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.

中sRANKL MC DNA 1。水动力传递<ol><li>通过小鼠尾静脉流体力学交货<ol><li>尾静脉注射前称量鼠标。中的小鼠体重的10％〜的总体积稀释sRANKL或绿色荧光蛋白（GFP）的MC在林格氏溶液（预暖，在37℃）。 </li><li>热身鼠标在一个笼子10分钟前注射，以扩张血管，使侧脉（LVS）可见。监视鼠标小心，以避免脱水和高热。 </li><li>只要LV的被扩张，可见，鼠标转移到阻挡并将插头远远不够倒入桶内，以抑制运动。监视鼠标正常活动如呼吸。如果需要调整限制器的插头。 </li><li>从尾尖用酒精擦拭消毒注射区的3/4。使用27-30号针头的小鼠尾静脉注射。紧紧握住尾巴用一只手插入针到尾静脉。施加压力的注射器，并在5-7秒完成注射。 </li><li>从尾静脉中取出针头并用棉球将压力施加于注射部位止血。监视鼠标15-30分钟，再用鼠标转回动物饲养，一旦呼吸率降低到正常速度。 </li></ol></li><li>的小鼠血清TRACP5b的帖子基因转移系统性鼠标sRANKL的表达和定量确认<ol><li>热身鼠标在一个笼子里5-10分钟，以扩张血管，使的LV可见。监视鼠标小心，以避免脱水和高热。 </li><li>作一小切口用刀片将鼠标的尾部在一个站点1列从尾部的尖端。 </li><li>收集约100微升的血液中的血清分离管中。 </li><li>孵育血清分离管在室温下放置30分钟。 </li><li>以10,000×g离心5分钟，离心样品并收集血清。 STOR在-80°C，E无血清作进一步的分析。 </li><li>使用市售的试剂盒血清样本进行sRANKL ELISA和小鼠血清TRACP5b的检测。 </li></ol></li></ol> 2，体外破骨细胞生成的鼠标桥梁养护管理<ol><li>隔离和桥梁养护管理的培养<ol><li>胫骨和股骨骨隔离：安乐死的供体小鼠与CO 2，消毒腿用70％乙醇。从耻骨解剖到跟骨，以除去股骨和胫骨完好无损。 </li><li>小心地取出皮肤和肌肉不破坏骨。放置处理股骨和胫骨中含有磷酸盐缓冲盐水（PBS）的培养皿中。 </li><li>去除骨髓：切断对股骨和胫骨的骨骼的一侧的前端和冲洗出骨髓入50毫升的管中，使用1ml注射器用25号针头，装有含1％青霉素/链霉素的α-MEM （破骨细胞培养基/ OCM）。 </li><l我&gt;过程骨髓：混合并通过一个70微米尼龙细胞过滤器传递给它的骨髓悬浮液转移至新的50ml管中。离心将细胞以300×g离心并重新悬浮在α-MEM中含有1％青霉素/链霉素和10％胎牛血清（破骨细胞培养基/ OCM（+））。 </li></ol></li><li>巨噬细胞的培养<ol><li>使用血球或自动细胞计数器计数细胞。 </li><li>板1×10 6每孔细胞的6孔板或3×10 5细胞在玻璃盖玻片（5毫米）或牙本质片放置在96孔板中孵育在37℃下</li><li>吸出物含有非贴壁细胞和培养细胞在37℃下用含有25纳克/毫升鼠M-CSF 48小时新鲜OCM（+）的所有介质。 </li><li>含有25毫微克/毫升鼠标M-CSF和30毫微克/毫升小鼠sRANKL启动破骨细胞吸出所有媒体和培养细胞在37℃的新鲜OCM（+）的媒体。根据需要补充媒体每3天。 </li><li>细胞生长约6-8天以形成能够再吸收骨巨多核细胞。只要多核细胞出现，执行TRAP（抗酒石酸酸性磷酸酶），使用市售的试剂盒染色，而当完全成熟，进行功能分析。直观上用鬼笔环肽染色和图像附着用扫描电子显微镜（SEM）如先前19描述牙本质片的细胞的盖玻片的F-肌动蛋白环。 </li></ol></li></ol> 3， 在体外破骨细胞生成从人PBMC <ol><li>人外周血单个核细胞的分离<ol><li>加入10 ml预热的Histopaque-1077的入50毫升的试管中。 </li><li>从用无菌PBS血库获得到50ml试管（1:1比例的血用PBS）空白细胞过滤器。层中的稀释血液在HISTOPAQUE溶液非常缓慢，并确保血液不与HISTOPAQUE混合。 </li><li>在1000×g离心20分钟，离心样品在18°C。跟随ING离心，小心分离含有白细胞的白色血沉棕黄层和层转移到新的50ml管中。 </li><li>在650×g离心5分钟，再稀释细胞，用PBS离心以收集细胞。 </li><li>在OCM去除上清，重悬细胞（ - ）介质，并使用细胞计数器计数细胞。 </li></ol></li><li>电镀与外周血单个核细胞培养<ol><li>重悬的细胞在OCM（+）介质和板8×10 6个细胞/ ml，每孔中×10 6个细胞/ ml，每孔在96孔板的6孔板或1。根据需要为免疫荧光成像，以及对骨片适于骨吸收测定法作为在玻璃盖玻片（5毫米）的板的细胞先前所描述20。 </li><li>改变培养基，补充细胞与人M-CSF，每2-3天或按要求。然后通过添加人sRANKL的30纳克/毫升连同25毫微克/毫升的人M-CSF在OCM（+）媒体发起osteoclastogenes进行到第5天的分化步骤是。 </li><li>生长细胞为约14至21天，以形成巨型多核细胞。只要多核细胞出现，这些细胞可以被标记为TRAP，并且当完全成熟，功能分析可以被执行。 F-肌动蛋白环可以可视化使用鬼笔环肽染色盖玻片。细胞的形态，可以检查连接在用SEM对牙本质切片的细胞​​。 </li></ol></li></ol>

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The authors have nothing to disclose.

