Le travail a été financé par une bourse de l&#39;Institut pour la médecine translationnelle et de thérapeutique, et une bourse de formation de l&#39;Institut de médecine et d&#39;ingénierie (T32HL007954) à l&#39;Université de Pennsylvanie et de l&#39;Marlene L. Cohen et Jérôme H. Fleisch Scholar Subvention à la University of Connecticut Health Center (LHS) et NIH NS-029331 (FAW).

1. Préparation du matériel <ol><li> Essuyez le cadre stéréotaxique et de la pompe avec de l&#39;éthanol à 75% afin de minimiser la contamination bactérienne. </li><li> Stériliser seringue Hamilton et l&#39;aiguille de silice fondue. Remarque: Si la stérilisation chimique est utilisé, n&#39;oubliez pas de rincer plusieurs fois dans de l&#39;eau stérile avant utilisation. </li><li> Essuyez la surface du papier de paraffine avec de l&#39;éthanol 75% et laisser sécher. </li></ol> 2. Préparation de la souris pour l&#39;injection Note: Avez-souris livré à votre animalerie au moins 7 jours avant la chirurgie afin de leur permettre de s&#39;acclimater à leur nouvel environnement et à réduire le stress. <ol><li> Peser la souris pour le pré-opératoire de base. </li><li> Induire une anesthésie avec 30% d&#39;oxygène, 70% d&#39;oxyde nitreux, et 4% d&#39;isoflurane jusqu&#39;au insensibles à pincement de la queue </li><li> Injecter de la souris avec la buprénorphine par voie intrapéritonéale pendant 0.1mg/kg analgésie postopératoire </li><li> Rasage du cuir chevelu </li><li> Manteau avec les yeux de la vaseline stérile </li><li> Préparer le cuir chevelu avec de la Bétadine x 3 lingettes, puis laisser sécher le cuir chevelu </li><li> Faire une incision médiane sagittale cm du cuir chevelu à l&#39;aide stériles scalpel chirurgical Remarque: Une incision généreuse permettra une exposition complète de repères crâne. </li><li> Rasage de 1 cm de la surface ventrale de la queue commence à 1 cm de base et préparer avec la bétadine x 3 lingettes </li><li> Placez la souris sur le cadre de stéréotaxie Note: Il est important de s&#39;assurer de la souris est en sécurité dans le cadre avec la surface du crâne parallèles avec la base du châssis, avec une excellente visibilité du bregma et au moins 3 mm à la droite du bregma. </li></ol> 3. Chirurgie de l&#39;hémorragie intracérébrale Notes: Durant toute la chirurgie de la souris est anesthésié avec 30% d&#39;oxygène, 70% d&#39;oxyde nitreux, et 1-3% isoflurane, constamment maintenue à 37 ± 0,5 ° C à l&#39;aide d&#39;une thermistance contrôlée coussin chauffant et surveillé par un thermomètre rectal. <ol><li> Fixez stériles aiguille 27 G sur une seringue de cc sur le châssis. </li><li> Ajustez le bras stéréotaxique jusqu&#39;au aiguille est exactement sur bregma. </li><li> Ajustez le bras de telle sorte que l&#39;aiguille est à 2,5 mm latéralement à bregma et inférieure à la surface du crâne. </li><li> Faites tourner manuellement seringue pour faire trou de trépan sur la surface du crâne tout en appliquant douce mouvement de baisse de frame-en prenant soin de ne pas complètement perforer le crâne. </li><li> Retirer l&#39;aiguille et remplir trou de trépan manuellement avec une seringue / aiguille Remarque: Remplir le trou de trépan à la main permet la reconnaissance immédiate lorsque vous avez perforé la table interne du crâne et minimise les risques d&#39;inadvertance pousser l&#39;aiguille dans le parenchyme cérébral. </li><li> Faire une incision transversale sur la surface ventrale de la queue à l&#39;aide stériles lame chirurgicale et permettre 2-3 gouttes de sang artériel grandes pour tomber sur du papier de paraffine. Puis rapidement arrêter le saignement avec la pression à l&#39;aide de gaze stérile. </li><li> Retirer 17 uL de sang dans la seringue et la seringue Hamilton place sur la pompe. </li><li> Ajustez le bras stéréotaxique au point 5 ° médial par rapport à l&#39;axe vertical. </li><li> Ajustez soigneusement le bras stéréotaxique de telle sorte que la pointe de l&#39;aiguille est sur le trou de trépan dans le crâne, puis abaissez l&#39;aiguille de 3,5 mm. </li><li> Attendre 2 minutes puis retirer l&#39;aiguille de 0,5 mm (de sorte que la pointe est de 3 mm de profondeur) </li><li> Attendre 5 minutes pour permettre au cerveau de re-développer autour de l&#39;aiguille et de minimiser les risques de reflux de sang jusqu&#39;à la piste insertion de l&#39;aiguille lors de l&#39;injection. </li><li> Injecter du sang 1 ul / minute pour 7,5 uL. </li><li> Attendez 5 minutes pour permettre au sang de la coagulation et initiales pour les quarts de tissus de se produire pour minimiser les élévations de la pression intracrânienne. </li><li> Injecter le reste 7,5 pi à 1 ul / minute </li><li> Laisser l&#39;aiguille de rester en place pendant 25 minutes pour permettre la coagulation du sang Remarque: Si vous n&#39;attendez pas la coagulation du sang se traduira dans le sang reflux jusqu&#39;à l&#39;emplacement insertion de l&#39;aiguille lors du retrait de l&#39;aiguille </li><li> Lentement retirer l&#39;aiguille et rincer immédiatement avec de l&#39;eau chaude, pour éviter toute trace de sang résiduel dans l&#39;aiguille de la coagulation et d&#39;assurer la réutilisabilité de l&#39;aiguille. </li><li> Retirez la souris du cadre et à proximité de la queue et les incisions du cuir chevelu avec de la colle chirurgicale vétérinaire. </li><li> Éteignez l&#39;anesthésie. </li><li> Laisser la souris pour réveiller tout en étant constamment réchauffé avec libre accès à la nourriture humide. </li><li> Retour à la souris en cage avec la même portée une fois pleinement éveillé. Placer la nourriture humide granulés sur le fond de la cage pour aider les animaux dans l&#39;accès à la nourriture. </li></ol> 4. Les résultats représentatifs: <img src="/files/ftp_upload/2618/2618fig1.jpg" alt="Figure 1"/> Figure 1. Coupe coronale de cerveau de souris 15 minutes après la chirurgie ICH. Immédiatement après le sacrifice du cerveau a été inspecté pour la réussite CIH basée sur l&#39;inspection brute d&#39;une coupe coronale au site d&#39;insertion de l&#39;aiguille. Les hémorragies qui a suivi jusqu&#39;à la base du cerveau, le trajet de l&#39;aiguille passé le corps calleux, ou dans les ventricules ont été jugées infructueuses et que la souris a été éliminée de toutes les analyses. Globalement ICH succetaux de SS étaient à 75-85% dans les 50 souris avec une mortalité de 0%. <img src="/files/ftp_upload/2618/2618fig2.jpg" alt="Figure 2"/> Test Figure 2. Cylindre démontre une hémiparésie gauche après l&#39;ICH ganglions droit basale. (A) à l&#39;arrière de la souris échantillon après la chirurgie ICH. Notez que le placement de la patte avant droite sur la paroi du cylindre, après l&#39;ICH ganglions laissé basale. (B) Graphique des tests de cylindre 1 des résultats de la cohorte de souris après l&#39;ICH chirurgie (n = 5) par rapport à fictive (n = 4). Souris Sham avait toutes les procédures d&#39;injection, sauf le sang (aiguille a été insérée dans le cerveau). Chaque souris a été placée dans un cylindre de 12 cm de diamètre en verre clair et observés pendant 20 arrières. Le placement initial des membres antérieurs sur la paroi du cylindre a été marqué par l&#39;arrière. Mouvements ultérieurs (tels que l&#39;exploration latérale) ne sont pas marqué tant que la souris retournée dans le sol et l&#39;arrière à côté marqué. L&#39;indice a été calculé comme la latéralité (# droite stages forelimb sur le côté du cylindre - # laissé stages forelimb) / (# # droite + gauche + # fois), où 0 indique pas de préférence des membres antérieurs et une indication que la patte avant droite a été utilisé .

<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>

Aucun conflit d&#39;intérêt déclaré.

Ce modèle chirurgical de l&#39;hémorragie intracérébrale chez des souris en utilisant les résultats autologues de sang dans l&#39;artère la queue un modèle reproductible d&#39;hémorragie spontanée ganglions basaux. Un modèle chez la souris CIH offre l&#39;avantage de la disponibilité des animaux transgéniques pour étudier la physiopathologie, mais leur petite taille les rend interventions neurochirurgicales techniquement plus difficile que dans les grands animaux. Le modèle de ...

Cadre stéréotaxique pour la neurochirurgie de la souris (Stoelting, 51925) Pompe microperfusion et le processeur (UMP-3 et Micro4, Instruments de précision mondiale, Sarasota, Floride) Plus chaudes de la souris (Stoelting, 50300) L&#39;anesthésie par inhalation de la souris (Braintree scientifique, EZ-AF9000) 25 uL étanche borosilicate Hamilton seringue avec piston enduits et aucune aiguille (Société de Hamilton, Reno, NV, une seringue 1702RN: 765401, ferrule: 30949, entretoise: 30946) l&#39;aiguille de silice fondue coupé à 2 cm de longueur (Hamilton, 17739) * Note Hamilton seringue et l&#39;aiguille de silice fondue peut être réutilisée pour des chirurgies multiples, si stérilisés avant chaque opération. Ces matériaux sont essentiels pour éviter la coagulation du sang. Gants chirurgicaux stériles Blouse chirurgicale, bonnet et un masque Betadine 75% d&#39;éthanol 27 g d&#39;aiguilles stériles (à usage unique) seringue stérile cc 1 (usage unique) stériles lame chirurgicale Cidex l&#39;eau stérile buprénorphine et l&#39;isoflurane gaze stérile carrés de papier de cire de paraffine Vétérinaire colle chirurgicale (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

Le modèle d&#39;injection de sang autologue d&#39;hémorragie intracérébrale chez la souris décrites dans ce protocole utilise la technique de double injection pour minimiser le risque de reflux du sang jusqu&#39;à la piste d&#39;aiguille, pas d&#39;anticoagulants dans le système de pompage, et élimine tous les espaces morts et tuyaux extensibles dans le système.

Injection de sang autologue au modèle spontané Hémorragie intracérébrale chez la souris

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

14.6K vues.  University of Connecticut Health Center. Le modèle d'injection de sang autologue d'hémorragie intracérébrale chez la souris décrites dans ce protocole utilise la technique de double injection pour minimiser le risque de reflux du sang jusqu'à la piste d'aiguille, pas d'anticoagulants dans le système de pompage, et élimine tous les espaces morts et tuyaux extensibles dans le système.

Vidéo: 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.

Méthodes pour caractériser spontanée et induite par Locomotion sursaut dans un modèle de la maladie de Parkinson induite de la roténone-de<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, ...

Recherche

Neurosciences

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.

Établir des modèles de souris pour troubles neurologiques Zika Viro-induite à l’aide de stratégies d’Injection intracérébrale : embryonnaires, néonatals et adultes

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.

Modèle de rat de démyélinisation corticale cérébrale généralisée induite par une injection intracérébrale de cytokines pro-inflammatoires

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.

Une méthode volumétrique pour la Quantification du vasospasme cérébral dans un modèle murin d’hémorragie sous-arachnoïdienne

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.

Évacuation de l’hémorragie intracérébrale endoscopique mini-invasive

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 Reprogrammation directe des cellules gliales résidentes en interneurones par injection intracérébrale de vecteurs viraux

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.

Double injection directe de sang dans la Cisterna Magna comme modèle d’hémorragie subarachnoïde

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.

Hémorragie pontine massive par double injection de sang autologue

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.

Injection unique pré-chiasmatique de sang autologue pour induire une hémorragie sous-arachnoïdienne expérimentale chez un rat

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.

Surveillance de la pression intracrânienne dans un modèle de rongeur hémorragique intraventriculaire non traumatique

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