Die Arbeit in diesem Papier enthaltenen Informationen wurden von der Dr. Douglas H. Johnson Award für Glaukomforschung vom American Health Assistance Foundation (XL), die William &amp; Mary Greve Besondere Scholar Award von der Forschung zur Verhütung Blindheit (XL), unterstützt die Illinois Gesellschaft zur Verhütung von Blindheit (HC) und NIH R01EY019034 (XL).

Verfahren C57BL/6J-Mäusen (Jackson Laboratory, Bar Harbor, ME) sind an der Northwestern University Animal Care Facility angehoben. Alle Tiere werden in Übereinstimmung mit den Protokollen von der Northwestern University Institutional Animal Care und Verwenden Ausschuss gestellt und den Leitlinien für die Verwendung von Tieren in Neuroscience Forschung vom NIH genehmigt werden. 1. Laserphotokoagulation Das Verfahren der Laserkoagulation von zuvor veröffentlichten Protokollen 5-7 modifiziert. <ol><li> Anesthetize eine 40-60 Tage alten Maus durch eine intraperitoneale Injektion von Ketamin (100 mg / kg, Butler Schein Tiergesundheit, OH) und Xylazin (10 mg / kg, Lloyd Inc. von Iowa, Shenandoah, IA). </li><li> Dilate die Pupille des rechten Auges des Versuchstieres durch topische Behandlung mit einem oder zwei Tropfen von 1% Atropin-Sulfat-Lösung (Alcon Labs, Inc., Fort Worth, TX). </li><li> Nach Mydriasis, glätten die einnterior Kammer zu verbessern Laserinduktion 6. Legen Sie eine Glasmikropipette mit scharfen Spitze (World Precision Instruments Inc, Sarasota, FL) in den vorderen Raum unter der Spaltlampe (SL-3E, Topcon, Oakland, NJ) ablaufen lassen die Flüssigkeit in die vordere Kammer. </li><li> Haltet die Maus in einem Kunststoff-Kegel Halter (Braintree Sci Inc., MA) und bis auf eine hausgemachte Plattform gebunden (siehe Abbildung 1a). Halten Sie die Maus mit Verzögerer und macht das rechte Auge der Maus auf die Lichtquelle hinter der Spaltlampe. Richten des rechten Auges des narkotisierten Maus unter der Spaltlampe. </li><li> Halten Sie die Maus Restrainer mit beiden Händen, gelten die Laserbeleuchtung der Hornhaut Limbus mit einem Argon-Laser (Ultima 2000SE, Coherent, Santa Clara, CA). Liefern 80-100 Laserspots (514 nm, 100 mW, 50 ms Puls und 200 um spot) senkrecht um den Umfang des trabekulären Netzwerks. Die C57BL / 6 Mäuse pigmentierten Iris, die als Barriere für alle p dientotential Streuenergie 7. </li><li> Instill aktuell 0,5% Moxifloxacin (Alcon Labs, Inc., Fort Worth, TX) auf der Augenoberfläche, um den Laser-behandelte Fläche und 0,5% Proparacaine (Bausch &amp; Lomb, Rochester, NY), Schmerzen zu lindern desinfizieren. </li><li> Halten Sie das Tier auf einem Heizkissen (Sunbeam Products Inc, Boca Raton, FL) für die Verwertung für etwa eine Stunde, bis er ganz wach ist. </li><li> Das linke Auge ist unbehandelt als Kontrolle zu dienen. </li></ol> 2. IOP Maße <ol><li> Legen Sie die Maus wach in eine Röhre, in der Kunststoff-Kegel Halter laden und dann zurückhalten es auf der Plattform (siehe Abbildung 2A). </li><li> Lassen Sie fünf bis zehn Minuten, damit die Maus an den Inhaber Position angepasst bekommen. Nähern Sie sich dem Rebound-Tonometer (TonoLab, Colonial Medical Supply, Franconia, NH) an der Maus, bis das Auge Sondenspitze ist 2-3 mm von der Oberfläche der Hornhaut 14. </li><li> Drücken Sie die Taste, um die Messung lassen sich die Sondenspitze traf die Mitte Oberflächevon Hornhaut sanft. Drei aufeinanderfolgende Sätze von sechs Messungen der IOP desselben Auges erfasst und gemittelt als IOP des Auges. Die unbehandelten Kontrolle Auge wird immer zuerst eine Basislinie für die Laser-behandelten Auge, das nächste wird gemessen bekommen gemessen. </li></ol> 3. Optomotorischen-Test Sehschärfe und Kontrastempfindlichkeit sind 14,15 getestet. Die beiden Augen einzelner Mäuse werden einzeln durch die Umkehrung der Richtung driftet Gitter untersucht, dh im Uhrzeigersinn Driften Gitter verwendet wird, um die visuelle Funktion des linken Auges und des gegen den Uhrzeigersinn treibt Gitter für das rechte Auge 16 zu identifizieren. Jeder Test dauert etwa 15 min und wird durch zwei Beobachter unabhängig wiederholt. <ol><li> Platzieren Sie die Maus und lassen Sie die Maus frei bewegen auf einer erhöhten Plattform von vier Computermonitore (3A-B) umgeben. </li><li> Stellen Sie die Monitore so dass sie horizontal treiben Display sinusförmigeGittern als visuelle Reize zur mittleren Leuchtdichte von 39 cd / m 2. Die Bewegungsrichtung des Gitters sollten sich abwechseln nacheinander zwischen Uhrzeigersinn und gegen den Uhrzeigersinn. </li><li> Analysieren Sie die Bewegungen des Tieres. Bewegungen des Tieres in Abstimmung mit den treibenden Gitter werden als &quot;positive&quot; innerhalb von 15 Sekunden nach der visuelle Reiz ist und dann allmählich erhöht. Die höchste Antwort-auslösenden visuellen Stimulus als das Tier die Sehschärfe 17 definiert. </li><li> Untersuchen Sie die Kontrastempfindlichkeit bei drei vorgewählten räumlichen Frequenzen: 0,075, 0,16 und 0,3 Zyklen pro Grad (cpd). Der Kontrast Schwelle für jedes Auge wird als die niedrigste dagegen die visuelle Reaktionen hervorruft bei der Pre-festen Frequenz definiert. Der Kontrast-Empfindlichkeit ist der reziproke Wert der Schwelle 17. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben. Die Autoren sind Vollzeit-Mitarbeiter von der Northwestern University. Die Autoren erhielten keine Mittel, die von Unternehmen, die Reagenzien und Instrumente in diesem Artikel verwendet produzieren bereitgestellt.

