Name: a simple and inexpensive method for determining cold sensitivity and adaptation in mice
Uploaded: 2015-03-17T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

Tüm fare protokolleri Sağlık kurallar National Institutes uygun ve Washington Üniversitesi Tıp Fakültesi Hayvan Çalışmaları Komitesi (St. Louis, MO) tarafından onaylanmıştır. 1. Test Plakası ve Kutuları hazırlanması <ol><li> Cam yüzeyini temizleyin. </li><li> Laboratuvar bant ile cam tabak ortasında yüzeyine T-tipi lif termokupl probu sabitleyin. </li><li> Plaka ortasında boyunca tek bir satır cam plaka üzerine hayvan muhafazaları yerleştirin. </li><li> Merkez hayvan muhafaza ile termokupl probu Konu ve veri kaydedici takın. Otomatik kapanma özelliği devreden ise veri logger açın ve sağlanan kablo ile bilgisayara veri logger takın. <ol><li> Deney sırasında plaka sıcaklığı kayıt varsa, plaka sıcaklıkları kaydetmeye başlamak için veri kaydedici yazılımı açın. </li><li> Gerekirse, yeniden yazılım ayarlayınkord plaka sıcaklığı kez her saniye. </li><li> Termal data logger ile paketlenmiş yazılımı kullanarak sıcaklıkları kayıt başlayın. </li></ol></li><li> Fareler arasındaki görsel etkileşimi önlemek için siyah ekler ile muhafaza ayırın. </li><li> Pozisyon muhafazaları alt rahat bir oturma konumundan görünür, öyle ki, cam altında yansıtır. </li></ol> 2. Isıtma / Cam Tabak Soğutma <ol><li> Isıtılmış su, ıslak buz ya da kuru buz alüminyum kutular doldurun ve cam plaka üzerine uygun bir şekilde konumlandırmak (kuru buz dolu alüminyum folyo paketler aynı zamanda cam soğutmak için de kullanılabilir; Şekil 1) 2. <ol><li> 30 ° C&#39;de test için, uzak hayvan muhafazaları (Şekil 2B) 2 &#39;yaklaşık 0.25 alüminyum kutuları yerleştirin. <ol><li> Cam levhasının her iki tarafında bir ısıtılmış su sirkülatör ayarlayın. 45 sirkülatörü ayarlayın - 60 ° C, ve bizeSıcak su (Şekil 1C) 2 sabit bir akım ile alüminyum kutuları doldurmak için e. </li><li> Sirkülatör yerleştirin alüminyum kutuları sıcak su doğrudan geri her iki tarafında (Şekil 1C) üzerindeki sirkülatörün rezervuar içine drene olduğu 2. </li></ol></li><li> Oda sıcaklığında test için, kutuları boş (Şekil 2) 2 bırakın. </li><li> (Şekil 2) 17 ° C&#39;de test için, uzakta iki tarafında hayvan muhafazaları gelen &#39;&#39; yaklaşık 0.25 kutuları yerleştirin ve ıslak buz ile doldurun 2. </li><li> (Şekil 2) 12 ° C&#39;de test edilmesi için, uzak iki tarafında muhafazalar ile ilgili &#39;&#39; yaklaşık olarak 1.25 kutuları pozisyon ve kuru buz ile doldurun 2. </li><li> (Şekil 2) 5 ° C&#39;de test için yaklaşık 0,25 &#39;&#39; uzak iki tarafında muhafazaları kutuları yerleştirin ve kuru buz ile doldurun 2.<ol><li> Kuru buz ile cam soğutma yaparken, odada CO 2 birikmesini önlemek için yeterli havalandırma olduğundan emin olun. </li></ol></li></ol></li><li> İstenilen sıcaklık aralığı ulaşması cam bekleyin. </li><li> Plaka üzerinde muhafazaları fareler ekleyin. NOT: Bir beyaz gürültü jeneratör gürültü bozuklukları azaltmak için kullanılabilir. </li><li> Fareler gelmesini bekleyin. NOT: Tesisimizde bu yaklaşık 2.5 saat sürer, ancak bu hayvan barınağı ve taşıma koşullarına göre önemli ölçüde değişebilir. </li><li> Kutular ısındı su, ıslak buz veya kuru buz dolu tutulmasını sağlayarak istenilen sıcaklık aralığında cam koruyun. NOT: Bizim aparat ile kutuları buz kabaca her 90 dakika ile doldurulmuş olması gerekir. NOT: 17 ° C koşulu için, buz ile