Questo studio è stato in parte sostenuto da NIH sovvenzione RO1NS053407 a JH Zhang. Vorremmo ringraziare il signor Damon Klebe per i suoi preziosi contributi.

Tutte le procedure sono state effettuate in conformità con la Guida NIH per la cura ed uso di animali da laboratorio e approvato dalla cura degli animali e del Comitato uso della Loma Linda University. 1. Preparazioni prechirurgica Tecniche asettiche sono raccomandati per tutte le procedure chirurgiche. Disinfettare l&#39;apparato stereotassico e preparare sterili strumenti chirurgici prima dell&#39;intervento. Indossare dispositivi di protezione individuale (DPI) durante tutto il trattamento degli animali. Utilizzare una piastra elettrica durante l&#39;intervento chirurgico per mantenere la temperatura fisiologica del corpo dell&#39;animale. <ol><li> Pesare il 8-12 vecchio mouse settimana utilizzando una tripla scala animale fascio. </li><li> Co-iniezione di ketamina (100 mg / kg) e xilazina (10 mg / kg) per via intraperitoneale quindi consentire 7-10 minuti per l&#39;anestesia abbia effetto (monitor per un sedazione adeguata). </li><li> Posizionare il mouse su una coperta termica e radersi il cuoio capelluto. </li><li> Applicare una pomata oftalmica a entrambi gli occhi. </li><li> Fissare ilvie aeree, delicatamente muovere la lingua lateralmente, e con attenzione fissare la testa del mouse sul dispositivo stereotassico. Nota: La testa deve essere fissato orizzontalmente alla base del telaio stereotassico. </li><li> Disinfettare l&#39;area chirurgica con Betadine, e risciacquare con etanolo al 70%. Ripetere alternando applicazioni di Betadine e etanolo al 70% per un totale di tre volte. Punte di cotone può essere utilizzato per questo scopo. </li></ol> 2. Sangue iniezione Modello <ol><li> Fai un cm 1 lunga incisione mediana del cuoio capelluto con una lama di bisturi # 10. </li><li> Utilizzare punte di cotone per rimuovere il tessuto molle che copre il cranio, in modo da esporre il punto di intersezione perpendicolare della sutura coronale e sagittale (bregma). </li><li> Montare la siringa Hamilton (250 microlitri) sulla pompa di iniezione, e stereotaxically dirigere l&#39;ago (26 Gauge) sopra bregma. </li><li> Quindi, regolare i bracci manipolatori stereotassica per posizionare l&#39;ago di 0,2 millimetri anteriore e 2millimetri lateralmente a destra. A queste coordinate fare un piccolo foro cranica bave, utilizzando un trapano a velocità variabile, con una punta da 1 mm. </li><li> Sospendere coda dell&#39;animale e disinfettare la superficie inferiore con etanolo 70%. </li><li> Puntura l&#39;arteria centrale della coda con un ago sterile (ad esempio, 26 Gauge) e raccogliere il sangue arterioso in un tubo capillare unheparinized. </li><li> Trasferire il sangue rapidamente dal tubo capillare nel cilindro in vetro della siringa Hamilton, quindi inserire il pistone. </li><li> Ricollegare il 30 microlitri ora o più di sangue arterioso contenente una siringa Hamilton sulla pompa di iniezione e inserire l&#39;ago (con il bordo smussato rivolto verso la sutura sagittale) attraverso il foro cranico solo fino alla sua smussatura non è più visibile. </li><li> Da questo punto anticipo l&#39;ago 3 millimetri ventralmente e iniettare 5 ml di sangue autologo ad una velocità di 2 ml / min. </li><li> Dopo il completamento della prima iniezione anticipo l&#39;ago di 0,7 millimetri ulteriormente in profondità. </li><li> Attendere5 min quindi iniettare 25 pl di sangue nello striato destro. </li><li> Al termine della seconda iniezione, lasciare l&#39;ago in posizione per altri 10 minuti, prima di prelevare ad un tasso di 1 mm / min. </li><li> Sigillare il foro cranico con cera ossea e suturare la pelle. </li><li> Per l&#39;analgesia postoperatoria iniettare 0,05 mg / kg di buprenorfina per via sottocutanea in pre-riscaldate liquidi (soluzione fisiologica). </li></ol> 3. Collagenasi iniezione Modello <ol><li> Dopo i preparativi preoperatori, ripetere i passaggi 1-4 come descritto per il modello iniezione sangue. </li><li> Riempire la siringa Hamilton (10 pl) con 0.075U di batteri (clostridi) collagenasi VII-S disciolti in 0,5 ml di soluzione fisiologica. Evitare la formazione di bolle d&#39;aria. </li><li> Ricollegare la siringa Hamilton sulla pompa di iniezione e inserire l&#39;ago (26 Gauge), attraverso il foro cranico solo fino alla sua smussatura non è più visibile. </li><li> Far avanzare il 3,7 millimetri ago ventralmente e iniettare il 0.075U di collagenasi into striato destra ad una velocità di 2 ml / min. </li><li> Al termine dell&#39;iniezione, lasciare l&#39;ago in posizione per altri 10 minuti, prima di prelevare ad un tasso di 1 mm / min. </li><li> Sigillare il foro cranico con cera ossea e suturare la pelle. </li><li> Iniettare 0,05 mg / kg di buprenorfina per via sottocutanea in preriscaldate postoperatorie fluidi. </li></ol> 4. Sham Funzionamento <ol><li> Dopo i preparativi preoperatori, ripetere i passaggi 1-4 come descritto per il modello iniezione