Bu çalışmada Dr. Bai-Chuang Shyu (MGK 99-2320-B-001-016-My3, MGK 100-2311-B-001-003-My3 ve MGK 102-2320- Ulusal Bilim Konseyi hibe ile desteklenmiştir B-001-026-My3). Bu çalışma Academia Sinica fon aldı Biyomedikal Bilimler, Enstitüsünde yürütülmüştür.

Bu çalışmada protokol Tayvan Academia Sinica Kurumsal Hayvan Bakım onay ve Kullanımı Komitesi aldı. 1. Hayvan Hazırlıklar <ol><li> (- 400 gr yaklaşık 300) erkek Sprague-Dawley sıçanları edinin. yiyecek ve suya serbest erişimi (8:00 ışıklar), 12 saat / 12 saat aydınlık / karanlık döngüsü altında - (22 ° C,% 50 nem 21) klimalı bir odada sıçan koruyun. </li></ol> 2. Deneysel Prosedür <ol><li> Bütün sıçan deney önce 1 hafta boyunca kendi kafeslerine ortama uyum sağlar. adaptasyon sırasında, taban kurmak von Frey ve plantar testleri gerçekleştirmek. </li><li> von Frey testi <ol><li> yerleşim 30 dakika boyunca bir akrilik muhafaza (30 cm x 30 cm x 80 cm) sıçan yerleştirin. </li><li> (. Hayır filamanın 11 - 20) von Frey filamentleri elde aynı uzunluğa sahip, ancak değişen çaplarda bir dizi sağlamak için100 g 2 - güçlerinin. </li><li> hayvanın pençesinin cinsinden pençe çekme eşiğini belirlemek için, 5 dakika intertrial aralıklarla ile akrilik plaka üzerinde ağ benzeri bir bağlantı noktası üzerinden pençelerin merkezi uyarma von Frey filamentleri kullanın. Maksimum uygulanan basınç kaydedilir kadar, düşükten yükseğe, küçükten büyüğe iplikçikler kullanmak, maksimum uygulanan basıncı kaydetmek için. 10 </li><li> Sıçanlar stimülasyon bir pençe çekme yanıtı sergilemek zaman filament numarasını kaydedin. en düşük uyaran göre bu denemede çekilme yanıtı belirleyin. </li><li> Aynı sıçan arkaya üç kez olmak üzere toplam, hemen aynı işlemi tekrarlayın. (Gram olarak) karşılık gelen yürürlüğe iplik numarasını dönüştürmek ve değerlerinin ortalamasını alın. </li></ol></li><li> plantar test <ol><li> yerleşim 30 dakika boyunca (dört kare, 80 cm x 30 cm x 15 cm ayrılmıştır) saydam pleksiglas kutuya sıçan yerleştirin. </li><li> Bir inf kullanındefa rar ile sıkıştırılmış çubuk, bir cam levha ile arka ayağın merkezi uyarmak için. yaklaşık 10 saniye ortalama pençe çekme yanıtı gecikme elde etmek için kızılötesi ışık yoğunluğunu ayarlayın. kızılötesi ışık kaynağı yanar ve dijital solid-state zamanlayıcı başlatır bir tuşa basarak bir deneme yapmak. kızılötesi ışık demetinin süresini işleyin. </li><li> sıçanlar bir pençe geri çekme yanıtı arzeden kızılötesi ışık süresini kaydedin. En uzun süre doku hasarını önlemek için her denemede 20 saniye geçmemelidir. birbirini takip eden uyarımı engellemek için en az 5 dakika bir intertrial aralığını kullanın. </li><li> Sol ve sağ pençelerin üç çalışmalar ile testi tekrarlamak ve her sıçan için her hindpaw için ortalama hesaplamak. </li></ol></li><li> Kollajenaz Lezyon Cerrahisi <ol><li> Cerrahi uyaranlara oluştuğu ayak-tutam yanıt kaybı ve somatik tepkiler kadar% 4 izofluran ile sıçan anestezisi. % 2 isoflur - 1.5 anestezi korumakAmeliyat süresince ane. </li><li> 37.5 ° C - 36.5 vücut ısısını korumak için basit bir ısıtma yastığı ile bir stereotaksik cihazda sıçan yerleştirin. anestezi altında iken kuruluğunu önlemek için gözleri göz kremi uygulayın. </li><li> sterilize elektrik kesme makineleri ile kürk Tıraş ve kafa derisi orta hat boyunca bir neşter ile düzgün bir kesi (yaklaşık 2-2.5 cm) yapmak. providence iyot ve dezenfeksiyonu için% 75 alkol solüsyonu alternatif ile deri ve kafatası temizleyin. Cerrahi aşamasında, tüm cihazlar, aletler sterilize etmek% 75 alkol kullanımı ve tezgah steril koşulları sağlamak için. Bundan önce, tüm cerrahi malzemelerin buhar otoklavda sterilize edilmelidir. </li><li> (Talamus (ventral posteromedial talamik nükleus [VPM] ve ventral posterolateral talamik çekirdek [VPL] dahil) VB üzerinde elektrikli matkap ile kafatasında 3.0 küçük bir delik (3 mm çapında), steril eldiven ve aletler kullanın ve matkap -3.5 mm arka, 3,0-3,5 mm yanal) Bregma ve kafatası yüzeyi 5,5-5,8 mm derinlik için. </li><li> Microinject serum fizyolojik 0.5 ul veya sırasıyla deney ve kontrol gruplarında tip 4 kollajenaz çözüm 0.125 U. </li><li> İlaç dağılımı için izin vermek için ek 5 dakika boyunca bir yerde bir iğne tutun. </li><li> Kafatasında boşluğu doldurmak için diş çimento kullanın ve kesi sütür. sütür insizyon sonra yaraya lokal analjezik anestezikler (lidokain merhem) uygulamak ve evlerinde kafeslerde onları geri. </li><li> Ameliyattan sonra, tek başına da sternum yatma korumak kadar plastik kafeslerde sıçan ev ve anestezi kurtarıldı kadar sıcak tutuyor. </li></ol></li><li> Cerrahi Kurtarma sonrasında davranışsal Testler <ol><li> Cerrahi işlemden 7 gün sonra, bölümler test aşamasında 2.2 ve 2.3 işlemleri tekrarlayın. sağlık ve hayvanların gelişim durumunu izlerken 4 hafta boyunca davranışsal testleri gerçekleştirin. </li><li&gt; Ameliyat sonrası aşaması için kontrol ve deney grupları arasında hayvanların sağlık koşulları (örn, vücut ağırlığı, beslenme miktarı ve serbest dolaşımı) izleyin. </li></ol></li><li> PE-50 Boru ile Kanül