doldurmadan önce tahliye deliklerinden alüminyum kutuları su en boşaltmak için yardımcı olur. Bu sıcaklık daha iyi ve pr stabilize edecekOlay taşması NOT: Kuru buz kesin miktarı mevsimsel değişiklik, ancak genel olarak sıcaklık sabit tutacak kutusunun tüm uzunluğu boyunca tam daha fazla kutu ¼ tutmak olacaktır. </li></ol> Sabit Sıcaklıklarda 3. Test Fareler <ol><li> Davranışsal oda dışında, kuru buz dolu yarısı bir buz kovası doldurun. </li><li> Bir çekiç ya da tokmak kullanarak, ince bir toz haline kuru buz ezmek. NOT: kova aşırı doldurulması zor tamamen toz haline kuru buz ezmek için yapacaktır. </li><li> Düz jilet veya makas kullanarak, 3 ml şırınga üst kesti. </li><li> Bir 21 G iğne kullanılarak, şırınga (toplam 6 delik), karşı uçlarında 3 delik karıştırmak. NOT: Kuru buz sıkıştırarak ederken bu delikler yüceltme tarafından oluşturulan basınç azalacak. Kesme şırınga çok deneyler için yeniden kullanılabilir. </li><li> Şırınga, kuru buz tozu ve davranışsal odasına el kronometre al. </li><li> Kuru buz tozu yarısı dolu şırınga odasına doldurun. Düz bir nesneye karşı şırınga kesme ucunu tutun ve sıkıca pistonu kullanarak tozu sıkıştırmak. Dikkatli ol; plastik piston bükmeyin veya basınç kırılabilir. Bu durumda, yeni bir şırınga pistonu değiştirin. </li><li> Şırınga kenarına geçmiş sıkıştırılmış kuru buz pelet ucunu uzatın. </li><li> Istirahatte tamamen olan test fareler. <ol><li> 30 ° C, 23 ° C ve 17 ° C sıcaklıkta, 4 camına pençeleri ve sahip deney fareleri hareketli, ama 19 tam uykuya değil. </li><li> 12 ° C ve 5 ° C de, hareketli ya da atlama 2 pençeler veya 4 pençe olup Hangi Test fareleri. </li></ol></li><li> Yavaşça, hedefleme için aynalar kullanarak ama sıkıca fare hindpaw (Şekil 1A) 2 altındaki cam yüzeyine karşı düz pelet floş basın. El-Sayacı başlatmak. </li><li> Zamanlayıcı durdurun ve fare uzakta soğutmalı cam hareket ettiğinde pelet çıkarın. NOT: çekilme hareketi, dikey veya yatay olabilir. <ol><li> Fare çok kısa bir süre pençe taşır ve daha sonra soğutma yüzeye dönerse fare uzak bir kalıcı hamle yapana kadar, zamanlama ve uyarıcı devam edin. NOT: Bizim laboratuvar olguların çoğunda fareler için 20 sn maksimum uyaran saatini kullanır. </li></ol></li><li> Her hayvanın her pençe en az 3 değerleri toplanana kadar bu test prosedürü tekrarlayın. En az 15 dakika herhangi bir pençe üzerinde en az 7 dakika, ve ayrı ardışık denemeler aynı fare zıt pençeleri test Ayrı denemeler. </li><li> Gerektiğinde, soğutma (Şekil 3) 1, farklı oranlarını üretmek üzere, çeşitli cam kalınlığı kullanır. NOT: soğutma oranı ters cam kalınlığı ile ilişkilidir. </li></ol> 4. Soğuk Uyum sırasında Fare Test NOT: Bu cam plat olarak test sağlayan bir alternatif protokoldürE ziyade plaka stabilize ve fareler tamamen soğuk ortama adapte kez daha, soğutur. <ol><li> Aparatı kurmak için Bölüm 1&#39;de listelenen talimatları uygulayın. </li><li> RT (Şekil 7A) 2 de temel ölçümleri almak için Bölüm 3&#39;te listelenen talimatları uygulayın. </li><li> Kuru buz ile alüminyum kutuları Öncesi serin. </li><li> Başlangıç ​​geri çekme gecikmeleri ölçülmüştür sonra, uzak muhafazaları ile ilgili her iki tarafında plaka, yaklaşık 1.25 &#39;ise önceden soğutulmuş kutuları pozisyon (Şekil 7A etiketli ok, &quot;kuru buz ilave&quot;) 2&#39;dir. </li><li> Cam plaka soğurken mümkün olduğunca sık ölçümleri alarak, ölçümler almak için Bölüm 3&#39;te listelenen talimatları uygulayın. </li></ol>

