Die Autoren sind dankbar für den Vizekanzler für Forschung der Ardabil Universität der medizinischen Wissenschaften (Ardabil, Iran) für die finanzielle Unterstützung (No zu gewähren: 9607).

Alle Verfahren wurden nach den Richtlinien der Ardabil University of Medical Sciences Research Council für die Durchführung von Untersuchungen an Tieren durchgeführt (ethische ID-Nummer: IR. ARACEAE. REC.1394.08). In dieser visualisierte Studie verwendeten wir Erwachsene männlichen Sprague-Dawley Ratten (300-350 g) aus Weide Institut (Teheran, Iran) gewonnen.
1. Anästhesie und Flowmetry
<ol><li>Induzieren Sie Anästhesie mit 4 % Isofluran und pflegen Sie es mit Isofluran (1-1,5 %) in einer Mischung von Lachgas (70 % V/V) und Sauerstoff (30 % V/V) für die Dauer der Operation.</li>
	<li>Ein narkotisierter Tier in die anfällig Haltung auf ein Feed-Back kontrollierter Wärme-Unterbett und halten Sie die Körpertemperatur bei 37±0.5 ° C durch eine rektale Sonde an dieser Heizung angeschlossen.</li>
	<li>Wenden Sie eine kleine Menge des ophthalmologischen Salbe auf beiden Augen, Trockenheit zu verhindern.</li>
	<li>Rasieren Sie die chirurgische Region auf der linken Seite des Schädels mit elektrischen Haarschneider. Verwenden Sie Betadine Lösung mit einem Tupfer, um die Haut zu desinfizieren. Spülen Sie dieser Region mit einem sterilen Pad mit 70 % igem Ethanol und wiederholen Sie die beiden Schritte für drei Zyklen17.</li>
	<li>Injizieren Sie für Analgesie 0,2 mL 0,5 % Bupivacain subkutan in die Chirurgie Region17.</li>
	<li>Machen Sie einen 1 cm langen Hautschnitt am Schädel (Verlängerung von den seitlichen Augenwinkel des linken Auges auf der Basis des linken Ohr) und sezieren des linken Temporalis Muskels um den Schädel zu entlarven. Erstellen Sie nach der Bregma18 einfache Platzierung der Doppler Durchflussmesser Bleistift Sondenspitze dann einen kleinen Grat Loch 5 mm seitlich, und 1 mm.</li>
	<li>Drehen Sie die Ratte aus der Rückenlage anfällig, und dann die Lasersonde Bleistift in die zuvor gebohrten Schädel Blind-Bohrung, die regionalen zerebralen Blutflusses (rCBF) zu überwachen.</li>
	<li>Zeichnen Sie jedes Tier Grundlinie rCBF auf und betrachten Sie dies als 100 %. Insbesondere ist mittlere zerebrale Arterie Okklusion (MCAO) gestartet, wenn eine Abnahme der rCBF von mehr als 80 % erkannten19,20ist.</li>
</ol>(2) Induktion von fokalen Cerebra Ischämie
<ol><li>Rasieren Sie Halsbereiches zu und desinfizieren Sie die Haut mit Betadine Lösung und 70 % Ethanol. 0,2 mL 0,5 % Bupivacain subkutan in Chirurgie Website für Analgesie17zu injizieren.</li>
	<li>Machen Sie eine 2 cm langen chirurgischen Inzision in der ventralen Oberfläche des Halses auf die linke gemeinsame Halsschlagader (LCCA) zugreifen. Isolieren Sie die LCCA aus der benachbarten Faszie und den Nervus Vagus zur Bifurkation der äußeren Halsschlagader (ECA) und der inneren Halsschlagader (ICA) zugreifen.</li>
	<li>Dauerhaft verbinden Sie, entweder die LCCA oder ECA mit einer 5-0 Seide Naht und sezieren Sie ICA frei auf das Niveau der Pterygopalatine Arterie zu.</li>
	<li>Lose stellen eine Krawatte Seide Naht 5-0 rund um LCCA, und dann vorübergehend ICA Klemmen mit einem vaskulären Mikro-Clip.</li>
	<li>Machen Sie einen kleinen Schnitt auf der LCCA vor der zuvor platzierte lose Krawatte und dann legen Sie eine 4-0-Silikon beschichteten Nylon-Naht in der luminalen Raum von ICA und ziehen Sie den Faden um die LCCA, die Nylon-Naht zu sichern und zu verhindern, dass Blut austritt.</li>
	<li>Entfernen Sie den vaskulären Mikro-Clip von der ICA. Dann vorab ein Silikon beschichteten Intraluminal Filament bis beobachten einen deutlichen Rückgang der rCBF, die Okklusion der MCA Herkunft angibt. Am Ende der ischämischen Periode (90 min) starten Sie Reperfusion durch den Rückzug der Intraluminal Naht21.</li>
</ol>(3) Halsschlagader Kanülierung und Evans blau (EB) Injektion