İmplantasyonu gerçekleştir <ol><li> % 4 izofluran ile sıçan anestezisi ve 36.5 de vücut ısısını korumak - Basit bir ısıtma yastığı ile 37.5 ° C. </li><li> bir neşter ile sırasıyla sol omuz ve göğüs boşluğu, karın kısmı dorsal ön ayakları kısmı ile kesişme orta hatta iki delik (2 cm çaplı her) kesti. </li><li> İki delik arasındaki bir makas ile deri ve kas ayırmak. </li><li> ventral delikten dış juguler ven PE 50 tüp (20 cm uzunluğunda) bir ucunu. dorsal deliğine aynı PE 50 boru terminal ucunu ve cilde yapıştırın. </li><li> doğrulamak için şah damara tuzlu enjekte etmek için bir şırınga kullanın olduğunu PE 50 boruengellenmez. </li><li> kesi dikiş, 6 mg / kg gentamisin (intraperitonal) farelere enjekte edilir. </li><li> 20 U / ml heparin ile tuzlu su, 0.1 ml, ardından, 0.3 ml% 0.9 tuzlu su ile boru ameliyat sonrası her gün yıkayın. </li></ol></li><li> Nihai Davranış Testi <ol><li> Tekrarlayın bu davranışı onaylamak için 2.2-2.3 adımları adım 2.6 bir hafta sonra cerrahi kurtarma sonra adım 2.5 ile karşılaştırıldığında kararlıdır. </li></ol></li><li> Radyoaktif Enjeksiyon <ol><li> uyum için 10 dakika - 5 için bir dinlenme kafeste sıçan yerleştirin. </li><li> Bir ayırıcı kullanarak, iki adet 1 ml&#39;lik şırınga PE 50 boru bağlayın. Normal tuzlu su bir kez şırınga doldurun ve (- 0.5 ml 0.3 bir hacimde 125 uCi / kg) [14C] -IAP çözeltisi ile diğer dolgu. </li><li> Eksternal juguler ven içine radyotracer enjekte ve 3 M potasyum klorür ile dolu başka bir şırınga ile şırınga değiştirin. </li><li> On saniye Radyoaktif inj sonraection, standart ötenazi yöntemlerine göre hayvanların izofluran aşırı dozda altında 3 M potasyum klorür enjekte ve kurban (yani, 50 dakika boyunca bir buharlaştırıcı gelen izofluran) Tayvan Academia Sinica Kurumsal Hayvan Bakım yönergelerine ve Kullanımı Komitesi dayalı. </li><li> 1 dakika sonra, kafatası maruz ve kalan kas keserek. rongeurs kullanılarak, beyinden kafatası dorsal yüzeyini soyularak. rongeurs kullanılarak kafatası kenarlarını uzak kesin. Sonra, bir spatula kullanarak, beynin ventral yüzeyi boyunca koku ampuller ve sinir bağlantılarını kesti ve beyin kaldırmak. </li><li> kuru buz ve metilbütana beyin dondurmak için optimum kesim sıcaklık (OKT) bileşik kullanın (yaklaşık -55 ° C). Dondurucuda beyin dokusunu saklayın. 11, 12 </li></ol></li><li> beyin Dilimleme <ol><li> Beyin aşağıya bakacak şekilde, bir mikrotom doku yönlendirin. 20 &amp; halinde beyin dilim bir Kriyostat kullanın# 181; m kalınlığındaki kesitler. </li><li> Her bir dilim arasında bir 240 um aralığında olan, -20 ° C&#39;de, bir kriyostat mikroskop lamları üzerinde beyin dilimleri yerleştirin. </li><li> mikroskop slaytlar ve -20 ° C&#39;de 3 gün boyunca poz kasetleri içine kademeli radyoaktivite ile beş standart filtre kağıtları yerleştirin. beyin dilimleri sırasına göre, yukarıdan aşağıya doğru mikroskop slaytlar düzenlemek. Son olarak, Kasetlerin alt filtre kağıtları yerleştirin. 12 </li><li> Pozlama kasetlerden fosfor ekran çıkarın ve beyin dilimleri için [14C] -IAP alımını göstermek için görüntüleri fosfor ekran okuma ve üretmek için bir değişken mod görüntüleyici kullanın. </li></ol></li></ol> 3. Veri Analizi <ol><li> Adım 2.9.4 sonra İstatistiksel Parametrik Haritalama (SPM) ve ImageJ yazılımı kullanarak görüntüleri ayarlayın. seri koronal kesitler kullanarak tüm görüntüleri yeniden yapılandırma. Pürüzsüz ve referans sıçan beyin modeline göre görüntüleri normalleştirmek.12, 13 </li><li> Nicel değerlendirmeler için, piksel sinyal yoğunluğunu belirlemek ve analiz için istatistiksel yazılımı kullanmak için ImageJ yazılımı kullanarak bölgeyi-faiz beyin görüntüleri (YG) ölçün. 12, 13 </li><li> Farklı beyin çekirdekleri arasındaki bağlantıları araştırmak için, bir bölgelerarası korelasyon matrisi radyoaktivite oranını görüntülemek için MATLAB korelasyon analizi yazılımı kullanmak ve renk haritaları gibi matrisleri görselleştirmek. Son olarak, ağ analizi için Pajek yazılımını kullanın. 12, 13 </li><li> Başlangıçta sahte ve CSKA gruplarında von Frey testi plantar testi ve mekanik kuvvet ısı tolerans süresini karşılaştırmak için, 2 × Varyans 5 çift yönlü karışık analizi (ANOVA), grup ve haftalar faktörler olarak kullanın ve hafta 1 - 5. ANOVA grubu ve beyin alanına göre radyoaktivite oranını ölçmek için 2 × 31 iki yönlü. Uygun olduğunda, Tukey Dürüst Önemli Fark (HSD) post hoc testleri yürütmek. Calhesaplanmalıdır Pearson korelasyon sham ve CSKA gruplarında seçilen beyin alanlarının tümü arasında korelasyon değerlendirmek katsayıları. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Davranışsal testlerde, CSKA grubu başlangıçta von Frey testi termal ağrı testi ve mekanik kuvvet pençe çekme eşiği azalmaları ve 1 hafta sergilendi -. 5. bulguları önceki bir çalışmada uyumluydu 14 [14 C] -IAP yöntem farklı beyin dilimleri kantitatif analiz için beyin görüntülerinin piksel yoğunluğuna dayanır. beyin görüntüleri verileri değerlendirmek için, piksel sinyal yoğunluğu tanımlanmıştır. 46,979 CPM - Bu çalışmada, çevre ...

