Bu araştırma Chungnam Ulusal Üniversitesi, Kore Enstitüsü Oriental Tıp (KIOM) ve vermek için Kore sağlık teknoloji R tarafından desteklenmiştir &#38; D proje ile Kore sağlık sanayi geliştirme Enstitüsü (Sağlık Bakanlığı tarafından finanse edilen KHIDI), &#38; Refah, Güney Kore (sayı vermek: HI15C0007).

tüm deneylerin Uluslararası Birliği etik kurallarına uygun olarak ağrı çalışma için yapılmıştır ve kurumsal hayvan bakım ve kullanım Komitesi tarafından onaylanmış Chungnam Ulusal Üniversitesi () Daejeon, Güney Kore).
 1. indüksiyon CCI siyatik sinir üzerindeki <ol><li>ev erkek ICR fareler ile kontrollü oda sıcaklığında (20-25 ° C'de muhafaza) ve nem (40-%60), bir 12 h açık/koyu döngüsü altında 20-25 g ağırlığında ücretsiz Gıda erişim ve su. Ameliyattan önce fareler hayvan bakım odasında bir calıştıkları süre en az 1 hafta izin.
	<ol><li>Konut sırasında fareler davranışını ve görünümünü gözlemlemek ve anormal Lokomotor aktivite gösteren fareler kullanmayın. 
		Not: Bir gün CCI ameliyat öncesi von Frey filamentler uygulayarak arka pençe mekanik duyarlılığı ölçmek ve normal temel değerlerini elde etmek için otomatik yürüyüş analizi gerçekleştirin. Ardından, fare rasgele kontrol ve deneysel gruplara izin atama.</li>
	</ol></li> <li>2,2,2-tribromoethanol (250 mg/kg, mayi (IP)) enjekte edilerek ameliyat günü, fareler anestezi. 
	Not: Cerrahi önlük, eldiven ve maske gibi kişisel koruyucu ekipman giymek.
	<ol><li>2,2,2 tribromoethanol 2.5 g tartmak ve 2-metil-2-buthanol 5 mL son hacim için ekleyin. Çözüm ışıktan uzak tutmak (örneğin, karanlık bir kapsayıcı kullanın veya şal folyo).</li>
		<li>40 ° C'de Isıtma ve 10-30 dk. karıştırarak tarafından tamamen erimesi için izin ver reaktifler</li>
		<li>Distile su 200 mL son hacmi ekleyin ve iyi kadar karışık karıştırın.</li>
		<li>Aliquots 4 ° C'de depolayın ve karanlıkta tut. İki hafta sonra anestezi ile yeni aliquots değiştirilmelidir.</li>
		<li>1 g vücut ağırlığı başına 20 µL hacmi enjekte. Vücut ağırlığı fare ile 25 g ise, örneğin, çözeltinin 500 µL ver. Bu anestezi tipik süresi 1 h. olduğu 
		Not: Bu çözüm ışığa maruz zaman zehirli olacak ve/veya ısı ışık ve ısı maruz önlemek dikkatli olun. IP enjeksiyon yolu önerilir.</li>
	</ol></li> <li>Fare derin anestezi düştüğünde, dorsal yüzü (Yani, yüzükoyun) kadar ameliyat masasında yerleştirin ve sağ tarafta bir % 70 alkol bez ile dış orta uyluk bölgeyi sterilize. 
	Not: derin anestezi yanıt-e doğru tutam veya uyarıcı arka paws veya kuyruk basınç eksikliği kontrol ederek onaylayın.</li>
	<li>Üzerinde fare sağ siyatik sinir CCI neden.
	<ol><li>Bir neşter bıçakla orta uyluk bölgesinin kalbinde 1.0-1.5 cm uzunluk çevresindeki deri bir kesi yapmak.</li>
		<li>Açık açık siyatik sinir ortaya çıkarmak için mikro-sivrisinek hemostat ile uyluk kasları teşrih.</li>
		<li>4-0 fotokromik akorduna dikiş kullanarak 3 kez 1.0-1.5 mm aralıkları ile maruz siyatik sinir ligate. 
		Not: hafif sallayarak Ipsilateral arka pençe üzerinde görünene kadar siyatik sinir bağlayarak gevşek d alın. Sham grup farelerin aynı cerrahi açılış sinir ligasyonu olmadan dışında benzer koşullarda alınan.</li>
	</ol></li> <li>5-0 ipek kullanarak 3-4 basit kesintiye uğramış dikiş ile cerrahi açılış kapatın ve cerrahi dış çevrenin dezenfeksiyonunda povidone iyot ile sterilize.</li>
	<li>Ameliyattan sonra bir Isıtma yastığını temiz bir kafeste fareyi getirin. Anesteziden hayvanlar kurtarıldı sonra onları ev onların kafes dönün. 