<ol><li>Machen Sie einen 1 cm längs-Schnitt in den Hals auf der linken Seite der Mittellinie und dann unverblümt sezieren Sie entfernt oberflächliche Faszie um externe Ortsverbandes der linken Halsschlagader (LGV) zuzugreifen. Dann dauerhaft verbinden Sie kraniale Ende der LGV mit 5: 0 Seide Naht. Legen Sie locker zwei Verbindungen rund um die Vene, und dann vorübergehend Klemmen Sie kardiale Ende der LGV mit einer vaskulären Mikro-Klemme.</li>
	<li>Machen Sie einen kleinen Schnitt auf der LGV zwischen zwei Nähte, und legen Sie heparinisierten Serum gefüllt Katheter in den luminalen Raum von der LGV und Kanüle ca. 10 mm. danach, ziehen Sie die Ligaturen um die Vene zur Sicherung des Katheters und Vorbeugung von Blutungen. Kleine Mengen von Serum zu vermeiden, reduzieren die Vene zu injizieren.</li>
	<li>Zu Beginn der Reperfusion Periode injizieren Sie langsam EB Farbstoff (1 mL/kg 2 % EB Lösung in Kochsalzlösung) über einen Zeitraum von 5 Minuten. Anschließend waschen Sie die Kanüle durch Injektion von 0,5 mL normale Kochsalzlösung und entziehen Sie die Vene12,22der Injektionskanüle.</li>
	<li>Schließlich Naht der Hals und Kopf Einschnitte und 0,05 mg/kg Buprenorphin intraperitoneale Injektion (IP) und wiederholen Sie der Injektion alle 6-8 Std. Reperfusion Zeitraum für postoperative Analgesie17.</li>
	<li>5 mL vorgewärmten saline(IP) Flüssigkeitszufuhr in der Wiederherstellung Käfig17zu injizieren.</li>
	<li>Ort das Tier in einem Käfig spezielle Recovery ausgestattet mit Temperatur und Klimaregelung und die Erholung des Tieres aus der Narkose zu überwachen.</li>
</ol>4. Bewertung der Blut Hirn-Schranke Durchlässigkeit
<ol><li>Nach 24 Stunden der Reperfusion tief betäuben des Tieres mit Natrium Thiopental. Öffnen Sie dann die Brusthöhle durch ein kleines Loch unter dem Brustbein.</li>
	<li>Machen Sie eine kleine Öffnung in den rechten Vorhof und injizieren Sie 250 mL vorgewärmten 0,9 % Kochsalzlösung (37 ° c) bei einem Druck von 110 MmHg durch die linke Herzkammer für 15 min, EB von der Zirkulation zu waschen, bis die normale Kochsalzlösung Exits vom Atrium wird farblos23zu.</li>
	<li>Sofort entfernen Sie das Gehirn aus dem Schädel zu und legen Sie es in der Gehirn-Matrix. Die Riechkolben und Kleinhirn zu sezieren, und dann mit einer sauberen Rasierklinge trennen die rechte Hemisphäre des Gehirns von der linken Seite (Lesioned) entlang der Mittellinie.</li>
	<li>Wiegen Sie jede Hemisphäre separat in 2,5 mL Phosphat gepufferte Kochsalzlösung zu homogenisieren Sie und mischen Sie diese mit 2,5 mL Trichloressigsäure Säure (60 %) für 2 min mit einem Vortex-Maschine.</li>
	<li>Zentrifugieren Sie Gehirn Proben für 30 min bei 1.322 x g und lassen Sie die Proben im Kühlschrank 4 ° C für 10 min. abkühlen lassen.</li>
	<li>Verwenden Sie die Überstände die EB-Extinktion von einem Spektrophotometer 610 nm23schätzen.</li>
	<li>Berechnen Sie EB Konzentrationen gegen eine Standardkurve und ausdrückliche Ergebnisse als µg/g des Hirngewebes.</li>
</ol>5. Herstellung von der EB-Standardkurve
<ol><li>Bereiten Sie 10 Beispiellösungen von EB mit Konzentrationen von 1 bis 10 µg von EB in 5 mL Phosphat gepufferte Kochsalzlösung.</li>
	<li>Messen Sie die Absorption Werte jeder einzelnen Probe bei 610 nm mit einem Spektralphotometer.</li>
	<li>Machen Sie ein Punktdiagramm mit einer Tabellenkalkulation und vorliegenden EB-Konzentrationen auf der x-Achse und die Extinktion Werte auf der y-Achse (Abbildung 1).</li>
	<li>Definieren Sie eine lineare Trendlinie für die Kurve mit der Gleichung. Dann können sie um die EB-Konzentration der Proben zu erhalten.</li>
	<li>Melden Sie die endgültigen Ergebnisse der EB Extravasation als µg/g Gehirn Gewebe Gewicht.</li>
</ol>6. Sham-Betrieb
<ol><li>Durchführen Sie der gleichen chirurgischen Verfahren mit Ratten in Sham betriebene Gruppe (einschließlich macht einen Einschnitt in der Halsregion und EB-Injektion), aber schließen Sie die MCAO aus.</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Bottiger, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cerebral+ischemia+due+to+cardiocirculatory+arrest+in+mice+causes+neuronal+degeneration+and+early+induction+of+transcription+factor+genes+in+the+hippocampus.">Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus.</a> Brain Res Mol Brain Res. 65 (2), 135-142 (1999).</li></ol>

