Çalışma Linköping Üniversitesi ve Östergötland ve İlçe Belediyesi tarafından finanse edildi.

1. Dorsal Rota aracılığıyla Ovariektomi <ol><li> Bir oksijen / nitrik oksit,% 30 /% 70 karışımı içinde% 1.5 izofluran (indüksiyonu için% 4) kullanılarak, örneğin hayvansal uyuşturmak. Bir rektal termometre bağlı bir ısıtma plaka üzerinde pozisyonda hayvan yerleştirin. Bu hayvan ve plaka arasında izolasyon ince bir tabaka olması önemlidir ve bu termometre geri besleme sistemi 37 içinde vücut ısısını korur ± 0.5 ° C Göz koruması için jel sürün ve postoperatif ağrı için hayvanın boyun lateral üzerindeki deri altına serum fizyolojik ile sulandırılmış 5 mg Rimadyl / kg vücut ağırlığı yönetmek. </li><li> Iliak kanattan cephally bir 4 * 4 cm (3 fareler için * 3 cm) alan Tıraş. Iyice bir 4 * 4 cm açıklık cerrahi sac ile hayvan kapsayan önce Örneğin Jodopax için ile traş yıkayın. </li><li> 2-3 cm&#39;lik orta hat kesi olun ve açık açık yatan fasya karşı cildi incelemek. Orta hat lateral Bir cm, başka bir kesi yapmak uçtan bir ucafasya ugh. Açık açık karın boşluğuna ulaşana kadar yüzeysel olarak mümkün olduğunca bu fasya altında yanal incelemek. </li><li> Cımbız kullanarak, karın boşluğunda over çevreleyen yağ dokusu kapmak ve yavaşça dışarı çekin. Over belirlemek ve uterin boynuz ve bu yapının proksimalde damarları 0.5-1 cm ligate. Over ve bağlanan yağ dokusu kesin ve karın boşluğuna kalan doku geri koydum. Kontralateral fasya başka bir kesi yapmak ve işlemi tekrarlayın. </li><li> Tercihen bir monofilament sütür kullanılarak, yaraları kapatmak ve anesteziden uyanık hayvan izin verin. Cerrahisinden sonra, en azından 2 saat boyunca ısıtılmış bir kafes (25-27 ° C) içinde hayvan yerleştirin. Hayvanlar birinci gün postoperatif ayrı muhafaza edilmelidir. Bu kafes sık iyileşme döneminde temizlenir / değiştirilir çok önemlidir. Yirmi dört saat cerrahisinden sonra, bir başka ile seyreltilir 5 mg Rimadyl / kg vücut ağırlığı ile deri altından enjekte hayvanlarıtuzlu. </li></ol> 2. Silastik Kapsül ile 17β-östradiol Subkutan İdaresi <ol><li> Iyice susam yağı 17β-östradiol istenilen miktarda karıştırın. Sıçanlar ve fareler fizyolojik konsantrasyonları için, sırasıyla 180 mcg / mL ve mcg / mL 18-36 öneririz. Plasebo kapsüller saf susam yağı ile doldurulur. </li><li> 3 cm (fareler için 2 cm) silastik boru uzunlukları ve 5 mm (fareler için 3 mm) takmak için ahşap aplikatör sopalarla uzunlukları kesin. Forseps kullanarak, tüm uzunluğu doldurmak için 17β-estradiol/sesame yağ solüsyonu enjekte önce silastik kapsül içine bir ahşap fiş sıkın. Yavaşça ahşap bir fiş (fişler kapsül kalıcı kalması beklenen) ile açık sonunda silastik kapsül kap. Implantasyondan önce, bir gece boyunca geri kalan 17β-östradiol çözelti içinde kapsül inkübe. </li><li> Kapsülün implantasyon sıçanlarda ovariektomi kullanılan anestezi gerçekleştirilebilir. Mi içince, ancak ovariektomi sonra 14 günlük bir iyileşme süresi kuvvetle narin deri yoluyla çıkıntılı kapsül riskini en aza indirmek için tavsiye edilir. </li><li> 1.1 gibi, ya ovariektomi insizyon kapatıldıktan sonra cerrahi hazırlıklardan sonra, tıraş ve hayvanın boyun dorsalinde 2 * 2 cm yıkayın. 1 cm&#39;lik bir kesi yapmak ve pervasızca kapsül için geniş bir alan olan, caudolaterally bir deri altı cebe incelemek. Silastik kapsül (bu zaman noktaya kadar 17β-östradiol çözüm inkübe edildiği) silin ve yavaşça sonradan bir dikişle kapatılır kesi yoluyla yükleyin. Bu emin olmak için çok önemli olduğu, özellikle yara yakın alanda mümkün olduğunca az kapsül suşları cilt. Implantasyon kapsül üretimi bakıldığında, kirlenmenin önlendiği önemlidir. </li></ol> 3. Nut-krem Nutella ile 17β-östradiol peroral İdaresi <ol><li> Iyice erimesi 17β-sonra susam yağı östradiol ve Nutella ile susam yağı karıştırın. Her günlük kısmı 17β-östradiol 28 mikrogram, 5 uL susam yağı ve bir kilogram vücut ağırlığı ve sıçanlar için 17β-estradiol 1.12 mg, 0,312 uL susam yağı ve her 30-g fare için 60 mg