	Not: Bu çalışmada, antibiyotik kullanılmadı. Bir doz 50 mg/kg pozitif kontrol olarak günde bir kez, CCI + GBP alınan grup IP gabapentin (GBP).</li>
</ol> 2. Mekanik Allodynia ölçümü (von Frey Test) Not: 1 g Ipsilateral arka pençe plantar yüzeyine von Frey filament kullanarak para çekme yanıt mekanik uyaranlara karşı sıklığını değerlendirmek.
<ol><li>Her test günü, fareler davranış test odasına getir ve en az 30 dk önce test için kendi ev kafeste fareler alışmana 
	Not: Cerrahi önlük, eldiven ve maske gibi kişisel koruyucu ekipman giymek.</li>
	<li>Şeffaf Akrilik kutu metal kafes yerde fare koymak ve fareyi 30 dakika süreyle alışmana</li>
	<li>Filaman virajlı kadar yavaşça von Frey filament 1 g arka pençe plantar yüzeyine uygulamak.</li>
	<li>Uygula filaman uyarmaya Ipsilateral hind pençe ile 10 kez en az 10 s aralıklarını ve sonuç kayıt. 
	Not: Bu çalışmada, pençe çekilme yanıt--dan 10 deneme sayısını pençe çekilme frekans (PWF, %) yüzdesi olarak gösterilir. Von Frey test iki günde CCI ameliyat sonrası gerçekleştirildi.</li>
</ol> 3. Yürüyüş çözümlemesini otomatik Not: hayvan yürüyor ve otomatik olarak çeşitli yürüyüş parametreleri çözümler yürüyüş analiz sistemi her pençe baskı görüntüler, böyle paw baskı, pençe gibi yoğunluk, adım uzunluğu, duruş faz adım dizisi, salıncak, salıncak, hız vb bu çalışmada pençe yazdırma alanı ve yüzde veri dönüştürme sonra tek tutumunu değiştirmek kontralateral arasında sola karşı Ipsilateral sağ arka pençe gösterdik. Böylece, bir sonuç % 50 pençe baskı boyutunu ve pençe alan kat, sağ ve sol arasında temas süresi aynı olduğunu gösterir. Buna ek olarak, temas süresi ve boyutu ile karşılaştırıldığında kontralateral yan Ipsilateral arka pençe içinde azalma % 0 yaklaşan alt veri değerlerini belirtmek (bkz: Şekil 2 Masası B ve Şekil 3).
<ol><li>Calıştıkları için günde bir 5 gün CCI ameliyattan önce başlayan yürüyüş analiz cihazı 10 dk içinde fareler devam. 
	Not: yürüyüş analizi testi calıştıkları dahil olmak üzere karanlıkta gerçekleştirilmelidir.</li>
	<li>Fareler için yürüyüş analizi test odası getirmek ve en az 30 dk önce test için onların ev kafeste alışmana
	<ol><li>Kur sekmesinde program menüsünü &#34; geçit uzunluğu &#34; 30 cm ve küme için &#34; maksimum süresi çalıştırmak &#34; 5 s ve &#34; maksimum varyasyon çalıştırmak &#34; % 50'ye.</li>
		<li>Kayıtlı bir kameradan seçin &#39; kurulum &#39; sekmesinde &#39; programı &#39; menü.</li>
		<li>Seçin &#34; açın edinme &#34; üzerinden &#39; elde &#39; sekmesinde &#39; programı &#39; menü.</li>
		<li>Durum iletisi tıklatın &#39; snap arka plan &#39; boş (Yani, boş) geçit arka plan resmini çekmek için düğmesini.</li>
	</ol></li> <li>'I tıklatın &#39; başlamak satın alma &#39; düğme ve yer bir fare üzerinde geçit; kaydı otomatik olarak fare hareket göre başlar. 
	Not: fare açıkça geçit arasında yürürken, program otomatik olarak bu hareket olarak sınıflandırır &#34; çalıştırmak uyumlu &#34; yeşil bir simge ile. Yazılım ayak basmak algılamak başarısız olduysa, deneyci kırmızı bir simge göreceksiniz ve kayıt yinelemeniz gerekir. En az beş başarılı uyumlu çalışır analiz için ihtiyaç vardır.</li>
	<li>Testten sonra seçin &#39; ishal sınıflandırmak &#39; edinme sekmesinden &#39; programı &#39; menü.</li>
	<li>Analizi ve tıklama için çalışır seçin &#39; otomatik sınıflandırmak &#39; düğmesini. 
	Not: sınıflandırma sonra tüm istatistik parametreleri otomatik olarak kaydedilir ve deneyci tıklatarak sonuçları bulabilirsiniz &#34; görünüm istatistikleri çalıştırmak &#34; Çözümle menüsünde.</li>
</ol>

