Name: modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats
Uploaded: 2015-11-20T14:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Yazarlar Dan Firl, Chris Corbett ve Jesse Denson, doktora müteşekkiriz. Harçlar SR NIH NINDS R01 NS060765 tarafından sağlandı, LJ için P30 COBRE Pilot Programı ve New Mexico Üniversitesi&#39;nde LJ için Çocuk Sağlığı İmza Programı.

Kurumsal Bakım ve Kullanım Komiteleri hem Boston Çocuk Hastanesi&#39;nde ve New Mexico Sağlık Bilimleri Merkezi Üniversitesi, tüm deneysel prosedürleri onayladı. NOT: Ön işlem, mühür başlamadan, sterilize ve tüm cerrahi aletler ve cerrahi örtüler otoklav. Buna ek olarak,% 0.125 bipivucaine ve 0.1 mg / kg dahil olmak üzere, buprenorfin, steril şişeler içinde post-operatif ilaç hazırlanması. Ayrıca steril lipopolisakarit (LPS) bir çözelti hazırlamak: 0,04 mg / ml LPS (0111: B4) Evan mavisi seyreltik ihtiva eden steril tuzlu su içinde. 1. Anestezi <ol><li> % 3 izofluran dengeli bir% 70 azot ve% 30 oksijen karışımı ile embriyonik gün 18 (E18) hamile Sprague Dawley sıçan anestezi neden. </li><li> Indüksiyon odasından sıçan çıkarın ve 37 ° C&#39;de set dökümlü cerrahi dolaşan su battaniye sıçan sırtüstü yerleştirin. Burun konisi anestezi aktarın ve isoflura azaltmak% 2 ne düzeyde. </li><li> Yavaşça kornea kurumasını önlemek için her gözün oftalmik merhem uygulayın. İşlem sırasında sürekli sıcaklık, solunum hızı ve hayvan kalp atışlarının izlenmesi. Anne fizyolojisi prosedür boyunca sabit kalması gerekir. </li></ol> 2. Cerrahi Hazırlık ve Scrub <ol><li> Kullanımı küçük hayvan kesme alt karın bölgesindeki bütün tüylerden. Meme nicklemek veya canlı doğmuş yavruların gelecekteki hemşirelik için rahatsız edici olabilir jilet döküntü üreten önlemek için dikkatli bir dikdörtgen desen Tıraş. </li><li> Steril pamuklu çubukla povidon iyot ve% 70 etanol fırçalayın uygulama değiştirerek karın cildi hazırlayın. Povidon-iyot ve% 70 etanol her moda alternatif 3x uygulanan olacak şekilde fırçalayın tekrarlayın. Kurumaya bırakın. </li><li> Ayak-tutam refleksinin yokluğu yoluyla anestezi derinliği onaylayın. Ağrı refleksi uyaranların yokluğunda,% 1 izofluran seviyesini azaltır. </li><li> Steril cerrahi havlu kullanarak,hayvan asmak. Onlar rahim boynuzları kan akışını engellediği değil iken absorbe sulama sıvı miktarını maksimize şekilde uygun bir açıyla perdeler yerleştirmek için özen gösterin. </li></ol> 3. Abdominal Laparotomi <ol><li> Bir neşter kullanılarak hazırlanan karın derisinde 3 cm&#39;lik orta hat kesi yapmak. Açık açık makas ile karın fasya deri tabakasını incelemek. Forseps ve cerrahi makas kullanarak, karın fasya tabakası yükseltmek ve periton boşluğuna erişmek için avasküler linea alba bir kesi yapmak. </li><li> Kesi dış cerrahi gazlı bez yerleştirin ve steril tuzlu su ile nemlendirin. Karın künt forseps ve dış basınç kullanarak, yavaşça periton boşluğuna gelen rahim boynuzları kaldırmak ve nemlendirilmiş gazlı bez üzerine yerleştirin. </li><li> Dikkatle dolanması önlemek ve bağırsak ile temasa geçin. Fetusun bireysel amnion kesesi arasındaki tek kas dokusu ile temasa geçerek forseps kullanarak düzenleyin.Açığa ve künt diseksiyon ile 4 uterin arterler izole. NOT: Bakım rahim arterleri incelemek için alınmalıdır. Ortam doku ve damarları kendilerini son derece hassastır. Anne gemilere hasar kanamaya yol ve ciddi vakalarda, fetal ve