مقدمة في معظم الحالات سوف يتم تحديد مجالات allodynia الجلدي في dermatomes من البطن والعجان أن يعصب الهياكل داخل الحوض -- T11 - L1 وS3 ، 4،5 على التوالي. وallodynia مصطلح يشير إلى وجود شعور مؤلم نتيجة حافزا ليست مؤلمة عادة ، في هذه الحالة عصا الثقافة القطن ذات الرؤوس. 1. بطن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويجري تفسيرا للاختبار ويتم الحصول على اذن للمضي قدما. </li><li style=";text-align:right;direction:rtl"> امرأة تكشف البطن من القفص الصدري للمنطقة من عظم العانة. </li><li style=";text-align:right;direction:rtl"> ويرد عصا الثقافة القطن يميل إلى المرأة ونحى ضد يدها لشرح كيفية الضغط الخفيف سوف يشعر. </li><li style=";text-align:right;direction:rtl"> يطلب من امرأة لاحظ النقطة التي تطبق على ضوء الضغط يصبح الجلد الحادة مثل ثقافة عصا القطن ذات الرؤوس ويوجه باستمرار على جدار البطن. </li><li style=";text-align:right;direction:rtl"> وسوف يتم بعد ذلك عصا الثقافة القطن ذات الرؤوس طفيفة تعادل أسفل البطن وامرأة المذكرة إما وجود أي تغيير في الإحساس أو سوف مذكرة الحدة المفاجئة ومنفصلة في الإحساس. يتم تكرار العملية حتى تم التعرف على المنطقة بأكملها من allodynia الجلدية. ويعرف الاختبار إيجابية عن وجود هذا الإحساس الحاد المفاجئ. </li><li style=";text-align:right;direction:rtl"> في بعض الحالات مجالات كبيرة ولكن في البعض الآخر أنها ستكون صغيرة. يمكن أن تكون من جانب واحد ولكن عادة سيتم الثنائية. </li></ol> 2. عجان <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويجري تفسيرا للاختبار ويتم الحصول على اذن للمضي قدما. </li><li style=";text-align:right;direction:rtl"> المرأة يفضح العجان في موقف استخراج الحصاة. </li><li style=";text-align:right;direction:rtl"> ويرد عصا الثقافة القطن يميل إلى المرأة ونحى ضد يدها لشرح كيفية الضغط الخفيف سوف يشعر. </li><li style=";text-align:right;direction:rtl"> يطلب من امرأة لاحظ النقطة التي تطبق على الضغط الخفيف على الجلد يصبح حادا كما هو وضع العصا الثقافة القطن ذات الرؤوس عبر العجان. </li><li style=";text-align:right;direction:rtl"> ثم يوجه العصا الثقافة القطن ذات الرؤوس عبر الأرداف أفقيا في اتجاه من الجانب الوحشي من الأرداف إلى نحو فتحة الشرج لخفض عبر مبشار S3. يتم تكرار العملية على الجانب المقابل. بالإضافة إلى ذلك يمكن استخلاصها من قضيب من القطن ذات الرؤوس من منطقة الشفرين الكبيرين في الاتجاه الخلفي مباشرة ، وتجنب المنطقة المهبلية. ويعرف الاختبار إيجابية عن وجود إحساس حاد مفاجئ. </li><li style=";text-align:right;direction:rtl"> على العجان ، ومنطقة وعادة ما يصف # S مبشار عندما إيجابية. ويمكن رؤية نتائج إيجابية من جانب واحد كذلك ، ولكن عادة أقل. </li></ol>

<ol>
	<li>Giamberardino, M. A., Affaitati, G., Lerza, R., Lapenna, D., Costantini, R., Vecchiet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+pain+symptoms+and+referred+sensory+and+trophic+changes+in+patients+with+gallbladder+pathology.">Relationship between pain symptoms and referred sensory and trophic changes in patients with gallbladder pathology.</a> Pain. 114 (1-2), 239-249 (2005).</li><li>Giamberardino, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Women+and+visceral+pain:+are+the+reproductive+organs+the+main+protagonists?+Mini-review+at+the+occasion+of+the+&amp;quot;European+Week+Against+Pain+in+Women+2007.">Women and visceral pain: are the reproductive organs the main protagonists? Mini-review at the occasion of the &amp;quot;European Week Against Pain in Women 2007.</a> Eur J Pain. 12 (3), 257-260 (2008).</li><li>Baranauskas, G., Nistri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+pain+pathways+in+the+spinal+cord:+cellular+mechanisms.">Sensitization of pain pathways in the spinal cord: cellular mechanisms.</a> Prog Neurobiol. 54 (3), 349-365 (1998).</li></ol>