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Yazarlar ifşa gerek yok.

Von Frey filamentler kullanarak mekanik allodynia şu anda, en yaygın ağrı hayvan modellerinde dokunsal aşırı duyarlılık göstermek için kullanılan yöntem ölçümdür. Nöropatik ağrı için hayvan modeller geliştirilmeye devam etmiş gibi duyusal işlev için değerlendirme metodolojisi de geliştirilmiş8,9,10,15olmuştur. Bu raporları, bu değişiklikler eski yöntemleri daha ...

Sinir sisteminin patolojik değişiklikler travma, metabolik fonksiyon bozukluğu, iltihap, enfeksiyon, iskemi indüklenen veya Otoimmün hastalık bazen bir lezyon ya da hastalığı doğrudan bir sonucu ortaya çıkan bir ağrı olarak tanımlanan nöropatik ağrı neden somatosensor sistem1etkileyen. Nöropatik ağrı genellikle dayanılmaz ve ne yazık ki, geleneksel analjezikler genellikle yeterli ağrı Rölyef2üretmek değildir. Nöropatik ağrı önemli bir özelliği spontan ve stimülasyon uyarılmış (Yani, allodynia ve hyperalgesia) ağrıları içerir. Allodynia Kısaca bastırmak veya sıcak stimülasyon gibi normalde ağrılı uyaranlara karşı oluşan nosiseptif bir yanıttır. Hyperalgesia mekanik ve/veya termal zararlı uyaranlara3için bir gelişmiş acı tepki gösterir. Her ne kadar bu iki belirtiler her iki kritik hastanın yaşam kalitesini bozabilir, yumuşak Kontakt günlük yaşamda önlemek zor olduğu için mekanik allodynia nazik dokunsal uyarım tarafından uyarılmış en sinir bozucu semptomdur.
Nöropatik ağrı tedavisinde yeni analjezikler ve temel mekanizması araştırmak için hassas ölçüm ağrı yanıtının esastır. Çok sayıda nöropatik ağrı hayvan modelleri geliştirdik hind nosiseptif yanıtları pençe alan onun yüksek erişilebilirlik4,5,6,7yüzünden. Böylece, çoğu ağrı yanıt Değerlendirmeler arka pençe plantar veya dorsal yüzeyi mekanik uyaranlara von Frey filamentler gibi özel araçlar kullanarak uygulama tarafından gerçekleştirilmiş. En sık kullanılan yöntem biri yukarı ve aşağı Dixon tarafından açıklanan yöntemi sürümleri9,108 ve daha sonra değiştirilebilir. Ancak, çok yetenekli, deneyimli Denemecileri von Frey testi gerçekleştirmek için gerekli olan ve sonuçları öznel olabilir.
Otomatik yürüyüş analiz sistemi nörolojik ve nöromüsküler hastalıklar kemirgenler serbestçe hareket yürüyüş çeşitli parametreler ölçerek araştırabilirsiniz. Sinir yaralanma hayvan modelleri çeşitli, duyusuna derecesini ve çeşitli tedaviler antinociceptive etkisini bir ağrı stimülasyon11,12,13, eklemeden değerlendirilebilir 14. bu analiz sistemi statik ve dinamik yürüyüş parametreleri, aşağıdaki gibi algılayabilir: yazdırma alanı (zemin ile bağlantı kurduğunda tam pençe baskı alanı) pençe, yoğunluğu (kontak pençe alanının ortalama yoğunluğu) pençe, adım uzunluğu ( Art arda gelen yerleşimleri aynı pençe arasındaki mesafe), duruş (süresi yere temas için bir tek arka pençe), adım dizisi (içinde dört paws sipariş yere yerleştirilir) faz (salıncak faz süresi) salıncak ve salıncak hız (hesaplanan uzunluğu stride ve salıncak süresi ve piksel / saniye olarak ifade). Bu kağıt bir analiz sistemi kullanımı gösterilmiştir ve kronik daralma yaralanma (CCI) nöropatik fare kullanarak von Frey testi ile veri kısa bir karşılaştırma sağlar.