<ol>
	<li>Brenner, D. S., Golden, J. P., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Behavioral+Assay+for+Measuring+Cold+Sensation+in+Mice.">A Novel Behavioral Assay for Measuring Cold Sensation in Mice.</a> Plos ONE. 7 (6), 8 (2012).</li><li>Brenner, D. S., Vogt, S. K., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+measure+cold+adaptation+in+freely+behaving+mice.">A technique to measure cold adaptation in freely behaving mice.</a> Journal of Neuroscience Methods. , (2014).</li><li>Choi, Y., Yoon, T. W., Na, H. S., Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+signs+of+ongoing+pain+and+cold+allodynia+in+a+rat+model+of+neuropathic+pain.">Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain.</a> Pain. 59 (3), 369-376 (1994).</li><li>Gauchan, P., Andoh, T., Kato, A., Kuraishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+increased+expression+of+transient+receptor+potential+melastatin+8+in+oxaliplatin-induced+cold+allodynia+in+mice.">Involvement of increased expression of transient receptor potential melastatin 8 in oxaliplatin-induced cold allodynia in mice.</a> Neuroscience letters. 458 (2), 93-95 (2009).</li><li>Carlton, S. M., Lekan, H. A., Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+manifestations+of+an+experimental+model+for+peripheral+neuropathy+produced+by+spinal+nerve+ligation+in+the+primate.">Behavioral manifestations of an experimental model for peripheral neuropathy produced by spinal nerve ligation in the primate.</a> Pain. 56 (2), 155-166 (1994).</li><li>Pizziketti, R. J., Pressman, N. S., Geller, E. B., Cowan, A., Adler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cold+water+tail-flick:+A+novel+analgesic+test+that+distinguishes+opioid+agonists+from+mixed+agonist-antagonists.">Rat cold water tail-flick: A novel analgesic test that distinguishes opioid agonists from mixed agonist-antagonists.</a> European Journal of Pharmacology. 119 (1-2), 23-29 (1985).</li><li>Pinto-Ribeiro, F., Almeida, A., Pego, J. M., Cerqueira, J., Sousa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+unpredictable+stress+inhibits+nociception+in+male+rats.">Chronic unpredictable stress inhibits nociception in male rats.</a> Neuroscience letters. 359 (1-2), 73-76 (2004).</li><li>Karashima, Y., 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=TRPA1+acts+as+a+cold+sensor+in+vitro+and+in+vivo.">TRPA1 acts as a cold sensor in vitro and in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (4), 1273-1278 (2009).</li><li>Knowlton, W. M., Bifolck-Fisher, A., Bautista, D. M., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM8,+but+not+TRPA1,+is+required+for+neural+and+behavioral+responses+to+acute+noxious+cold+temperatures+and+cold-mimetics+in+vivo.">TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo.</a> Pain. 150 (2), 340-350 (2010).</li><li>Allchorne, A. J., Broom, D. C., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cold+pain,+cold+allodynia+and+cold+hyperalgesia+in+freely+behaving+rats.">Detection of cold pain, cold allodynia and cold hyperalgesia in freely behaving rats.</a> Molecular pain. 1, 36 (2005).</li><li>Colburn, R. W., 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=Attenuated+cold+sensitivity+in+TRPM8+null+mice.">Attenuated cold sensitivity in TRPM8 null mice.</a> Neuron. 54 (3), 379-386 (2007).</li><li>Dhaka, A., Murray, A. N., Mathur, J., Earley, T. J., Petrus, M. J., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM8+is+required+for+cold+sensation+in+mice.">TRPM8 is required for cold sensation in mice.</a> Neuron. 54 (3), 371-378 (2007).</li><li>Bautista, D. M., 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=The+menthol+receptor+TRPM8+is+the+principal+detector+of+environmental+cold.">The menthol receptor TRPM8 is the principal detector of environmental cold.</a> Nature. 448 (7150), 204-208 (2007).</li><li>Obata, 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=TrpA1+induced+in+sensory+neurons+contributes+to+cold+hyperalgesia+after+inflammation+and+nerve+injury.">TrpA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury.</a> The Journal of Clinical Investigation. 115 (9), 2393-2401 (2005).</li><li>Tang, Z., 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=Pirt+functions+as+an+endogenous+regulator+of+TRPM8.">Pirt functions as an endogenous regulator of TRPM8.</a> Nature communications. 4, 2179 (2013).</li><li>Lee, H., Iida, T., Mizuno, A., Suzuki, M., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+thermal+selection+behavior+in+mice+lacking+transient+receptor+potential+vanilloid+4.">Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 25 (5), 1304-1310 (2005).</li><li>Pogorzala, L. A., Mishra, S. K., Hoon, 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=The+cellular+code+for+Mammalian+thermosensation.">The cellular code for Mammalian thermosensation.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 33 (13), 5533-5541 (2013).</li><li>Yalcin, I., Charlet, A., Freund-Mercier, M. -. J., Barrot, M., Poisbeau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+thermal+allodynia+and+hyperalgesia+using+dynamic+hot+and+cold+plate+in+rodents.">Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents.</a> The journal of pain official journal of the American Pain Society. 10 (7), 767-773 (2009).</li><li>Callahan, B. L., Gil, A. S., Levesque, A., Mogil, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mechanical+and+thermal+nociceptive+sensitivity+in+the+laboratory+mouse+by+behavioral+state.">Modulation of mechanical and thermal nociceptive sensitivity in the laboratory mouse by behavioral state.</a> The journal of pain: official journal of the American Pain Society. 9 (2), 174-184 (2008).</li><li>Brenner, D. S., Golden, J. P., Vogt, S. K., Dhaka, A., Story, G. M., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+set+point+for+thermal+adaptation+requires+phospholipase+C-mediated+regulation+of+TRPM8+in+vivo.">A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo.</a> Pain. , (2014).</li><li>Patwardhan, A. M., Scotland, P. E., Akopian, A. N., Hargreaves, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+in+the+spinal+cord+by+oxidized+linoleic+acid+metabolites+contributes+to+inflammatory+hyperalgesia.">Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18820-18824 (2009).</li><li>Fujita, F., Uchida, K., Takaishi, M., Sokabe, T., Tominaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Temperature+Affects+the+Temperature+Threshold+for+TRPM8+Activation+through+Interaction+of+Phosphatidylinositol+4,5-Bisphosphate.">Ambient Temperature Affects the Temperature Threshold for TRPM8 Activation through Interaction of Phosphatidylinositol 4,5-Bisphosphate.</a> Journal of Neuroscience. 33 (14), 6154-6159 (2013).</li><li>Rohacs, T., Lopes, C. M., Michailidis, I., Logothetis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI(4,5)P2+regulates+the+activation+and+desensitization+of+TRPM8+channels+through+the+TRP+domain.">PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain.</a> Nature neuroscience. 8 (5), 626-634 (2005).</li><li>Daniels, R. L., Takashima, Y., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+the+neuronal+cold+sensor+TRPM8+is+regulated+by+phospholipase+C+via+the+phospholipid+phosphoinositol+4,5-bisphosphate.">Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate.</a> The Journal of biological chemistry. 284 (3), 1570-1582 (2009).</li><li>Zhang, H., 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=Neurokinin-1+receptor+enhances+TRPV1+activity+in+primary+sensory+neurons+via+PKCepsilon:+a+novel+pathway+for+heat+hyperalgesia.">Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 27 (44), 12067-12077 (2007).</li><li>Wang, H., Zylka, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mrgprd-expressing+polymodal+nociceptive+neurons+innervate+most+known+classes+of+substantia+gelatinosa+neurons.">Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 29 (42), 13202-13209 (2009).</li></ol>

