1. تحضير المريض <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويشار إلى المرضى من قسم علم الأورام الطبية لاستثارة العصب الأساس اختبار قبل بدء في العلاج الكيميائي. </li><li style=";text-align:right;direction:rtl"> يجب أن تحدد مدى ملاءمة المريض لاختبار استثارة. ينبغي استبعاد المرضى إذا كان لديهم تاريخ طويل من خط الأساس أو أدلة العصبية من الأعصاب الطرفية، وتلقى أعصاب قبل العلاج الكيماوي أو وجود أي الاستطباب استثارة للاختبار. </li></ol> 2. إجراءات الإثارة محواري <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تقوم بروتوكولات استثارة الحسية والحركية في العصب المتوسط، وذلك باستخدام نصف الآلية QTracS نظام محوسب (معهد علم الأعصاب، كوين سكوير، والمملكة المتحدة)، وهي معزولة خطي ثنائي القطب ومشجعا على تيار مستمر (Digitimer، يلوين جاردن سيتي، المملكة المتحدة)، ومكبر للصوت ( الياقوت IIA، Medelec، المملكة المتحدة). </li><li style=";text-align:right;direction:rtl"> إعداد سطح الجلد في المعصم والساعد مع هلام جلخ أو وسادة للحد من الجلدمقاومة، ثم طلب من القضاء على الكحول. </li><li style=";text-align:right;direction:rtl"> إعداد الموقع تسجيل للتسجيلات، مع محرك كهربائي غير قطوب وضعت على بطن عضلة المبعدة لإبهام اليد القصيرة والبعيدة 4cm الإلكترود المرجعي لتسجيل مجمع السيارات إمكانات العمل (CMAPs). </li><li style=";text-align:right;direction:rtl"> إعداد الموقع لتسجيل التسجيلات الحسية - باستخدام أقطاب عصابة وضعت في المفاصل بين السلامية القريبة والبعيدة لتسجيل وأقطاب المرجعية على التوالي، ليسجل إمكانات مركب عمل حسي (CSAPs). </li><li style=";text-align:right;direction:rtl"> وضع لوحة الكهربائية الأرض المحايدة في راحة، مع جل موصل. </li><li style=";text-align:right;direction:rtl"> وينبغي إزالة الضجيج الكهربائي في الإعداد تسجيل أكبر قدر ممكن، وذلك باستخدام هراء 50/60 هرتز الضوضاء مزيل (البحث العلمي الآلات، شمال فانكوفر، كندا). </li><li style=";text-align:right;direction:rtl"> تحفيز العصب المتوسط ​​في الرسغ. وينبغي اختيار موقع التحفيز كموقع لأدنى حد، وذلك باستخدام قطب كهربائي ثنائي القطب repositionable وبعد ذلك غير فolarizable الكهربائي. </li><li style=";text-align:right;direction:rtl"> يجب أن يوضع القطب القطب الموجب 10 سم القريبة من القطب خلال تحفيز العظام. </li><li style=";text-align:right;direction:rtl"> رصد درجة الحرارة في موقع التحفيز في جميع أنحاء اختبار والتأكد من أن درجة الحرارة أكبر من 32 درجة مئوية. </li></ol> 3. بروتوكولات الإثارة محواري <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تسجيل منحنى حافز للاستجابة عن طريق زيادة الحوافز تدريجيا حتى تكون الاستجابة القصوى ولا زيادة ولا عند زادت كثافة التحفيز. </li><li style=";text-align:right;direction:rtl"> تم تعيين السعة هدف لتتبع تلقائيا إلى عتبة 30٪ -40٪ من السعة القصوى، المقابلة لمنطقة من أشد منحدر على منحنى التحفيز والاستجابة. </li><li style=";text-align:right;direction:rtl"> وفقا لطريقة بوستوك وآخرون، 1998، والتغيرات في العتبة الحالية اللازمة لتحقيق ويتم تتبع اتساع الهدف عبر الإنترنت. </li><li style=";text-align:right;direction:rtl"> تسجيل المعلمات استثارة متعددة، بما في ذلك توترية كهربية عتبة (TE)، دورة الانتعاش (RC) والجاري عتبة (I / V) علاقةكما هو موضح في كيرنان وآخرون، 2000 وكيرنان وآخرون، 2001. </li><li style=";text-align:right;direction:rtl"> ويتم تقييم عتبة توترية كهربية باستخدام التيارات دون العتبي 100ms الاستقطاب، مع استقطاب المجموعة الحالية إلى 40٪ ± من عتبة مراقبة (الشكل 1). التغيير في العتبة الحالية اللازمة للحفاظ على الهدف سعة استجابة بعد كل من دي وسجلت وفرط الاستقطاب. عتبة توترية كهربية يقدم تقييما للالمواصلة internodal وإمكانات غشاء، مع الاستجابات في الاتجاه hyperpolarizing في نهاية نبض الاستقطاب (90-100 MS) يرتبط بقوة مع غشاء المحتملة. </li><li style=";text-align:right;direction:rtl"> ويتم تقييم دورة انتعاش باستخدام نموذج نبض الاقتران (الشكل 2)، مع وجود حافز تكييف فوق الأعظمي الأولي تليها على فترات مختلفة من التحفيز الاختبار (من 2.5 مللي ثانية إلى 200 مللي ثانية). في أعقاب التحفيز فوق الأعظمي، وهو أكثر صعوبة لتوليد استجابة لاحقة، ووصف &#39;حران&#39;، الأمر الذي يعكس تعطيل ز من الجهدated نا + القنوات. بعد فترة