1. הכנת החולה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החולים מופנים מהמחלקה לאונקולוגיה רפואי רגישות הבסיס העצבי בדיקה לפני תחילתו על כימותרפיה. </li><li style=";text-align:right;direction:rtl"> התאמת המטופל לבדיקת רגישות צריכה להיקבע. חולים צריכים להיות שליליים, אם יש להם היסטוריה של ראיות או בסיס נוירופיזיולוגי של נוירופתיה היקפית, קיבלו טיפול כימותרפי לפני neurotoxic או קיימים קונטרה אינדיקציות למוצרי בדיקות רגישות. </li></ol> 2. רגישות נהלים axonal <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מתחייבים פרוטוקולים רגישות חושית ומוטורית על העצב המדיאני, באמצעות חצי אוטומטיות QTracS מערכת ממוחשבת (המכון לנוירולוגיה, מלכה בכיכר, אנגליה), ממריץ מבודד ליניארי דו קוטבי זרם קבוע (Digitimer, Welwyn Garden City, בריטניה) מגבר ( ספיר IIA, Medelec, בריטניה). </li><li style=";text-align:right;direction:rtl"> הכן את פני העור בשורש כף היד והאמה עם ג&#39;ל משטח מחוספס או להפחית העורהתנגדות, ולאחר מכן יישום של אלכוהול למחוק. </li><li style=";text-align:right;direction:rtl"> להכין את האתר עבור הקלטה הקלטות למוצרי עם רכב שאינו polarizable אלקטרודות המונחות על הבטן השריר של pollicis brevis החוטף ואת אלקטרודה 4 ס&quot;מ התייחסות דיסטלי להקליט המנוע מתחם פעולה פוטנציאלים (CMAPs). </li><li style=";text-align:right;direction:rtl"> להכין את האתר עבור הקלטה הקלטות חושית - באמצעות אלקטרודות המונחות על טבעת המפרקים interphalangeal הפרוקסימלית ומ דיסטלי להקלטה אלקטרודות התייחסות בהתאמה, כדי לרשום חושיות מתחם פעולה פוטנציאלים (CSAPs). </li><li style=";text-align:right;direction:rtl"> מניחים צלחת נייטרלי electrosurgical האדמה בכף, עם ג&#39;ל מוליך. </li><li style=";text-align:right;direction:rtl"> רעש חשמלי הגדרת הקלטה יש להסיר עד כמה שניתן, באמצעות בלוף 50/60 הרץ רעש Eliminator (Quest מכשירים מדעיים, צפון ונקובר, קנדה). </li><li style=";text-align:right;direction:rtl"> לגרות את העצב המדיאני בשורש כף היד. האתר גירוי יש לבחור כאתר הסף הנמוך ביותר, באמצעות אלקטרודה ביפולרית repositionable ולאחר מכן לא עמ &#39;olarizable האלקטרודה. </li><li style=";text-align:right;direction:rtl"> האלקטרודה האנודה יש ​​למקם 10 ס&quot;מ הפרוקסימלי של האלקטרודה מגרה את העצם. </li><li style=";text-align:right;direction:rtl"> מעקב אחר הטמפרטורה במקום הגירוי לאורך כל הבדיקה ולוודא כי הטמפרטורה עולה על 32 ° C. </li></ol> 3. רגישות הפרוטוקולים axonal <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הקלט את עקומת התגובה לגירוי על ידי הדרגתי להגדיל את הגירוי עד התגובה היא מקסימלית ולא להגדיל את עוצמת הגירוי, כאשר הוא גדל עוד יותר. </li><li style=";text-align:right;direction:rtl"> משרעת יעד למעקב סף מוגדר באופן אוטומטי ל -30% -40% של משרעת מקסימלית, המתאימה לאזור המדרון התלול על עקומת התגובה לגירוי. </li><li style=";text-align:right;direction:rtl"> על פי השיטה של בוסטוק et al., 1998, שינויים הסף הנוכחי הנדרש כדי להשיג את היעד משרעת מתבצע מעקב אחר באינטרנט. </li><li style=";text-align:right;direction:rtl"> הקלט פרמטרים רבים, כולל רגישות סף electrotonus (TE), מחזור ההתאוששות (RC) ו סף השוטף (I / V) מערכת היחסיםכמתואר קירנן et al., 2000 ו קירנן et al., 2001. </li><li style=";text-align:right;direction:rtl"> סף electrotonus נבחנת באמצעות 100ms הזרמים התת הקיטוב, עם מקטב סט הנוכחי 40% ± סף הביקורת (איור 1). השינוי הנוכחי הסף הנדרש כדי לשמור על תגובת האמפליטודה היעד הבא הן דה ו Hyper-הקיטוב נרשם. סף electrotonus מספק הערכה של conductances internodal ופוטנציאל הממברנה, עם תשובות בכיוון hyperpolarizing בסוף הדופק מקטב (90-100 MS) קשור מאוד עם פוטנציאל הממברנה. </li><li style=";text-align:right;direction:rtl"> מחזור ההחלמה מוערך באמצעות פרדיגמה הדופק יחד (איור 2), עם מיזוג הגירוי הראשוני supramaximal בעקבות במרווחים שונים של גירוי המבחן (מ -2.5 ל MS 200 MS). בעקבות גירוי