Die Arbeit wurde durch ein Stipendium des Institut for Translational Medicine and Therapeutics, sowie einer Trainings-Zuschuss aus dem Institut für Medizin und Technik (T32HL007954) an der University of Pennsylvania und der Marlene L. Cohen und Jerome H. Fleisch Scholar Grant an der Finanzierung University of Connecticut Health Center (LHS) und NIH NS-029331 (FAW).

1. Vorbereitung der Ausrüstung <ol><li> Wischen Sie die stereotaktischen Rahmen und die Pumpe mit 75% Ethanol, um bakterielle Verunreinigungen zu minimieren. </li><li> Sterilisieren Hamilton Spritze und Fused-Silica-Nadel. Hinweis: Wenn chemische Sterilisation verwendet wird, achten Sie darauf, mehrmals in sterilem Wasser spülen vor Gebrauch. </li><li> Wischen Sie die Oberfläche von Paraffinwachs Papier mit 75% Ethanol und trocknen lassen. </li></ol> 2. Vorbereitung der Maus für die Injektion Hinweis: Haben Mäuse geliefert, Ihr Tier Anlage mindestens 7 Tage vor der Operation, damit sie an die neue Umgebung zu gewöhnen und Stress abzubauen. <ol><li> Wiegen Sie mit der Maus für die präoperative Ausgangswert. </li><li> Induce Anästhesie mit 30% Sauerstoff, 70% Lachgas und 4% Isofluran, bis nicht mehr auf Schwanz kneifen </li><li> Inject Maus mit Buprenorphin 0.1mg/kg intraperitoneal zur postoperativen Analgesie </li><li> Shave Kopfhaut </li><li> Coat Augen mit einer sterilen Vaseline </li><li> Bereiten Kopfhaut mit betadine x 3 wischt, dann erlauben Kopfhaut zu trocken </li><li> Machen Sie 1 cm Mittellinie sagittal Schnitt der Kopfhaut mit sterilen chirurgischen Skalpell Hinweis: Ein großzügiger Schnitt wird vollständige Freilegung des Schädels Sehenswürdigkeiten ermöglichen. </li><li> Shave 1 cm von Ventralfläche des Schwanzes ab 1 cm von der Basis und bereiten mit betadine x 3 Tücher </li><li> Bewegen Sie die Maus auf stereotaktischen Rahmen Hinweis: Es ist wichtig, um sicherzustellen, Maus ist in den Rahmen mit der Oberfläche des Schädels verläuft parallel mit der Basis des Rahmens zu sichern, mit hervorragenden Belichtung Bregma und mindestens 3 mm rechts von Bregma. </li></ol> 3. Intrazerebrale Blutungen Chirurgie Hinweise: Während der gesamten Operation die Maus narkotisiert mit 30% Sauerstoff, 70% Lachgas und 1-3% Isofluran, kontinuierlich bei 37 ± 0,5 ° C mit einem Thermistor-gesteuerte Heizkissen und überwacht durch rektale Thermometer. <ol><li> Bringen Sie sterile 27 g Nadel auf 1 cc Spritze auf dem Rahmen. </li><li> Passen stereotaktischen Arm, bis die Nadel genau über Bregma. </li><li> Passen Sie den Arm so, dass die Nadel ist bei +2,5 mm lateral Bregma und unteren an die Oberfläche des Schädels. </li><li> Manuell drehen Spritze Bohrloch auf der Oberfläche des Schädels zu machen, während leichtem Abwärtsbewegung des Frame-Betreuung nicht vollständig perforieren Schädel. </li><li> Entfernen Sie die Nadel und vollständige Bohrloch manuell mit Spritze / Nadel Hinweis: Die Vollendung des Bohrloch von Hand ermöglicht die unmittelbare Anerkennung, wenn Sie perforiert die innere Tabelle des Schädels und minimiert das Risiko eines unbeabsichtigten Drücken Nadel in Hirnparenchym haben. </li><li> Machen Querinzision am ventralen Schwanz mit sterilen Skalpell und lassen 2-3 große Tropfen des arteriellen Blutes, um auf Paraffin Papier fallen. Dann schnell zu stoppen Blutungen mit Druck mit steriler Gaze. </li><li> Ziehen Sie 17 ul Blut in Hamilton Spritze und Ort Spritze an der Pumpe. </li><li> Passen