We would like to thank Prof David Walker for his technical advice and provision of equipment in the development of this technique. This work is supported by the Victorian Government&#8217;s Operational Infrastructure Support Program. This work was partly supported by the Victorian Government&#8217;s Operational Infrastructure Support Program.

<ol>
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The authors declare that they have no competing interests. The authors have no conflicts to disclose.

تقنية الموضحة هنا هي طريقة غير الغازية لتقييم المعلمات الجهاز التنفسي من الفئران غير المقيد وunanesthetized. وتشمل نقاط القوة في هذا البروتوكول بساطته والدقة لقياس وظائف الرئة طوليا مع الحد الأدنى من القطع الأثرية. هناك، ومع ذلك، بعض القيود والخطوات الحاسمة لأن يلاحظ حول ?...

ضعف الرئة هو واحد من الأسباب الرئيسية للمراضة والوفيات في العالم. وتتميز حالة عدم كفاية الأكسجين الصرف، مرادفا السعال، وآلام الصدر وضيق التنفس. حسابات أمراض الجهاز التنفسي ل~ 10٪ من الوفيات في جميع أنحاء العالم 1. وفقا لمنظمة الصحة العالمية، يتم تعيين معدلات الوفيات في الارتفاع بسبب التدخين المستمر، والتلوث والمهيجات المهنية. UWBP هو إضافة مفيدة لدراسة علم وظائف الأعضاء الرئة، التي تكمل بقوة البيوكيميائية والنسيجية التقليدية تحليلات 2. الإجراءات الأخرى المستخدمة لتقييم الرئة لا توفر نفس المزايا على النحو UWBP. تخطيط التحجم الغازية هي تقنية تستخدم عادة يتطلب أن يكون تخدير الحيوان 3،4 وبالتالي، مما أدى قياسات الجهاز التنفسي لا يعكس بالضرورة حالة طبيعية. علاوة على ذلك، شرط التحديات التهوية الميكانيكية والكيميائية يمنع القياسات المستقبلية 3،4.طريقة أخرى لجمع البيانات عن طريق الجهاز التنفسي هو التذبذب القسري، التي هي أكثر حساسية للتغيرات في ادق المعلمات الجهاز التنفسي مقارنة UWBP 5. التذبذب القسري، ومع ذلك، تقنية الغازية ويتطلب إنهاء الحيوان لجمع البيانات 5-7. UWBP ينطوي على وضع الحيوان داخل غرفة المتخصصة. خلال الإلهام، وارتفعت درجة حرارة الهواء المد والجزر وترطيب داخل الرئتين زيادة ضغط بخار الماء ويسبب التمدد الحراري من الغاز 8. هذا التأثير يسبب التغير في حجم الهواء يؤدي إلى حدوث ارتفاع في الضغط داخل غرفة مخطاط التحجم 8. يحدث العكس أثناء انتهاء خلق الموجي التنفسي من الحيوان. ثم يتم استخدام التحليل الموجي لقياس من تتبع الجهاز التنفسي: معدل التنفس (الأنفاس / دقيقة)، ومجموع وقت دورة التنفس (ثانية)، إلهام / وقت انتهاء الصلاحية (تي / تي، وثانية) والتغيرات في الضغط بسبب كل حجم المد والجزر (P T). <stroنانوغرام&gt; ويوضح الشكل 1 أصل كل القياسات من تتبع الجهاز التنفسي. هذه القياسات هي بسيطة لحساب ويمكن اشتقاق معلمات الجهاز التنفسي متعددة من هذه القياسات. وتشمل هذه المعايير: حجم المد والجزر (نقل حجم الهواء بين استنشاق وزفير طبيعي)، حجم الدقيقة (حجم الغاز المستنشق من الرئتين في الدقيقة)، دورة عمل الشهيق (النسبة المئوية للوقت إلهام لمجموع التنفس مدة دورة) و معدل تدفق الشهيق (كمية الهواء مستوحاة في وقت معين). يوفر UWBP دقيق، غير الغازية، والتحليل الكمي للفسيولوجيا الجهاز التنفسي في النماذج الحيوانية، ويمكن استخدامها لقياس تطور أمراض الجهاز التنفسي وظيفة الرئة 6،9. خلافا للتقنيات تخطيط التحجم أخرى، UWBP يتجنب استخدام التخدير، والقيود والتلاعب الغازية التي تنتج المصنوعات اليدوية والتجريبية تقلب 6،9. التخدير يمكن قمع التنفس،تغيير معدل ضربات القلب ويمكن أن يكون تحديا لتنظيم 10. القيود تحفز زيادة في التنفس بسبب الإجهاد الإضافي عن طريق الكورتيزون والادرينالين الافراج 11،13. ويتكرر سمة أساسية من سمات UWBP تقييم الفسيولوجية مما يجعلها قابلة للدراسات طولية. ويوصى بشدة UWBP لتقييم الطولي للعلم وظائف الأعضاء الرئة ويقدم مهارة قيمة لتقييم المخدرات التنفسي في المستقبل. وقد استخدمت بليوميسين، ألبومين البيض، ونقص الأكسجة للحث على التحديات الجهاز التنفسي في العديد من الدراسات وUWBP ويقاس بنجاح الرئة دقيقة تقييم الفسيولوجية 7،9،13-16. تم تصميم بروتوكول صفها للفئران المختبر الكبار القياسية. ومع ذلك، فقد تم تكييف UWBP إلى الحيوانات الأخرى مثل الفئران والخنازير الغينية، والرئيسيات غير البشرية 17-20. لا يقتصر UWBP فقط لتقييم الخلل الوظيفي الرئوي ولكن كما استخدمت لتقييم نضج الرئة 3.وقد أنشأت براعة والبساطة واستنساخ UWBP تقنية ممتازة لتقييم وظائف الرئة في الحيوانات. سيطلب البرامج المختلفة (انظر الجدول المواد والمعدات) لمتابعة هذا الإجراء. سيكون عالم ذوي الخبرة تكون قادرة على أداء هذا البروتوكول مع ماوس في حدود 1 ساعة.

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">LabChart 7 software (for Macintosh)</td>
			<td style="width:139px;">ADINSTRUMENTS</td>
			<td style="width:155px;">MLU60/7</td>
			<td style="width:475px;">used in protocol step 4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerLab 8/30 (model ML870)</td>
			<td>ADINSTRUMENTS</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Octal Bridge Amp (model ML228)</td>
			<td>ADINSTRUMENTS</td>
			<td>FE228</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Black BNC to BNC cable (1m)</td>
			<td>ADINSTRUMENTS</td>
			<td style="width:155px;">MLAC01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Macintosh OS&#160;</td>
			<td>Apple Inc.&#160;</td>
			<td>-</td>
			<td style="width:475px;">Mac OS X 10.4 or later</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgipack Digital Rectal Thermometer&#160;</td>
			<td>Vega Technologies</td>
			<td>MT-918</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Grass volumeteric pressure transducer PT5A</td>
			<td>Grass Instruments Co.</td>
			<td style="width:155px;">Model number PT5A; serial No. L302P4.</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1ml Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">5ml serological syringe pipettes</td>
			<td>Greiner Bio One</td>
			<td>606160</td>
			<td style="width:475px;">Connected via plastic tubing</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Balance/Scales</td>
			<td>VWR International, Pty Ltd</td>
			<td>SHIMAUW220D</td>
			<td style="width:475px;">Any weighing balance with of 0.1 gram resolution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HM40 Humidity &#38; temperature meter&#160;</td>
			<td>Vaisala&#160;</td>
			<td>HM40A1AB</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Barometer</td>
			<td>Barometer World</td>
			<td>1586</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Laboratory tubing</td>
			<td>Dow Corning&#160;</td>
			<td>508-101</td>
			<td style="width:475px;">Used to connect water column to the syringe and pressure transducer</td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;width:263px;">Cylindrical Perspex Chamber</td>
			<td>Dynalab Corp.</td>
			<td></td>
			<td style="width:475px;">Custom built cylindrical chamber with internal dimensions as follows: 50mm(w) x 1500mm(l). There are two lids for each side, with dimensions 80mm(l) x 80mm(w). Each lid has a 60mm wide circular hole cut on the face of the lid 50mm deep. This allows the chamber to fit into the lid. A rubber ring is fitted around each hole of the lid where the chamber will fit. For attachment of syringe and pressure transducer, the openings are 5mm in diameter. For attachment of humidity probe, the openings are 25mm in diameter.&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">80% Ethanol (4L)</td>
			<td>VWR International, Pty Ltd</td>
			<td>BDH1162-4LP</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring respiratory function in mice using unrestrained whole-body plethysmography

ضعف الجهاز التنفسي هي واحدة من الأسباب الرئيسية للمراضة والوفيات في العالم، وتستمر معدلات الوفيات في الارتفاع. التقييم الكمي وظائف الرئة في نماذج القوارض هو أداة هامة في تطوير العلاجات المستقبلية. التقنيات المستخدمة عادة لتقييم وظيفة الجهاز التنفسي بما في ذلك تخطيط التحجم الغازية والتذبذب القسري. وبينما توفر هذه التقنيات المعلومات القيمة، ويمكن جمع البيانات تكون محفوفة المصنوعات اليدوية وتقلب التجريبية نظرا للحاجة للتخدير و / أو الأجهزة الغازية من الحيوان. في المقابل، غير المقيد تخطيط التحجم كامل الجسم (UWBP) يقدم غير الغازية، وطريقة دقيقة، والكمية التي يمكن من خلالها تحليل المعلمات الجهاز التنفسي. هذا الأسلوب يتجنب استخدام التخدير والقيود، وهو أمر شائع لتقنيات تخطيط التحجم التقليدية. وهذا الفيديو يظهر الإجراء UWBP بما في ذلك معدات انشاء والمعايرة وظيفة الرئة تسجيل. ذلكسوف يشرح كيفية تحليل البيانات التي تم جمعها، وكذلك تحديد القيم المتطرفة والتحف الفنية التجريبية التي تنتج من حركة الحيوان. وتشمل المعلمات الجهاز التنفسي تم الحصول عليها باستخدام هذه التقنية حجم المد والجزر، وحجم دقيقة، دورة العمل الشهيق، ومعدل تدفق الشهيق ونسبة الوقت إلهام لوقت انتهاء الصلاحية. UWBP لا تعتمد على المهارات المتخصصة وغير مكلفة لأداء. ومن السمات الرئيسية لUWBP، والأكثر جاذبية للمستخدمين المحتملين، هو القدرة على أداء التدابير المتكررة من وظائف الرئة في نفس الحيوان.

عندما اتبعت هذا الإجراء بشكل صحيح، يتم إنشاء أي أثر تتأرجح يتفق على برنامج تحليل البيانات. يوفر الإجراء أثر التنفسي في غضون بضع دقائق بعد الإعداد مع حسابات الحوسبة بسيطة لتحديد المعلمات الجهاز التنفسي المدرجة. الشكل 5 يمثل أثر التنفس مناسبة من عنصر تحكم (صحي...

Watch this Scientific Journal Video about Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography at JoVE.com

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

وقد اعتمد تقييم الفيزيولوجيا التنفسي تقليديا على التقنيات، التي تتطلب ضبط النفس أو تخدير للحيوان. غير المقيد تخطيط التحجم كامل الجسم، ومع ذلك، يوفر دقيق، غير الغازية، والتحليل الكمي للفسيولوجيا الجهاز التنفسي في النماذج الحيوانية. بالإضافة إلى ذلك، تسمح تقنية تكرار تقييم الجهاز التنفسي في الفئران مما يسمح للدراسات طولية.

قياس وظيفة الجهاز التنفسي في الفئران عن طريق تخطيط التحجم غير مقيد كامل الجسم

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

measuring-respiratory-function-mice-using-unrestrained-whole-body

Research

JoVE Journal

Biology

30.0K Views.  Monash Institute of Medical Research.  وقد اعتمد تقييم الفيزيولوجيا التنفسي تقليديا على التقنيات، التي تتطلب ضبط النفس أو تخدير للحيوان. غير المقيد تخطيط التحجم كامل الجسم، ومع ذلك، يوفر دقيق، غير الغازية، والتحليل الكمي للفسيولوجيا الجهاز التنفسي في النماذج الحيوانية. بالإض...

Video: Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

قياس إيماس في الخلايا العصبية عن طريق وصفها وزير الخارجية

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

قياس ضغط الدم في الفئران باستخدام تسجيل حجم الضغط ، وهو أسلوب الذيل الكفة

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

تصور وظيفة الجهاز التنفسي الميتوكوندريا باستخدام السيتوكروم C أوكسيديز / سكسينات نازعة (كوكس / SDH) مزدوجة وضع العلامات الكيمياء النسيجية

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

تقييم ميكانيكا الجهاز التنفسي في الفئران باستخدام تقنية التذبذب القسري

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

كامل الجسم جسيمات متناهية الصغر الهباء التعرض بالاستنشاق

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

قياس الاستجابة التغذية الناجم عن الجلوتاثيون في حيدرة

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

إعادة إعماره الوظيفية وآخر قناة قياسات تنقية Wildtype والمسخ CFTR البروتين

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

قياس صلابة من<em&gt; فيفو السابقين</em&gt; ماوس Aortas باستخدام مجهر القوة الذرية

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

قياس سمية الببتيد RAN في C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

في قياس الجسم الحي لوظيفة العضلات إكستنور الركبة في الفئران

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...