この作品は、自然科学とカナダの工学研究評議会（RGPIN 238382から2011）およびヘルスリサーチ（MOP115076）のカナダの研究所からの助成金を介してサポートされていました。ヘザー・ポールは、自然科学とカナダの工学研究審議会大学院奨学金とアルバータ革新健康ソリューション奨学金によってサポートされていました。ミーガン・ハラムは、自然科学と工学研究評議会大学院奨学金、フレデリック・バンティングとチャールズベストカナダ大学院奨学金、およびアルバータ州の小児病院研究所研修遺伝学賞、子どもの発達、および健康によってサポートされていました。

このプロトコルは、カルガリー大学の動物実験委員会によって承認され、 実験動物の管理と使用に関する指針に準拠しました。 子孫から1.個別のダム<ol><li> 12を搾乳する前に、5分の最小のための彼女の子孫からダムを区切ります。 注：ダムは、しかし長い4時間以上の分離の期間は、乳組成物14 を変更することができる 、分離1,6,13後5-6時間までに搾乳することができます。分離時間はミルク収集量12に影響を与えるようには見えないが、それは、一貫した分離時間は、研究を通じて維持されることをお勧めします。授乳15全体に変更されることがあり乳組成物は、そのための試みは、一貫性のあるミルクのコレクションの一日を維持するためになされるべきです。最大の牛乳生産は、泌乳14日目12で発生することが示唆されています。 </li><li>温暖化室を使用して、子犬が適切な体を維持することができる確実temperatu搾乳手順の間、母親の存在なしに再。 注：以下に示す研究では、搾乳は、したがって、何の温暖化室は使用されなかった、ダムは約22週齢で子孫21日齢時、離乳で実施しました。 </li></ol> 2.セットアップと準備<ol><li>搾乳手順に必要なすべての材料を収集します。 注：すべての材料は、材料や機器の表に記載されています。 </li><li>搾乳が行われ、アンダーパッド吸収性ベンチでパッドをカバーするベンチに加熱パッドを配置します。 </li><li>麻酔システムを設定します。システムに十分な酸素や開始前イソフルランを持っていることを確認してください。マシンへの最初の麻酔導入のために使用される麻酔マスクを取り付けます。近くの初期麻酔マスクと異なる場合、麻酔の維持のために使用されるマスクを配置します。 </li><li>オキシトシン1 mlシリンジに、25G針を取り付け無菌技術を用いて注入。 </li><li>母体の体温は搾乳手順の間に維持されるように、加熱パッドをオンにします。注：あまりにも高温になり、やけどを起こさないパッドを確実にするために加熱パッドの温度を監視します。あるいは、特定の温度に設定することができる加熱手術台などの熱源を使用します。 </li></ol> 3.イソフルランを使用してダムを麻酔<ol><li>酸素タンクを開き、毎分1リットル（千CC）への流れを向けます。イソフルランの流れをオンにして、5％に設定されています。注意：麻酔薬の直接吸入を避け、麻酔蒸気の蓄積を防ぎます。 </li><li>ダムを麻酔。 </li><li>必要に応じて、吸収ベンチパッドの上にダム仰向けに配置し、メンテナンスマスクに切り替え。ペダル反射の欠如によって麻酔を確認してください。 </li><li>麻酔の維持のために2〜3％のイソフルランの流れを減らします。継続ドを確保するための手順を通じてダムを監視呼吸のPRESSIONは発生しません。注：一度麻酔下で、ダムの目が乾燥や傷​​になってから目を防ぐために、無菌の目の潤滑剤を使用して保護する必要があります。 </li></ol> 4.オキシトシン注射液<ol><li>オキシトシンを確認します（20 USP単位/ ml）を、その有効期限を経過していません。 /アルコールクレンジングパッドを拭き、滅菌アルコールでオキシトシンのバイアルを消毒します。 </li><li>無菌技術を使用して、シリンジ内にオキシトシンの2 IU（0.1 ml）を描きます。搾乳される各ダムのための新しい針と注射器を使用してください。 注：オキシトシンの投与量は、一般的に1の単回注射から5 IU 1,6,12,16の範囲。あるいは、4 IU / kg体重の投与量は、12を使用することができます。難易搾乳が発生した場合2 IUの単回投与は、一回繰り返すことができます。 </li><li>腹腔内にオキシトシンを注入。角度で、針が頭の方を向いて腹部の右下の象限に針を挿入します15〜30°の、約0.5cmの深さです。 </li><li>注射前に、負圧を確保するために、プランジャーを後方に引いて。任意の流体（血液、尿、腸の内容物など ）が注射器内に吸引されている場合は、針を削除し、新しい針と注射器で注入を試みます。何の流体が吸引されていない場合はオキシトシンを注入し、すぐにバイオハザード容器に針と注射器を廃棄します。 </li><li>ミルクレットダウンを刺激するオキシトシンのために約5〜15分待ちます。 </li></ol>搾乳サイトの5準備<ol><li>牛乳が収集されるからサイト/乳首を選択してください。乳は、任意の乳頭12から回収することができます。 </li><li>毛皮は、牛乳の吸い上げによるサンプル収集の難しさを引き起こす可能性があるとして慎重に、トリマーで搾乳される乳首の周りに毛皮を削除します。穏やかな - 乳首の周りの皮膚は非常に敏感で、乾燥していてもよく、のような傷や涙の影響を受けやすいです。 </li><li>乳首iの滅菌必要じゃないが、毛を除去した後、必要に応じて、ぬるま湯で乳首をきれいに。複数のサイトのような少なくとも二つの部位を調製するミルクコレクションのために必要とされてもよいです。 注：ミルク分析は、微生物プロファイリングが含まれる場合は、乳頭領域は、ヨウ素5に滅菌を必要とするかもしれません。 </li></ol> 6.ミルクコレクション<ol><li>静かに手動でコレクションのミルクを排出、乳首の根元を絞ります。 注：ミルク分析は、微生物プロファイリングが含まれている場合は、ミルクの最初の数滴は廃棄されるべきです。 </li><li>毛細管を充填し、毛細管内にミルク液滴を集めます。大容量（例えば、50μl）を収容するキャピラリーチューブは、プロセスを容易にします。 注：ミルクを収集することが困難に遭遇した場合には、オキシトシンの第二の用量を投与することができます。これは、投与量は4 IUの合計を超えないことをお勧めします。 </li><li>により滅菌マイクロチューブに毛細管からミルクを分配します毛管作用を介して毛細管から引き出されているミルクを観察する - マイクロ遠心チューブの側面に乳首からミルクを引き出すために使用された管の端部に触れます。 注：1 mlシリンジに取り付けた18 G針を使用してチューブに引き込まれていないミルクを「吹き飛ばし」。 </li><li>継続的に痛みや呼吸抑制の兆候がダムを監視し、それに応じてイソフルランの流れを調整します。 </li><li>このセクションで説明するように、十分なミルクが選ばれたミルク分析のために収集されるまで、ミルクを収集し続けます。 creamatocritとタンパク質濃度の決意は、以下に説明するために、ミルクの0.25ミリリットルを収集します。 注：ミルクレットダウンが遅くなるか、選択したサイトが十分に牛乳を追放しない場合、別の搾乳サイトを使用してください。動物当たりの乳の約2.5 mlで最大17、または乳頭あたり0.5 mlまで収集することができます。著者は、O、麻酔下の合計時間は約45〜60分に制限されることをお勧めします搾乳の45分rは。 </li><li>新鮮な牛乳サンプルからcreamatocrit測定用マイクロヘマトクリット管にミルクを収集し、粘土シーラントでチューブの端部をシールします。ダムIDでミクロヘマトクリットチューブにラベルを付け、直立ミルク試料を保存します。 </li><li>搾乳が完了すると、イソフルランと酸素の流れをオフにします。ダムからマスクを外し、目を覚ましまでダムの監視を継続します。それは完全に意識されていない場合、ダムが吸引さまたは復元中のダムの目を傷から寝具を防止するために、ケージの寝具ではなく直接よりも、回復期間中に、吸収ベンチパッドの上に置かれるべきであることをお勧めします。 注：それは胸骨recumbancyを維持するのに十分な意識を取り戻したまで無人ダムに放置しないでください。 </li><li>更なる分析が必要とされない場合は、-80℃でミルクを凍結。他の人はミルクが-20℃18で3時間まで4℃で、または5ヶ月間保存することができることを示唆しています。 </li></ol> 7. Creamatocrit測定<ol><li>ミルクcreamatocrit（ミルク試料中のクリームの割合）19を計算することにより、ミルク中の脂肪含量を推定します。 注：母乳で測定が新鮮または凍結のいずれかミルクがcreamatocrit測定のために使用することができることを実証している、しかし、新鮮な牛乳は、より高度に脂質濃度11（R = 0.90対R = 0.92）相関しています。解凍したミルクはcreamatocrit 11値でわずかな減少と関連しているようcreamatocrit測定のための新鮮または凍結したミルクの使用は、研究全体の一貫性が保たれるべきです。 </li><li>新鮮な牛乳の場合、ミクロヘマトクリット管に乳首からミルクのサンプルを収集します。少なくとも¾フル（約15〜20μl）を記入してください。また、よく混合した後、キャピラリーチューブに採取した試料からの新鮮な牛乳を描きます。粘土シーラントで終了をシール。 </li><li>で、ヘマトクリットスピナーに毛細管を配置密封端遠心機はバランスが取れて確保し、外側に向かって指しています。 </li><li>ヘマトクリットスピン（13,700×gで120秒）を開始します。 注：スピン時間や速度は使用遠心分離機のモデルに応じて変更されることがあります。 </li><li>スピンが完了した後に遠心分離機からチューブを外し、creamatocritを算出するための測定を行います。クリーム層と透明層内に試料の分離を観察します。 </li><li>測定し、チューブ内の流体の合計の長さと脂肪（クリーム）の長さノギスや定規を使用したレイヤを記録します。 注：（クリーム層/ミルク列の全長の長さ）として算出したミルク試料19内のクリーム層の割合は、100（ 図1A）xとcreamatocritが発現されます。次のようにcreamatocrit測定から脂肪濃度及びエネルギー値を計算する：脂肪濃度（g / L）=（creamatocrit（％） - 0.59）/0.146（ 図1B）19。エネルギー値（KCアル/ L）+ 290（66.8 * creamatocrit（％）（ 図1C）= 19。 </li></ol> 8.タンパク質濃度の決意<ol><li>タンパク質標準としてウシ血清アルブミンを用いて、例えば、ローリー法6のような標準的なタンパク質アッセイを用いて、全乳タンパク質の濃度を決定します。 注：乳タンパク質の測定値がアッセイの標準曲線内に入るようにするためのミルクの希釈が必要であり得ます。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+and+energy+contents+of+expressed+human+breast+milk+in+prolonged+lactation.">Fat and energy contents of expressed human breast milk in prolonged lactation.</a> Pediatrics. 116 (3), e432-e435 (2005).</li></ol>