stereotaktischen Arm Punkt 5 ° nach medial relativ zum vertikalen Achse. </li><li> Sorgfältig stellen stereotaktischen Arm so, dass die Spitze der Nadel ist über das Bohrloch im Schädel und dann untere Nadel 3,5 mm. </li><li> Warten Sie 2 Minuten, dann ziehen Sie die Nadel 0,5 mm (so dass die Spitze ist 3 mm tief) </li><li> 5 Minuten warten, damit das Gehirn um die Nadel wieder zu erweitern und das Risiko zu minimieren Blut siedendem die Nadeleinführung Titel während der Injektion. </li><li> Inject Blut 1 ul / Minute für 7,5 ul. </li><li> 5 Minuten warten, damit anfängliche Blutgerinnung und für das Tissue Verschiebungen auftreten, um Erhebungen in Hirndruck zu minimieren. </li><li> Spritzen Sie die verbleibenden 7,5 l bei 1 ul / Minute </li><li> Lassen Nadel, um im Platz für 25 Minuten bleiben, um für die Blutgerinnung ermöglichen Hinweis: Die Nichtbeachtung warten für die Blutgerinnung im Blut Rückfluss bis die Nadel Insertionsstelle Ergebnis wird beim Herausziehen der Nadel </li><li> Langsam ziehen Sie die Nadel und sofort mit heißem Wasser spülen, um restliche Blut in die Nadel aus Blutgerinnung verhindern und Wiederverwendbarkeit der Nadel. </li><li> Entfernen Sie die Maus aus dem Rahmen und schließen Sie den Schwanz und die Kopfhaut Einschnitte mit veterinärmedizinischen chirurgischen Klebstoff. </li><li> Schalten Sie Anästhesie. </li><li> Lassen Sie die Maus zu wecken, während sie kontinuierlich mit freiem Zugang zu Nahrung befeuchtet erwärmt. </li><li> Zurück Maus Käfig mit Geschwistern, wenn ganz wach. Legen Sie Nassfutter Pellet am Boden des Käfigs, Tiere in den Zugang zu Nahrungsmitteln zu helfen. </li></ol> 4. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2618/2618fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Frontalschnitt von Gehirngewebe der Maus 15 Minuten nach ICH Chirurgie. Unmittelbar nach der Tötung des Gehirns für ICH Erfolg auf grobe Inspektion eines koronalen Abschnitt an der Nadel Insertionsstelle beruhte inspiziert. Blutungen, die bis auf die Basis des Gehirns verfolgt, bis die Nadel track Vergangenheit des Corpus Callosum, oder in die Ventrikel wurden als nicht erfolgreich und die Maus wurde aus allen Analysen ausgeschlossen. Insgesamt ICH success lagen 75-85% in 50 Mäuse mit 0% Mortalität. <img src="/files/ftp_upload/2618/2618fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Zylinder Test zeigt links Hemiparese nach rechts Basalganglien ICH. (A) Beispiel Maus hinten nach ICH Chirurgie. Beachten Sie die Platzierung der nur das Recht Vorderbein an der Wand des Zylinders nach links Basalganglien ICH. (B) Graph des Zylinders Test 1 ergibt sich aus Kohorte von Mäusen nach ICH Chirurgie (n = 5) im Vergleich zu den schein (n = 4). Sham-Mäuse hatten alle Verfahren mit Ausnahme von Blut-Injektion (Nadel in Gehirn eingesetzt). Jede Maus wurde in einem 12-cm Durchmesser klare Glaszylinder gelegt und beobachtet seit 20 hinten. Die anfängliche Platzierung der Vorderbeine an der Wand des Zylinders wurde per hinten erzielt. Nachfolgende Bewegungen (z. B. laterale Exploration) wurden nicht gewertet, bis die Maus wieder auf den Boden und die nächste hinten erzielt. Die Lateralität Index wurde berechnet als (# rechte Vorderbein Platzierungen auf der Seite des Zylinders - # linke Vorderbein Platzierungen) / (# rechts + # linke + # beides), wobei 0 zeigten keine forelimb Vorlieben und 1 angegeben nur das Recht forelimb verwendet wurde .

