La obra contenida en este documento ha sido apoyado por el premio Douglas Johnson H. Dr. de Glaucoma Research de la Fundación Panamericana de la Salud Asistencia (XL), la Greve Premio Académico Especial William &amp; Mary de la investigación para prevenir la ceguera (XL), el Sociedad de Illinois para la Prevención de la Ceguera (HC) y NIH subvención R01EY019034 (XL).

Procedimientos C57BL/6J ratones (Jackson Laboratory, Bar Harbor, ME) se crían en la Instalación de Cuidado de Animales de la Universidad de Northwestern. Todos los animales se utilizan de acuerdo con los protocolos aprobados por la Universidad de Northwestern Institucional Cuidado de Animales y el empleo Comisión y conforme a las directrices sobre el uso de animales en Investigación de Neurociencia de la NIH. 1. La fotocoagulación con láser El procedimiento de fotocoagulación con láser es una modificación de los protocolos previamente publicados 5-7. <ol><li> Anestesiar a un ratón viejo 40-60 días por una inyección intraperitoneal de ketamina (100 mg / kg, Butler Schein Animal Health, OH) y xilazina (10 mg / kg, Lloyd Inc. de Iowa, Shenandoah, IA). </li><li> Dilatar la pupila del ojo derecho del animal experimental por el tratamiento tópico con una o dos gotas de solución de sulfato de atropina 1% (Alcon Laboratorios, Inc., Fort Worth, TX). </li><li> Después midriasis, aplanar la unacámara de nterior para mejorar la inducción láser 6. Inserte una micropipeta de vidrio con punta afilada (World Precision Instruments Inc., Sarasota, FL) en el espacio anterior bajo la lámpara de hendidura (SL-3E, Topcon, Oakland, NJ) para drenar el fluido en la cámara anterior. </li><li> Restringir el ratón en un soporte de cono de plástico (Braintree Sci Inc., MA) y atado en una plataforma hecha en casa (Ver Figura 1). Mantenga el ratón con inmovilización y expone el ojo derecho del ratón para la fuente de luz detrás de la lámpara de hendidura. Alinear el ojo derecho del ratón anestesiado bajo la lámpara de hendidura. </li><li> Mientras sostiene el retenedor de ratón con las dos manos, aplicar la iluminación láser para el limbo corneal utilizando un láser de argón (Ultima 2000SE, coherente, Santa Clara, CA). Entregar aproximadamente 80-100 puntos de láser (514 nm, 100 mW, 50 ms de pulso, y 200 micras punto) perpendicularmente alrededor de la circunferencia de la malla trabecular. Los ratones C57BL / 6 tienen iris pigmentada que sirve como una barrera para cualquier potencial energía perdida 7. </li><li> Inculcar tópica 0,5% moxifloxacino (Alcon Labs, Inc., Fort Worth, TX) en la superficie ocular para desinfectar el área tratada con láser y el 0,5% Proparacaine (Bausch &amp; Lomb, Rochester, NY) para aliviar el dolor. </li><li> Mantenga al animal en una almohadilla eléctrica (Sunbeam Products Inc, Boca Raton, FL) para la recuperación durante aproximadamente una hora hasta que esté completamente despierto. </li><li> El ojo izquierdo es sin tratar para servir como un control. </li></ol> 2. Las mediciones de PIO <ol><li> Coloque el ratón despierto en un tubo de carga en el soporte de cono de plástico y luego siente en la plataforma (véase la Figura 2A). </li><li> Espere cinco a diez minutos para dejar que el ratón consigue adaptarse a la posición de soporte. Acérquese al tonómetro de rebote (TonoLab, Colonial Medical Supply, Franconia, NH) en el ojo del ratón hasta que la punta de la sonda es de 2 a 3 mm de la superficie de la córnea 14. </li><li> Presione el botón de medición para que la punta de la sonda alcanzó la superficie centralde la córnea suavemente. Tres series consecutivas de seis mediciones de PIO del mismo ojo se adquieren y se promediaron como el IOP del ojo. El ojo control no tratado se mide siempre primero para conseguir una lectura de referencia para el ojo tratado con láser que se mide a continuación. </li></ol> 3. Prueba optomotor La agudeza visual y la sensibilidad al contraste se prueban 14,15. Los dos ojos de ratones individuales se examinan por separado mediante la inversión de la dirección de rejilla a la deriva, es decir, una rejilla de la deriva las agujas del reloj se utiliza para identificar la función visual del ojo izquierdo y una rejilla a la deriva en sentido antihorario para el ojo derecho 16. Cada prueba dura aproximadamente 15 minutos y se repite por dos observadores de forma independiente. <ol><li> Coloque el ratón y dejar que el ratón se mueva libremente sobre una plataforma elevada rodeada por cuatro monitores de ordenador (Figura 3A-B). </li><li> Configurar los monitores para que se muestren a la deriva horizontal sinusoidalrejillas como estímulos visuales con luminancia media de 39 cd / m 2. La dirección de movimiento de la rejilla debe alternar consecutivamente entre las agujas del reloj y en sentido contrario. </li><li> Analizar los movimientos del animal. Los movimientos de los animales en-concierto con las rejillas de la deriva se consideran &quot;positivo&quot; dentro de 15 segundos después de que el estímulo visual está encendido y luego se aumenta gradualmente. La respuesta más alta inductora de estímulo visual se define como la agudeza visual del animal 17. </li><li> Examine la sensibilidad al contraste en tres frecuencias preseleccionadas espaciales: 0,075, 0,16 y 0,3 ciclos por grado (cpd). El umbral de contraste para cada ojo se define como el contraste más bajo que provoca respuestas visuales a la frecuencia prefijada. La sensibilidad al contraste es el recíproco del umbral 17. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia. Los autores son empleados de tiempo completo de la Universidad Northwestern. Los autores no recibieron ninguna financiación que fue proporcionada por las empresas que producen los reactivos e instrumentos utilizados en este artículo.