肌肉骨骼条件，导致发病率和致残的主要原因，并包含超过150个疾病和证候;影响大约90万美国人。关节的炎症和骨质破坏是骨骼肌肉条件的主要功能，包括关节炎和骨质疏松症。骨质疏松症是骨弱化完整性，常常导致骨的骨折的情况。关节炎是一种慢性的，衰弱的疾病，其特征是变得肿胀，招标和僵硬，限制正常运动，并可能导致残疾的关节发炎。虽然肌肉骨骼疾病的发病机制大大不同于彼此，?...

肌肉骨骼疾病影响到千百万人在美国和现在的严重后果为国家和地方卫生系统1。这些疾病的特点是骨损失和关节功能，需要广泛的治疗和恢复很长时间。通常，在破骨细胞的数量和/或活性的相对增加，细胞专门的重吸收骨骼，在骨质疏松和关节炎，观察2。在生理条件下的破骨细胞的数量和活性是通过核因子κ-B配体（RANKL），它是由成骨细胞产生的受体活化调节。护骨素（OPG），诱饵受体RANKL由成骨细胞3也产生涉及sRANKL全身过度表达，或删除OPG的体内动物模型是非常有价值的骨质疏松症的研究。然而，这些方法需要转基因小鼠4,5的产生。这里，一种新型的替代过表达sRANKL为肌肉骨骼相关的疾病的研究的方法进行说明。具体地，小环（MC）DNA技术和流体动力传递方法被用来实现sRANKL的体内基因转移和过表达小鼠sRANKL全身6。 这种方法也补充其他体内模型的骨质疏松，如由低钙饮食8以下卵巢切除7和饮食干预破骨细胞的激素调节。这些模型是研究的但是他们需要外科手术，可能需要长达数月，在一个显著的成本9肌肉骨骼相关疾病的不同方面非常有用。卵巢切除（OVX）啮齿动物模型的实验动物模型，其中卵巢切除导致雌激素缺乏，从而模仿人类绝经后骨质疏松症10。人绝经期后骨质疏松症，一个条件，其中的雌激素deficiENCY导致骨折的风险增加和骨质疏松症影响约800万妇女在美国就有。虽然绝经后骨质疏松症的去势模型是非常有用的它提供了有限的优势，在一般的研究骨质疏松症。雌激素可以抑制骨质流失，通过诱导破骨细胞，抑制成骨细胞凋亡，因此，在其没有增加破骨细胞活性观察10-12。一个RANKL-OPG比例失衡有利于骨吸收也观察到13。然而， 在体内的雌激素缺乏也伴随着降低的转化生长因子β（TGFβ），增加白细胞介素-7（IL-7）和肿瘤坏死因子，IL-1和IL-6的水平14,15。由于这些细胞因子已知的骨重塑调节功能独立于RANKL通路，这是不可能的属性的任何破骨细胞的活化，仅与RANKL-RANK轴。在本文中描述的模型使研究人员在VIV研究ØRANKL-RANK轴的破骨细胞和骨量丢失无促炎细胞因子相比，去卵巢啮齿类动物模型。 此外， 体外破骨细胞技术是必不可少的工具来研究破骨细胞活化肌肉骨骼疾病的潜在治疗方法。以前的研究还表明，与小鼠巨噬细胞集落刺激因子（M-CSF）和小鼠sRANKL培养小鼠骨髓衍生的巨噬细胞（桥梁养护）能导致破骨细胞分化3,16,17。在此，协议来生成从小鼠骨髓以及从人外周血单核细胞（PBMC） 在体外 18多核破骨细胞样细胞进行了描述。都定义一个成熟的终末分化和功能齐全的破骨细胞所需的基于细胞的测定也简单说明。这些体外技术补充体内的方法小说一起用于为powerful调查工具，研究破骨细胞分化和活化。使用这些系统中，科学家们能够产生在体内和体外的破骨细胞和确定所需的增殖和活化的刺激和信号，以及测试的药理和生物抑制剂的功效。

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			<td height="21" style="height:21px;width:320px;">Tailveiner Restrainer for mice</td>
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			<td height="21" style="height:21px;">Acid Phosphatase, Leukocyte (TRAP) Kit</td>
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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.

这里， 在体内和细胞培养协议破骨细胞分化的新基因转移技术，用于差分前体细胞在体外破骨细胞作为研究细胞因子对破骨细胞形成的影响的方法进行了描述。在图1中，绿色荧光蛋白和小鼠sRANKL MC小鼠成功的基因转移的代表性结果示。在图2中，示出的小鼠骨髓或前体细胞向破骨细胞通过明场显微术的人PBMC细胞分化时程文化的代表性图像， 图3?...

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.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

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

a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

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 次观看.  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. 

视频: 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.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

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, ...

科研

医学

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.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

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.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

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

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

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.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

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.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

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

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

基于音频的虚拟游戏环境的发展，以帮助盲人的导航技巧

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

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.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

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.

肌肉骨骼超声成像的一种新应用

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.

特发性脊柱侧弯的使用蜂窝介电谱的预后预测细胞为基础的检测协议

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