sayımlarını içerir.
Aletli omurga enfeksiyonunun önceki hayvan modelleri invaziv, ex vivo doku analizini içerirken, bu yazıda sunulan SII'nin fare modeli, statik doku çalışmasının yerini almak için biyolüminesan bakterilerin invaziv olmayan, gerçek zamanlı in vivo optik görüntülemesinden yararlanır. Modelin uygulamaları geniştir ve alternatif biyolüminesan bakteri suşlarının kullanılmasını, konakçı bağışıklık tepkisini eşzamanlı olarak incelemek için diğer genetik olarak tasarlanmış fare türlerini dahil etmeyi ve antibiyotikler veya implant kaplamaları gibi mevcut veya yeni tanı ve tedavi yöntemlerini değerlendirmeyi içerebilir.

Burada sunulan prosedür, SII'nin in vivo fare modelinde antibiyotik rejimlerinin etkinliğini değerlendirmek için kullanıldı. Spesifik olarak, vankomisin ve rifampin antibiyotik kombinasyonunun etkinliği, vankomisin monoterapisi ve tedavi edilmemiş enfekte kontrollerle karşılaştırıldı.
Ameliyattan önce, fareler kombinasyon tedavisine, monoterapiye veya enfekte kontrole randomize edildi. Örneklem büyüklüğünü hesaplamak için istatistiksel güç analizi yapılmıştır. Örn...

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

Protokol, paslanmaz çelik bir k-tel implantının biyolüminesan Staphylococcus aureus Xen36 ile enfekte olduğu yeni bir in vivo spinal implant enfeksiyonu fare modelini açıklar. Bakteri yükü biyolüminesan görüntüleme ile uzunlamasına izlenir ve ötenazi sonrası koloni oluşturan birim sayıları ile doğrulanır.

Spinal İmplant Enfeksiyonunun İn Vivo Fare Modeli

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

in-vivo-mouse-model-of-spinal-implant-infection

Research

JoVE Journal

Medicine

2.2K Görüntüleme.  University of California Los Angeles. Protokol, paslanmaz çelik bir k-tel implantının biyolüminesan Staphylococcus aureus Xen36 ile enfekte olduğu yeni bir in vivo spinal implant enfeksiyonu fare modelini açıklar. Bakteri yükü biyolüminesan görüntüleme ile uzunlamasına izlenir ve ötenazi sonrası koloni oluşturan birim sayıları ile doğrulanır.

Video: In Vivo Mouse Model of Spinal Implant Infection

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Vivo Enfeksiyon Oncolytic Virüsler Canlı Doku

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Tıp

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Roman kullanarak Spinal Kord Yaralanması sonra Fare Hind Ekstremite Kemik Kaybı Boyuna değerlendirilmesi, In vivo, Metodolojisi

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrofik Lateral Skleroz için Fare Modeli (ALS) Klinik Test ve Omurilik Kaldırma

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Sıçanlarda Şiddetli Spinal Kord Yaralanması bir bere modeli

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombine<em&gt; In vivo</em&gt; Optik ve uCT Görüntüleme Farelerde bir Ortopedik İmplant Enfeksiyon Enfeksiyon, Enflamasyon ve Kemik Anatomisi Monitör

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Travmatik Beyin Hasarı için Kontrollü Kortikal Etki Modeli

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Rahim içi Telemetri Fare Kontraktil Basıncını Ölçmek<em&gt; İn Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Bir Yenidoğan Fare Omurilik Sıkıştırma Yaralanması Modeli

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

Insanlaşmış Fare Modelinde HIV enfeksiyonuna karşı kök hücre Tabanlı Engineered Bağışıklık

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

İnsan Tümör ksenogratflarının gelen Fare Hücrelerinin tükenmesi Anlamlı Hedef Hücre Alt Analizi geliştirir

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...