supramaximal, קשה יותר ליצור תגובה לאחר מכן, כינה &quot;refractoriness&quot;, המשקף את איון של מתח-Gated Na + ערוצים. לאחר תקופת עקשן, תקופה של סיוע המכונה superexcitability מתרחשת. </li><li style=";text-align:right;direction:rtl"> השוטף סף יחסים נבחנת באמצעות זרמים הקיטוב של 200 ms המשתנות של כוח 50% ל -100% על הסף. </li></ol> 4. המטופל הערכה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש בשיטות אלה, חולים מוערכים הן בחריפות ו longitudinally פני טיפול כימותרפי. כדי להעריך neurotoxicity חריפה, בחולים לחזור להערכה שלאחר טיפול כימותרפי תוך 48 שעות מיום קבלת הטיפול. </li><li style=";text-align:right;direction:rtl"> לבחון neurotoxicity כרונית, הערכות שנעשו לפני עירוי כימותרפיה מושווים longitudinally פני מחזורי טיפול. </li><li style=";text-align:right;direction:rtl"> בנוסף בדיקות רגישות axonal, קונבנציונאלי סולמות הדירוג קליניים יש להשתמש כדי להעריך כימותרפיה neurotoxicity, כולל סרטן - של Common Criteria עבור שלילית אירועים נוירופתיה בתת חושי, ציון נוירופתיה סה&quot;כ ופאטient דיווח תוצאות ההערכה. </li></ol> 5. ניתוח ופרשנות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לזהות neurotoxicity חריפה, והתוצאות לעומת טיפול לפני ואחרי כימותרפיה. כדי להעריך את ההתפתחות של שינויים ארוכי טווח בתפקוד העצבי, התוצאות מושוות על הטיפול. פרמטרים מרכזיים להערכת כוללים refractoriness, superexcitability, במידה של שינוי סף electrotonus הסף (hyperpolarizing 90-100 MS), בנוסף לפרמטרים המקובלים כגון משרעת חביון שיא. </li><li style=";text-align:right;direction:rtl"> כדי להעריך את השינוי הכולל פרמטרים של רגישות על פני הטיפול, ציון רגישות המשולב מחושב. שינוי בשלושה פרמטרים -, superexcitability refractoriness ועל סף electrotonus (hyperpolarizing 90-100 MS) - מסתכמת מ הראשונית לטיפולים האחרונים כדי לתת ציון כולל של שינוי. </li></ol> 6. נציג תוצאות דוגמאות לתוצאות רגישות של פטient מטופלים עם אוקסליפלטין מסופקים. מיד לאחר אוקסליפלטין עירוי, שינויים חריפים ביותר הן רגישות חושית מוטורית לפיתוח, המרמזים על התפתחות תפקודי channelopathy Na + 10 -13. עם זאת, שינוי משמעותי בפרמטרים רגישות מרובים מפתחת בהדרגה על פני טיפול אוקסליפלטין רק אקסונים חושית, עם אקסונים מוטוריים מושפעים (איור 3), המשקף נזק נרחב axonal חושי פוטנציאל שינוי הממברנה. דפוס זה תואם את הביטוי הקליני של סימפטומים הנגרמת neurotoxicity אוקסליפלטין כרונית. רגישות לשינויים ב אקסונים חושית קודם הפחתת המשרעת שיא כפי שנבחנו באמצעות קונבנציונאלי הולכה טכניקות עצב, וסבורים כי טכניקות רגישות axonal עשויים לספק כלי אבחון רגיש neurotoxicity אוקסליפלטין הנגרמת מוקדם. <img alt="איור 1" src="/files/ftp_upload/3439/3439fig1.jpg" /> באיור 1. </strong&gt; סף electrotonus, המתארים גל בתגובה התת ממושכת הקיטוב הנוכחי (100 MS), עם כיוון hyperpolarizing להתוות ברבע התחתון (כחול) וכיוון depolarizing להתוות ברבע העליון (אדום). להלן גירוי waveform להחיל על מנת ליצור את התגובה electrotonus הסף. <img alt="איור 2" src="/files/ftp_upload/3439/3439fig2.jpg" /> איור 2. שחזור מחזור של רגישות, הוכחת את רצף אופייני שינויים רגישות לאחר הולכה הדחף, עם תקופה של רגישות מופחתת (refractoriness) עד 3 MS בעקבות גירוי supramaximal, ואחריה תקופה של רגישות מוגברת (superexcitability) לשיא של 5 MS -7 ולאחר מכן רגישות מופחתת (subexcitability). הפרדיגמה לזווג הדופק פרוטוקול גירוי הוא הבלעה. <img alt="איור 3" src="/files/ftp_upload/3439/3439fig3.jpg" /> תאנהיור 3. שינויים רגישות חושית של אקסונים אוקסליפלטין שטופלו, עם הקלטות הבסיס המוצגים הקלטות שחור שלאחר הטיפול המוצגים לבן, לאחר 4-6 חודשי טיפול אוקסליפלטין. שינויים אלה נחשבים לשקף נזק axonal רחבה ופוטנציאל שינוי הממברנה. תמונה של רגישות הגדרת בסביבה האונקולוגית הוא מופיע בצד שמאל. <a href="http://www.jove.com/files/ftp_upload/3439/3439fig3large.jpg" target="_blank">לחץ כאן כדי להציג תמונה גדולה יותר</a> .

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כימותרפיה נוירופתיה היא תופעת לוואי חמורה של טיפול בסרטן, אשר עשויים להשפיע במהלך הטיפול לייצר לטווח ארוך נכות למטופל. קיים מחסור של אמצעי אבחון רגישים אובייקטיבי במיוחד למדוד תפקוד העצב בחולים שטופלו בכימותרפיה. הפיתוח הקליני של טכניקות רגישות axonal סיפקה מידע שימושי ניבוי להערכת neurotoxicity כימותרפיה. על ידי מתן מידע על תפקוד ערוץ יונים, פוטנציאל מנוחה, ותפקוד קרום axonal, טכניקות אלו מאפשרים תובנה התהליכים שבבסיס פתופיזיולוגיים axonal תפקוד אצל חולי סרטן המטופלים בכימותרפיה. בנוסף, טכניקות רגישות axonal הוכחו להיות ריאלי בסביבה לאונקולוגיה קלינית, בדיקה אחד יכול להסתיים ב 5-10 דקות. ב אוקסליפלטין המטופלים, טכניקות רגישות axonal לספקסמן רגיש המאפשר זיהוי מוקדם של חולים בסיכון neurotoxicity קשה. בהשוואה קונבנציונליים הולכה מחקרים העצב אשר לזהות אוקסליפלטין הנגרמת מנזק עצבי רק לאחר אובדן axonal כבר התרחש, מחקרים רגישות axonal לספק סמנים חזוי של חוסר תפקוד עצבי לפני אובדן axonal. לפיכך, מחקרים רגישות axonal עשוי להיות מנוצל על מנת לספק הערכה של התפקוד העצבי בניסויים קליניים של אסטרטגיות neuroprotective של פוטנציאליים, כדי לקבוע באופן אובייקטיבי יעילות neuroprotective.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> ציוד וחומרים </td><td> חברה </td></tr><tr><td> QTracS התוכנית </td><td> Digitimer, המכון לנוירולוגיה, מלכה בכיכר, בריטניה </td></tr><tr><td> DS5 מבודד ליניארי דו קוטבית ממריץ זרם קבוע </td><td> Digitimer </td></tr><tr><td> ספיר מגבר IIA </td><td> Medelec </td></tr><tr><td> בלוף 50/60 הרץ רעש Eliminator </td><td> Quest מכשירים מדעיים </td></tr><tr><td> ללא polarizable אלקטרודות </td><td> Unomedical </td></tr><tr><td> Electrosurgical נייטרלי צלחת הארץ </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.

