وقد تم دعم العمل الواردة في هذه الورقة من قبل دوغلاس جائزة الدكتور H. جونسون لأبحاث الزرق من مؤسسة الصحة الأمريكية المساعدة (XL)، وجائزة الباحث غريف الخاص وليام وماري من البحوث للوقاية من العمى (XL)، في جمعية إلينوي للوقاية من العمى (HC) والمعاهد الوطنية للصحة منح R01EY019034 (XL).

الإجراءات تثار C57BL/6J الفئران (مختبر جاكسون، بار هاربور، ME) في منشأة رعاية الحيوان جامعة نورث وسترن. وتستخدم كل الحيوانات وفقا للبروتوكولات المعتمدة من جامعة نورث وسترن المؤسسي رعاية الحيوان واللجنة الاستخدام ومطابقة للمبادئ التوجيهية بشأن استخدام الحيوانات في البحوث العصبية من المعاهد الوطنية للصحة. 1. الليزر الضوئي يتم تعديل هذا الإجراء من الليزر الضوئي من البروتوكولات نشرت سابقا 5-7. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير ماوس القديمة 40-60 يوم عن طريق الحقن داخل الصفاق الكيتامين (100 ملغ / كغ، بتلر شين صحة الحيوان، OH) وزيلازين (10 ملغم / كغم، لويد وشركة من ولاية ايوا، شيناندواه، IA). </li><li style=";text-align:right;direction:rtl"> تمدد التلميذ من العين اليمنى للحيوانات التجارب عن طريق العلاج الموضعي مع واحد أو اثنين من قطرات الأتروبين 1٪ محلول كبريتات (ألكون مختبرات، وشركة، فورت وورث، تكساس). </li><li style=";text-align:right;direction:rtl"> بعد توسيع حدقة العين، تتسطح علىغرفة nterior لتعزيز الاستقراء ليزر 6. إدراج micropipette الزجاج مع طرف حاد (عالم الآلات الدقيقة شركة، ساراسوتا، فلوريدا) في الفضاء الأمامي تحت المصباح الشقي (SL-3E، توبكون، أوكلاند، NJ) لتجفيف السوائل في الغرفة الأمامية. </li><li style=";text-align:right;direction:rtl"> كبح الماوس في حامل مخروط من البلاستيك (برينتري الخيال العلمي وشركة، MA) وقيدوا على منصة محلية الصنع (انظر الشكل 1A). عقد الماوس مع كابح ويفضح العين اليمنى من الماوس لمصدر الضوء وراء المصباح الشقي. محاذاة العين اليمنى من الفأرة تخدير تحت المصباح الشقي. </li><li style=";text-align:right;direction:rtl"> في حين الضغط على كابح الماوس بكلتا يديه، وتطبيق إضاءة الليزر إلى حوف القرنية باستخدام الليزر الأرجون (ألتيما 2000SE ومتماسكة، سانتا كلارا، كاليفورنيا). تسليم حوالي 80-100 اماكن ليزر (514 نانومتر، و 100 ميغاواط، 50 ميللي ثانية النبض، و 200 بقعة ميكرون) عموديا حول محيط الشبكة التربيقية. وC57BL / 6 الفئران ديك قزحية المصطبغة التي هي بمثابة حاجز لأي عالطاقة الضالة otential 7. </li><li style=";text-align:right;direction:rtl"> غرس موضعي 0.5٪ موكسيفلوكساسين (ألكون مختبرات، وشركة، فورت وورث، تكساس) على سطح العين لتطهير المنطقة المعالجة بالليزر و 0.5٪ بروباراكايين (بوش ولومب، روتشستر، نيويورك) لتخفيف الألم. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الحيوان على وسادة التدفئة (شعاع الشمس المنتجات المؤتمر الوطني العراقي، بوكا راتون، فلوريدا) لاسترداد لمدة ساعة تقريبا حتى يصبح مستيقظا تماما. </li><li style=";text-align:right;direction:rtl"> العين اليسرى هو غير المعالجة لتكون بمثابة السيطرة. </li></ol> 2. القياسات IOP <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع الماوس مستيقظا في أنبوب لتحميل في حامل مخروط من البلاستيك ومن ثم كبح جماح ذلك على منصة (انظر الشكل 2A). </li><li style=";text-align:right;direction:rtl"> تسمح خمس إلى عشر دقائق للسماح للحصول على تكييفها الماوس إلى موقف حامل. نقترب من مقياس توتر العين انتعاش (TonoLab، المستعمرة التموين الطبي، فرانكونيا، NH) للعين الماوس حتى تلميح التحقيق هو 2-3 ملم بعيدا من سطح القرنية 14. </li><li style=";text-align:right;direction:rtl"> اضغط على زر القياس لترك ضرب تلميح التحقيق سطح مركزمن القرنية بلطف. يتم الحصول على ثلاث مجموعات متتالية من ستة قياسات IOP من نفس العين وبلغ متوسط ​​كما IOP من العين. يتم قياس العين السيطرة غير المعالجة دائما في المقام الأول للحصول على قراءة خط الأساس للعين المعالجة بأشعة الليزر التي يتم قياسها المقبل. </li></ol> 3. اختبار Optomotor ويتم اختبار حدة البصر وحساسية النقيض 14،15. يتم فحص عيون اثنين من الفئران على حدة عن طريق عكس اتجاه الانجراف صريف، أي يتم استخدام صريف الانجراف في اتجاه عقارب الساعة لتحديد وظيفة البصرية من العين اليسرى وصريف الانجراف عكس اتجاه عقارب الساعة لعينه اليمنى 16. كل اختبار يستغرق حوالي 15 دقيقة، ويتكرر من قبل اثنين من المراقبين بشكل مستقل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع الماوس والسماح الماوس على التحرك بحرية على منصة مرتفعة تحيط بها أربع شاشات الكمبيوتر (الشكل 3A-B). </li><li style=";text-align:right;direction:rtl"> إعداد مراقبين حتى يتمكنوا العرض أفقيا الانجراف الجيبيةحواجز شبكية ومحفزات بصرية مع الإنارة متوسط ​​39 شمعة / م 2. يجب الاتجاه تتحرك من صريف بالتناوب على التوالي بين اتجاه عقارب الساعة وعكس. </li><li style=";text-align:right;direction:rtl"> تحليل حركات الحيوان. وتعتبر حركات الحيوان في الحفل مع حواجز شبكية الانجراف &quot;ايجابية&quot; خلال 15 ثانية بعد التحفيز البصري على وثم تزداد تدريجيا. يتم تعريف أعلى استجابة انتزاع التحفيز البصرية وحدة البصر الحيوان 17. </li><li style=";text-align:right;direction:rtl"> دراسة حساسية التباين في ثلاثة ترددات محددة مسبقا المكانية: 0.075، 0.16، و 0.3 دورة في درجة (CPD). يتم تعريف عتبة النقيض لكل عين وأقل النقيض الذي يتسبب الاستجابات البصرية في وتيرة ما قبل الثابتة. حساسية النقيض هو متبادلة من عتبة 17. </li></ol>