<ol>
	<li>Hua, Y., Schallert, T., Keep, R. F., Wu, J., Hoff, J. T., Xi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Tests+After+Intracerebral+Hemorrhage+in+the+Rat.">Behavioral Tests After Intracerebral Hemorrhage in the Rat.</a> Stroke. 33, 2478-2484 (2002).</li><li>James, M., Warner, D., Laskowitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Models+of+Intracerebral+Hemorrhage:+A+Translational+Perspective.">Preclinical Models of Intracerebral Hemorrhage: A Translational Perspective.</a> Neurocritical Care. 9, 139-152 (2008).</li><li>Foerch, C., Arai, K., Jin, G., Park, K. -. P., Pallast, S., van Leyen, K., Lo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Model+of+Warfarin-Associated+Intracerebral+Hemorrhage.">Experimental Model of Warfarin-Associated Intracerebral Hemorrhage.</a> Stroke. 39, 3397-3404 (2008).</li><li>Nakamura, T., Xi, G., Hua, Y., Schallert, T., Hoff, J. T., Keep, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+hemorrhage+in+mice:+model+characterization+and+application+for+genetically+modified+mice.">Intracerebral hemorrhage in mice: model characterization and application for genetically modified mice.</a> Journal of Cerebral Blood Flow and Metabolism. 24, 487-494 (2004).</li><li>Rynkowski, M. A., Kim, G. H., Komotar, R. J., Otten, M. L., Ducruet, A. F., Zacharia, B. E., Kellner, C. P., Hahn, D. K., Merkow, M. B., Garrett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+intracerebral+hemorrhage+using+autologous+blood+infusion.">A mouse model of intracerebral hemorrhage using autologous blood infusion.</a> Nat. Protocols. 3, 122-128 (2008).</li><li>Wang, J., Fields, J., Dor&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+an+improved+preclinical+mouse+model+of+intracerebral+hemorrhage+using+double+infusion+of+autologous+whole+blood.">The development of an improved preclinical mouse model of intracerebral hemorrhage using double infusion of autologous whole blood.</a> Brain Research. 1222, 214-221 (2008).</li><li>Zhao, X., Sun, G., Zhang, J., Strong, R., Song, W., Gonzales, N., Grotta, J. C., Aronowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoma+resolution+as+a+target+for+intracerebral+hemorrhage+treatment:+Role+for+peroxisome+proliferator-activated+receptor+&#947;+in+microglia/macrophages.">Hematoma resolution as a target for intracerebral hemorrhage treatment: Role for peroxisome proliferator-activated receptor &#947; in microglia/macrophages.</a> Annals of Neurology. 61, 352-362 (2007).</li></ol>

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Dieses chirurgische Modell intrazerebralen Blutungen in Mäusen unter Verwendung autologer Schwanzarterie Blutwerte in ein reproduzierbares Modell der spontanen Basalganglien Blutung. Eine ICH-Modell bei Mäusen hat den Vorteil der Verfügbarkeit von transgenen Tieren zu Pathophysiologie untersuchen, jedoch macht ihre geringe Größe neurochirurgische Verfahren technisch schwieriger als in größeren Tieren. Die Collagenase-Modell und die Eigenblut Injektion Modell sind zwei etablierte Model...

Stereotaktischen Rahmen für Maus Neurochirurgie (Stölting, 51925) Mikroinfusion Pumpe und Prozessor (UMP-3 und Micro4, World Precision Instruments, Sarasota, FL) Maus wärmer (Stölting, 50300) Inhalationsanästhesie Maus Anästhesie (Braintree Scientific, EZ-AF9000) 25 ul gasdicht Borosilikat Hamilton-Spritze mit beschichteten Kolben und ohne Nadel (Firma Hamilton, Reno, NV, 1702RN Spritze: 765401, Aderendhülse: 30949, spacer: 30946) Fused-Silica-Nadel geschnitten 2 cm Länge (Hamilton, 17739) * Hinweis Hamilton Spritze und Fused-Silica-Nadel kann für mehrere Operationen verwendet werden, wenn sterilisiert vor jeder Operation. Diese Materialien sind entscheidend, um zu vermeiden Blutgerinnung. Sterile OP-Handschuhe OP-Kittel, Haube und Maske Betadine 75% Ethanol sterile 27 g Nadel (single use) sterile 1 ml-Spritze (single use) sterile OP-Messer Cidex sterilem Wasser Buprenorphin und Isofluran steriler Gaze Paraffin Papier Quadrate Veterinary chirurgische Kleber (Vetbond, 3M, St. Paul, MN)

autologous blood injection to model spontaneous intracerebral hemorrhage in mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

Watch this Scientific Journal Video about Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice at JoVE.com

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Die Eigenblut Injektion Modell der intrazerebralen Blutung bei Mäusen in diesem Protokoll beschriebenen Verwendungen der doppelten Einspritzung Technik, um das Risiko von Blut Reflux bis die Nadel Spur, keine Antikoagulanzien in das Pumpsystem zu minimieren, und eliminiert alle Totraum und erweiterbar Schlauch in das System.

