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の1。流体力学的送達<ol><li>マウス尾静脈を介して流体力学的送達<ol><li>尾静脈注射の前に、マウスを秤量する。リンゲル液中の希釈sRANKL又は緑色蛍光タンパク質（GFP）MC（37℃で予備加温）マウスの体重の約10％の総体積。 </li><li>血管を拡張し、横方向の静脈LVS（）が表示されるようにするために、注射の前に10分間ケージにマウスを温める。脱水と高体温を避けるために、慎重にマウスを監視します。 </li><li>とすぐのLVが拡張したと表示されているように、制止にマウスを移動し、動きを拘束するバレルに十分プラグを挿入します。このような呼吸などの正常な活動のためのマウスを監視します。必要に応じて、拘束具のプラグを調整します。 </li><li>拭いてアルコールで尾の先端から注入領域3月4日を消毒。マウス尾静脈注射用の27〜30 Gの針を使用してください。片手でしっかりと尾を持ち、針を挿入尾静脈へ。注射器に圧力を適用し、5〜7秒以内に注入を完了します。 </li><li>尾静脈から針を外し、出血を止めるために注射部位にコットンボールに圧力をかける。呼吸数は、通常の速度に低下した後、バックビバリウムにマウスを移動して、15〜30分間マウスを監視します。 </li></ol></li><li>マウス血清TRACP5bはポスト遺伝子導入の全身マウスsRANKLの発現および定量の確認<ol><li>血管を拡張と論理ボリュームが見えるようにするために5〜10分間ケージにマウスを温める。脱水と高体温を避けるために、慎重にマウスを監視します。 </li><li>尾の先端からサイト1項のマウスの尾の刃で小さな切開を行います。 </li><li>血清分離チューブ内の血液の約100μLを収集します。 </li><li> 30分間室温で血清分離管をインキュベートする。 </li><li> 5分間万×gで遠心分離サンプルと血清を収集します。 STORさらなる分析のために-80℃でのE血清。 </li><li>市販のキットを用いて血清サンプルにsRANKL ELISAおよびマウス血清TRACP5bはアッセイを行う。 </li></ol></li></ol> 2マウス BMMSからin vitro破骨細胞世代で <ol><li> BMMSの単離​​と培養<ol><li>脛骨および大腿骨の骨の単離：CO 2をドナーマウスを安楽死させると、70％エタノールで足を消毒。無傷で大腿骨と脛骨の骨を除去するために、踵骨の骨に恥骨から解剖。 </li><li>骨を損傷することなく、慎重に皮膚や筋肉を取り外します。リン酸緩衝生理食塩水（PBS）を含有するペトリ皿に処理し、大腿骨と脛骨を置く。 </li><li>骨髄の除去：1％ペニシリン/ストレプトマイシンを含有するα-MEMを装填し、大腿骨と脛骨の骨の一方の側に先端をカットし、25 G針を1ミリリットル注射器を用いて50ミリリットルチューブに骨髄を洗い流す（破骨細胞培養培地/ OCM）。 </li><lI&gt;プロセス骨髄：70ミクロンのナイロンセルストレーナーを通過させることにより、新しい50ミリリットルチューブに骨髄懸濁物を混合し、転送します。 300×gで遠心細胞及び1％ペニシリン/ストレプトマイシン及び10％FBS（破骨細胞培養培地/ OCM（+））を含有するα-MEM中に再懸濁する。 </li></ol></li><li>マクロファージの文化<ol><li>血球計または自動セルカウンターを用いて細胞を数える。 </li><li>プレート6ウェルプレート、ウェルあたり1×10 6個の細胞または96ウェルプレートに入れ、ガラス製カバースリップ（5mm）を又は象牙質切片に3×10 5個の細胞を、37℃でインキュベートする。 </li><li>吸引物のすべてのメディアは、非接着細胞を含有し、48時間、25 ng / mlのマウスM-CSFを含有する新鮮なOCM（+）37℃で細胞をインキュベートする。 </li><li>吸引物は、すべてのメディアと新鮮なOCM（+）メディアは、破骨細胞形成を開始する25 ng / mlのマウスのM-CSF、および30 ng / mLのマウスsRANKLを含む、37℃で細胞を培養する。必要に応じて、3日おきにメディアを補充する。 </li><li>細胞を育てる約6〜8日間、骨を再吸収することのできる巨大な多核細胞を形成する。とすぐに多核細胞が出現するように、市販のキットを用いて染色し、TRAP（酒石酸抵抗性酸性ホスファターゼ）を行い、完全に成熟したときに、機能的アッセイを行う。ファロイジン染色および画像先に説明したように19、走査電子顕微鏡（SEM）によってスライスを象牙質に付着細胞を使用してカバースリップ上でF-アクチンを可視化リング。 </li></ol></li></ol>ヒトPBMCから3。 インビトロ破骨細胞の生成<ol><li>ヒトPBMCを単離<ol><li> 50ミリリットルチューブに予め温めヒスト-1077の10ミリリットルを追加します。 </li><li>空の白血球フィルターは50mlのチューブ（PBSで血液を1:1の比）中に滅菌PBSで血液バンクから得た。血がヒストと混合しないことを確認しながら、非常にゆっくりとヒストソリューションより希釈した血液をレイヤー。 </li><li> 18℃で20分間1000×gで遠心分離サンプルフォロー遠心ING、慎重に白血球および新しい50mlチューブへの転送を含む白色バフィーコート層を単離する。 </li><li>細胞を回収し、5分間650×gで再度遠心PBSで細胞を希釈する。 </li><li>メディアや細胞カウンターを用いて細胞を数える - （）OCMで上清と再懸濁細胞を除去する。 </li></ol></li><li>メッキおよび培養したPBMC <ol><li> OCMにおける細胞（+）培地およびプレート8×10 6細胞/ ml当たり6ウェルプレートまたは1×10 6細胞/ ml当たりに96ウェルプレートにウェル再懸濁する。免疫蛍光イメージングのため、ならびに骨吸収アッセイに適した骨切片上のカバーガラス（5mm）をプレート上の細胞を、必要に応じて以前に20に記載のように。 </li><li> 2〜3日毎に又は必要に応じてヒトM-CSFで細胞を補充変化培養培地。その後、OCMにおける25 ng / mlのヒトM-CSF（+）と一緒に、人間のsRANKLの30 ng / mlのを追加して5日目の分化のステップに進んでosteoclastogenesを開始するメディアです。 </li><li>巨大多核細胞を形成するために約14〜21日間、細胞を増殖させる。できるだけ早く多核細胞が出現ように、これらの細胞は次いで、TRAPのために標識することができ、完全に成熟したときに、機能的アッセイを行うことができる。 F-アクチンリングはファロイジン染色を用いてカバーガラス上で可視化することができる。細胞形態は、SEMにより象牙質切片に付着した細胞において試験することができる。 </li></ol></li></ol>