The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

Kemirgenlerde soğuk cevabın ölçülmesi normal ve patolojik koşullar altında, insanlarda soğuk duyarlılık potansiyel mekanizmaları anlaşılmasına yönelik olarak önemlidir. Soğuk Plantar Test (CPA), başlangıçta önce 1 birkaç yıl oda sıcaklığında teslim soğuk uyarana tekrar üretilebilir, açık sıçangil davranışsal tepkileri üretmek için tasarlanan geliştirmiştir. Bu deneyde, bir daha yeni geliştirmeler sıcaklıkta 2 geniş bir soğuk duyarlılık tekrarlanabilir ölçümünü mümkün kılmıştır. Her iki versiyon, nispeten yüksek verimli, ve kullanımı ucuz olması için tasarlanmıştır. Ilerleme bir hayli diğer davranışsal yöntemler kullanılarak soğuk hassasiyeti mekanizmalarını anlamada yapılmıştır. Bir yöntem dabbing veya fare pençe üzerinde aseton püskürtme ve fare pençe 3,4 Flicking harcadığı süreyi ölçen içerir aseton buharlaşma testidir. Ne yazık ki,aseton buharlaşma tepkiler ıslak duyu ve aseton kokusu tarafından eleştirilmiştir edilir. Ayrıca, aseton buharlaştırma testte uygulanan soğuk uyarıcı uygulanmış aseton miktarına bağlı olarak ve ölçmek zordur değişebilir. Son olarak, zarar görmemiş farelerde, başlangıçta aseton minimum bir yanıt sahip imkansız bu yöntemle aşırı duyarlılık yokluğunda analjezi ölçmek için adrestir. Soğuk yanıtlar için başka klasik tahlili Kuyruk soğuk su 5,6 batırılır sonra geri çekilmesi için gecikme ölçülür Kuyruk titremesi tahlili vardır. Bu deneyde davranışsal tepkileri kesin ve deney, belirli bir sıcaklığa yanıtları ölçen birlikte, hayvanlar iyi tarif edilen stres kaynaklı analjezik mekanizmaları 7 boyunca soğuk yanıt değiştirebilir ki, test sırasında tespit edilmelidir. Başka yaygın kullanılan bir araçtır davranışsal ölçen soğuk plaka testi, birfarelerin tepkileri bir Peltier soğutulmuş plaka 8-10 yerleştirilirler. Bu araç, belirli sıcaklıklarda hayvan yanıtları hakkında bilgi sağlarken, aynı zamanda tutarsız kullanılmıştır; farklı gruplar atlar 8,11 sayısı, ilk tepki 8,11- 13 gecikme dahil yanıtların farklı ölçüldü ve pençe sayısı çok farklı sonuçlar 11,13,14 kaldırır. Soğuk plaka deneyi de bir seferde test edilebilir tek bir hayvan gibi nispeten düşük kapasiteli olduğunu ve pahalı ve kırılgan Peltier cihazı gerektirir. 2-plaka sıcaklığı tercih testi hayvanlar farklı sıcaklıklarda 9,15- 17 2 bağlantılı plakalar üzerinde harcama zaman göreceli miktarını ölçen soğuk plaka testi yaygın olarak kullanılan bir türevidir. Bir başka benzer yaygın olarak kullanılan tahlil termal gradyan tahlil, fareler, farklı sıcaklık bölgelerinde geçirmek zaman miktarıdıruzun bir metal plaka ile, 5 ° C ile 45 ° C arasında değişen 16 ölçülür. Bu deneyler sıcaklık karşılaştırılmasına olanak iken, davranış sıcaklığı kaçınma veya sıcaklık tercihine temsil belirsizdir. Son olarak, dinamik soğuk plaka tahlil fareler ortam sıcaklıkları 18 değişen nasıl tepki ölçmek için kullanılır olmuştur. Bu yöntem, bir RT Peltier cihazda fareler yerleştirerek ve fareler atlamak veya farklı plaka sıcaklıklarda pençeleri yalamak ne kadar ölçüm yaparken 1 ° C aşağı rampa içerir. Bu fareler, bir soğutma ortamına uyum nasıl test ederken, bu fareler bir soğutucu ortam sıcaklığının ortamda soğuk bir uyarana cevap nasıl test etmek için bir yol sağlamaz. Ayrıca, bunu gerçekleştirmek için pahalı ekipman gerektirir ve soğuk hassasiyeti ölçmeden önce test ekipmanları fareler gelmesini için bir yol sağlamaz. Bu analizleri tamamlamak için, EBM acclima testleriSıcaklık aralıkları çeşitli de iyi tanımlanmış bir soğuk uyarıcıya Ted tepkileri ya da soğuk ortam sıcaklıklarında adapte işlemi esnasında uygulanabilir. Bu ucuz yüksek verimli testi için ölçeklendirilebilir potansiyeline sahip, bizim şimdiki aparatı ile bir seferde 14 farelere kadar test edebilirsiniz.