İnme kanama nöropatik ağrı muzdarip hastaların% 8&#39;den fazla meydana geldiği görülmüştür olarak merkezi felç sonrası ağrı (CSKA) atıfta bulundu. 1-3 CSKA somatosensör disfonksiyonu, böylece aşırı duyarlılık ve allodini uyaran neden olabilir. 4 Ancak , CSKA somatosensoriyel disfonksiyonu patofizyolojik mekanizmaları belirsizdir. Örneğin, somatik duyu kaybının hemorajik beyin bölgesinde nöronal deafferentasyonu kaynaklanır. Hiperaljezi merkezi nosiseptif sinir hücrelerinin ya da merkezi disinhibisyon, 5, 6, ancak CSKA semptomlar yer sinirsel substratlar halen bilinmemektedir hiper uyarılabilirliğine neden olabilir. Bazı çalışmalar dorsolateral prefrontal korteks (dPFC), rostral anterior singulat korteks (ACC), amigdala, hipokampus, periakuaduktal gri (PAG), rostral ventromedyal medulla ve birbirleriyle olan bağlantılarını nosiseptif işleme aracılık ileri sürmüşlerdir. Ek 7ly, medial prefrontal korteks (MPFC) -amygdala devreleri CSKA patofizyolojik mekanizmaları üzerinde 8 Veri çeşitlidir. ağrıya bağlı algısında rol oynadığı gösterilmiştir ve CSKA nöral substratların aktivasyonu daha fazla inceleme ihtiyacı vardı. [14 C] -Iodoantipyrine (IAP) alımı beyin aktivitesi ve CBF arasında bir ilişki varsayarak, dolaylı olarak bölgesel serebral kan akımı (rCBF) gözlemlemek için kullanılır. Her ne kadar [14 C] -IAP gibi fonksiyonel manyetik rezonans görüntüleme (fMRI) ile olduğu gibi, gerçek zamanlı olarak beyin aktivitesini değerlendirmek olamaz, bu çeşitli avantajları vardır. Örneğin, [14C] -IAP kendiliğinden ölçüm patolojik durumları sırasında beyin olay meydana için uygundur. 9 Ayrıca, [14C] -IAP alım anestezi olmadan ölçülür. Aynı zamanda fMRI ve pozitron emisyon tomografisi (PET) gibi diğer görüntüleme yöntemleri, daha az maliyeti. [14C] -IAP yöntemi Ölçme ve uygun olduğu ileri sürülmüştürtalamusun ventral bazal nükleus (VB) lezyonlarının indüklenir g spontan ağrı (örneğin, CSKA). 9 Mevcut protokol hayvan modelinde talamus VB lezyonları ile indüklenir CSKA nöral yüzeylerde katılımını değerlendirmek için [14C] -IAP yöntemi gerçekleştirmek açıklamaktadır. teknik, davranışsal ve nöronal düzeyde CSKA belirtileri altında yatan patofizyolojik mekanizmaları belirleme yol sunar.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