المقاومة للحرارة، وهي فترة من تيسير المعروفة باسم superexcitability يحدث. </li><li style=";text-align:right;direction:rtl"> ويتم تقييم الجاري عتبة علاقة باستخدام التيارات من استقطاب 200 مللي ثانية والتي تختلف في قوتها من +50٪ إلى -100٪ من عتبة. </li></ol> 4. تقييم المريض <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام هذه التقنيات، ويتم تقييم المرضى على حد سواء بشكل حاد، وطوليا عبر العلاج الكيماوي. لتقييم السمية العصبية الحادة، والمرضى العودة لمرحلة ما بعد العلاج الكيميائي تقييم في غضون 48 ساعة من تلقي العلاج. </li><li style=";text-align:right;direction:rtl"> لدراسة السمية العصبية المزمنة، وتتم مقارنة التقييمات التي اتخذت قبل العلاج الكيميائي التسريب طوليا عبر دورات العلاج. </li><li style=";text-align:right;direction:rtl"> وبالإضافة إلى اختبار استثارة محور عصبي، يجب استخدام المقاييس التقليدية الدرجات السريرية لتقييم العلاج الكيميائي الناجم عن العصبية، بما في ذلك المعهد الوطني للسرطان - معايير مشتركة لأحداث الضارة Subscale العصبي الحسي، ومجموع نقاط الاعتلال العصبي وباتient المبلغ عنها نتائج التقييم. </li></ol> 5. تحليل وتفسير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتحديد السمية العصبية الحادة، وتتم مقارنة نتائج العلاج قبل وبعد العلاج الكيميائي. لتقييم التنمية من التغييرات الطولي في وظيفة العصب، وتتم مقارنة النتائج عبر العلاج. المعايير الأساسية لتقييم وتشمل حران، superexcitability، مدى التغيير في عتبة عتبة توترية كهربية (hyperpolarizing 90-100 مللي ثانية)، بالإضافة إلى المعايير التقليدية مثل السعة القصوى والكمون. </li><li style=";text-align:right;direction:rtl"> لتقييم التغيير الشامل في المعلمات عبر استثارة العلاج، ويتم احتساب النتيجة استثارة مركب. تتلخص من الأولي إلى العلاجات النهائية لإعطاء علامة الشاملة التغيير - التغيير في المعلمات الثلاث - superexcitability، حران، وعتبة توترية كهربية (hyperpolarizing 90-100 مللي ثانية). </li></ol> 6. ممثل النتائج أمثلة على النتائج في استثارة باتوتقدم ient تعامل مع اوكزالبلاتين. فورا بعد اوكزالبلاتين التسريب، والتغيرات الحادة في استثارة كل من الحسية والحركية تطوير، موحية للتطوير وظيفي channelopathy + نا 10 -13. ومع ذلك، تغير كبير في استثارة المعلمات متعددة تتطور تدريجيا عبر معالجة اوكزالبلاتين فقط في المحاور العصبية الحسية، مع المحاور العصبية الحركية تتأثر (الشكل 3)، والتي تعكس على نطاق واسع ضرر محور عصبي حسي وغشاء تغيير محتمل. هذا نمط يطابق التعبير السريري للأعراض العصبية في اوكزالبلاتين التي يسببها المزمنة. تغيرات في استثارة محاور عصبية حسية تسبق تخفيضات في السعة الذروة وفقا لتقييم استخدام التقنيات التقليدية التوصيل العصبي، وتشير إلى أن تقنيات استثارة محواري قد توفر أداة تقييم حساسة بالنسبة اوكزالبلاتين العصبية التي يسببها في وقت مبكر. <img alt="الشكل 1" src="/files/ftp_upload/3439/3439fig1.jpg" /> الشكل 1. </stرونغ&gt; عتبة توترية كهربية، والتي تصور الطول الموجي في استجابة لفترات طويلة دون العتبي الاستقطاب الحالي (100 مللي ثانية)، مع اتجاه hyperpolarizing المرسومة في الربع السفلي (الأزرق) واتجاه depolarizing المرسومة في الربع العلوي (الحمراء). أقل من التحفيز الموجي تطبيقها لتوليد استجابة توترية كهربية عتبة. <img alt="الشكل 2" src="/files/ftp_upload/3439/3439fig2.jpg" /> الشكل 2. دورة الانتعاش من استثارة، مما يدل على تسلسل سمة من التغييرات استثارة بعد التوصيل دفعة، مع فترة من استثارة مخفضة (حران) تصل إلى 3 مللي بعد حافزا فوق الأعظمي، تليها فترة من استثارة زيادة (superexcitability) وتبلغ ذروتها في 5 -7 واستثارة مرض التصلب العصبي المتعدد في وقت لاحق خفضت (subexcitability). النموذج نبض إقران بروتوكول التحفيز هو أقحم. <img alt="الشكل (3)" src="/files/ftp_upload/3439/3439fig3.jpg" /> تينلدى عودتهم 3. التغييرات الإثارة في محاور عصبية حسية اوكزالبلاتين معاملة المرضى، مع تسجيلات الأساس هو مبين في التسجيلات أسود والمعالجة اللاحقة هو مبين في أبيض، وبعد 4-6 أشهر من العلاج اوكزالبلاتين. ويعتقد أن هذه التغييرات لتعكس ضرر محواري نطاق واسع وغشاء تغيير محتمل. ويظهر صورة لاستثارة مجموعة المتابعة في تحديد الأورام في اليسار. <a href="http://www.jove.com/files/ftp_upload/3439/3439fig3large.jpg" target="_blank">اضغط هنا لمشاهدتها بشكل اكبر</a> .

