This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

行ったすべての実験は、眼科と視覚研究における動物の使用に関するARVO声明に従った、とラクイラの動物倫理委員会の大学とオーストラリア国立大学動物実験倫理委員会の承認を得て。成体ラット（P100-200）は、この研究を通して使用した。 1共重合樹脂ペレットを作成<ol><li>通風室の小さなガラスビーカーに20エルバックス40Wペレットを配置します。 </li><li>全てのペレットをカバーするために確保し、100％エタノールでビーカーを埋める。 </li><li>パラフィルムでビーカーを覆い、及び7〜10日間室温で100％エタノールでペレットを浸す。注：これ以降のステップから、樹脂を処理するか、保存するために、金属やプラスチックを使用しないでください。 </li></ol> 2のロードのための医薬品の溶液を調製<ol><li> 0.1％ジメチルスルホキシド（DMSO）で選択される薬物を溶解する。ボリューム内の40μlに薬液を構成している。単回投与で与えられる薬物の3倍の濃度で樹脂をロードします。 </li></ol> 3。薬剤 - 樹脂複合体を準備し、ロード<ol><li>別の小さなガラスビーカーに20洗浄したペレットを転送します。約45分間、4ミリリットルのジクロロメタン中のペレットを溶解する。ペレットが溶解している間パラフィルムでビーカーをカバー。 </li><li> 0.001 mg / mlの濃度に0.1％DMSOに溶解してファストグリーンFCFの溶液を調製する。 </li><li> 1ピペットを取り、以前の準備の薬剤溶液40μlを策定。ファストグリーン溶液40μlを持つ別のピペットをロードします。 </li><li>ビーカーに同時に2つのソリューションを追加します。グリーン色素が均一に混合全体に分散されるまで攪拌棒ガラスを用いて急速に混合する。 </li><li>速やか樹脂を凍結する高速に10分間ドライアイスの上にビーカーを転送します。 </li><li>溶媒を蒸発させるために &quot;蒸発室」を設置。埋める硫酸カルシウム小石と三分の一に大きなコンテナをバックアップします。小石でよくを作成し、慎重に小石をチャンバー内にビーカーを安定させるために、ビーカーの半分まで到達するようにウェル内にビーカーを置く。 </li><li>パラフィルムを有するより大きな外側のコンテナをカバー。 </li><li>冷凍庫に蒸発チャンバーを転送します。 2〜3週間、-20℃で保管してください。 </li></ol> 4。外科的移植のための薬剤 - 樹脂複合体を準備します<ol><li>冷凍庫から蒸発室を削除します。ガラス皿に固体薬剤 - 樹脂ブロックの転送​​を氷上に保持。 </li><li>顕微鏡やルーペの助けを借りて、トレパンやパンチツールを使用して、ブロックから直径約0.05ミリメートル、長さが0.1ミリメートルの部分をカット。 </li><li>凍結防止の材料（ 例えば 、アルミ箔）のブロックの残りの部分をラップし、必要になるまで-20℃で保管してください。 </li></ol> 5。外科的インプラントのラットの目への薬物 - 樹脂複合体</P&gt; <ol><li>手術のために動物を準備します。ケタミン（100 mg / kg体重）およびキシラジン（12 mg / kg体重）の腹腔内注射を用いて麻酔する。完全な麻酔が達成されるまで、動物を観察します。注：動物は（呼吸を除く）運動の喪失、および角膜とつま先ピンチ反射の損失を持っている必要があります。 </li><li>手術台に完全に麻酔をかけた動物を転送します。 （ 例えば 、硫酸アトロピン）mydriaticumを使用し、局所麻酔薬（ 例えば 、塩酸テトラカイン）手術を受け、眼（複数）に低下します。手術中に湿った角膜を維持するために人工涙液を適用します。手術全体の使い捨て注射針および滅菌器具を使用してください。 </li><li>瞳孔が完全に拡張された後に鈍い湾曲したピンセットで目を安定させます。 25 Gの針を用いて縁から硝子体、約2mmに到達するために、完全な深刺し傷を作るために手術用顕微鏡を使用してください。 </li><li> tは準備された部分を挿入します穿刺部位に浸透することができます罰金、先のとがった鉗子（ 例えば 、ピンセット）のペアを使用して刺し傷への彼の薬物-樹脂複合体。 </li><li>細菌感染の予防としては、動物が回復している間に角膜の乾燥を防ぐだけでなく、炎症を防ぐために、目に抗生物質軟膏を適用します。 </li><li>完全に回復するまで動物を観察します。それは意識を取り戻したまでは無人の動物を放置しないでください。完全に回復するまで、他の動物の会社に動物を返さないでください。動物がストレスや不快感の兆候を示した場合、鎮痛薬は承認された倫理プロトコルに従って使用してください。 </li><li>組織の収集と分析のための通常の実験手順に従ってください。 </li><li>実験の終了時に、ペントバルビタール（60 mg / kg体重）を2％リドカイン塩酸塩溶液の腹腔内注射で動物を安楽死させる。 </li></ol>注：リグノカイン塩酸分地元の不快感をimizes。