Nutella başına 1 gr Nutella içerir öneririz. Placebo ceviz krem ​​17β-östradiol olmadan hariç aynı hazırlanır. </li><li> Deney öncesinde beş gün için hormon içermeyen ceviz krem ​​yemeği için hayvanlar eğitin. Eğitimin ilk üç gün, fareler optimal evlerinde kafeslerde, (yaklaşık 0.5 gr Nutella 2-5 hayvanlar için) grupları konusunda eğitilmelidir. Eğitimin dördüncü ve beşinci günlerde, hayvanların deneysel duruma benzemeye günlük kısmını almadan önce ayrı kafeslere konur. </li><li> Deneme sırasında, hayvanların Nutella kısmı küçük seramik karoların üzerinde servis edilir sonra, her beslenmeden durum için ayrı kafeslere konur. Eğitim yukarıda, o gibi yapıldıysaur deneyim sıçan ve farelerin fazla% 95 fındık-krem kabul, ve bir kez tam habitüe, hayvanların çoğu saniye içinde tüketmek olmasıdır. </li></ol> 4. Temsilcisi Sonuçlar Mevcut yöntemlerin yeniden hedefliyoruz serum fizyolojik 17β-östradiol konsantrasyonu, yaklaşık olarak 5-140 pg / sıçan ve fareler 4,6 için 5-35 pg / mL için ml. Subkutan silastik kapsülleri yukarıdaki talimatlara göre, 300 gram dişi Sprague-Dawley sıçanlara zaman, yavaş yavaş pg / mL günde yaklaşık 40 adet 2 ila 10 pg / mL günde 35 17β-östradiol serum konsantrasyonları azalmakta üretirler. Ancak, hemen uygulamadan sonra, 1000 pg / ml civarında bir serum pik konsantrasyonu 4,5 (Şekil 1A) 8 saat içinde fizyolojik aralığına düşen beklenebilir. 25 g dişi C57BL / 6 fare uygulanmıştır 36 ug 17β-estradiol/mL, yukarıda tarif fareler için silastik kapsüller, serum kons işlemekyavaş yavaş yaklaşık 90 pg / mL günde 2 ila 25 pg / mL günde 35 düşmesi entrations. Bu (Şekil 1B) 6 incelenmemiştir rağmen, sıçanlarda bulundu gibi Bu muhtemelen benzer bir pik önce gelmelidir. Dozlarda peroral Nutella yöntemi 300 g dişi Sprague-Dawley farelerinin uygulandığı zaman, yukarıda anlatılan 10-70 pg / ml (Şekil 2A) 4 arasında günlük dalgalanmalarla 17β-estradiol fizyolojik seviyeleri vermektedir. Yukarıda belirtilen dozlar kullanılarak fareler için, günde yaklaşık 20 seviyeleri ve 120 pg / ml (Şekil 2B) 6 arasında değişen. <img alt="Şekil 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> . sıçanlarda 17β-östradiol seviyelerini gösteren rong&gt; Şekil 1 Stilize grafikleri; 4,5 ve fareler (Bir sonraki zaman noktalarında n zaman puan = 10 en çok 24 saat, n = 15) (B n = 10) kapsül implantasyon sonrası 6. Silastik kapsülleri 1000 serum konsantrasyonları kısa bir başlangıç ​​zirveye kadar üretmek pg / ml, ama 100 pg / ml altındaki seviyelere saat içinde azalır. Giderek azalmakta olsa da önümüzdeki 4-5 hafta için, kapsül, serum konsantrasyonları istikrarlı verim. Gölgeli alanlar sırasıyla sağlam dişi sıçanlar ve fareler fizyolojik 17β-östradiol serum karşılık gelir. Görsel grafikler gösterilememiştir rağmen, inter-bireysel değişkenliği (çok düşük ortalama 27 sıçanlarda% 183 ve 28 silastik kapsül yöntemi ile farelerde% 115 değişkenlik katsayıları render 17β-östradiol konsantrasyonu vardır konsantrasyonları genellikle) varyasyon katsayıları yüksek oluşturur. iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; Şekil 2 sıçanlarda 17β-östradiol konsantrasyonu sunan Stilize grafikler (A; n = 15). 4 ve fareler (B n = 10) Nutella günlük hormonu verilmesinin bir sonucu olarak 6. Peroral Nutella yöntem subkutan veya intravenöz yöntemlerle üretilen kadar yüksek değil ancak açık farkla, günlük pik vermektedir. Bu insan hormon replasman tedavisi farmakokinetiği taklit eden ve oral tedavi sadece yukarıdaki örnekte ilk 96 saat sunmak bile, gerekli olduğu sürece için devam edebilirsiniz. Gölgeli alanlar sırasıyla sağlam dişi sıçanlar ve fareler fizyolojik 17β-östradiol serum karşılık gelir. Rağmen görsel grafikler gösterildiği değil, inter-bireysel değişkenliği (çok düşük ortalama konsantrasyonları Nutella yöntemi ile farelerde sıçanlarda 40-% 132 den ve 13 219% ile varyasyon katsayıları render 17β-östradiol konsantrasyonu vardır genellikle yüksek üretmekvaryasyon katsayıları).