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ليس لدينا ما يكشف.

الناجم عن العلاج الكيماوي الاعتلال العصبي هو أحد الآثار الجانبية الخطيرة لعلاج السرطان، والتي قد تؤثر على مسار العلاج وطويلة الأمد عجز المريض. هناك عدم وجود تدابير تقييم حساسة وموضوعية لقياس تحديدا خلل الأعصاب لدى مرضى العلاج الكيميائي العلاج. وقد وفرت التنمية السريرية لتقنيات استثارة محواري معلومات مفيدة والتنبؤية لتقييم العلاج الكيميائي الناجم عن العصبية. من خلال توفير المعلومات حول وظيفة القناة الايونية، وإمكانات يستريح، ومحور عصبي وظيفة الغشاء، وهذه التقنيات تمكن التبصر في العمليات الفيزيولوجية المرضية الكامنة وراء الخلل في محواري مرضى السرطان للعلاج الكيماوي. وبالإضافة إلى ذلك، ثبت عمليا أن تقنيات استثارة محواري أن يكون ذلك ممكنا في وضع علم الأورام السريرية، ويمكن الانتهاء من اختبار واحد في 5-10 دقائق. في اوكزالبلاتين المرضى الذين عولجوا، وتقنيات توفير استثارة محواريالعلامات البيولوجية الحساسة التي يمكن التعرف المبكر على المرضى المعرضين للخطر من العصبية الشديدة. بالمقارنة مع التقليدية دراسات التوصيل العصبي الذي يحدد اوكزالبلاتين الناجم عن تلف الأعصاب فقط بعد خسارة محواري حدث بالفعل، والدراسات استثارة محواري توفير علامات تنبئ عن ضعف العصب قبل فقدان محور عصبي. على هذا النحو، يمكن استخدام دراسات استثارة محواري لتقديم تقييم وظيفة العصب في التجارب السريرية لاستراتيجيات اعصاب محتمل، لتحديد فعالية بموضوعية اعصاب.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> المعدات والمواد </td><td> شركة </td></tr><tr><td> QTracS برنامج </td><td> Digitimer، معهد علم الأعصاب، كوين سكوير، والمملكة المتحدة </td></tr><tr><td> DS5 معزولة خطي ثنائي القطب ومشجعا على تيار مستمر </td><td> Digitimer </td></tr><tr><td> الياقوت IIA مكبر للصوت </td><td> Medelec </td></tr><tr><td> هراء 50/60 هرتز مزيل الضوضاء </td><td> السعي أجهزة علمية </td></tr><tr><td> غير قطوب الأقطاب </td><td> Unomedical </td></tr><tr><td> الكهربائية لوحة محايدة الأرض </td><td> Unomedical </td></tr></tbody></table>

