Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

SRANKL MC DNA 1. Hidrodinamik Gönderme <ol><li> Fare Tail Ven yoluyla hidrodinamik Teslim <ol><li> Kuyruk damarından enjekte edilmesinden önce fare tartılır. Fare vücut ağırlığının ~% 10 bir toplam hacim içinde sRANKL veya Ringer çözeltisi yeşil floresan protein (GFP) MC (ön sıcak, 37 ° C&#39;de) seyreltin. </li><li> Kan damarlarını genişletmek ve görünür yan damarlar (LVs) yapmak için enjeksiyondan önce 10 dakika boyunca bir kafeste fare ısıtın. Dehidratasyon ve hipertermi önlemek için dikkatli fare izleyin. </li><li> En kısa sürede LVs dilate ve görünür gibi, süzgeç için fare aktarmak ve hareketi dizginlemek için varil içine yeterince fişini takın. Solunum gibi normal bir aktivite için fare izleyin. Gerekirse süzgeç fişini ayarlayın. </li><li> Silin bir alkol ile kuyruğun ucundan enjeksiyon alanı 3/4 dezenfekte. Fare kuyruğu ven enjeksiyon için 27-30 G iğne kullanın. Bir elinizle sıkıca tutun kuyruk ve iğneyikuyruk toplardamarına. Şırınga üzerinde baskı uygulayın ve 5-7 saniye içinde enjeksiyon tamamlayın. </li><li> Kuyruk damarından gelen iğne kaldırmak ve kanamayı durdurmak için enjeksiyon yerinde bir pamuk ile basınç uygulayın. Solunum hızı normal hızına düşürülür sonra bir kez vivari geri fare aktarmak, 15-30 dakika boyunca fare izleyin. </li></ol></li><li> Fare Serum TRACP5b Mesaj Gen Transferi Sistemik Fare sRANKL İfade ve Nicelik Onay <ol><li> Kan damarlarını genişletmek ve LVs görünür yapmak için 5-10 dakika için bir kafes içinde fare ısıtın. Dehidratasyon ve hipertermi önlemek için dikkatli fare izleyin. </li><li> Kuyruk ucundan ileri bir site bir de fare kuyruğuna bir bıçak ile küçük bir kesi olun. </li><li> Serum ayırma tüplerine kan yaklaşık 100 ul toplayın. </li><li> 30 dakika boyunca oda sıcaklığında serum ayırma tüpleri inkübe edin. </li><li> Santrifüj 5 dakika boyunca 10,000 x g&#39;de ve serum numuneleri toplamak. Stordaha fazla analiz için -80 ° C e serum. </li><li> Bir ticari olarak temin edilebilen bir kit kullanılarak serum örneklerinde sRANKL ELISA ve fare serum TRACP5b deneyi yapın. </li></ol></li></ol> Fare BMMs 2. In vitro Osteoklast Üretimi <ol><li> İzolasyon ve BMMs ekimi <ol><li> Tibia ve femur kemikleri İzolasyonu: CO 2 ile donör fare Euthanize, ve% 70 etanol ile dezenfekte bacaklar. Sağlam femur ve tibia kemikleri kaldırmak için, kalkaneus kemik kasık kemiğinden ayır. </li><li> Kemik zarar vermeden dikkatli bir cilt ve kas çıkarın. Fosfat tamponlu tuzlu su (PBS) ihtiva eden bir Petri çanağı içinde işlenmiş femur ve tibia kemiği yerleştirin. </li><li> Kemik iliği çıkarılması: femur ve tibia kemikleri bir tarafındaki ucu kesin ve α-MEM,% 1 penisilin / streptomisin içeren yüklü bir 25 G iğne ile bir 1 ml şırınga ile bir 50 ml tüp içine, kemik iliği atılmasına (Osteoklast Kültür Orta / OCM). </li><li&gt; işlem kemik iliği: karıştırın ve 70 um naylon hücre süzgecinden geçirilerek, yeni bir 50 ml tüp, kemik iliği süspansiyon aktarın. Santrifüj 300 xg ve en hücreler,% 1 penisilin / streptomisin ve% 10 FBS (osteoklast kültür ortamı / OCM (+)) ihtiva eden α-MEM içinde yeniden askıya. </li></ol></li><li> Makrofajlar Kültür <ol><li> Hemasitometre veya otomatik hücre sayıcı kullanarak hücreleri saymak. </li><li> 96 oyuklu bir plaka içerisine yerleştirildi ve 37 ° C de inkübe cam kapak slipleri (5 mm) ve diş kemiği dilimleri 6 kuyulu bir levha ya da 3 x 10 5 hücre için oyuk başına Plaka 1 x 10 6 hücre </li><li> Aspire yapışmayan hücreleri ihtiva eden ve 48 saat boyunca 25 ng / ml fare M-CSF ihtiva eden taze OCM (+) ile 37 ° C&#39;de inkübe hücreleri tüm medya. </li><li> 25 ng / ml fare M-CSF ve osteoklastojenezi başlatmak için 30 ng / ml fare sRANKL ihtiva eden aspire tüm medya ve taze OCM ile 37 ° C&#39;de inkübe hücreleri (+) media. Gerektiği gibi her 3 günde bir ortam doldurun. </li><li> Hücrelerin büyümesineyaklaşık 6-8 gün kemik eritme kapasitesine sahip çok çekirdekli dev hücrelerin oluşturulması için. En kısa sürede, çok çekirdekli hücreler görünür gibi, ticari bir kit kullanılarak boyanan TRAP (tartarat dirençli asit fosfataz) yapmak, ve tam olgunlaşmış zaman, fonksiyonel testleri gerçekleştirmek. Phalloidin leke ve resim daha önce tarif edildiği gibi 19 elektron mikroskopi taraması (SEM) ile dentin dilimleri bağlanmış hücreler kullanılarak lamelleri F-aktin halkaları görselleştirme. </li></ol></li></ol> İnsan PBMC &#39;den 3. In vitro Osteoklast Üretimi <ol><li> İnsan PBMC İzolasyonu <ol><li> Bir 50 ml tüp içine önceden ısıtılmış Histopaque-1077 10 ml ilave edilir. </li><li> Boş lökosit filtre 50 ml tüp (PBS ile kan 1:1 oranında) içine steril PBS ile kan bankasından elde. Kan histopaque karışmadığı emin, çok yavaş histopaque çözüm üzerinde seyreltilmiş kan Katman. </li><li> 18 ° C&#39;de 20 dakika boyunca 1000 x g&#39;de santrifüjleyin örnekleri Takip etmeksantrifüj ing dikkatlice beyaz kan hücreleri içeren beyaz ince beyaz tabaka tabaka izole edilmesi ve yeni bir 50 ml&#39;lik tüpe transfer. </li><li> Hücreleri toplamak için 5 dakika boyunca 650 x g hızında tekrar PBS ve santrifüj ile hücreler seyreltin. </li><li> Medya ve hücre sayacı kullanarak hücrelerin sayısı (-) OCM süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li></ol></li><li> PBMC&#39;Ierde Kaplama ve kültüre <ol><li> OCM hücrelerin tekrar (+) ve ortam levha 8 x 10 ug / ml göz başına 6 çukurlu bir levha ya da 1 x 10 6 hücre / ml her bir kuyucuk için 96 oyuklu bir plaka içerisinde 6 hücre. Gibi immünofloresan görüntüleme gibi kemik erimesi deneyleri için uygun kemik dilimleri üzerinde için gerekli olan cam kapak slipleri (5 mm) levhalar 20 hücreleri daha önce tarif edildiği. </li><li> Değişim kültür ortamı, insan M-CSF, her 2-3 günde bir ile ya da gerektiğinde hücreleri ilave etme. Daha sonra osteoclastogenes başlatmak için OCM (+) ortamı içinde 25 ng / ml insan M-CSF ile birlikte insan sRANKL 30 ng / ml ilave etmek suretiyle, 5. günde farklılaşma aşamasına geçinolduğunu. </li><li> Çok çekirdekli dev hücreler oluşturmak için yaklaşık olarak 14-21 gün için hücreler büyür. En kısa sürede çok çekirdekli hücreler olarak görünür, bu hücreler daha sonra TRAP için etiketlenebilir ve tam olarak olgunlaşmış zaman, işlevsel tahliller gerçekleştirilebilir. F-aktin halkaları phalloidin leke kullanarak lamelleri üzerinde görülebilir. Hücre morfolojisi, SEM ile dentin dilimleri bağlı hücrelerde incelenebilir. </li></ol></li></ol>

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The authors have nothing to disclose.

Kas-iskelet koşulları morbidite ve sakatlık nedenleri lider vardır ve 150&#39;den fazla hastalık ve sendromların oluşmaktadır; Bugün yaklaşık 90 milyon Amerikalıyı etkileyen. Eklem enflamasyonu ve kemik yok artrit ve osteoporoz dahil olmak üzere kas-iskelet sistemi koşulları baskın özellikleri vardır. Osteoporoz genellikle kemik kırıkları yol açan kemik bütünlüğünü zayıflatan bir durumdur. Artrit şiş, hassas ve sert kısıtlayan normal hareket olmak ve sakatlığa yol açabilir eklem ilti...

Kas-iskelet sistemi hastalıkları, ulusal ve yerel sağlık sistemleri için 1 Amerika Birleşik Devletleri ve mevcut ciddi sonuçlara milyonlarca insanı etkiler. Bu bozukluklar arasında kemik kaybı ve geniş bir tedavi ve kurtarma uzun süre gerektiren eklem fonksiyonu ile karakterize edilir. Genellikle, osteoklastların sayısı ve / veya aktivitesinde bir nispi artış, hücre osteoporozda, kemik emmek için özel ve artrit 2 görülmektedir. Fizyolojik koşullar altında, osteoklastların sayısı ve etkinliği osteoblastlar tarafından üretilen nükleer faktör κ-B ligandının (RANKL) reseptör aktivatörü düzenlenir. Osteoprotegerin (OPG), RANKL için bir yem reseptörü de 3 osteoblastlar tarafından üretilen sistemik sRANKL aşın veya OPG silinmesini içerir in vivo hayvan modelleri, osteoporoz araştırmalarında, çok değerlidir.; Bununla birlikte, bu yöntemler, transgenik farelerin 4,5 üretilmesini gerektirir. Burada, yeni bir alternatifkas-iskelet sistemi ile ilgili hastalıkların çalışma için sRANKL aşırı ifade eden bir yöntem tarif edilmektedir. Özellikle, minicircle (MC) DNA teknolojisi ve hidrodinamik dağıtım yöntemler, canlı ortam içinde sRANKL arasında gen transferini elde etmek ve sistemik 6 fare sRANKL aşırı ifade etmek için kullanılmıştır. Bu yöntem, aynı zamanda, düşük kalsiyum diyeti 8 ile ovariektomi 7 ve diyet müdahalesi şu osteoklast hormonal düzenlenmesi gibi osteoporoz ve diğer in vivo modeller, tamamlayıcıdır. Bu modeller cerrahi gerektiren ve önemli bir maliyetle 9 at, birkaç aya kadar sürebilir ancak kas-iskelet sistemi ile ilgili bozuklukların farklı yönlerini incelemek için çok yararlıdır. Ovariektomi uygulanmış olan (OVX) kemirgen modeli yumurtalıkların çıkarılması böylece insan menopoz sonrası osteoporoz 10 taklit eden estrojen eksikliğine yol açar deneysel bir hayvan modelidir. İnsan menopoz sonrası osteoporoz, östrojen yetersizliklerinin bir durumdurency kemik kırılmaları yönünden artan bir riske yol açar ve osteoporoz sadece Amerika Birleşik Devletleri&#39;nde yaklaşık sekiz milyon kadınları etkiler. OVX modeli, menopoz sonrası osteoporoz için faydalı olmasına rağmen, genel olarak osteoporoz okuyan sınırlı avantajlar sunmaktadır. Östrojen yokluğunda bu nedenle de artmış osteoklast aktivitesi 10-12 görülmektedir, osteoklast uyarılması ve osteoblast apoptoz inhibe ederek, kemik kaybını önler. Kemik erimesini yana A RANKL&#39;ın-OPG oranı dengesizlik ayrıca 13 görülmektedir. Büyüme faktörü β (TGF β), yüksek interlökin-7 (IL-7) ve TNF, IL-1 ve IL-6 14,15 dönüşüm seviyelerinin uzaması Bununla birlikte, in vivo koşullarında östrojen eksikliği de eşlik etmektedir. Bu sitokinler RANKL&#39;ın yolunun kemik yeniden düzenleyici fonksiyonları bağımsız bilinen gibi, sadece RANKL-RANK eksenine herhangi bir osteoklast aktivasyonunu atfetmek mümkün değildir. Bu yazıda anlatılan modeli viv çalışma için araştırmacılar sağlaro pro-enflamatuar sitokinler olmaksızın osteoclastogenez ve kemik kaybına RANKL-RANK eksen OVX kemirgen modelleri ile karşılaştırıldığında. Buna ek olarak, in vitro osteoklastojenezisi teknikleri kas-iskelet sistemi hastalıklarının tedavisi için potansiyel terapötik osteoklast aktivasyonunu çalışma için gerekli araçlardır. Daha önceki çalışmalar, aynı zamanda, fare makrofaj koloni uyarıcı faktör (M-CSF) ve fare sRANKL ile fare kemik iliğinden elde edilen makrofajların (BMMs) kültürlenmesi osteoclast farklılaşmasında 3,16,17 yol göstermiştir. Burada, fare kemik iliği hem de in vitro 18, insan periferal kan tek-çekirdekli hücreleri (PBMC) için çok çekirdekli osteoklast-benzeri hücreleri oluşturmak için protokolleri açıklanmıştır. Olgun terminal açıdan farklılaşmış ve tamamen işlevsel osteoklast tanımlamak için gerekli olan hücre bazlı deneyler, aynı zamanda kısaca açıklanmıştır. Bu in vitro teknikler vivo yaklaşım romanı tamamlamak ve p olarak hizmetowerful soruşturma araçlar osteoklast farklılaşmasını ve aktivasyonunu incelemek. Bu sistemleri kullanarak, bilim adamları, in vivo ve in vitro olarak osteoklastlar üretmek ve bunların proliferasyon ve aktivasyon için gerekli olan uyarıcılar ve sinyaller tespit hem de farmakolojik ve biyolojik inhibitörlerinin sonuç verirliğinin test edilmesi mümkün bulunmaktadır.