<ol>
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Yazarlar ifşa hiçbir şey yok.

Daha önceki çalışmalarda var Yukarıda sunulan yönetim modları 17β-östradiol fizyolojik serum üretimi açısından ticari yavaş salımlı tablet ve enjeksiyon üstün olduğunu kanıtladı. Yavaş salınımlı pelet çok yüksek ve uzun süreli serum konsantrasyonu zirveleri üretirken, enjeksiyon aşırı, günlük hormon dalgalanmalara neden ve hayvanların 4-6 tekrarlanan stresli ele alınmayı gerektirir. Yeterli yönetim modları seçmenin önemi düşük doz 17β-östradiol yönetim rejimleri ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Rimadyl, 50 mg / ml </td><td> Pfizer </td><td> 462986 </td><td> Ovariektomi postoperatif analjezi. </td></tr><tr><td> Lubrithal göz jeli </td><td> Leo Laboratuvarları </td><td></td><td> Ameliyat sırasında göz koruması. </td></tr><tr><td> Jodopax veteriner </td><td> Pharmaxim </td><td></td><td> Cerrahi dezenfeksiyon. </td></tr><tr><td> Vicryl 4-0 sütür </td><td> Johnson &amp; Johnson </td><td></td><td> Sütür ve over ligasyonu Yaralanması. </td></tr><tr><td> 17β-estradiol,&gt;% 98 </td><td> Sigma </td><td> E8875-1G </td><td> Silastik kapsül ve Nutella için susam yağı içinde. </td></tr><tr><td> Susam yağı </td><td> Sigma </td><td> S3547-1L </td><td> 17β-östradiol dağıtılması. </td></tr><tr><td> Silastik tüp, ID / OD: 1.575/3.175 mm </td><td> Dow Corning </td><td> 508-008 </td><td> Silastik kapsül üretimi. </td></tr><tr><td> Ahşap aplikatör çubuk </td><td> Selefatrade </td><td> 12-0783 </td><td> Silastik kapsül Kapatma. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Peroral 17β-östradiol için Orta. </td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