sangue. </li><li> Inserire il (26 Gauge) ago 3,7 millimetri ventralmente attraverso il foro cranico. L&#39;ago deve rimanere in posizione per 10 min prima di essere ritirato ad una velocità di 1 mm / min. </li><li> Sigillare il foro cranico con cera ossea e suturare la pelle. </li><li> Iniettare 0,05 mg / kg di buprenorfina per via sottocutanea in preriscaldate postoperatorie fluidi. </li></ol> 5. Risultati rappresentativi Sperimentale emorragia intrastriatal evoca morfologicocosì come cambiamenti comportamentali nei roditori. Queste modifiche possono essere valutati per garantire una adeguata esecuzione della procedura, o per studiare gli effetti di possibili trattamenti. Generazione di spurgo in una zona del cervello mirata (ad esempio nuclei della base) è più essenziale per un approccio riproducibile, e può essere verificata su sezioni di cervello lordi o istologicamente tinto (Figura 1-2). Lesioni ai risultati dei gangli della base in deficit sensomotori, che possono essere quantificati attraverso varie valutazioni comportamentali. Risultati della prova turn angolo mostrato che, dopo sperimentale lato destro ICH, topi trasformato significativamente più spesso ipsilaterale e dalla ridotta controlaterale (sinistro), di animali sham operati a 24 e 72 ore dopo l&#39;intervento (Figura 3). Inoltre, la possibilità di posizionare adeguatamente la compromissione (sinistra) forelimb su una superficie, dopo stimolazione vibrisse, è stata valutata mediante il test forelimb immissione. A 24 e 72 ore dopo l&#39;intervento, i topi subproiettate a destra lato ICH ha mostrato un numero significativamente inferiore di posizionamenti animali finti operati. Misura di edema cerebrale è spesso impiegato per quantificare l&#39;entità del danno cerebrale dopo ICH sperimentale. Iniezioni intracerebrali di sangue autologo (30 microlitri) o collagenasi batterica (0,075 U) ha portato ad un notevole aumento del contenuto di acqua nel cervello corteccia ipsilaterale e gangli basali a 24 (Figura 4 A) e 72 ore (Figura 4 B) dopo l&#39;intervento ( rispetto alla sham). Il risultato delle prove di comportamento (Figura 3) e il grado di edema cerebrale (Figura 4) ha mostrato alcuna differenza tra il sangue e modelli iniezione collagenasi in volumi dati. <img alt="Figura 1" src="/files/ftp_upload/4289/4289fig1.jpg" /> Figura 1. ICH modellazione nei topi. (A) Lo schema semplificato di una sezione coronale cervello 0,2 millimetri anteriore bregma illustra la proposed posizione di sangue autologo o iniezione collagenasi. Il ventricolo laterale è segnato LV. CPu sta per caudato-putamen, una parte del corpo striato, e GP identifica il globo pallido. Entrambi, il corpo striato e il globo pallido appartiene ad un gruppo di sub-corticale nuclei, noto anche come gangli basali. (B) microfotografia Rappresentante di una sezione del cervello coronale anteriore 0,2 millimetri di bregma, ottenuto a 24 ore dopo l&#39;iniezione di sangue autologo intrastriatal sangue intero. <img alt="Figura 2" src="/files/ftp_upload/4289/4289fig2.jpg" /> Figura 2. Manifestazione istologica dell&#39;ematoma. Rappresentante ematossilina ed eosina (H &amp; E) tinto cryosection coronale (10 micron) di un cervello di topo, illustrando dimensione ematoma a 24 ore dopo l&#39;iniezione di collagenasi batterica intrastriatal (0,075 U). LH = emisfero sinistro, RH = emisfero destro. &lt;<html> img alt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot; /&gt; Figura 3. Valutazioni neurofunzionali seguenti ICH sperimentale nei topi. Intrastriatal iniezione di sangue autologo (30 pl) o collagenasi batterica (0,075 U) causato riproducibili deficit neurofunzionali. (A) I topi dopo ICH sperimentale ha mostrato curve molto più a destra di quelle degli animali finti operati a 24 e 72 ore dopo l&#39;intervento chirurgico. (B) capacità zampa anteriore ponendo l&#39;arto sinistro è stato alterato dopo la ICH a 24 e 72 ore dopo l&#39;intervento chirurgico. I valori sono espressi come media ± SEM e analizzati con Kruskal-Wallis Una analisi della varianza su ranghi, seguita dalla Student-Newman-Keuls Method. Un valore di p &lt;0.05 è stato considerato statisticamente significativo; n = 6-12 per gruppo, * P &lt;0.05 rispetto al sham. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . igura 4 &quot;src =&quot; / files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; Figura 4. Valutazione del contenuto di acqua in seguito ICH cerebrale sperimentale nei topi. Iniezione intracerebrale di sangue autologo (30 microlitri) o collagenasi batterica (0,075 U) ha portato ad un significativo aumento del contenuto di acqua nel cervello corteccia ipsilaterale e gangli basali a 24 (A) e 72 hr (B) dopo ICH-induzione. I valori sono espressi come media ± SEM ed analizzate con una analisi della varianza, seguita da test di Tukey post hoc. Un valore di p &lt;0.05 è stato considerato statisticamente significativo; n = 6-10 per gruppo, * P &lt;0.05 rispetto al sham. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

