Cette étude a été financée en partie par NIH RO1NS053407 à JH Zhang. Nous tenons à remercier M. Damon Klebe pour ses précieuses contributions.

Toutes les procédures ont été menées en conformité avec le Guide du NIH pour le soin et l&#39;utilisation des animaux de laboratoire et approuvé par le Comité de protection des animaux et l&#39;utilisation à Loma Linda University. 1. Les préparatifs préopératoires Des techniques aseptiques sont recommandés pour toutes les interventions chirurgicales. Désinfecter l&#39;appareil stéréotaxique et préparer les outils chirurgicaux stériles avant la chirurgie. Porter un équipement de protection individuelle (EPI) pendant toute la manipulation des animaux. Utilisez un coussin chauffant pendant la chirurgie pour maintenir la température du corps de l&#39;animal physiologique. <ol><li> Peser la souris 8-12 semaine vieux en utilisant une échelle triple faisceau animal. </li><li> Co-injection de kétamine (100 mg / kg) et de xylazine (10 mg / kg) par voie intrapéritonéale puis laisser 7-10 min pour l&#39;anesthésie de prendre effet (moniteur pendant une sédation adéquate). </li><li> Placez la souris sur une couverture thermique et raser le cuir chevelu. </li><li> Appliquer une pommade ophtalmique à deux yeux. </li><li> Fixez ledes voies aériennes, par un léger mouvement de la langue latéralement, et sécuriser soigneusement la tête de la souris sur l&#39;appareil stéréotaxique. Remarque: La tête doit être fixé horizontalement à la base du cadre stéréotaxique. </li><li> Désinfecter la zone chirurgicale avec de la Bétadine, et rincer avec de l&#39;éthanol à 70%. Répétez alternance applications de Bétadine et de l&#39;éthanol à 70% pour un total de trois fois. Coton-tige peut être utilisé à cette fin. </li></ol> 2. Modèle injection de sang <ol><li> Faire un 1 cm longue incision médiane du cuir chevelu avec une lame de bistouri n ° 10. </li><li> Utiliser le coton-tige pour nettoyer les tissus mous recouvrant le crâne, de manière à exposer le point d&#39;intersection perpendiculaire de la suture coronale et sagittale (bregma). </li><li> Monter la seringue Hamilton (250 pi) sur la pompe d&#39;injection, et diriger l&#39;aiguille stéréotaxique (26 Gauge) sur bregma. </li><li> Ensuite, ajuster les bras manipulateurs stéréotaxiques pour positionner l&#39;aiguille 0,2 mm antérieure et 2mm latéralement vers la droite. A ces coordonnées faire un petit trou crânienne bavures, à l&#39;aide d&#39;une perceuse à vitesse variable avec un foret de 1 mm. </li><li> Suspendre la queue de l&#39;animal et de désinfecter sa surface inférieure avec de l&#39;éthanol à 70%. </li><li> Ponction de l&#39;artère caudale centrale avec une aiguille stérile (par exemple 26 Gauge) et recueillir le sang artériel dans un tube capillaire unheparinized. </li><li> Transférer le sang rapidement du tube capillaire dans le tube de verre de la seringue Hamilton, puis insérez le piston. </li><li> Remettez le ul maintenant 30 ou plus de sang artériel contenant une seringue Hamilton sur la pompe à injection et insérer l&#39;aiguille (avec son bord biseauté face à la suture sagittale) à travers le trou de trépan jusqu&#39;à ce que son biseau n&#39;est plus visible. </li><li> De ce point de l&#39;avance de l&#39;aiguille 3 mm ventralement et injecter 5 ul de sang autologue à un taux de 2 pi / min. </li><li> Après l&#39;achèvement de la première injection avance l&#39;aiguille de 0,7 mm plus en profondeur. </li><li> Attendre5 min puis injecter 25 ul de sang dans le striatum droit. </li><li> À la fin de la seconde injection, laissez l&#39;aiguille en position pour 10 minutes supplémentaires, avant de le retirer à une vitesse de 1 mm / min. </li><li> Sceller le trou de trépan avec de la cire d&#39;os et de suturer la peau. </li><li> Pour l&#39;analgésie postopératoire injecter 0,05 mg / kg de buprénorphine sous-cutanée dans préchauffées liquides (sérum physiologique). </li></ol> 3. Modèle injection de collagénase <ol><li> Après les préparatifs préopératoires, répétez les étapes 1-4 comme décrit pour le modèle d&#39;injection de sang. </li><li> Remplir la seringue Hamilton (10 pi) avec 0.075U des bactéries (Clostridium) collagénase VII-S dissous dans 0,5 ul de solution saline. Eviter la formation de bulles d&#39;air. </li><li> Remettez la seringue Hamilton sur la pompe à injection et insérer l&#39;aiguille (26 Gauge), à ​​travers le trou de trépan jusqu&#39;à ce que son biseau n&#39;est plus visible. </li><li> Avancer l&#39;aiguille 3,7 mm ventralement et injecter le 0.075U de la collagénase into le striatum droit à un taux de 2 pi / min. </li><li> À la fin de l&#39;injection, laissez l&#39;aiguille en position pour encore 10 minutes, avant de le retirer à une vitesse de 1 mm / min. </li><li> Sceller le trou de trépan avec de la cire d&#39;os et de suturer la peau. </li><li> Injecter 0,05 mg / kg par voie sous cutanée à la buprénorphine préchauffées post-opératoires fluides. </li></ol> 4. Opération Sham <ol><li> Après les préparatifs préopératoires, répétez les étapes 1-4 comme décrit pour le modèle d&#39;injection de sang. </li><li> Insérez l&#39;aiguille (26 Gauge) 3,7 mm ventralement à travers le trou de trépan. L&#39;aiguille