Şekil 1A deneysel zaman çizelgesi göstermektedir. Sıçanlar davranış testleri için sahte ve CSKA gruplar (örneğin, von Frey testi ve plantar testi) tahsis edilmiştir. Deneyin ilk gün taban olarak görev yaptı ve testler 1 hafta tekrarlandı - 4. Heparin (20 U / ml, 0.1 ml / gün) boyunca enjekte edildi haftada 5. PE-50 kateterizasyon dış juguler ven yapıldı hafta 4 ve heparin enjeksiyonundan sonra 5. Beş dakika, [14 C] -IAP enjekte ed...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

otoradyografik Ölçümler [<sup&gt; 14</sup&gt; Orta İnme Sonrası Ağrı ardından Rat Beyin C] -Iodoantipyrine

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

autoradiographic-measurements-14c-iodoantipyrine-rat-brain-following

Research

JoVE Journal

Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K Görüntüleme.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Video: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2-Gemi Tıkanıklığı / Hipotansiyon: Küresel Beyin İskemi Rat Modeli

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Tıp

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Farelerde inme sonrası depresyon Inducing için orta serebral arter tıkanıklığı tekniği

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

Davranış

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

Rat Bölgesel Beyin Enerji Yüzeyleri Alımına Kantitatif Tedbir için A Çift Tracer PET-MR Protokolü

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Nörobilim

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Biyokimya

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Pozitron Emisyon Tomografi ile kullanma Non-invaziv Görüntüleme ve Serebral iskemi Analizi Yaşam Sıçanlarda<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Kafa içi farmakoterapi ve ağrı deneyleri Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Serebral protein sentezi In vivo bölgesel oranlarının belirlenmesi için nicel Autoradiographic yöntemi

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Görüntüleme [11C] DPA-713 iskemik inme bir fare Model kullanma Neuroinflammation evde beslenen hayvan

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Görselleştirme ve farmakolojik hedefleri karakterizasyonu için basit ve güçlü yöntemi olarak autoradiography

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Tek Bir Kemirgen Beyin Örneklerinde İnme Sonrası Serebral Ödem, Enfarktüs Bölgesi ve Kan-Beyin Bariyeri Kırılımının Ölçülmesi

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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