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The authors have nothing to disclose.

筋骨格条件は、罹患率と障害の原因をリードしているし、150以上の疾患や症候群で構成されます。今日は約90万人のアメリカ人に影響を与える。関節の炎症や骨の破壊性関節炎や骨粗しょう症などの筋骨格症状の主な機能です。骨粗しょう症は、多くの場合、骨の骨折につながる骨の整合性を弱める条件である。関節炎は腫れ、圧痛およびスティッフ制限通常の移動となり、障害につながる?...

筋骨格系疾患は、米国および国や地域の保健システムの1のために存在深刻な結果に何百万人もの人々に影響を与えます。これらの疾患は、骨の損失および広範な治療および回復の長い期間を必要とする関節機能によって特徴付けられる。一般に、数および/ ​​または骨粗鬆症および関節炎では、骨を吸収する破骨細胞特化し、細胞の活性の相対的増加が観察される2。生理学的条件下で破骨細胞の数および活性は、骨芽細胞により産生される核因子κ-Bリガンド（RANKL）の受容体活性化によって調節される。オステオプロテゲリン（OPG）、RANKLのためのデコイ受容体はまた、骨芽3によって生成される全身sRANKLの過剰発現、又はOPGの欠失を含むインビボ動物モデルは、骨粗しょう症の研究に非常に有用である。;しかしながら、これらの方法は、トランスジェニックマウス、4,5の発生を必要とする。ここでは、新たな代替筋骨格関連疾患の研究のためのsRANKLを過剰発現させる方法が記載されている。具体的には、ミニサークル（MC）DNA技術および流体力学的送達方法は、インビボで sRANKLの遺伝子導入を達成し、全身6マウスsRANKLを過剰発現するために使用した。 この方法はまた、低カルシウム食8により卵巣切除7および食事介入後の破骨細胞のホルモンの変調のような骨粗鬆症の他のin vivoモデルに対して相補的である。これらのモデルは、しかし、彼らは外科手術を必要とし、大幅なコスト9で、数ヶ月かかることがあり筋骨格関連障害のさまざまな側面を研究することは非常に便利です。卵巣摘出（OVX）げっ歯類モデルは卵巣の除去は、それによって人間の閉経後骨粗しょう症10を模倣するエストロゲン欠乏につながる実験動物モデルである。人間の閉経後骨粗しょう症、条件エストロゲン欠乏症encyは骨折のリスクの増加につながり、骨粗しょう症は、米国だけで約8万人の女性に影響を与えます。 OVXモデルは、閉経後の骨粗しょう症のために有用であるが、それは一般的に骨粗しょう症の研究に限定された利点を提供しています。エストロゲンは、したがって、その非存在下で破骨細胞活性の増加が観察される10〜12、破骨細胞を誘導し、骨芽細胞のアポトーシスを阻害することによって、骨損失を抑制する。骨吸収に有利RANKL-OPG比の不均衡13も観察される。しかしながら、 インビボでのエストロゲン欠乏はまた、を伴う成長因子β（TGFのβ）のレベルを減少させ、インターロイキン-7（IL-7）、TNF、IL-1およびIL-6 14,15が増加した。これらのサイトカインは、RANKL経路の骨リモデリングの調節機能が独立した知っていたとして、それは単に、RANKL-RANK軸に任意の破骨細胞活性化を帰属させることは不可能である。このホワイトペーパーで説明されたモデルは、VIVに留学する研究者を可能にしますO前炎症性サイトカインのない破骨細胞形成および骨量の減少におけるRANKL-RANK軸は、OVX齧歯類モデルと比較。 また、in vitroでの破骨細胞形成技術は、筋骨格疾患の潜在的な治療的処置のために、破骨細胞の活性化を研究するために不可欠なツールです。以前の研究は、マウスのマクロファージコロニー刺激因子（M-CSF）でマウス骨髄由来マクロファージ（BMMS）を培養し、マウスsRANKLが破骨細胞分化3,16,17につながることが示されている。ここで、マウスの骨髄から、ならびにインビトロ18ヒト末梢血単核細胞（PBMC）からの多核破骨細胞様細胞を生成するためのプロトコルが記載されている。成熟した終末分化して完全に機能する破骨細胞を定義するために必要な、細胞ベースのアッセイも簡単に説明している。これらのインビトロ技術は、生体内のアプローチで小説を補完し、一緒にPとして機能owerful調査ツールは、破骨細胞の分化および活性化を研究する。これらのシステムを使用して、科学者は、 インビボおよびインビトロで破骨細胞を生成し、それらの増殖および活性化に必要な刺激および信号を定義するだけでなく、薬理学的および生物学的阻害剤の有効性を試験することができる。

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			<td height="63" style="height:63px;width:320px;">alpha-MEM</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td style="width:119px;">12561-056</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Human M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-096-492</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-643</td>
			<td></td>
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			<td height="42" style="height:42px;width:320px;">Human RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td>130-094-631</td>
			<td></td>
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			<td height="42" style="height:42px;width:320px;">Mouse RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-076</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tailveiner Restrainer for mice</td>
			<td style="width:71px;">Braintree</td>
			<td style="width:119px;">TV-150 STD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&#160;</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">MTR00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">387A</td>
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			<td height="63" style="height:63px;width:320px;">MouseTRAP assay&#160;</td>
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			<td style="width:119px;">SB-TR103</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.

ここで、破骨細胞形成に対するサイトカインの効果を研究するための方法として、in vitroで破骨細胞への前駆細胞を分化するためのインビボおよび細胞培養プロトコルにおける破骨細胞の分化のための新規な遺伝子導入技術が記載されている。 図1では、マウスにおけるGFPおよびマウスsRANKL MCの成功した遺伝子導入の代表的な結果を示す。 図2にお...

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.

小説<em&gt; in vivoで</em&gt;遺伝子導入技術と<em&gt; in vitroで</em筋骨格障害における骨損失の検討のための&gt;細胞ベースのアッセイ

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 ビュー.  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. 

ビデオ: 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.

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

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, ...

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.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

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.

小説を使用して、脊髄損傷後のマウス後肢の骨の損失の経時的評価、 in vivoで、方法論

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.

がんの生産と活性酸素種の検出（ROS）

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.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

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.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

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.

ブラインドのナビゲーションスキルを支援するためにオーディオベースのバーチャル·ゲーム環境の開発

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.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

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.

筋骨格超音波画像の新規応用

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.

携帯誘電分光法による特発性側弯症の予後予測のための細胞ベースのアッセイプロトコール

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