doit rester en place pendant 10 min avant d&#39;être retiré à une vitesse de 1 mm / min. </li><li> Sceller le trou de trépan avec de la cire d&#39;os et de suturer la peau. </li><li> Injecter 0,05 mg / kg par voie sous cutanée à la buprénorphine préchauffées post-opératoires fluides. </li></ol> 5. Les résultats représentatifs Expérimentale hémorragie intrastriatale évoque morphologiqueainsi que des changements de comportement chez les rongeurs. Ces changements peuvent être évalués pour assurer une bonne exécution de la procédure, ou pour enquêter sur les effets des traitements potentiels. Génération de purge dans une région du cerveau ciblée (par exemple, les noyaux gris centraux) est le plus essentiel d&#39;adopter une approche reproductible et peut être vérifiée sur des coupes de cerveau brut ou teinté histologique (figure 1-2). Blessures aux résultats des noyaux gris centraux des déficits sensori-motrices, qui peuvent être quantifiés par l&#39;intermédiaire de diverses évaluations comportementales. Les résultats du test ont montré que son tour d&#39;angle, après expérimentale du côté droit ICH, souris s&#39;est significativement plus souvent ipsilatérale et loin de l&#39;altération controlatéral (à gauche), à une opération fictive animaux à 24 et 72 h après la chirurgie (Figure 3). En outre, la capacité de bien placer le affaiblies (à gauche) membre antérieur sur une surface, suite à une stimulation des vibrisses, a été évaluée par le test de patte avant de placer. À 24 et 72 heures après la chirurgie, les souris sousrejeté à droite face ICH a montré placements nettement moins nombreuses que les animaux une opération fictive. Mesure de l&#39;œdème cérébral est fréquemment utilisée pour quantifier l&#39;étendue des lésions cérébrales après ICH expérimentale. Injections intracérébrales de sang autologue (30 pi) ou la collagénase bactérienne (0,075 U) conduit à une augmentation significative de la teneur en eau du cerveau dans le cortex ipsilatéral et ganglions de la base à 24 (Figure 4) et 72 h (figure 4 B) après la chirurgie ( par rapport à fictive). Les résultats des tests de comportement (figure 3) et l&#39;étendue de l&#39;œdème cérébral (figure 4) n&#39;a montré aucune différence entre le sang et les modèles à injection de collagénase à des volumes donnés. <img alt="Figure 1" src="/files/ftp_upload/4289/4289fig1.jpg" /> Figure 1. ICH modélisation chez la souris. (A) Le schéma simplifié d&#39;une section coronale du cerveau antérieur de 0,2 mm du bregma illustre la proposed emplacement de sang autologue ou l&#39;injection de collagénase. Le ventricule latéral est marqué LV. CPu signifie caudé-putamen, une partie du striatum, et GP identifie le globus pallidus. À la fois, le striatum ainsi que le globus pallidus appartiennent à un groupe de sous-corticale noyaux, également connu sous le nom ganglions de la base. Microphotographie représentant (B) d&#39;une section coronale du cerveau antérieur de 0,2 mm du bregma, a obtenu 24 h après l&#39;injection intrastriatale de sang total autologue. <img alt="Figure 2" src="/files/ftp_upload/4289/4289fig2.jpg" /> Figure 2. Manifestation histologique de l&#39;hématome. Représentant hématoxyline et l&#39;éosine (H &amp; E) teinté cryosection coronale (10 um) d&#39;un cerveau de souris, illustrant la taille hématome à 24 h après l&#39;injection de collagénase bactérienne intrastriatale (0,075 U). LH = hémisphère gauche, HR = hémisphère droit. &lt;<html> img alt = &quot;Figure 3&quot; src = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot; /&gt; Figure 3. Neurofonctionnels évaluations suivantes ICH expérimentale chez la souris. Intrastriatale injection de sang autologue (30 pi) ou la collagénase bactérienne (0,075 U) a provoqué des déficits neurofonctionnels reproductibles. (A) Les souris après ICH expérimentale a montré tour à tour nettement plus à droite que les animaux sham fonctionnant à 24 et 72 heures après la chirurgie. (B) la capacité de placer des pattes avant de la jambe gauche a été altéré après l&#39;ICH à 24 et 72 heures après la chirurgie. Les valeurs ont été exprimées en moyenne ± SEM et analysées avec Kruskal-Wallis Une analyse de variance sur les rangs, suivis par la méthode de Student-Newman-Keuls. Valeur P &lt;0,05 était considérée comme statistiquement significative, n = 6-12 par groupe, * p &lt;0,05 par rapport au trompe-l&#39;œil. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . igure 4 &quot;src =&quot; / files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; Figure 4. Évaluation de la teneur en eau du cerveau après l&#39;ICH expérimentale chez la souris. Injection intracérébrale de sang autologue (30 pi) ou la collagénase bactérienne (0,075 U) conduit à une augmentation significative de la teneur en eau du cerveau dans le cortex ipsilatéral et ganglions de la base à 24 (A) et 72 h (B) après ICH-induction. Les valeurs ont été exprimées en moyenne ± SEM et analysées avec une analyse de variance, suivie par Tukey test post hoc. Valeur P &lt;0,05 était considérée comme statistiquement significative, n = 6-10 par groupe, * p &lt;0,05 par rapport au trompe-l&#39;œil. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> .

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Aucun conflit d&#39;intérêt déclaré.

Les modèles animaux d&#39;hémorragie intracérébrale (HIC) contribuent largement à une compréhension approfondie de la physiopathologie de la maladie, et sont couramment utilisés pour développer et évaluer de nouvelles stratégies thérapeutiques dans un cadre préclinique. Injections intra-parenchymateuses de sang autologue ou la collagénase bactérienne sont bien établies méthodes pour générer ICH chez les rongeurs. Les deux méthodes ont été initialement développées chez le rat, mais en raison de l&#...

<table border="1"><tbody><tr><td> Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commenter </td></tr><tr><td> Cadre Tête stéréotaxique </td><td> Stoelting Co. </td><td> 51600 </td><td></td></tr><tr><td> Pousse-seringue Nanomite </td><td> Harvard Apparatus </td><td> PY2 70-2217 </td><td></td></tr><tr><td> Seringue Hamilton </td><td> Entreprise Hamilton </td><td> 1725RN (250 pi) RN 1701 (10 pl) </td><td> Aiguille de 26 Gauge pour 250 pi et 10 pi de seringues. </td></tr><tr><td> Micro-foret </td><td> Outils Fine Science </td><td> 18000-17 </td><td></td></tr><tr><td> Micro-foret fraise </td><td> Outils Fine Science </td><td> 19007-09 </td><td> 0,9 mm de diamètre </td></tr><tr><td> Collagénase de type VII-S </td><td> Sigma-Aldrich </td><td> C2399 </td><td></td></tr><tr><td> Microhématocrite Tubes capillaires </td><td> Fisher Scientific </td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td> Cire osseuse </td><td> Ethicon </td><td> W31 </td><td></td></tr><tr><td> Suturer </td><td> Ethicon </td><td> 1676G </td><td></td></tr><tr><td> La kétamine </td><td> JHP Pharmaceuticals </td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td> Xylazine </td><td> Laboratoires LLOYD </td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