Eigenblut Injection zu Spontane intrazerebrale Blutungen in Mäuse-Modell

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

autologous-blood-injection-to-model-spontaneous-intracerebral

Research

JoVE Journal

Neuroscience

14.7K Aufrufe.  University of Connecticut Health Center. Die Eigenblut Injektion Modell der intrazerebralen Blutung bei Mäusen in diesem Protokoll beschriebenen Verwendungen der doppelten Einspritzung Technik, um das Risiko von Blut Reflux bis die Nadel Spur, keine Antikoagulanzien in das Pumpsystem zu minimieren, und eliminiert alle Totraum und erweiterbar Schlauch in das System.

Serie: Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Methoden zur Charakterisierung Spontan und Startle-induzierte Locomotion in einer Rotenon-induzierten Parkinson-Krankheit Modell der<em&gt; Drosophila</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

Forschung

Neurowissenschaften

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Mausmodelle für Zika-Virus-induzierte neurologische Erkrankungen mit intrazerebrale Injektion Strategien etablieren: embryonale, Neugeborene und Erwachsene

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Rattenmodell der weit verbreiteten zerebralen kortikalen Demyelinisierung, induziert durch eine intrazerebrale Injektion von entzündungsfördernden Zytokinen

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Eine Volumetrische Methode zur Quantifizierung der zerebralen Vasospasmus in einem Mausmodell der Subarachnoidalblutung

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based treatment options for this devastating disease process. In the past decade, a number of minimally invasive surgeries have emerged to address this issue, one of which is endoscopic evacuation. Stereotactic ICH Underwater Blood Aspiration (SCUBA) is a novel endoscopic evacuation technique performed in a fluid-filled cavity using an aspiration system to provide an additional degree of freedom during the procedure. The SCUBA procedure utilizes a suction device, endoscope, and sheath and is divided into two phases. The first phase involves maximal aspiration and minimal irrigation to decrease clot burden. The second phase involves increasing irrigation for visibility, decreasing aspiration strength for targeted aspiration without disturbing the cavity wall, and cauterizing any bleeding vessels. Using the endoscope and aspiration wand, this technique aims to maximize hematoma evacuation while minimizing collateral damage to the surrounding brain. Advantages of the SCUBA technique include the use of a low-profile endoscopic sheath minimizing brain disruption and improved visualization with a fluid-filled cavity rather than an air-filled one.

This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique.

Minimalinvasive endoskopische intrazerebrale Blutungsevakuierung

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

In Vivo Direkte Reprogrammierung von Resident Glial Cells in Interneurons durch Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Doppelte Direkteinspritzung von Blut in die Cisterna Magna als Modell der subarachnoiden Blutung

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

Massive Pontinenblutung durch Doppelinjektion von Eigenblut

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Prächiasmatische, einmalige Injektion von Eigenblut zur Induktion einer experimentellen Subarachnoidalblutung in einem Rattenmodell

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Intracranial Pressure Monitoring In Nontraumatic Intraventricular Hemorrhage Rodent Model

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage animal models is essential. In this study, we sought out ways to measure intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during nontraumatic intraventricular hemorrhage in rats. The experimental design included three Sprague Dawley groups: sham, standard 200 &#181;l intraventricular hemorrhage, and vehicle control groups. By introducing an intraparenchymal fiberoptic pressure sensor, precise intracranial pressure measurements were obtained in all groups. Cerebral perfusion pressures were calculated with the knowledge of intracranial pressure and mean arterial pressure values. As expected, the intraventricular hemorrhage and vehicle control groups both experienced a rise in the intracranial pressure and subsequent decline in cerebral perfusion pressure during intraventricular injection of autologous blood and artificial cerebrospinal fluid, respectively. The addition of an intraparenchymal fiberoptic pressure sensor is beneficial in monitoring precise intracranial pressure changes.

Monitoring intracranial pressure in rodent&#160;models of nontraumatic intraventricular hemorrhage is not common in the current literature. Herein, we demonstrate a technique for measuring intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during intraventricular hemorrhage in a rat&#160;animal model.

Intrakranielle Drucküberwachung im nichttraumatischen intraventrikulären Blutungsmodell

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage ...