Il lavoro è stato finanziato da una borsa di studio presso l&#39;Istituto di Medicina Traslazionale e Therapeutics, e una borsa di formazione presso l&#39;Istituto di Medicina e Ingegneria (T32HL007954) presso l&#39;Università della Pennsylvania e della Marlene L. Cohen e Jerome H. Fleisch Scholar sovvenzione al University of Connecticut Health Center (LHS) e NIH NS-029331 (FAW).

1. La preparazione delle attrezzature <ol><li> Pulire la struttura stereotassica e la pompa con il 75% di etanolo per minimizzare la contaminazione batterica. </li><li> Sterilizzare siringa Hamilton e l&#39;ago di silice fusa. Nota: Se la sterilizzazione chimica è usato, assicuratevi di risciacquare più volte in acqua sterile prima dell&#39;uso. </li><li> Pulire la superficie della carta paraffina con il 75% di etanolo e lasciare asciugare. </li></ol> 2. Preparazione del mouse per iniezione Nota: i topi hanno trasportato al vostro impianto animale almeno 7 giorni prima dell&#39;intervento per consentire loro di acclimatarsi nel nuovo ambiente e ridurre lo stress. <ol><li> Pesare mouse per pre-operatoria di base. </li><li> Indurre l&#39;anestesia con il 30% di ossigeno, il 70% di ossido di azoto, e il 4% isoflurano fino a che non risponde a pizzico coda </li><li> Iniettare mouse con buprenorfina 0.1mg/kg intraperitoneale per analgesia post-operatoria </li><li> Rasatura del cuoio capelluto </li><li> Occhi cappotto con vaselina sterile </li><li> Preparare cuoio capelluto con betadine x 3 salviette, quindi lasciare asciugare cuoio capelluto </li><li> Fare 1 incisione mediana sagittale centimetro di cuoio capelluto usando bisturi chirurgici sterili Nota: Un generoso incisione permetterà l&#39;esposizione completa dei punti di riferimento cranio. </li><li> Shave 1 cm di superficie ventrale della coda a partire dal 1 cm dalla base e preparare con betadine x 3 salviette </li><li> Posizionare il mouse sul telaio stereotassico Nota: è importante garantire il mouse sia correttamente inserita nel telaio con superficie del cranio parallelo con la base del telaio, con ottima esposizione di bregma e almeno 3 mm a destra del bregma. </li></ol> 3. Emorragia intracerebrale chirurgia Note: Durante l&#39;intervento intero il mouse è anestetizzato con il 30% di ossigeno, il 70% di ossido di azoto, e 1-3% isoflurano, continuamente mantenuto a 37 ± 0,5 ° C con un termistore controllato piastra elettrica e monitorato da termometro rettale. <ol><li> Attaccare sterile 27 ago g su 1 siringa cc sul telaio. </li><li> Regolare il braccio fino a quando l&#39;ago stereotassica è esattamente sopra bregma. </li><li> Regolare il braccio in modo che l&#39;ago è a 2,5 millimetri di bregma laterale e inferiore alla superficie del cranio. </li><li> Ruotare manualmente siringa per fare buca bava sulla superficie del cranio durante l&#39;applicazione dolce movimento verso il basso del quadro facendo attenzione a non forare completamente cranio. </li><li> Rimuovere l&#39;ago e completo buco radica manualmente con siringa / ago Nota: Completare il buco bava a mano consente l&#39;immediato riconoscimento quando si ha perforato la tabella interna del cranio e minimizza il rischio di spingere inavvertitamente ago nel parenchima cerebrale. </li><li> Fai incisione trasversale sulla superficie ventrale della coda con lama chirurgica sterile e lasciare 2-3 gocce di sangue arterioso per cadere su carta paraffina. Poi rapidamente arrestare le emorragie con la pressione con garza sterile. </li><li> Prelevare 17 microlitri di sangue nella siringa Hamilton e siringa posto sulla pompa. </li><li> Regolare il braccio stereotassica al punto 5 ° medialmente rispetto all&#39;asse verticale. </li><li> Regolare con cura braccio stereotassica in modo che punta dell&#39;ago è sopra il foro nel cranio bave e quindi abbassare l&#39;ago da 3,5 mm. </li><li> Attendere 2 minuti e poi ritirare il 0,5 millimetri ago (in modo che punta è di 3 mm di profondità) </li><li> Attendere 5 minuti per permettere al cervello di ri-espandersi intorno all&#39;ago e minimizzare il rischio di reflusso di sangue la traccia inserimento dell&#39;ago durante l&#39;iniezione. </li><li> Iniettare sangue 1 ml / minuto per 7,5 microlitri. </li><li> Attendere 5 minuti per consentire iniziale coagulazione del sangue e per i turni di tessuto che si verifichi per ridurre al minimo innalzamento della pressione intracranica. </li><li> Iniettare il restante 7,5 microlitri di 1 ml / minuto </li><li> Lasciare l&#39;ago di rimanere in posizione per 25 minuti per consentire la coagulazione del sangue Nota: non aspettate la coagulazione del sangue si tradurrà nel sangue refluisce il sito di inserimento dell&#39;ago quando ritirare l&#39;ago </li><li> Estrarre lentamente l&#39;ago e subito risciacquare con acqua calda per evitare residui di sangue l&#39;ago dalla coagulazione e garantire riutilizzo di aghi. </li><li> Rimuovere il mouse dal telaio e chiudere la coda e incisioni cuoio capelluto con colla chirurgica veterinaria. </li><li> Spegnere l&#39;anestesia. </li><li> Permettono di mouse per risvegliare pur essendo continuamente riscaldato con accesso gratuito al cibo umido. </li><li> Ritorna mouse per gabbia con fratellini quando è completamente sveglio. Luogo umido cibo precipitato sul fondo della gabbia per aiutare gli animali in accesso al cibo. </li></ol> 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2618/2618fig1.jpg" alt="Figura 1"/> Figura 1. Sezione coronale di cervello di topo 15 minuti dopo l&#39;intervento chirurgico ICH. Immediatamente dopo il sacrificio del cervello è stato ispezionato per il successo ICH sulla base di un&#39;ispezione lorda di una sezione coronale nel punto di inserimento dell&#39;ago. Emorragie che rintracciato alla base del cervello, il percorso dell&#39;ago passato il corpo calloso, o nei ventricoli sono state ritenute successo e che il mouse è stato eliminato da tutte le analisi. Nel complesso ICH succetassi ss erano il 75-85% in 50 topi con 0% di mortalità. <img src="/files/ftp_upload/2618/2618fig2.jpg" alt="Figura 2"/> Test Figura 2. Cilindro dimostra emiparesi destra a sinistra dopo emorragia intracerebrale gangli della base. (A) topo posteriore del campione dopo l&#39;intervento chirurgico ICH. Nota la posizione del solo dell&#39;arto anteriore destro sulla parete del cilindro dopo sinistra ICH gangli della base. (B) Grafico di test cilindro 1 risultati di coorte di topi dopo ICH chirurgia (n = 5) rispetto alla farsa (n = 4). Topi Sham aveva tutte le procedure ad eccezione di iniezione del sangue (l&#39;ago è stato inserito nel cervello). Ogni mouse è stato posto in un cilindro di 12 cm di diametro in vetro chiaro e osservati per 20 posteriori. Il posizionamento iniziale degli arti anteriori sulla parete del cilindro è stato segnato al posteriore. Movimenti successivi (come l&#39;esplorazione laterale) non sono stati segnati con il mouse fino a quando tornò a terra e la parte posteriore prossimo segnato. L&#39;indice di lateralità è stato calcolato come (# tirocini zampa anteriore destra sul lato del cilindro - # sinistra tirocini forelimb) / (+ # # destra sinistra + # entrambi), dove 0 non hanno indicato alcuna preferenza zampa anteriore e 1 indicata solo la zampa anteriore destra è stata utilizzata .

<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>

Nessun conflitto di interessi dichiarati.

Questo modello chirurgico dell&#39;emorragia intracerebrale in topi con la coda risultati autologo del sangue dell&#39;arteria in un modello riproducibile di emorragia spontanea gangli della base. Un modello ICH nei topi offre il vantaggio della disponibilità di animali transgenici per studiare fisiopatologia, tuttavia, la loro piccola dimensione rende le procedure neurochirurgiche più tecnicamente difficile che in animali più grandi. Il modello di collagenasi e l&#39;iniezione di sangue ...

Telaio per neurochirurgia stereotassica mouse (Stoelting, 51925) Microinfusion pompa e processore (UMP-3 e Micro4, strumenti di precisione mondiale, Sarasota, FL) Il mouse più caldo (Stoelting, 50300) Topo anestesia per inalazione (Braintree scientifico, EZ-AF9000) 25 microlitri tenuta di gas borosilicato siringa Hamilton con pistone patinata e senza ago (Società Hamilton, Reno, NV, siringa 1702RN: 765401, ghiera: 30949, spacer: 30946) ago silice fusa tagliato a 2 cm di lunghezza (Hamilton, 17739) * Nota Hamilton siringa e l&#39;ago di silice fusa possono essere riutilizzati per interventi chirurgici multipli se sterilizzato prima di ogni intervento chirurgico. Questi materiali sono cruciali per evitare la coagulazione del sangue. Guanti chirurgici sterili Camice chirurgico, cofano e mascherina Betadine 75% di etanolo 27 g ago sterile (uso singola) 1 siringa sterile cc (uso singola) sterili lama chirurgica Cidex acqua sterile buprenorfina e isoflurano garza sterile paraffina carta piazze Colla chirurgica veterinaria (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

L&#39;iniezione di sangue autologo modello di emorragia intracerebrale in topi descritti in questo protocollo utilizza la tecnica di iniezione doppio per minimizzare il rischio di reflusso del sangue il percorso dell&#39;ago, non anticoagulanti nel sistema di pompaggio, ed elimina tutto lo spazio morto e tubi espandibili nel sistema.

Iniezione di sangue autologo Modello spontanea emorragia intracerebrale in topi

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 Views.  University of Connecticut Health Center. L'iniezione di sangue autologo modello di emorragia intracerebrale in topi descritti in questo protocollo utilizza la tecnica di iniezione doppio per minimizzare il rischio di reflusso del sangue il percorso dell'ago, non anticoagulanti nel sistema di pompaggio, ed elimina tutto lo spazio morto e tubi espandibili nel sistema.

Video: 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.

Metodi di caratterizzare spontanea e startle indotta Locomozione in un rotenone-indotta malattia di Parkinson Modello di<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, ...

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Neuroscienze

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.

Creazione di modelli murini per disturbi neurologici indotta da Virus di Zika utilizzando strategie di iniezione intracerebrale: embrionale, neonatale e adulto

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.

Modello di ratto di demielinizzazione corticale cerebrale diffusa indotta da un'iniezione intracerebrale di citochine pro-infiammatorie

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.

Un metodo volumetrico per la quantificazione di Vasospasm cerebrale in un modello murino di emorragia subaracnoidea

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.

Evacuazione endoscopica endoscopica minimamente invasiva dell'emorragia intracerebrale

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.

Riprogrammazione diretta in vivo delle cellule gliali residenti negli interneuroni mediante iniezione intracerebrale di vettori virali

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.

Doppia iniezione diretta di sangue nella Cisterna Magna come modello di emorragia subarachnoide

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.

Emorragia pontina massiccia per doppia iniezione di sangue autologo

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.

Pre-chiasmatico, singola iniezione di sangue autologo per indurre emorragia subaracnoidea sperimentale in un modello di ratto

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.

Monitoraggio della pressione intracranica nel modello di roditore emorragia intraventricolare non traumatico

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