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

Hémorragie intracérébrale spontanée (ICH) définit une maladie potentiellement mortelle neurologique qui représente 10-15% de toutes les hospitalisations liés à l&#39;AVC et pour laquelle aucun traitement efficace n&#39;est disponible à ce jour 1,2. En raison de l&#39;hétérogénéité de l&#39;ICH chez l&#39;homme, divers modèles précliniques sont nécessaires pour explorer en profondeur les stratégies thérapeutiques potentiels 3. ICH expérimentale est généralement induite chez des rongeurs par injection intra-parenchymateux de sang autologue soit la collagénase bactérienne ou 4. Le modèle approprié est choisi en fonction de la physiopathologie de l&#39;induction et la progression de l&#39;hémorragie des blessures. Le modèle simule l&#39;injection de sang d&#39;une hémorragie à progression rapide. Alternativement, la collagénase bactérienne par voie enzymatique perturbe la membrane basale des capillaires cérébraux, entraînant un saignement actif qui évolue généralement sur ​​plusieurs heures 5. Résultante oedème perihematomal et les déficits neurofonctionnels peuvent être quantifiés en arrièrem les deux modèles. Dans cette étude, nous avons décrit et évalué un modèle d&#39;injection double modifiée de sang total autologue 6, ainsi que d&#39;un modèle d&#39;injection de collagénase bactérienne ICH 7, les deux qui ciblent les noyaux gris centraux (corpus striatum) des hommes souris CD-1. Nous avons évalué les déficits neurofonctionnels et un œdème cérébral à 24 et 72 heures après l&#39;induction de l&#39;ICH. Injection intrastriatale de sang autologue (30 pi) ou la collagénase bactérienne (0.075U) a provoqué des déficits neurofonctionnels reproductibles chez les souris et un œdème cérébral significativement augmenté à 24 et 72 h après la chirurgie (p &lt;0,05). En conclusion, les deux modèles donnent des uniformes infarctus hémorragiques et représentent méthodes de base pour la recherche préclinique ICH.

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

Modèles animaux cliniquement pertinentes de l&#39;hémorragie intracérébrale (HIC) sont nécessaires pour étendre notre connaissance des accidents vasculaires cérébraux hémorragiques et d&#39;examiner de nouvelles stratégies thérapeutiques. Dans cette étude, nous décrivons et évaluons deux modèles ICH qui mettent en œuvre des injections unilatérales soit du sang total autologue ou la collagénase bactérienne dans les noyaux gris centraux (corpus striatum) de souris.

Hémorragie intracérébrale chez la souris Modélisation: L&#39;injection de sang autologue ou collagénase bactérienne

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 vues.  Loma Linda University School of Medicine. Modèles animaux cliniquement pertinentes de l'hémorragie intracérébrale (HIC) sont nécessaires pour étendre notre connaissance des accidents vasculaires cérébraux hémorragiques et d'examiner de nouvelles stratégies thérapeutiques. Dans cette étude, nous décrivons et évaluons deux modèles ICH qui mettent en œuvre des injections unilatérales soit du sang total autologue ou la collagénase bactérienne dans les noyaux gris centraux (corpus striatum) ...

Vidéo: 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.

Course de modélisation chez la souris - Moyen-occlusion artérielle cérébrale avec le modèle filamentaire

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

Recherche

Médecine

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.

Séquençage de la microflore bactérienne dans le sang périphérique: notre expérience avec des patients infectés par le VIH

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.

Délivrance d&#39;agents thérapeutiques à travers intracérébroventriculaire (ICV) et intraveineuse (IV) par injection chez la souris

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.

Évaluer les changements dans volatils anesthésie générale sensibilité des souris après l&#39;intervention pharmacologique local ou systémique

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.

Mesures microscopie intravitale Vidéo de la rétine débit sanguin chez les souris

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.

Injection intrastriatale de sang autologue ou Clostridium collagénase comme modèles murins de Hémorragie intracérébrale

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.

Modélisation spontanée carcinome rénal métastatique (MRCC) chez des souris après néphrectomie

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.

Guidée par échographie transthoracique intramyocardique injection chez la souris

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.

Modélisation des maladies chez les souris: Coagulation permanente de l&#39;artère cérébrale moyenne 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.