著者らは、開示すること、関心の競合がありません。全ての動物実験は、CCAC承認されたプロトコルに従って行いました。

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

ミルクは乳児の成長と発展の1,2のためのエネルギーや栄養素を提供し、新生児の哺乳動物のための唯一の栄養源です。ミルクは、主に、細胞、脂質、およびタンパク質1で構成されている間、それはまた、酵素、炭水化物、ホルモン、抗体、成長因子、サイトカイン、エキソソーム、マイクロベシクル、及びそのような低分子RNAを含む子孫の若年期の発達を調節する生物活性化合物の多数を含んでいますマイクロRNA 1,2として。幼児は病気2を受けにくい母乳証拠と相まって免疫子孫と腸の健康3の確立に母乳の基本的な役割は、人生の早い段階で病気のプロセスに関連付けられたミルク成分と関与する分子メカニズムを識別することの重要性を強調して彼らの行動です。開発ラットは早い段階で、栄養様々な生理学的、および化学的介入の効果を調べるための人気モデルであります-Life開発4。ラットのミルクの分析は、したがって、母体および子孫の健康に新たな洞察を提供してもよいです。 現在の科学の進歩は今健康と病気の特定の乳成分の効果の詳細な調査のための機会を増やしています。例えば、牛乳細菌プロファイルのシークエンスが幼児の腸5の初期の腸の植民地化におけるそれらの役割を解明した、ミルクオリゴ糖の質量分析は、母体の食事6、中に分泌マイクロRNAの深いシーケンシングを介しミルクオリゴ糖プロフィールの変更への洞察を提供してきました母乳の脂肪球は、遺伝子転写、代謝、および免疫機能7の可能な役割を強調しています。 ラットモデルは、母体の研究8,9で使用される最も人気のあるモデル生物の1つを表します。一つの利点は、唯一approximat持続が短い妊娠及び授乳期間であり、エリー21日各;したがって、授乳に妊娠の開始からの合計時間は貴重なデータを生成することができる時間の短い期間を示します。マウスと比較してラットの大きなサイズは、ミルクのコレクションとの関連で、ミルクおよびミルクの収集の容易さの体積に対して有意な利点を提供し得ます。マウスでの牛乳の生産は、例えば、より多くのミルク10を生成する重いマウスを用いた全体重に依存するように思われます。 ここでは、授乳中のラットからミルクを手動で収集するための一般的な説明が提供されています。このプロトコルは、最小限の設備を必要とする、非侵襲性で、安価で、さらに下流の分析のためにミルクの適切なボリュームを収集するために使用することができます。簡単に言えば、ダムはミルクレットダウンは、オキシトシンによって刺激され、イソフルランで麻酔され、ミルクはミルクのマニュアル発現によりキャピラリーチューブに回収されます。最後に、ミルクの2つの主要コンポーネントは、簡単なdescriptio脂肪とタンパク質であるとして、creamatocrit測定値11及び標準的なタンパク質アッセイを使用して総タンパク質濃度の定量化を用いて、乳脂肪含有量を推定するnが示されています。

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

ミルクは、コントロールを消費しウィスターダム（約22週齢、350〜400グラムを秤量）（AIN-93G、n = 5）を、高タンパク質から離乳時に説明したように収集された（40％カゼイン重量/重量、N = 5）しました、または高いプレバイオティクス繊維（21.6％重量/重量、1：オリゴフルクトース及びイヌリンの1の比率、N = 4）妊娠中や授乳を通してダイエット。オキシトシン投与量は、IU 2でした。ミルク?...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

キャピラリーチューブ、Creamatocritによって乳脂肪含量の推定を用いたラットにおける牛乳コレクション

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K ビュー.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

ビデオ: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

ラットポータル線維芽細胞の分離により、<em&gt;その場</em&gt;肝灌流

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

マウスおよびリーブス」キョン鹿のためのミルク収集方法

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

の収集と分析<em&gt;シロイヌナズナ</em&gt;師部はEDTA-促進法を用いた滲出

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

ハイコンテンツ顕微鏡を用いた細胞レドックスプロファイリング

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

野生型および肝臓の肝臓試料および牛乳試料中のシアル酸の定量<em&gt; CMAH</em&gt;ノックアウトマウス。

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

ショウジョウバエメラノガスター幼虫脂肪体における飢餓誘発オートファジーの解析

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

脱細胞化による骨格筋脂肪浸潤の定量化

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

著者スポットライト:骨格筋脂肪浸潤の脱細胞化に基づく定量化  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.