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة. الكتاب والعاملين بدوام كامل من جامعة نورث وسترن. تلقى الكتاب NO التمويل الذي تم توفيره من قبل الشركات التي تنتج الكواشف والأدوات المستخدمة في هذه المقالة.

نحن التقرير المذكور أن فرط ضغط العين المستمر يمكن أن يتسبب من خلال إضاءة الليزر في عيون الماوس. بالمقارنة مع نموذج الحقن بالمحلول الملحي 18 ونموذج الكي الوريد 11 وكلاهما تتطلب مهارات المجهرية واسعة، وإضاءة الليزر هو بسيط نسبيا وسهلة لتنفيذ. وعادة ما يمكن أ?...

<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

الزرق، ويرتبط في كثير من الأحيان مع ارتفاع ضغط العين (IOP)، هي واحدة من الأسباب الرئيسية للعمى. سعينا إلى إنشاء نموذج الفأر من فرط ضغط العين لتقليد الإنسان التوتر العالي الزرق. هنا يتم تطبيق إضاءة الليزر إلى حوف القرنية إلى photocoagulate تدفق مائي، الأمر الذي أدى إغلاق زاوية. ويتم رصد التغيرات في معهد جامعة هارفارد باستخدام مقياس توتر العين انتعاش قبل وبعد العلاج بالليزر. يتم استخدام اختبار السلوكية optomotor لقياس التغيرات المناظرة في القدرة البصرية. يظهر نتيجة ممثل من الماوس واحد التي وضعت حقت IOP الارتفاع بعد إضاءة الليزر. ويلاحظ وجود انخفاض حدة البصر وحساسية التباين في هذه العين الماوس ارتفاع ضغط الدم. معا، ويدخل دراستنا نظام نموذجا قيما للتحقيق تنكس الخلايا العصبية والآليات الجزيئية الكامنة في الفئران زرقي.

كما هو موضح في الإجراءات، ويهدف إضاءة الليزر في الشبكة التربيقية في المنطقة الحوفي إلى photocoagulate تدفق مائي، الأمر الذي أدى إغلاق زاوية (الشكل 1). عيون معظم lasered عرضت قوع أضرار مادية كبيرة، مفرزة الصباغ أو عدوى، بما يتفق مع النتائج السابقة 6. عندما قامت مجم?...

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

ويتسبب ارتفاع ضغط الدم المزمن العين باستخدام الليزر الضوئي من الشبكة التربيقية في عيون الماوس. هو ارتفاع ضغط العين (IOP) لعدة أشهر بعد العلاج بالليزر. ويتم رصد انخفاض حدة البصر وحساسية النقيض من حيوانات التجارب باستخدام اختبار optomotor.

نموذج الفأر الليزر التي يسببها ارتفاع ضغط الدم المزمن المقلة لتوصيف العيوب البصرية

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 Views.  Northwestern University. ويتسبب ارتفاع ضغط الدم المزمن العين باستخدام الليزر الضوئي من الشبكة التربيقية في عيون الماوس. هو ارتفاع ضغط العين (IOP) لعدة أشهر بعد العلاج بالليزر. ويتم رصد انخفاض حدة البصر وحساسية النقيض من حيوانات التجارب باستخدام اختبار 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.

القيادة المحاكاة في عيادة: اختبار السلوك الاستكشافي البصرية في أنشطة الحياة اليومية في المرضى الذين يعانون من عيوب المجال البصري

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

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 استئصال الكلية في تركيبة مع حمية عالية الملح والنيتريك أوكسيد تثبيط سينسيز للحث على مرض الكلى المزمن في الفئران لويس

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.

الكبار ماوس وريدي ارتفاع ضغط الدم نموذج: الشريان السباتي المشترك لجهات خارجية الوداجي الوريد التحام.

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.

تقييم وظيفة الأوعية الدموية في المرضى الذين يعانون من مرض الكلى المزمن

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.

نموذج الفأر ليزر بفعل اتساع الأوعية الدموية المشيمية

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.

وميكروبيدات انسداد المغناطيسي نموذج للحث على بصري ارتفاع ضغط الدم التي تعتمد الزرق في الفئران

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.

إجراء ميكانيكي بسيط لإنشاء الحوفي نقص الخلايا الجذعية في الفأر

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.

تحريض وتوصيف ظاهري لقصور القلب الأيمن الحاد في نموذج حيواني كبير لارتفاع ضغط الدم الرئوي الخثاري المزمن

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.

متساوي القياس كونتراكتيليتي القياس الشريان المساريقي العلوي الماوس باستخدام أسلاك ميوجرافي

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.

والسيليكون عكسها الناجمة عن النفط العين ارتفاع ضغط الدم النموذج في الفئران

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