maternal ölüm olabilir. </li></ol> Anevrizma Klipleri 4. Yerleştirme <ol><li> Her uterin arter üzerinde bir sıçan 30 G anevrizma klibi yerleştirin. Bireysel plasenta rahim dahil gemilerin proksimal ve distal bakliyat ve koyulaşması dahil kan akışının durması emin olun. Maruz boynuzları ve gazlı bez ile tüm cerrahi alanı kapsayacak ve steril tuzlu su ile yıkayın. Sulama yaklaşık her 10 dk ile nemli alan tutmaya özen gösterin. </li><li> 60 dakika sonra, gazlı bez kaldırmak ve alanını sulamak. Rahim boynuzları ve damarlar yeterince başarılı klip kaldırılması için ıslatılmış olduğundan emin olun. Yavaşça forseps kullanarak her anevrizma klibi kaldırın. Gemiye travma ve maint sebebiyet vermemek özen gösterinçıkarılması sırasında ain doku bütünlüğü. </li><li> İyice amnion kesesi gelen gazlı bez herhangi bir sokak Konuları kaldırmak için özen, rahim boynuzları ve alanını sulamak. </li></ol> Amniyon Sacs için Lipopolisakarit 5. Enjeksiyon <ol><li> Her amniyon kesesi dibinde, sadece plasental plaka ön amniyon sıvısına seyreltilmiş Evan mavi boya ile LPS (4 ug / kese) 100 ul enjekte edilir. Stabilize ve enjeksiyon için en uygun pozisyona her amniyotik kese döndürmek için künt forseps kullanın. Seyreltik Evan mavi boya uygun şırınga yerleştirme ve enjeksiyon teyit yararlı bir kontrast ajandır. NOT: intra-amniyotik enjeksiyonlar için ekli 8 mm 31 G iğne ile Yalnızca bir ultra-ince 0.3 ml insülin şırınga. Daha büyük göstergesi iğneler kullanılarak kronik amniyotik sıvı kaybı, fetal ölüm ve gebelik reabzorpsiyonunda neden olacaktır. Şırınga çıkartılması üzerine amniyotik sıvı sızıntı küçük miktarlarda mitig olabiliramniyon kesesi doğrudan basınç ile bir-. Bazı sıçan fetus oligohidramnios bir ölçüde tolere edebilir. Ancak, büyük göstergesi iğne veya kronik sıvı sızıntısı ile sonuçlanan kazayla ponksiyonu, fetal kayıp ve ciddi vakalarda sonuçları, komşu gebelik kaybı ile delinerek akut amniyotik sıvı kaybı. </li><li> Boynuzları steril serum fizyolojik ile 3x uterus yıkayın. </li></ol> 6. laparotomi Kapanış <ol><li> Forseps kullanarak, dikkatli periton boşluğuna rahim boynuzları geri dönün. Amnion kesesi ve orta hat kesi arasında yeterli boşluk sağlanması, çalışan 3-0 ipek sütür kullanılarak muskulofasyal katman kenarlarını yeniden yaklaşmaktadır. Kas kesi kapatırken amnion kesesi yerleştirme farkında olun. Içine veya kese ile sütür için değil dikkatli olun. </li><li> Cilt katmanı kapatarak, bir çalışan 3-0 ipek sütür kullanılarak, cilt tabakası yeniden yaklaşmaktadır. NOT: laparotomi izin devamlı sütür iki kat kapalı olmalıdırCilt ve gebelik artan kas genişleme. Sürekli dikişler eşit olarak dağıtılmış yara gerginliği için izin verir. Birden knot rahatsız edici ve kolayca anestezi kurtarma üzerine sıçan tarafından çiğnenmiş edilebilir Kesilen sütür az arzu edilir. Cerrahi zımba arzu edilmez. Cerrahi knot kuyrukları çok kısa (&lt;3 mm) kesilmelidir. </li><li> Bir 26 G iğne kullanılarak% 0.125 bupivakain deri altından yaklaşık yara kenarlarının 1 ml enjekte edilir. Ense de kg buprenorfin subkutan 0.1 mg tek doz / yönetmek. </li><li> Izofluran ve havlu kuru sıçan gerekirse kapatın. Temiz ev kafeste yerleştirin ve anestezi ayilmanizi. Hipotermik hale gelmez sıçan olun. </li></ol> 7. Ameliyat Sonrası Kurtarma ve Bakım <ol><li> Yavrular (yaklaşık E22 veya E23) doğarlar kadar günlük sonra 72 saat boyunca siçana her 