אנו מודים חנה וורנר לתרום את הנתונים עבור איור 3 ופרופ &#39;יונה Bergquist עבור קלט בעל ערך. שוודית המועצה למחקר (גרנט 522-2006-6416 (MA), 521-2007-5407 (MA); קרן Wiberg של אייק (MA, JH), האקדמיה המלכותית השוודית למדעים (MA, JH), ואת כימית שוודית החברה (JH) מוכרים בהכרת תודה על תמיכה כספית.

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החוקרים אין לי מה לחשוף.

ישנם מספר יתרונות של העסקת MALDI הדמיה ספקטרומטריית מסה במחקר של נוירופפטידים. ניתוח בלתי מוטה של ​​נתונים טרשת נפוצה יכולה לגלות כי גרעין מסוים במוח בלבד, או בתוצאות שהוצגו כאן, במקום רק את החלק דורסולטרלי של הסטריאטום קשורה בתנאי פתופיזיולוגיים מסוים. על ידי שמירה על מידע מרחבי זה אז ניתן להגדיר מחדש את האזורים המעניינים בביצוע ניתוחים סטטיסטיים עם רגישות גבוהה שונות נמוכה יותר לעומת ניתוח של חלקים במוח כולו או שימוש מסורתי מחקרים peptidomics על תמציות פפטיד. בנוסף, חשוב להבין MALDI IMS יכול לזהות בקלות ולא מוכרים שלאחר שינוי של תרגום, אבל ניתוח מבניות חייב לעקוב כדי לקבוע את המקומות המדויקים חומצות האמינו כי הם שונה. מהמלכודות הרגילות הטמונות לדמיין את ה-IMS MALDI הנתונים כוללים מיפוי עוצמת שיא בקנה מידה מירבית אופטי ליניארית מ BLACK (0%) לצבע (100%) עבור כל חלק וכל בסדרת הניסוי (איור 3), במקום למפות את כל החלקים כדי בקנה מידה מוחלטת משותף שבו 100% היא עוצמת שיא המרבי של כל חלקי (איור 5) . השיטה השנייה מאפשרת השוואה של נתונים קבוצתיים ויזואליזציה של הבדלים בין קבוצות הטיפול. המכשול העיקרי בניתוח IMS MALDI הוא הקצאה של פפטידים על פסגות המוניים ספציפיים. על רקמת בד בבד, ספקטרומטריית מסה לפעמים אפשרי, אבל בדרך כלל מוכיח די קשה 13,14. אנו מוצאים כי הגישה המסורתית יותר, כולל חלוקה preparative על חזקה קטיון חילופי כרומטוגרפיה, ואחריו LC-MS/MS שלב מהופכים ניתן להשתמש בהצלחה נוירופפטידים רצף רבים פפטידים אופיואידים במיוחד. זה עדיין לא נדיר לקבל איכות טובה MS / MS ספקטרום שאינם תואמים כל הערכים מסד נתונים באמצעות מנועי חיפוש נפוצים כגון הקמע. במקרים אלה דה נובו, סידור ביד הוא onlY אפשרות. ההוכחה הסופית של הזהות השיא ניתן להשיג על ידי MALDI IMS של סעיפים רקמה העכבר בנוקאאוט המתאים, אבל זה לא תמיד זמין או אפשרי. כחלופה לכך, כדי לאמת את התוצאות בשיטה שונה לחלוטין, למשל על ידי immunoblotting אימונוהיסטוכימיה המערבי או. זה יכול לעיתים קרובות כוללים העלאת נוגדנים כמות משמעותית של עבודת אימות נוגדנים חדשים. האסטרטגיה הכללית הציג בפרוטוקול זה מותאם במיוחד גדולים בקנה מידה ונוירופפטידים ניסויים MALDI ה-IMS, כולל כמה חלקים תנאי הניסוי. פרוטוקול כבר מותאם במיוחד עבור פפטידים אופיואידים ואת תהיה השפעה גדולה במחקרים עתידיים, כפי המועסקים בתחומים שונים של מחקר, כולל כאב מנגנוני הבסיס לבין התגובה אנדוגני לתרופות של התמכרות.