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著者らは、開示することは何もない。

本稿では、網膜への薬物の徐放性送達のための薬剤 - 樹脂複合体の使用を実証した。私たちは、比較的安価で小型の動物モデルにおいて適用が容易である方法を提供することを目的とした。 APBの機能は、グルタミン酸類似体として作用することを考えると、眼における網膜の応答をブロックする。結果は、APBは、1週間の着床後による網膜応答の閉塞を引き起こしたこと...

これらの疾患は、一般に、長期間の治療を必要とする糖尿病や高血圧などの慢性疾患の治療は、しばしば人生のために、多くの課題を提示する。これは、頻繁な投薬の必要性を減らす徐放性薬物送達システムの開発を求めている。これらの徐放方法の有効性は、糖尿病を治療するために必要なインスリン注射の数を減らすために、インスリンポンプの開発により実証されている。眼の慢性疾患、その内部層に影響を与え、特にものは、侵襲的な手続きを経て、薬物の頻繁な投与を必要とします。人間の目に影響を与える一つのこのような慢性疾患は、加齢黄斑変性症（AMD）です。それは、ビジョンを開始する責任眼球の奥に位置する神経組織の層である中心網膜に影響します。 AMDは、西側世界1における失明の主な原因である。再処理すると、特定の課題tinal疾患、薬物は、しばしば送達の侵襲的方法を必要とする眼、この深い層に到達するために必要とされることである。薬剤は通常、硝子体内注射を使用して、硝子体チャンバーと網膜に投与される。しかし、各注射で眼内炎、網膜剥離、白内障及び硝子体出血2後噴射を含む合併症の危険性がある。このリスクは、薬物のすべての再注入と乗算される。 複数回の注射の必要性を減少させることは、AMDの治療において大きな利点であろう。新しい血管の成長が特徴であり、AMDの湿潤型を治療するために、確立された治療戦略は、VEGF阻害剤3を使用して内皮増殖因子（VEGF）を標的とすることである。現在、これらを繰り返し、硝子体内注射を介して配信されます。同様に、黄斑浮腫、糖尿病性網膜症の一般的な合併症の治療において、コルチコステロイドは、反復注射を介して配信されている<s&gt; 4アップ。徐放方法を介してこれらの薬物の送達は、実際に後噴射の合併症のリスクを減らすことができる。 徐放性薬物送達システムの概念は、最初の動物組織5中に小分子を送達するためにシリコーンゴム車両を用いて説明した。それ以来、他の徐放方法は、眼において試験されたいくつかのそれらのより大きな分子を送達するために開発されている。そのような生分解性ミクロスフェア、ポリ-ラクチド-コ-グリコリド（PLGA）ナノ粒子およびリン脂質小胞（リポソーム）などの粒子担体は、送達ビヒクル6,7として有用であり得る。 PLGAナノ粒子およびリポソームは、時間7を介して強膜を横切って抗がん剤を送達するそれらの能力についてインビトロ環境で比較した。どちらの車がゆっくりと薬剤を放出するのに有効であった。しかし、研究は唯一のin vitro環境で行われた。 Bochot ら （2002）8は、試験しリポソームの有効性は、 インビボで網膜に分子を送達する。彼らは、リポソームが正常ウサギの網膜に小さなオリゴヌクレオチドを​​提供することを明らかにした。著者らは、リポソームは、網膜疾患8の処置に有益であり得ることを示唆した。しかし、硝子体中に浮遊するためにこれらの小胞の性質は、彼らが可能性が高いにじみやビジョン9を損なうことを意味します。 岡部ら （2003）、10はウサギにおいてβ-メタゾンを適用するために33％のエチレン-酢酸ビニルからなる非生分解性ポリマーディスクを使用する。彼らは、強膜ポケットにディスクを移植し、1ヶ月まで10硝子体および網膜への薬物の効果的な放出を示した。しかし、この特定のプロトコルでは、インプラントは、比較的大きな剛性であり、大規模な強膜切開や縫合など、より複雑な外科手術を必要とした。 