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Nessun conflitto di interessi dichiarati.

Modelli animali di emorragia intracerebrale (ICH) contribuiscono notevolmente a una conoscenza avanzata della fisiopatologia della malattia, e sono comunemente utilizzati per sviluppare e valutare nuove strategie terapeutiche in un ambiente preclinica. Iniezioni intraparenchimali di sangue autologo o collagenasi batterica sono consolidati metodi per generare ICH nei roditori. Entrambi i metodi sono stati sviluppati inizialmente nel ratto, tuttavia, a causa della disponibilità rapida crescita di ceppi transgenici e knoc...

<table border="1"><tbody><tr><td> Materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Testa telaio stereotassica </td><td> Stoelting Co. </td><td> 51600 </td><td></td></tr><tr><td> Nanomite pompa a siringa </td><td> Harvard Apparatus </td><td> PY2 70-2217 </td><td></td></tr><tr><td> Hamilton Siringa </td><td> Hamilton Azienda </td><td> 1725RN (250 microlitri) 1701 RN (10 microlitri) </td><td> 26 gauge per 250 microlitri e 10 siringhe pl. </td></tr><tr><td> Microdrill </td><td> Strumenti Scienza Belle </td><td> 18000-17 </td><td></td></tr><tr><td> Microdrill bava </td><td> Strumenti Scienza Belle </td><td> 19007-09 </td><td> 0,9 millimetri di diametro </td></tr><tr><td> Collagenasi di tipo VII-S </td><td> Sigma-Aldrich </td><td> C2399 </td><td></td></tr><tr><td> Microematocrito tubi capillari </td><td> Fisher Scientific </td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td> Bone Wax </td><td> Ethicon </td><td> W31 </td><td></td></tr><tr><td> Suturare </td><td> Ethicon </td><td> 1676G </td><td></td></tr><tr><td> Ketamina </td><td> JHP Pharmaceuticals </td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td> Xylazina </td><td> LLOYD Laboratories </td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