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a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Burada, osteoklastojenez sitokinlerin etkilerini incelemek için bir yöntem olarak, in vitro olarak osteoklast öncü hücreleri ayırt etmek için in vivo ve hücre kültürü protokollerinde osteoklast farklılaşması için yeni bir gen transfer tekniği tarif edilmektedir. Şekil 1, farelerde GFP ve fare sRANKL MC başarılı gen transferi için temsili sonuçları gösterilmiştir. Şekil 2&#39;de, fare kemik iliği ya da parlak alan mikroskopi ile osteo...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Bir Roman<em&gt; In vivo</em&gt; Gen Transferi Tekniği ve<em&gt; In vitro</emİskelet Hastalıkları Kemik Kaybı Çalışmaları&gt; Hücre Tabanlı Tahliller

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K Görüntüleme.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Ortak Bozuklukları de Novo Mutasyonlar, otizm ve şizofreni gibi tanımlayın Bir Strateji

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Tıp

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Bağışıklık Eksik Fare Kuruluş ve İnsan Retinoblastom Tümörler yayılımı

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Roman kullanarak Spinal Kord Yaralanması sonra Fare Hind Ekstremite Kemik Kaybı Boyuna değerlendirilmesi, In vivo, Metodolojisi

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

TSSB, Nöral Hematolojik ve Davranış Bağlantılı Revealing için Hayvan Modelinde Biyobelirteçler

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Bir Fetal Fare Modelinde Biyoaktif Ajan intratrakeal Yönetimi İçin Yeni Bir Cerrahi Yaklaşım

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Yeni Bir Yüksek çözünürlüklü<em&gt; In vivo</emTümör Büyüme ve Tedavi için İntrakranial Yapıların Dinamik Tepki Eğitim&gt; Görüntüleme Tekniği

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Kas-iskelet Ultrason Görüntüleme Bir Roman Uygulama

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Hücresel Dielektrik Spektroskopisi kullanarak İdiopatik Skolyoz Prognostik Tahmininde Hücre-tabanlı Deney Protokolü

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

Hücre Takip için vivo 19 F MRG

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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