Se presenta por encima de que la hipertensión ocular sostenida puede ser inducida por iluminación con láser en ojos de ratón. En comparación con el modelo de inyección de solución salina 18 y el cauterio modelo de vena 11 ambos de los cuales requieren amplios conocimientos de microcirugía, la iluminación con láser es relativamente simple y fácil de realizar. Por lo general, se puede realizar la iluminación láser para 4-6 ratones en 2-3 horas. Los pasos críticos para lograr la elevaci?...

<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

El glaucoma, con frecuencia asociado con la presión intraocular (PIO) elevada, es una de las principales causas de ceguera. Se pretende establecer un modelo de ratón de la hipertensión ocular para imitar glaucoma alta tensión humana. Aquí iluminación láser se aplica al limbo corneal para fotocoagular la salida del humor acuoso, la inducción de ángulo cerrado. Los cambios en la PIO se controlan usando un tonómetro de rebote antes y después del tratamiento con láser. Un ensayo de comportamiento optomotor se utiliza para medir los cambios correspondientes en la capacidad visual. Se muestra el resultado representativo de un ratón que desarrolla PIO elevación sostenida después de la iluminación láser. Una disminución de la agudeza visual y la sensibilidad al contraste se observa en este ocular ratón hipertensiva. Juntos, nuestro estudio introduce un sistema modelo valioso para investigar la degeneración neuronal y los mecanismos moleculares subyacentes en ratones glaucomatosos.

Como se describe en los Procedimientos, iluminación con láser está dirigido a la malla trabecular en la región limbar a fotocoagular la salida del humor acuoso, la inducción de ángulo cerrado (Figura 1). La mayoría de los ojos lasered mostraron ningún daño físico significativo, desprendimiento de pigmento o infección, de acuerdo con los resultados anteriores 6. Cuando un pequeño grupo de ratones (menos del 5% de todos los animales lasered) mostraron signos físicos de daños grave...

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

Hipertensión ocular crónica se induce mediante fotocoagulación con láser de la malla trabecular en los ojos de ratón. La presión intraocular (PIO) es elevada durante varios meses después del tratamiento con láser. La disminución de la agudeza visual y la sensibilidad al contraste de los animales experimentales se vigila por medio de la prueba de optomotor.

Un modelo de ratón inducida por láser de la hipertensión ocular crónica a Caracterizar los defectos visibles

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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Research

JoVE Journal

Medicine

12.9K vistas.  Northwestern University. Hipertensión ocular crónica se induce mediante fotocoagulación con láser de la malla trabecular en los ojos de ratón. La presión intraocular (PIO) es elevada durante varios meses después del tratamiento con láser. La disminución de la agudeza visual y la sensibilidad al contraste de los animales experimentales se vigila por medio de la prueba de optomotor.

Video: 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.

Simulación de conducción en la clínica: Pruebas de Comportamiento Visual exploratoria en las Actividades de la vida diaria en pacientes con defectos del campo visual

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

Investigación

Medicina

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 nefrectomía en combinación con dieta alta en sal y el óxido nítrico sintasa inhibición para inducir la enfermedad renal crónica en la rata Lewis

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.

Adultos Mouse hipertensión venosa: Modelo: arteria carótida común a la vena yugular externa anastomosis.

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.

Evaluación de la función vascular en pacientes con enfermedad renal crónica

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.

Un modelo de ratón para inducida por láser neovascularización coroidea

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.

A Magnetic Microbead Oclusión Modelo para inducir hipertensión ocular dependiente de glaucoma en ratones

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.

Un procedimiento mecánico simple de crear deficiencia de células madre limbares en ratón

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.

Inducción y fenotipado de la insuficiencia cardíaca derecha aguda en un modelo animal grande de hipertensión pulmonar tromboembólica crónica

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.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

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.

Un modelo de hipertensión ocular inducida por aceite de silicio reversible en ratones

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