Östrojenler etkileri istisnai olarak geniş bir yelpazeye sahip kadın cinsel hormonların bir aile vardır. Sıçan ve farelerde östrojen araştırmalarında kullanılan ettiklerinde genellikle 17β-östradiol Eksojen yerine, hızla bisiklete hormon üretimini baskılamak için ovarektomi edilir. Hormonu fizyolojik serum elde etmek için uygulanmalıdır nasıl olduğu konusunda görüş birliği eksikliği Ancak, vardır. 17β-östradiol seviyesi / uygulama yöntemi derinden deneysel sonuçlar 1-3 etkilediğinden Bu çok önemlidir. Biz subkutan silastik kapsül ve başına sözlü somun-krem Nutella ticari yavaş salımlı granül (Amerika Yenilikçi Araştırma şirketi tarafından üretilen) üstün olduğu bulma, 17β-östradiol yönetimin farklı modları karakterize bir dizi çalışma var ve 17β-estradiol 4-6 fizyolojik serum konsantrasyonları üretimi açısından günlük enjeksiyonlar. Adv arasındaDaha önce buprenorfin idaresi 7 için kullanılır olmuştur fındık dondurma yöntemi, bir antages östrojen yönetimi için kullanılan zaman oral hormon replasman tedavisi benzer ve non-invaziv olmasıdır. Subkutan silastik kapsüller uygundur ve çok kararlı serum konsantrasyonları üretirler. Bu video, makale ovariektomi adım adım gösteri ve silastik kapsül ve yönetim prosedürlerinin önemli yönlerini bir tartışma izler sıçan ve farelerde yapılan oral Nutella tarafından 17β-östradiol hormon replasman içerir.

Watch this Scientific Journal Video about Ovariectomy and 17Î²-estradiol Replacement in Rats and Mice: A Visual Demonstration at JoVE.com

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Silastik kapsüller ve peroral Nutella vasıtası ile overlerin alınması ve 17β-estradiol müteakip değiştirilmesi sıçan ve farelerin gösterilmiştir.

Sıçan ve Farelerde Ovariektomi ve 17β-östradiol Değiştirme: Görsel Gösteri

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

ovariectomy-17-estradiol-replacement-rats-mice-visual

Research

JoVE Journal

Biology

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

56.7K Görüntüleme.  Linköping University. Silastik kapsüller ve peroral Nutella vasıtası ile overlerin alınması ve 17β-estradiol müteakip değiştirilmesi sıçan ve farelerin gösterilmiştir.

Video: Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Drosophila Görsel ve Koku aracılığı ile Uçuş Kontrol Araştırma Manyetik Tether Sistemi

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Biyoloji

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Gösterme Deri allodini Kronik Pelvik Ağrı Derneği

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Genetik Đndüklenebilir Kader Haritalama Pratik Bir Yaklaşım: Hücreler Mark ve Track Görsel Bir Rehberi In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Manuel Tutucu ve Fare ve Sıçanlarda Ortak Bileşik İdaresi Rotalar

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Tanı Otopsi ve Sıçanlar ve Fareler Seçilmiş Doku ve Örnek Toplama

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Drosophila'da Sıcaklık Tercih Davranışının ve Sirkadiyen Ritminin Tasarımı ve Analizi

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Fizyoloji Laboratuvarı Gösteri: Bir Sıçan Glomerüler Filtrasyon Hızı

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Fare Modeli Doğrulanması Hücre-spesifik şekilde LINC kompleksler bozacak

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

Vivo Algılama ve Rb Protein SUMOylation insan hücrelerinde analizi

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...