Watch this Scientific Journal Video about Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity at JoVE.com

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

תקציר זה מתאר שיטה חדשה כדי להעריך את התפתחות neurotoxicity בחולים המקבלים טיפול כימותרפי. בעוד שיטות הערכה קונבנציונליות מוגבלים ביכולתם לזהות שינויים מוקדמים בתפקוד העצבים, רגישות טכניקות העצב לספק זיהוי מוקדם של חולים בסיכון neurotoxicity תובנה קשה לתוך פתופיזיולוגיה.

רגישות העצב הערכה ב neurotoxicity כימותרפיה

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

18.1K Views.  University of New South Wales. תקציר זה מתאר שיטה חדשה כדי להעריך את התפתחות neurotoxicity בחולים המקבלים טיפול כימותרפי. בעוד שיטות הערכה קונבנציונליות מוגבלים ביכולתם לזהות שינויים מוקדמים בתפקוד העצבים, רגישות טכניקות העצב לספק זיהוי מוקדם של חולים בסיכון neurotoxicity תובנה קשה לתוך פתופיזיולוגיה.

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.

עכבר לשחזור Sciatic עצב קראש והערכה לאחר ההתחדשות של ניתוח הר שלם שריר

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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

השתלה של תאי ensheathing חוש ריח להערכת החלמה תפקודית לאחר פגיעה עצבית היקפית

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

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).

הערכה של תפקוד העצבי באמצעות גירוי עצבי חשמלי Percutaneous

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.

Microglia בתור biosensor סרוגייט לקביעת Nanoparticle neurotoxicity

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.

ברחם Electroporation גישות לחקר רגישות של העצבית subpopulations וקישוריות תא יחיד

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.

אין ויוו מדידות אלקטרופיזיולוגיות של עצב הגומד עכברוש עם הבדיקות דעתנית עצב

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

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

הכוח של מרווח Interstimulus להערכה של זמני עיבוד בחולדות

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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.

הערכת השלב המוקדם פתיחת גלאוקומה בחולים על ידי מבודדים-בדוק פוטנציאל ויזואלי מעורר

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.

טיהור של פיברותקיעות ותאי Schwann מפני חושים ומוטוריים עצבים בחוץ גופית

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.

הערכה רב-גורמית של התנהגות מוטורית בחולדות לאחר פציעת ריסוק עצבית סיאטית חד-צדדית

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 ...