وجود allodynia الجلدي يدل على امرأة وكان ألم شديد في الماضي إلى حد أن التغييرات التي حدثت الأعصاب. على الرغم من أن المشكلة قد نشأت من عسر الطمث أو بطانة الرحم ، وقد ظهرت حالة جديدة -- ألم مزمن في الحوض. قد تكون نشطة حالتها الأصلية أو هادئة. تقديرا لهذا التطور الجديد المهم لتث...

demonstration of cutaneous allodynia in association with chronic pelvic pain

ألم الحوض هو حالة شائعة مقترن عسر الطمث وبطانة الرحم. في بعض النساء النوبات الشديدة لتغيير دوري الألم والألم الناتجة تصبح مستمرة وهذا الشرط يصبح يعرف الألم الحوضي المزمن. وهذا يمكن أن تكون الدولة موجودة حتى بعد وضعت العلاج الطبي أو الجراحي المناسب. يمكن أن تترافق مع الألم والألم في عضلات البطن وجدار الحوض داخل العضلات مما يؤدي إلى عسر الجماع شديدة. أعراض إضافية من القولون العصبي والتهاب المثانة الخلالي شائعة. وهناك علامة مشتركة لتنمية هذه الدولة هو ظهور allodynia الجلدي الذي يخرج من الأحشاء ، المنعكس يسمى جسدية. ويرد اختبار بسيط السرير لوجود allodynia الجلدية التي لا تتطلب وقتا مفرطا أو معدات خاصة. هذا الاختبار يعتمد على الأعمال السابقة المرتبطة بالتغيرات في الإحساس وتتعلق وظيفة المرارة والمنعكس الأحشاء ، الجسدية (1 ، 2). ويجري اختبار هذا الموضوع بإذن ق بعد تفسيرا للكيفية التي سيتم إجراء الاختبار. Allodynia يشير إلى الحالة التي يتم تفسير حافزا غير مؤلمة عادة من الموضوع ، كما مؤلمة. سيكون في هذه الحالة لا ترتبط مع لمسة خفيفة من القطن قضيب ذات الرؤوس من المتوقع أن تكون مؤلمة. ويلاحظ مع ذلك اختبار إيجابية من جانب المرأة ومؤلمة أو حادة فجأة فجأة. أنماط هذا الإحساس عادة في نمط منفصلة لقطاع جلدي من الأعصاب التي يعصب الحوض. ويفسر الآن الباثولوجيا الكامنة كدليل على المرونة العصبية نتيجة الألم الشديد وتكرار مع تغييرات في وظائف من القرون الظهرية للنخاع الشوكي أن النتائج تتغير في وظيفة الأنسجة الحشوية والناتجة عن أعراض جسدية (3). على أهمية الاعتراف شرط يكمن في الوعي بأن هذه العملية قد الحالي بالتزامن مع بدء أو شرط بعد أن تم التعامل معها. كما أنه يسمح الطبيب لتقييم الوضع من منظور أن تفسيرات بديلة للألم قد تكون موجودة والتي قد لا تتطلب جراحة إضافية.

Watch this Scientific Journal Video about Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain at JoVE.com

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

مظاهرة للاختبار عن السرير<em&gt; allodynia الجلدي</em&gt; وآثارها السريرية.

مظاهرة Allodynia الجلدية في الشراكة مع ألم مزمن في الحوض

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

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Biology

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

19.6K Views.  University of Calgary. مظاهرة للاختبار عن السرير allodynia الجلدي وآثارها السريرية.

Video: Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

المناعية على أقسام البارافين للبشرة باستخدام الماوس أجسام نيون

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

تظاهرة فنية من الجامعة التهجين الجينومي المقارن صفيف الجينوم

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

تحديد حرارة والميكانيكية عتبة الألم في الجلد ملتهبة من الموضوعات البشرية

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

زرع الأنسجة المهندسة في قلب فأر

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

عرض تاريخي والتظاهر في فسيولوجيا NMJ من جراد البحر العضلات فتاحة

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

استئصال المبيض و17β استراديول، مع تبديل في الجرذان والفئران: تظاهرة البصرية

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

متعدد المعلمة قياس نفاذية المسام افتتاح المرحلة الانتقالية في القلب ماوس متفرقة الميتوكوندريا

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

التصوير متحد البؤر واحدة من الميتوكوندريا الأكسيد ومضات في القلب سليمة أو<em&gt; في فيفو</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

مظاهرة من بروتين تفعيل قناة الصوديوم الظهارية (عناق) من خلال الجمع بين المقاييس الحالية مع الكشف عن الشق شظايا

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

علم وظائف الأعضاء مختبر مظاهرة: معدل الترشيح الكبيبي في الجرذ

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...