以前の研究では、TISSUを調査電子のウサギの眼の角膜にそれらを注入することにより、さまざまなポリマーの車両への応答、およびアルコール中で洗浄したエチレン - 酢酸ビニル共重合体は、炎症または刺激を引き起こさないことを見出した。これらの複合体は、いくつかの薬剤11に依存して100日までの超過、長期間動物組織へのより大きな化合物の送達を持続することが示された。共重合体樹脂のような一タイプはエルバックス40Wの形で工業的に開発された（ &#39;W&#39;アミド添加剤の重量のエチレン - ビニルアセテートコモノマー含量40重量％、ペレットの取り扱いを改善する）。この共重合体樹脂は室温と体温の両方で安定な不活性物質である。これは、生体組織におけるアレルギーまたは毒性を引き起こすことが示されていない。この樹脂は、効果的にそのような乳管システム12、一次聴覚野13,14 &lt;などのさまざまなシステムの機能を調査する別の実験モデルにおいて、薬の多種多様を提供してきました/ SUP&gt;、そしてカエルの視覚システム15。この樹脂はまた亀16,17、ニワトリ胚18,19の開発に薬物を送達するために眼に使用されており、大人のフェレットは20を網膜。ラット中枢神経系において、樹脂のみ脳21-23で使用されているが、ラットの眼におけるその使用が報告されていない。 他の方法よりもゆっくりと網膜に薬物を送達するために、この共重合体樹脂を使用する利点は、それが目に炎症または刺激を引き起こさず、安定な化合物であることである。それは、通常、代わりに硝子体中に浮遊の送達部位に留まるように、粒子担体とは異なり、薬剤 - 樹脂複合体は、移植後に視力を損なわない。それだけで、目の角膜輪部に近い硝子体腔に簡単な注入工程を必要とし、移植後の縫合を必要としない。近年、など、いくつかの新規な送達システムの出現があったカプセル化細胞技術（ECT）24,25、26、およびマイクロフィルム27をヒドロゲル。しかしながら、薬剤 - 樹脂複合体を製造し、送達するための現在の研究で使用される方法は、それによって基本的な研究環境で使用するための、より有利で、追従しやすく、安価でもある。長期の薬物治療を送達するためにこの複合体を使用することの課題は、より少ない硝子体内注射を持つことの治療上の利益を最大化する薬剤の最適濃度を決定することである。 本稿では、成体ラット網膜の長期治療の​​ための薬剤 - 樹脂複合体の使用を実証することを目的とする。この送達様式の有効性は、薬物としてのグルタミン酸類似体2 - アミノ-4 - ホスホノ（APB）を用いて試験する。 APBブロック網膜28にグルタミン酸、内因性神経伝達物質を模倣することによって、ON双極細胞の光応答。 APBは、その受容体のためのグルタミン酸と競合する、それブロックガーゼT応答。 APBは、網膜機能を制御し、そのような電図（ERG）のような電気生理学的方法を使用してその効果を測定するために生理学的研究に使用されている。以前の研究では、APBは、短期29と現像網膜の長期治療の両方に使用されている;後者は、30日間30毎日、眼内注射を介して、単回投与を与えることに関与。前作28,29,31で示唆されているように、APBと同じ量（50μMの濃度で滅菌生理食塩水中の0.092 mgを、）すべての注射のために使用した。私たちは、眼内に薬物を送達するために徐放車両等の薬剤 - 樹脂複合体をテストするために、APBを選びました。この研究で概説された方法は、16,32、複雑な薬-樹脂の製造に関わる前述の方法と似ています。しかし、私たちにもディテール特に成体ラットの眼での使用。眼内へのAPB-ロードされた樹脂の外科的移植の後、ERGは、APBをrを廃止かどうかを確立するために実施したetinal光応答は、従って、APBが正常硝子体および網膜中に放出されたかどうか。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