<table><tbody><tr><td>T-type thermocouple probe</td><td>Physitemp</td><td>IT-24p</td><td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td></tr><tr><td>Glass plate</td><td>Local glass company (in St. Louis, Stemmerich Inc)</td><td></td><td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#039;&#039;, but 3/16&#039;&#039; and 1/8&#039;&#039; are also useful</td></tr><tr><td>Thermal Data logger</td><td>Extech</td><td>EA15</td><td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&amp;amp;prodid=408)</td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td></tr><tr><td>Computer</td><td>If using Extech logger, any Pcwill work</td><td></td><td></td></tr><tr><td>Aluminum boxes</td><td>Washington University in St. Louis machine shop</td><td></td><td>boxes are 3&#039; long, 4.5&#039;&#039; wide, and 3&#039;&#039; tall with a sealed lid.&amp;nbsp; There is a 1/2&#039;&#039; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td></tr><tr><td>Heated water circulator</td><td>VWR</td><td></td><td>Any water circulator model with a pump will work</td></tr><tr><td>21 G needle</td><td>BD</td><td>305165</td><td>The exact needle size is not important</td></tr><tr><td>Hand timer</td><td></td><td></td><td>Any hand timer will work</td></tr><tr><td>Mirror</td><td></td><td></td><td>Any flat mirror will work</td></tr></tbody></table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

30 ° C, 23 ° C, 17 ° C ve 12 ° C &#39;de başlayan farelerden ortaya davranışsal tepkileri yüksek oranda tekrarlanabilir (Şekil 4A), 20 bulunmaktadır. Arka ayağın altında elde edilen soğuk uyarıcı ölçmek için, fareler ketamin / ksilazin / asepromazin kokteyli ile anestezi uygulandı ve pençeleri bir T-tipi filaman termokupl (Şekil 4B), 20 üst camın üzerine tespit edilmiştir. Cam soğuduktan veya arzu edilen test aralığına ısıtıldı. Pl...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

a-simple-inexpensive-method-for-determining-cold-sensitivity

Research

JoVE Journal

Neuroscience

14.7K Görüntüleme.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Video: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Basit ve Bilgisayar destekli Fareler için Koku Testi

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Davranış

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Farelerde Yerel Anafilaksi Ölçme

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

İmmünoloji ve Enfeksiyon

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Nörobilim

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Farelerde Işıktan Kaçınma Kullanarak Migren Benzeri Davranışların İncelenmesi

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Farelerde ve Sıçanlarda İskelet Kas Termogenezinin Ölçülmesi

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

Ürokinaz tipi Plazminojen Aktivatörü ile İndüklenen Fare Sırt Ağrısı Modeli

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Kahverengi Yağ Dokusunda Metabolit Değişiminin Araştırılması

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Yazar Spotlight: Yağ Dokusu Metabolizması ve Hastalıkların Önlenmesi için Yeni Yolların Aydınlatılması 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Kahverengi Yağ Dokusunda İn Vivo Metabolit Değişimini Ölçmek için Arteriyovenöz Metabolomikler

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Biyokimya

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Soğuk nosisepsiyon için Roman Tahlili<em&gt; Drosophila</em&gt; Larva

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Basit ve Bilgisayar destekli Fareler için Koku Testi

Farelerde Yerel Anafilaksi Ölçme

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Farelerde Işıktan Kaçınma Kullanarak Migren Benzeri Davranışların İncelenmesi

Farelerde ve Sıçanlarda İskelet Kas Termogenezinin Ölçülmesi

Ürokinaz tipi Plazminojen Aktivatörü ile İndüklenen Fare Sırt Ağrısı Modeli

Kahverengi Yağ Dokusunda İn Vivo Metabolit Değişimini Ölçmek için Arteriyovenöz Metabolomikler

Kahverengi Yağ Dokusunda İn Vivo Metabolit Değişimini Ölçmek için Arteriyovenöz Metabolomikler

Kahverengi Yağ Dokusunda İn Vivo Metabolit Değişimini Ölçmek için Arteriyovenöz Metabolomikler

Soğuk nosisepsiyon için Roman Tahlili