<table>
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td>JW Pharmaceutical</td>
			<td>N/A</td>
			<td>Vehicle for drugs</td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td>Injecting device</td>
		</tr>
		<tr>
			<td>2, 2, 2-tribromoethanol (97% purity)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>2-methyl-2-buthanol (99% purity)</td>
			<td>Sigma</td>
			<td>152463</td>
			<td>Solvent for 2, 2, 2-tribromoethanol</td>
		</tr>
		<tr>
			<td>Catwalk Automated gait analysis system</td>
			<td>Noldus</td>
			<td>N/A</td>
			<td>Automatic analysis software of aniaml gait</td>
		</tr>
		<tr>
			<td>Chromic catgut (4-0 thickness)</td>
			<td>AILEE</td>
			<td>C442</td>
			<td>Ligature to make chronic constriction injury on the sciatic nerve</td>
		</tr>
		<tr>
			<td>Gabapentin</td>
			<td>Sigma</td>
			<td>Y0001280</td>
			<td>Analgeisc, Used as a positive control drug in this study</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11051-10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>DAESHIN ELECTRONICS</td>
			<td>M-303AT</td>
			<td>Regulation of body temperature</td>
		</tr>
		<tr>
			<td>ICR Mouse</td>
			<td>Samtaco</td>
			<td>N/A</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Mersilk (3-0 thickness)</td>
			<td>ETHICON</td>
			<td>W598H</td>
			<td>Suture material for surgical closure of skin</td>
		</tr>
		<tr>
			<td>Micro-Mosquito</td>
			<td>F.S.T</td>
			<td>13010-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Micro-scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>F.S.T</td>
			<td>12002-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Firson</td>
			<td>N/A</td>
			<td>Disinfectant to prevent infection after surgery</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>F.S.T</td>
			<td>10010-00 (#10)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12 (#3)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Von-Frey filaments</td>
			<td>North Coast</td>
			<td>NC12775-99</td>
			<td>Measurement device to test sensory function for mechanical stimulation</td>
		</tr>
	</tbody>
</table>