nerve excitability assessment in chemotherapy-induced neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

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Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

هذا الملخص يصف طريقة جديدة لتقييم تطور العصبية في المرضى الذين يتلقون العلاج الكيماوي. في حين تقتصر وسائل التقييم التقليدية في قدرتها على الكشف عن التغييرات في وقت مبكر في وظيفة العصب، وتقنيات استثارة العصب توفير التعرف المبكر على المرضى المعرضين للخطر من العصبية الشديدة والتبصر في الفيزيولوجيا المرضية.

العصب تقييم الإثارة في العلاج الكيميائي الناجم عن العصبية

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

nerve-excitability-assessment-in-chemotherapy-induced-neurotoxicity

Research

JoVE Journal

Neuroscience

18.0K Views.  University of New South Wales. هذا الملخص يصف طريقة جديدة لتقييم تطور العصبية في المرضى الذين يتلقون العلاج الكيماوي. في حين تقتصر وسائل التقييم التقليدية في قدرتها على الكشف عن التغييرات في وقت مبكر في وظيفة العصب، وتقنيات استثارة العصب توفير التعرف المبكر على المرضى المعرضين للخطر من العص...

Video: Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

استنساخه سحق الفأر العصب الوركي وتقييم لاحق للتجديد من قبل الجامعة جبل تحليل العضلات

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

تقييم عصبي عضلي وظيفة عن طريق العصب عن طريق الجلد الكهربائية تحفيز

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

الخلايا الدبقية الصغيرة بمثابة جهاز الاستشعار البيولوجي الأب البديل لتحديد الجسيمات النانوية العصبية

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

في الرحم النهج Electroporation لللدراسة استثارة الخلايا العصبية القطعان والربط وحيدة الخلية

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

في فيفو القياس الكهربية للعصب الزندي الفئران مع اختبار استثارة محواري

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

تقييم المرحلة المبكرة من الزرق المفتوح الزاوية في المرضى عن طريق التحقق من إمكانات استحضار البصرية

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

تنقية الخلايا الليفية وخلايا شوان من الأعصاب الحسية والحركية في المختبر

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

تقييم متعدد العوامل للسلوك الحركي في الفئران بعد إصابة سحق العصب الوركي من جانب واحد

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

الحقن المجهري للبطين الدماغي لعديد السكاريد الشحمي في يرقات الزرد لتقييم الالتهاب العصبي والسمية العصبية

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

تقييم استثارة القشرية الشوكية أثناء سلوك الوصول الموجه نحو الهدف

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...