Spontanea emorragia intracerebrale (ICH) definisce una malattia potenzialmente pericolosa per la vita neurologica che rappresenta il 10-15% di tutti i ricoveri legati a ictus e per i quali non sono disponibili trattamenti efficaci ad oggi 1,2. A causa della eterogeneità delle ICH negli esseri umani, diversi modelli preclinici sono necessari per esplorare a fondo i potenziali strategie terapeutiche 3. ICH sperimentale è comunemente indotta in roditori da iniezione intraparenchimale sia di sangue autologo o collagenasi batterica 4. Il modello appropriato viene selezionato in base alla fisiopatologia di induzione emorragie e lesioni progressione. Il modello di iniezione sangue imita una emorragia rapida progressione. In alternativa, collagenasi batterica enzimaticamente sconvolge la lamina basale dei capillari cerebrali, causando un sanguinamento attivo che si evolve in genere per diverse ore 5. Risultante edema perihematomal e deficit neurofunzionali possono essere quantificati from entrambi i modelli. In questo studio abbiamo descritto e valutato un modello modificato doppia iniezione di sangue intero autologo 6 così come un modello ICH iniezione di collagenasi batterica 7, entrambi bersaglio gangli della base (corpo striato) di maschi topi CD-1. Abbiamo valutato deficit neurofunzionali ed edema cerebrale a 24 e 72 ore dopo l&#39;induzione ICH. Intrastriatal iniezione di sangue autologo (30 microlitri) o collagenasi batterica (0.075U) causata riproducibili deficit neurofunzionali in topi e edema cerebrale significativamente aumentata a 24 e 72 ore dopo l&#39;intervento (p &lt;0,05). In conclusione, entrambi i modelli producono consistenti infarti emorragici e rappresentano metodi di base per la ricerca preclinica ICH.

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Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modelli animali clinicamente rilevanti di emorragia intracerebrale (ICH) sono necessari per ampliare la nostra conoscenza di ictus emorragico e di esaminare nuove strategie terapeutiche. In questo studio, abbiamo descrivere e valutare due modelli ICH che implementano iniezioni unilaterali di ciascuna sangue autologo intero o collagenasi batterica nel gangli della base (corpo striato) di topi.

Modellazione emorragia intracerebrale in Mouse: iniezione di sangue autologo o collagenasi batterica

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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24.0K Views.  Loma Linda University School of Medicine. Modelli animali clinicamente rilevanti di emorragia intracerebrale (ICH) sono necessari per ampliare la nostra conoscenza di ictus emorragico e di esaminare nuove strategie terapeutiche. In questo studio, abbiamo descrivere e valutare due modelli ICH che implementano iniezioni unilaterali di ciascuna sangue autologo intero o collagenasi batterica nel gangli della base (corpo striato) di topi.

Video: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Corsa di modellazione in Mouse - Medio occlusione dell&#39;arteria cerebrale con il Modello Filament

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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Medicina

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

Sequenziamento di microflora batterica nel sangue periferico: la nostra esperienza con pazienti affetti da HIV

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Consegna di agenti terapeutici attraverso intracerebroventricolare (ICV) e per via endovenosa (ev) nei topi

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

Valutare Variazioni Volatile generale anestetico sensibilità dei topi dopo l&#39;intervento farmacologico locale o sistemica

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Microscopio video intravitale misurazioni della retina flusso sanguigno nei topi

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Iniezione intrastriatali di sangue autologo o Clostridial Collagenase come modelli murini di emorragia intracerebrale

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modellazione di Carcinoma renale metastatico spontanea (MRCC) in topi dopo nefrectomia

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Ultrasound-guided transtoracica intramiocardico iniezione nei topi

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Corsa di modellazione in Mouse: Coagulazione permanente dell&#39;arteria cerebrale media distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.