automated gait analysis in mice with chronic constriction injury

Von Frey test yaygın olarak nöropatik ağrı hayvanların duyusal işlev incelemek için kullanılan klasik bir yöntemdir. Ancak, bazı dezavantajları öznel verileri gibi ve yetenekli, deneyimli deneyci şartı vardır. Bugüne kadar değişiklikler çeşitli gelişmiş von Frey yöntemi, ama yine de birkaç sınırlaması vardır. Son raporlar yürüyüş analizi nöropatik hayvanlardan daha doğru ve objektif verileri üreten tavsiye ettiler. Bu iletişim kuralı, farelerde nöropatik ağrı derecesini belirlemek için otomatik yürüyüş analizi gerçekleştirmek gösterilmiştir. Calıştıkları birkaç gün sonra fareler serbestçe ayak izleri aydınlatmak için cam zemin üzerinde yürümek için izin verildi. O zaman, miktar ayak izleri ve yürüyüş gerçekleştirilen alan pençe baskı, swing zaman, Agiz pençe, vb gibi çeşitli yürüyüş parametreleri otomatik olarak analiz ile video klipler aracılığıyla
Bu çalışmanın temel amacı otomatik yürüyüş analizi yöntemi açıklamak ve kısaca klasik duyusal test von Frey filament kullanarak verilerle karşılaştırın etmektir.

Biz von Frey testi gerçekleştirilen ve CCI ameliyattan sonra 10 gün kadar yürüyüş analizi CCI farelerde otomatik. İstatistiksel analiz için iki yönlü Varyans analizi (ANOVA) tekrarlanan ölçüleri belirleyen genel etkileri ve Dunnett'ın post-hoc analizi deneysel grupları arasında p değeri belirlemek için gerçekleştirildi.
Şekil 1 ' de gösterilen sonuçları zamanlı Kur...

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

Ağrı yanıt nöropatik hayvan modeli olarak hassas bir değerlendirme ağrı hastalıkları Patofizyoloji araştırmak ve yeni analjezikler geliştirmek çok önemlidir. Biz bir otomatik yürüyüş analizi sistemi tarafından hind pençe kemirgen duyusal işlev belirlemek için hassas ve objektif bir yöntem mevcut.

Kronik daralma Yaralanmalı farelerde otomatik yürüyüş analizi

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

automated-gait-analysis-in-mice-with-chronic-constriction-injury

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.0K Görüntüleme.  Chungnam National University. Ağrı yanıt nöropatik hayvan modeli olarak hassas bir değerlendirme ağrı hastalıkları Patofizyoloji araştırmak ve yeni analjezikler geliştirmek çok önemlidir. Biz bir otomatik yürüyüş analizi sistemi tarafından hind pençe kemirgen duyusal işlev belirlemek için hassas ve objektif bir yöntem mevcut.

Video: Automated Gait Analysis in Mice with Chronic Constriction Injury

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Çoklu Ölçeklerde Sayısallaştırılmış Nöronal Morfoloji Otomatik Sholl Analizi

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Nörobilim

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Kronik Nikotin İdaresi fareler Knock-in içinde floresan etiketli Nikotinik Reseptör Spektral Konfokal Görüntüleme

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Sagittal düzlem kinematik yürüyüş analizi MOG35-55 için tabi C57BL/6 farelerde deneysel otoimmun ensefalomiyelit indüklenen

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Kronik yaygın Marmoset bütün kortikal Electrocorticographic dizide implantasyonu

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

Farelerde Travmatik Beyin Hasarı Sonrası Caecum Mikrobiyotasında Ki Değişikliklerin Araştırılması

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Periferik Sinir veya Spinal Kord Kontüzyonu Yaralanması olan Kemirgenlerde Fonksiyonel İyileşmeyi Değerlendirmek için Otomatik Yürüyüş Analizi

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

İnme sonrası hastalarda yürüme analizi ve değerlendirmesi için motor ikili görevler

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

Kemirgenlerde Hareketsizliğe Bağlı Yürüyüş Değişikliğinin Kapsamlı Anlaşılması

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Farelerde Hafif Travmatik Beyin Hasarının Elektromanyetik Kontrollü Kapalı Kafa Modeli

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...