8-12 saat Monitör ve. Buprenorfın q8-12 saat / 72 saat ek doz yönetmek veya IACUC tarafından belirlenen prn. </li><li> Ağrı, rahatsızlık, vajinal kanama ya da cerrahi sitesinden kanama belirtileri için sıçanları izleyin. Sütürler ve kesi kontrol edin ve sıçan çiğneme veya erken kendi sütür kaldırarak görmediğinden emin olmak için özen gösterin. Derece nadir olmasına rağmen, onların sütürler tehlikeye sıçanlar yara açılma riski altındadır. NOT: Bazen sıçanlar yatak ya da gıda dışı maddelerin aşırı miktarda yemek olabilir, buprenorfın yönetiminin bir yan etkisi olarak, pika denir. Çok nadir olmakla birlikte, sıçanlar pika ve potansiyel sonraki barsak tıkanıklığı için izlenmesi gerekir. </li></ol> 8. Doku İşleme ve Cryosectioning <ol><li> , Hematoksilen (H &amp; E) doğum sonrası beyin boyanması için hazırlık doğum sonrası gün 2 (P2) üzerine evlerine kafes yavrular kaldırmak için. Cerrahi makas hafifçe sıçan yavrular başını kesmek ve kullanma kafatası beyin kaldırmak. </li><li> Fosfat tamponlu tuzlu su içinde% 4 paraformaldehit 7 ml içeren 15 ml konik bir tüp içinde düzeltme beyin bırakın(PBS). 4 ° C &#39;de beyin yerleştirin ve 72 saat boyunca sabitlemek. </li><li> 72 saat sonra,% 30 sukroz ihtiva eden bir steril PBS çözeltisi beyinleri transferi (w / v) ve 4 ° C &#39;geri dönün. NOT: beyinleri sukroz çözeltisi damla kez onlar bir kriyostat üzerinde kesitli hazırdır. </li><li> Hızla beynini dondurma ve dondurulmuş koronal kesitlerin edinimi için kriyostat tokmak monte. 20 mikron dondurulmuş koronal bölümleri kesmek ve slaytlar monte. Emin olun bölümler seri toplanır. </li><li> Slaytlar gece boyunca oda sıcaklığında kurumaya bırakın. -20 ° C&#39;de de kayar. </li></ol> 9. Hematoksilen &amp; Eosin Boyama <ol><li> Oda sıcaklığına slayt monte donmuş bölümleri ve sıcak atın. NOT: Tüm çözümler taze hazırlayın. </li><li> 2 saat boyunca 50 ° C&#39;ye kadar bir slayt sıcak grubu yerleştirin kayar. </li><li> Transfer boyama raf kayar. Dip (GKD 2 O) çifte damıtılmış deiyonize su içinde 10x slaytlar. </li><li> 5 dakika boyunca% 100 hematoksilin slaytlar inkübe edin. Tihematoksilin beni mor boyama derecesine bağlı olarak optimize edilebilir. </li><li> Dip slaytlar musluk H 2 O 4x ve 1 dakika süreyle 2 O temiz musluk H bekletilir. </li><li> Dip (250 mi,% 70 etanol + 1 mi konsantre hidroklorik asit) asidi alkol içinde 15x kayar. </li><li> Dip slaytlar musluk H 2 O 4x ve 1 dakika süreyle 2 O temiz musluk H bekletilir. </li><li> 2 dakika için% 1 lityum karbonat inkübe edin. </li><li> Dip slaytlar musluk H 2 O 4x ve 1 dakika süreyle 2 O temiz musluk H bekletilir. </li><li> 1 dakika boyunca% 95 etanol içinde inkübe edin. </li><li> Dip slaytlar% 100 Eosin içinde 7x. Eosin batırma sayısı pembe boyama derecesine bağlı olarak modifiye edilebilir. </li><li> Dip slaytlar% 95 etanol içinde 5 kata. </li><li> % 95 etanol taze değişiklik Lamı 5x. </li><li> 1 dakika boyunca% 100 etanol içinde slaytlar inkübe edin. </li><li> 1 dakika boyunca% 100 etanol içinde taze değişiklik slaytlar inkübe edin. </li><li> 15 dakika% 100 ksilen slaytlar inkübe edin. NOT: Ksilen adımlar veBir çeker ocak içinde gerçekleşmelidir coverslipping. </li><li> 15 dakika boyunca ksilen taze değişiklikler slaytlar kuluçkaya yatmaktadır. </li><li> Permount ile lamel ve davlumbaz kurumaya bırakın. </li><li> Görüntü bir ışık mikroskobu ile kayar. </li></ol>

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N., 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=Arrested+oligodendrocyte+lineage+maturation+in+chronic+perinatal+white+matter+injury.">Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.</a> Ann Neurol. 63, 520-530 (2008).</li><li>Robinson, S., Li, Q., Dechant, A., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. 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=White+matter+injury+and+autistic+like+behavior+predominantly+affecting+male+rat+offspring+exposed+to+group+B+streptococcal+maternal+inflammation.">White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.</a> Devel Neurosci. 35, 504-515 (2013).</li><li>Uchida, K., 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=Effects+of+Ureaplasma+parvum+lipoprotein+multiple+banded+antigen+on+pregnancy+outcome+in+mice.">Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice.</a> J Reprod Immunol. 100, 118-127 .</li></ol>

Yazarlar ifşa hiçbir şey yok.

Prematüre Ensefalopati nedeniyle yaralanma mekanizmaları örtüşen etiyolojiler, nörogelişimsel zamanla, insan beyin ağ oluşumu karışıklık, karmaşık etkileşim hayvanlarda modellenmesi zordur ve MSS farklı fenotipleri insan erken doğmuş bebeklerde tezahür hakaretler. EOP hücre tipine özel güvenlik ile ilişkilidir (örneğin, olgunlaşmamış oligodendrositler) 21, hem de çok çeşitli gelişimsel olarak düzenlenmiş yolları (örneğin, Plaka yapısı, membran taşıy?...

37 hafta tahmini gebelik yaşı 1 önce Amerika Birleşik Devletleri&#39;nde doğan bebeklerin% 12&#39;nin üzerinde olan, prematürite perinatal beyin hasarı (PBI) kalıcı sakatlık önemli bir nedenidir. PBI prematürite, prematüre olarak da adlandırılır ensefalopati (EOP), tüm merkezi sinir sistemi (CNS) etkilemektedir. MSS yaralanması genellikle in utero başlar ve koriyoamniyonit ve hipoksi ve sepsis gibi doğum sonrası komplikasyonlar da dahil olmak üzere doğum öncesi süreçler tarafından şiddetlenir. Sistemik hakaret gelen PBI nöro-gelişimsel sürecini değiştirir ve duygusal regülasyonu, bellek ve yürütücü işlev 1,2 etkileyen serebral palsi, epilepsi, bilişsel gecikme ve çok sayıda nöropsikiyatrik bozukluklara yol açar. Çok ilerleme kaydedilmiş olmasına rağmen, sınırlı bir anlayış erken doğumdan itibaren MSS yaralanma hücresel ve moleküler sonuçları preterm doğan çocuklarda nörolojik sekel çokluk çevirmek nasıl kalır. Bilgi hind bu eksikliğiGerçek zamanlı MSS yaralanma şiddetinin tanı ve gelişmekte olan müdahalelerin haberdar dozaj ers. Ayrıca, bu savunmasız hasta popülasyonu için yaşa uygun tedavi stratejileri zor kalır. Rahim içi iltihabı, aşırı prematüre çok yaygın olan ve 3 karmaşık bir fetal-maternal-plasental inflamatuar dizisini içerir. Intrauterin enfeksiyon genellikle subklinik olduğunu. Akut inflamasyon veya histolojik koryoamniyonit ile uyumlu Belirli plasental bulgular, fetal enflamatuar yanıtın önemli belirleyicileri ve erken doğum 3-5 ile ilişkili beyin hasarı ile tesadüf. Nitekim, fetal inflamatuvar yanıt erken doğumdan itibaren uzun vadeli sonuçları için farklı klinik etkileri vardır. Gebelik yaşı (SGA) ya da kim enfeksiyon deneyim için küçük bebekler nörolojik defisit 3,4 ile son derece açıktır. Koryoamniyonit tipik patolojik tanı takip erken doğum olduğunu <sup&gt; 6,7 ve histolojik inceleme çok erken 4 doğan bebeklerin gelen plasenta% 70 iltihabın belirtileri ortaya koymaktadır. Dahası, korioamnionit iki yıl 8&#39;de bilişsel bozukluğu ile ilişkilidir. Son derece erken doğan bebeklerin plasentada maternal vasküler underperfusion kanıtı da çocukluk 9 serebral palsi ile ilişkilidir. Korioamnionit ve plasental perfüzyon defektlerinin sinerjik etkisi de yaş 10,11 iki yıl bu hasta popülasyonunda anormal nörolojik sonuçların oldukça yüksek risk gösterilmiştir. Patojen kaynaklı inflamasyon ile ilişkili insan sistemik plasental perfüzyon defektleri ve koryoamniyonit taklit etmek, sıçanlarda içi amniyotik lipopolisakarit (LPS) ile kombine prenatal geçici sistemik hipoksi-iskemi (TSHI) bir model geliştirdi. Amacımız, intrauterin iltihabı dahil yalnız sıçanlarda 12-16 TSHI bizim modeli adapte olduerken doğum ile ilgili CNS yaralanma preklinik modelleme kolaylaştırmak için. TSHI başına oligodendroglial soyu hücreleri, kortikal nöronlar kalıcı zarar ortaya koymuştur, prenatal beyin hasarı 16 ile uyumlu bir yaralanma kademeli kalıp giden aşamalı iskemik aralıklı, hücre ölümü artışı ve pro-enflamatuar sitokin seviyelerinin yüksek. Onlar 17-19 yaş olarak bu modelin iskemik bileşenlerine değişiklikler de sıçanlarda hafıza kodlama, kısa ve uzun süreli hafıza ve hafif kas-iskelet değişiklikler eksiklikler göstermiştir. Gerçekten de, daha önce TSHI + LPS kombinasyonu oligodendrosit ve nöronal kaybın, aksonal yaralanma, enflamasyon ve fonksiyonel hücre anormallikleri dahil olmak üzere, 20 EOP patofizyolojik işaretlerinden, tekrarlar olduğunu göstermiştir.