Die Autoren haben nichts preisgeben.

So weit, verschiedene Methoden wie Autoradiographie und Erkennung der radioaktiven Tracern24,25, Immunfluoreszenz-Mikroskopie26,27und EB Extravasation Technik20, 23 wurden verwendet, um die Blut - Hirn-Schranke Schaden zu bewerten. EB-Farbstoff kann sich stark an der Serum-Albumin gebunden und dient als Tracer für vaskuläre Leckagen Erkennung u...

Vasogenic Hirnödem wegen Störung der Blut - Hirn-Schranke (BBB) bleibt eine wichtige Komplikation des ischämischen Schlaganfalls und eine wichtige Determinante für die Überlebensrate in der Schlaganfall-Patienten1,2. Die Blut - Hirn-Schranke (BBB), die durch Kapillare endothelial Zellen im Gehirn (BCECs) gebildet und besteht aus verschiedenen neurovaskuläre Komponenten (z. B. enge Kreuzungen unter Perizyten, BCECs, Astroglial und neuronalen Zellen3), bietet eine spezialisierte und dynamische Schnittstelle zwischen dem zentralen Nervensystem (ZNS) und peripheren Durchblutung4,5. Beleidigungen wie Ischämie-Reperfusion Verletzungen könnte stören die funktionelle Integrität der BBB und führen nachträgliche Eindringen von zirkulierenden Leukozyten in das Gehirn Parenchym, die letztlich zerebrale Entzündung und primäre Hirnverletzungen auslösen 6 , 7. Tiermodelle für die genaue Erfassung der Dysfunktion des BBB nach Auftreten eines Schlaganfalls erforderlich sind. Solche Modelle sind von großer Bedeutung für die Untersuchung zugrunde liegenden pathophysiologische Mechanismen und Einführung von neuen neuroprotektive Strategien. In-vitro- Kultur-basierte Zellmodelle der BBB hoch entwickelt und verwendet für molekulare Studie der BBB Physiopathologie8,9,10. In Vivo Tiermodellen, die ischämische Schaden der BBB analog zu menschlichen klinischen Bedingungen zu produzieren, sind jedoch auch in dieser Hinsicht sehr lohnenswert. Quantitativen Nachweis der Extravasation von Evans blau (EB) ist eine gut angenommen und empfindliche Technik, die für die Beurteilung der BBB Integrität und Funktion bei neurodegenerativen Erkrankungen, einschließlich der ischämischen Schlaganfall11, verwendet worden ist 12 , 13 , 14. diese Methode ist kostengünstig, machbar, reproduzierbare und völlig zutreffend in einem experimentellen Labor. Ihre Umsetzung erfordert keine vorgerückte Ausrüstung, wie radioaktive Tracer15 oder Magnetresonanz-Bildgebung (MRI)16, die Voraussetzung für andere Methoden sind. In diesem Artikel zeigen wir umfassend grundlegende technische Prozesse der BBB Bewertung mit EB Extravasation in Ratte-Modelle des ischämischen Schlaganfalls.