Wir berichten über diesem anhaltenden Augenhypertension können durch Laser-Beleuchtung in Mäuseaugen induziert werden. Im Vergleich zur Injektion von Kochsalzlösung Modell 18 und der Vene cautery Modell 11 beide umfangreichen mikrochirurgischen Fähigkeiten erfordern, ist die Laserbestrahlung relativ einfach und leicht durchzuführen. Normalerweise können wir den Laser-Beleuchtung für 4-6 Mäuse in 2-3 Stunden durchzuführen. Die kritischen Schritte aufrechterhalten IOP Höhe erreichen sind d...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>moxifloxacin</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 0065-4013-03</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Proparacaine Hydrochloride</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Ophthalmic Solution USP</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>.5 %, Rx only</td>
 </tr>
 <tr>
 <td>ketamine</td>
 <td>Butler Schein Animal Health</td>
 <td>NDC 11695-0550-1</td>
 <td>100 mg / kg </td>
 </tr>
 <tr>
 <td>xylazine</td>
 <td>LLOYD Inc. of Iowa</td>
 <td>NADA 139-236</td>
 <td>10 mg / kg </td>
 </tr>
 <tr>
 <td>atropine sulfate solution</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 61314-303-02</td>
 <td>1 %, Rx only</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Slit Lamp, TOPCON </td>
 <td>Visual Systems Inc</td>
 <td>SL-3E</td>
 <td>powered by PS-30A</td>
 </tr>
 <tr>
 <td>OptoMotry 1.8.0 virtual</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>opto-kinetic testing system</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tonometer, TonoLab, for mice</td>
 <td>Colonial Medical Supply</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>Sunbeam Products Inc</td>
 <td>722-810</td>
 <td></td>
 </tr>
 <tr>
 <td>Argon laser </td>
 <td>Coherent Inc</td>
 <td>Ultima 2000SE</td>
 <td></td>
 </tr>
 <tr>
 <td>DECAPICONE Plastic cone holder </td>
 <td>Braintree Sci Inc.</td>
 <td>MDC-200</td>
 <td>for mouse</td>
 </tr>
 </tbody>
 </table>

a laser-induced mouse model of chronic ocular hypertension to characterize visual defects

Glaukom, häufig mit erhöhtem Augeninnendruck (IOP) verbunden sind, ist eine der führenden Ursachen für Erblindung. Wir versuchten, ein Maus-Modell von Augenhypertension die menschliche High-Tension Glaukom imitieren zu etablieren. Hier Laserbeleuchtung mit dem Hornhautrand, um die wässerige Abfluß photocoagulate, induzieren Engwinkel. Die Änderungen des IOP erfolgt über ein Rebound-Tonometer vor und nach der Laserbehandlung. Ein optomotor Verhaltens-Test wird verwendet, um die entsprechenden Änderungen in der visuellen Leistungsfähigkeit zu messen. Die repräsentatives Ergebnis von einer Maus, die nachhaltig IOP Erhebung nach der Laser-Beleuchtung entwickelt gezeigt. Eine verminderte Sehschärfe und Kontrastempfindlichkeit wird in diesem Augeninnendruck Maus beobachtet. Gemeinsam stellt unsere Studie einen wertvollen Modellsystem zur neuronalen Degeneration und die zugrunde liegenden molekularen Mechanismen in glaukomatöse Mäusen zu untersuchen.

Wie in den Verfahren beschrieben, ist die Laser-Beleuchtung an der Trabekelnetzwerk im Limbus Region, um die Kammerwasserabfluss photocoagulate Ziel, Induktion Winkel Verschluss (Abbildung 1). Die meisten gelaserten Augen zeigten keine erheblichen Sachbeschädigungen, Pigment Ablösung oder Infektion, im Einklang mit früheren Befunden 6. Wenn eine kleine Gruppe von Mäusen (weniger als 5% aller gelaserten Tiere) körperliche Anzeichen von schweren Schäden wie entleert Augäpfel, schwere Kat...

Watch this Scientific Journal Video about A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects at JoVE.com

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Chronische Augenhypertension induziert wird mittels Laserkoagulation der Trabekelnetzwerk in Maus Augen. Der Augeninnendruck (IOP) wird für mehrere Monate nach der Laser-Behandlung erhöht. Die Abnahme der Sehschärfe und Kontrastempfindlichkeit von Versuchstieren werden anhand des optomotor Test.

Ein Laser-induzierten Maus-Modell der chronischen Ocular Hypertension zu Sehfehler Charakterisierung

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

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JoVE Journal

Medicine

12.9K Aufrufe.  Northwestern University. Chronische Augenhypertension induziert wird mittels Laserkoagulation der Trabekelnetzwerk in Maus Augen. Der Augeninnendruck (IOP) wird für mehrere Monate nach der Laser-Behandlung erhöht. Die Abnahme der Sehschärfe und Kontrastempfindlichkeit von Versuchstieren werden anhand des optomotor Test.

Serie: A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.

Fahrsimulation in der Klinik: Testen von Visual Exploratory Behavior in Aktivitäten des täglichen Lebens bei Patienten mit Gesichtsfeldstörungen

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

Forschung

Medizin

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th Nephrektomie in Kombination mit hohen Salz-Diät-und Stickstoffmonoxid-Synthase-Hemmung zu Chronic Kidney Disease in der Lewis-Ratte induzieren

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Erwachsene Maus venöse Bluthochdruckmodell: A. carotis communis, um äußere Jugularvene Anastomose.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Beurteilung der Gefäßfunktion bei Patienten mit chronischer Nierenerkrankung

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Ein Mausmodell für Laser-induzierten CNV

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

Ein Magnet Microbead Occlusion Modell Ocular Hypertension Abhängige Glaucoma in Mäusen zu induzieren

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Eine einfache mechanische Verfahren Limbusstammzellinsuffizienzen in der Maus zum Erstellen

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

Induktion und Phänotypisierung der akuten Rechtsherzinsuffizienz in einem Großtiermodell der chronischen thromboembolischen pulmonalen Hypertonie

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Ein reversibles Siliziumöl-induziertes Okularhypertonie-Modell bei Mäusen

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...