網膜は、眼内の深い位置するように網膜の疾患は治療が困難である。薬物送達の侵襲的方法は、多くの場合、これらの疾患を治療するために必要とされる。そのような網膜浮腫や新生血管などの慢性網膜疾患は、通常、効果的に症状を治療するために、複数の眼内注射を必要とします。しかし、これらの注入に関連したリスクは、薬物の再配達に伴って増加。したがって、代替的な送達方法は、再注入のリスクを最小化するために確立される必要がある。他のいくつかの研究は、眼の中にゆっくり化学物​​質を放出することが可能な材料を通って、長時間にわたって薬物を送達するための方法を開発した。この研究では、成体ラット網膜への薬物送達のためのビヒクルとして機能するのElvax 40W、共重合樹脂の使用を概説する。樹脂を作製し、薬物がロードされる。薬剤 - 樹脂複合体は、それがゆっくりとtiの上に薬物を放出する硝子体腔に移植される私。この方法は、2 - アミノ-4 - ホスホノ（APB）、ブロック網膜の光反応をグルタミン酸類似体を用いて試験した。これは、APBをゆっくりと樹脂から遊離されることを実証した、と、移植後7日目までに、網膜応答をブロックすることができた。これは、この共重合体樹脂を用いた徐放性薬物送達は、網膜を治療するために有効であり、更なる試験で治療的に使用することができることを示している。

全視野電図（ERG）が網膜上APBの効果を検出するために使用された。 ERGが実行された方法については、以下の研究33,34を参照してください。簡単に述べると、動物を麻酔した後、金電極は、眼の角膜上に置き、参照電極は、網膜の電気的活動を記録するために、目の間の前方頭皮に入れた。刺激は、電子フラッシュユニットを使用して生成され、強度が10〜10ロッドごとに有効感光性分?...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

本論文では、エルバックス40Wは成体ラットの網膜への薬物送達のための徐放方法として使用することができる方法を詳しく説明します。調製ローディング、および眼への薬剤 - 樹脂複合体を送達するためのプロトコルが記載されている。

ラット網膜へのエルバックス40Wを通じて徐放性ドラッグデリバリー：慢性症状の治療のための含意

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.4K ビュー.  University of L'Aquila. 本論文では、エルバックス40Wは成体ラットの網膜への薬物送達のための徐放方法として使用することができる方法を詳しく説明します。調製ローディング、および眼への薬剤 - 樹脂複合体を送達するためのプロトコルが記載されている。 

ビデオ: Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

医学

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

ラットにおける慢性栄養注入のモデル

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

マイクロコンピュータ断層撮影イメージング用Microfilで虚血後肢と血管系の灌流に浸透ドラッグデリバリー

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

マウスの脳に直接薬剤送達のための異粘膜移植手順

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

マウスの動静脈瘻内皮への管腔内薬物送達

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

ラット腎臓への生物製剤の血管内デリバリー

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

薬剤耐性側頭葉てんかん患者における孔開存電極レコーディングのネットワーク解析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor、主要な代謝産物および嚢胞性線維症患者の体液中の Lumacaftor の臨床サンプルの高スループット分析のための最適化された LC-MS/MS 法

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

難聴の治療のためのトランス鼓膜ドラッグデリバリー

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...