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an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

Ischämischen Schlaganfall führt zu Vasogenic zerebrales Ödem und anschließende primäre Hirnverletzung, die durch Zerstörung der Blut - Hirn-Schranke (BBB) vermittelt wird. Ratten mit induzierter ischämischen Schlaganfall wurden gegründet und als in Vivo Modellen verwendet, um die Funktionsfähigkeit der BBB zu untersuchen. Photometrische Erkennung von Evans blau (EB) in den Gehirn-Proben mit ischämischen Verletzungen konnte die Forschung und Entwicklung von neuartigen therapeutischen Modalitäten zuverlässig begründet. Diese Methode liefert reproduzierbare Ergebnisse und gilt in jedem Labor ohne spezielle Ausrüstung. Hier präsentieren wir Ihnen eine visualisierte und technische Leitlinie zur Detektion der Extravasation von EB nach Induktion der ischämische Schlaganfall bei Ratten.

Es gab keinen signifikanten Unterschied in EB Ebenen in der rechten Hemisphäre im Vergleich zu der linken Hemisphäre der Schein betrieben Ratten (1,06 ± 0,1 µg/g und 1,1 ± 0,09 µg/g, beziehungsweise). Siehe Figuren 2A-2 b, Induktion von transienten Ischämie (90 min Ischämie / Reperfusion 24 h) verursacht einen signifikanten Unterschied in EB Ebenen (10,41 ± 0,84 µg/g, p < 0,001) in der linken Hemisphäre von ischämischen Ratten, im Vergleich zu den jew...

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Eine In-Vivo -Bewertung der Störung der Blut - Hirn-Schranke in einem Rattenmodell des ischämischen Schlaganfalls

Das übergeordnete Ziel dieses Verfahrens ist es, eine hoch reproduzierbare Technik für in Vivo Beurteilung der Blut - Hirn-Schranke Störung in Ratte-Modelle des ischämischen Schlaganfalls liefern.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

an-vivo-assessment-blood-brain-barrier-disruption-rat-model-ischemic

Research

JoVE Journal

Neuroscience

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

11.3K Aufrufe.  Ardabil University of Medical Sciences. Das übergeordnete Ziel dieses Verfahrens ist es, eine hoch reproduzierbare Technik für in Vivo Beurteilung der Blut - Hirn-Schranke Störung in Ratte-Modelle des ischämischen Schlaganfalls liefern.

Serie: An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Erzeugung eines Immortalisierte Murine Gehirn mikrovaskulären Endothelzellen Zelllinie als<em&gt; In Vitro</em&gt; Blut-Hirn-Barriere Modell

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Forschung

Neurowissenschaften

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Funktionelle Bildgebung mit Ultraschall-Blut-Hirnschranke Störung und Mangan-MRT

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Beurteilung der Durchlässigkeit der Blut - Hirn-Schranke durch intravenöse Infusion von FITC-markierte Albumin in einem Mausmodell der Neurodegenerative Erkrankung

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from hospital admission to reperfusion. Rapid restoration of cerebral blood flow is essential for the outcomes of patients with acute ischemic stroke. Endovascular treatment (EVT) has become the standard of care to accomplish this in patients with acute stroke due to large vessel occlusion (LVO). To achieve reperfusion of ischemic brain regions as fast as possible, all in-hospital time delays have to be carefully avoided. Therefore, management of patients with acute ischemic stroke was optimized with an interdisciplinary standard operating procedure (SOP). Stroke neurologists, diagnostic as well as interventional neuroradiologists, and anesthesiologists streamlined all necessary processes from patient admission and diagnosis to EVT of eligible patients. Target times for every step were established. Actually achieved times were prospectively recorded along with clinical data and imaging scores for all endovascularly treated stroke patients. These data were regularly analyzed and discussed in interdisciplinary team meetings. Potential issues were evaluated and all staff involved was trained to adhere to the SOP. This streamlined patient management approach and enhanced interdisciplinary collaboration reduced time from patient admission to reperfusion significantly and was accompanied by a beneficial effect on clinical outcomes.

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

Optimierte Verwaltung der endovaskulären Behandlung von akuten ischämischen Schlaganfall

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Eine In Vivo Blut - Hirn-Schranke Permeabilität Assay bei Mäusen mit Gewebekulturen beschriftet Tracer

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Die Macht der Interstimulus Intervall für die Beurteilung der zeitlichen Verarbeitung bei Nagern

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Ein präklinisches Modell zur Beurteilung der Gehirnerholung nach akutem Schlaganfall bei Ratten

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Ein Benchtop-Ansatz zur standortspezifischen Blut-Hirn-Barriere-Öffnung mit fokussiertem Ultraschall in einem Rattenmodell

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Messung des Abbaus von Hirnödem nach dem Schlaganfall, Infarktzone und Blut-Hirn-Barrierenabbau in einem einzigen Satz von Nagetier-Gehirnproben

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

Ein Rattenmodell der EcoHIV-Gehirninfektion

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...