<table><tbody><tr><td>Saline Solution, 0.9%</td><td>Sigma</td><td>S8776</td><td></td></tr><tr><td>LPS 011B4</td><td>Sigma</td><td>L2630</td><td></td></tr><tr><td>Evan&#039;s Blue Dye</td><td>Sigma</td><td>E2129</td><td></td></tr><tr><td>Surgical gloves</td><td>Biogel</td><td>40870</td><td></td></tr><tr><td>OR Towels</td><td>Cardinal Health</td><td>287000-008</td><td>Sterile</td></tr><tr><td>PDI Alcohol Prep Pads</td><td>Fisherbrand</td><td>06-669-62</td><td></td></tr><tr><td>Mini Arco Rechargeable Clippers</td><td>Kent Scientific Corp.</td><td>CL8787</td><td></td></tr><tr><td>Betadine surgical scrub</td><td>Purdue Products L.P.</td><td>67618-151-17</td><td></td></tr><tr><td>Eye Lubricant</td><td>Refresh Lacri Lube</td><td>00023-0312</td><td></td></tr><tr><td>Blunt Forceps</td><td>Roboz</td><td>RS-8100</td><td></td></tr><tr><td>Scissors</td><td>Roboz</td><td>RS-6808</td><td></td></tr><tr><td>Surgical Scissors</td><td>Roboz</td><td>RS-5880</td><td></td></tr><tr><td>Surgical Scissors</td><td>F.S.T.</td><td>14002-16</td><td></td></tr><tr><td>Syringe</td><td>BD</td><td>309628</td><td>1 ml</td></tr><tr><td>Needle</td><td>BD</td><td>305122</td><td>25G 5/8</td></tr><tr><td>Needle</td><td>BD</td><td>305128</td><td>30G 1</td></tr><tr><td>Cotton-tipped Applicators</td><td>Fisherbrand</td><td>23-400-114</td><td>Small, 6 inch sterile</td></tr><tr><td>Cotton Gauze Sponge</td><td>Fisherbrand</td><td>22-362-178</td><td></td></tr><tr><td>Needle Holders</td><td>Kent Scientific Corp.</td><td>INS600109</td><td>12.5 CM STR</td></tr><tr><td>Vessel Clips</td><td>Kent Scientific Corp.</td><td>INS600120</td><td>30G Pressure</td></tr><tr><td>3-0 Perma Hand Silk Sutures</td><td>Ethicon</td><td>1684G</td><td>Black braided, 3-0 (2 metric), 18&quot;, non-absorbable,&amp;nbsp; PS-1 24 mm needle, 3/8 circle</td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>328438</td><td>0.3 cc 3 mm 31G</td></tr><tr><td>Pentobarbital</td><td></td><td></td><td></td></tr><tr><td>Buprenorphine</td><td></td><td></td><td></td></tr><tr><td>Bupivacaine</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Lithium Carbonate</td><td>Acros Chemicals</td><td>554-13-2</td><td></td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Hematoxylin</td><td>Leica</td><td>3801521</td><td>Surgipath Gill II Hematoxylin</td></tr><tr><td>Eosin</td><td>Leica</td><td>3801601</td><td>Surgipath Eosin</td></tr><tr><td>Xylenes</td><td>Fisherbrand</td><td>X3S-4</td><td>Histological Grade</td></tr><tr><td>Permount</td><td>Fisherbrand</td><td>SP15-100</td><td></td></tr><tr><td>Coverglass</td><td>Fisherbrand</td><td>12-548-5P</td><td>Fisher Finest Premium Coverglass</td></tr></tbody></table>

modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Prematüre Ensefalopati (EOP) en ileri öteleme hedefleri belirleyin. Erken doğum ile ilişkili anormallikleri merkezi sinir sistemini (MSS) kapsayan bir terimdir ve erken doğum ile ilişkili beyin hasarı için yeni tedavi stratejileri ortaya çıkarmak, ÇOP&#39;un preklinik modelleri benzer mekanizmalar içermelidir Prenatal küresel yaralanma insanlarda görülen ve anne-plasental-fetal sistemin birden fazla bileşen içerir. İdeal olarak, modeller, olgun hayvan fonksiyonel açıklarının benzer spektrumunu üretmek ve patofizyoloji birden yönlerini özetlemek gerekir. Erken erken doğum patojen kaynaklı iltihap ile bağlantılı insan plasental perfüzyon sistemik bozuklukları, plasental underperfusion ve / veya koryoamniyonit taklit etmek için, içi amniyotik lipopolisakarit (LPS) ile bir araya prenatal geçici sistemik hipoksi, iskemi (TSHI) içindeki bir model geliştirmiştir. Hamile Sprague Dawley sıçanlarda, TSHI uterin arter tıkanıklığı o yoluylan embriyonik gün 18 (E18) fetusta MSS hasarı artan ilişkili bir kademeli plasental underperfusion kusur neden olur. Intra-amniyotik LPS enjeksiyonu ile kombine edildiğinde, plasental inflamasyon artar ve MSS hasar ilişkili beyaz cevher, yürüme ve manyetik rezonans görüntüleme bulguları ile karmaşıklaşır. Prenatal TSHI ve TSHI + LPS prenatal hakaret nöronlar, oligodendrositler ve akson, Plaka yapısı kaybı ve son derece doğan çocuklarda gözlenen taklit yetişkin hayvanlarda fonksiyonel açıklarının kaybına neden intrauterin hakaret recapitulating içeren bir ÇOP&#39;un modeli kriterlerine çeşitli karşılamak erken. Ayrıca, bu model farklı yaralanma türlerine neden olduğu inflamasyonun diseksiyon sağlar.

E18 de TSHI + LPS&#39;yi ardından, hematoksilen ve eozin boyama önemli histopatolojik plasenta hem anormallikler (Şekil 1) ve beyinde (Şekil 2) ortaya koymaktadır. E19 ve E21 üzerine incelenen plasenta desidua ve labirent boyunca mikro kanama ve nekroz ile fena halde ödemli bulunmaktadır. Önemli inflamatuar infiltrasyon ve artan damarlanma da görülmektedir. P2 incelenen beyinleri ventrikülomegali ortaya yanı sıra beyaz cevher ve Plaka yapısı nöron kaybı Shams göre. Da...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Prematüre Modelleme Ensefalopati Sıçanlarda İçi amniyotik Lipopolisakkarid ile Prenatal Hipoksi-iskemi kullanma

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Research

JoVE Journal

Medicine

11.3K Görüntüleme.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Video: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Periventriküler lökomalazi Fare Modelleri

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Nörobilim

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Perinatal İskemi Hipoksik İskemik Ensefalopati Modeli

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biyoloji

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Yenidoğan Hipoksik-İskemik Ensefalopati A Piglet Modeli

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Tıp

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Yenidoğan hipoksik-iskemik beyin hasarı, fare modeli neurobehavioral değerlendirmesi

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Microcoil stenoz ile hafif intrauterin hipoperfüzyon fare modeli

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

İnflamasyon duyarlıgeç Preterm Hipoksik-iskemik Beyin Hasarı Bir Gelincik Modeli

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

İntraventriküler Hemoglobin Enjeksiyonu ile Yenidoğan İntraventriküler Kanamasının Modellenmesi

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Sıçanlarda Prematüre Posthemorajik Hidrosefalisinin Modellenmesi

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://app.jove.com/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

Lipopolisakkarit Kullanılarak Bir Sıçan Modelinde Serebral Mikrokanamaların İndüksiyonu

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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