1.1 לדוגמה אוסף והכנת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Isoflurane (Apoteksbolaget AB, שבדיה) </li><li style=";text-align:right;direction:rtl"> כלים כירורגיים, שני זוגות מספריים, rongeur, מרית </li><li style=";text-align:right;direction:rtl"> קרח יבש כתוש דק </li><li style=";text-align:right;direction:rtl"> רדיד אלומיניום ושקיות פלסטיק לאחסון </li><li style=";text-align:right;direction:rtl"> Cryostat (Microm, היידלברג, גרמניה) </li><li style=";text-align:right;direction:rtl"> אינדיום בדיל תחמוצת (איטו) מצופים שקופיות MALDI (Bruker Daltonics, גרמניה) </li><li style=";text-align:right;direction:rtl"> חיתוך אופטימלי לחום בינוני (אוקטובר) (סאקורה Finetek אירופה, הולנד) </li><li style=";text-align:right;direction:rtl"> ואקום dessicator </li></ol> 1.2 יישום מטריקס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (ACN) אצטוניטריל, מתנול (MeOH), אתנול (EtOH) של תומכי ניתוח כיתה (Merck, גרמניה). </li><li style=";text-align:right;direction:rtl"> Trifluoroacetic חומצה (TFA) (סיגמא אולדריץ&#39;, סנט לואיס, מיזורי). </li><li style=";text-align:right;direction:rtl"> מים מטוהרים עם מערכת מילי-Q טיהור (Millipore, בדפורד, מסצ&#39;וסטס). </li><li style=";text-align:right;direction:rtl"> שבב 1000 (Shimadzu, יפן) או לאורך 630 הצופה (LabCyte, Sunnyvale, CA) </li></ol> 1.3 ספקטרומטריית מסה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ULtraflex II (Bruker Daltonics) </li><li style=";text-align:right;direction:rtl"> תוכנות FlexImaging (v 2.0, Bruker Daltonics) </li><li style=";text-align:right;direction:rtl"> פפטיד או חלבון כיול סטנדרטי (Bruker Daltonics) </li></ol> 1.4 עיבוד נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מקור v.8.1 (OriginLab קורפ, Norhampton, MA) </li><li style=";text-align:right;direction:rtl"> Matlab גירסה 7.9.0.529 (MathWorks, Natick, MA) </li><li style=";text-align:right;direction:rtl"> pBin ( <a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a> ) </li></ol> 1.5 neuropeptide זיהוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אולטראסאונד sonicator </li><li style=";text-align:right;direction:rtl"> 10 kDa מולקולרית חתך משקל המסנן (Millipore, בדפורד, מסצ&#39;וסטס) </li><li style=";text-align:right;direction:rtl"> SP Sephadex C 25 ג (Sigma, גרמניה) </li><li style=";text-align:right;direction:rtl"> Hypersil עמודה (ThermoFisher) </li><li style=";text-align:right;direction:rtl"> פירידין, חומצה פורמית (FA), אצטוניטריל (ACN), מתנול (MeOH), אתנול (EtOH) של כיתה בעד ניתוח (Merck, גרמניה). </li><li style=";text-align:right;direction:rtl"> Trifluoroacetic חומצה (TFA) סיגמא אולדריץ&#39; (סנט לואיס, מיזורי, ארה&quot;ב). </li><li style=";text-align:right;direction:rtl"> Agilent 1100 nanoflow LC, microWPS, טור C18 (0.075x150mm) </li><li style=";text-align:right;direction:rtl"> Agilent 1100 Micro-fractioncollector, צלחת Prespotted anchorchip היעד (Bruker Daltonics) </li><li style=";text-align:right;direction:rtl"> BioTools תוכנה (v 3.1 SR2, Bruker Daltonics) </li><li style=";text-align:right;direction:rtl"> ביו תוכנת Works (v.3.3, תרמו פישר; <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ) </li><li style=";text-align:right;direction:rtl"> הקמע תוכנה (v 2.2, MatrixScience; <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ) </li><li style=";text-align:right;direction:rtl"> Swepep מסד נתונים ( <a href="http://www.swepep.org" target="_blank">www.swepep.org</a> ) </li></ol>

maldi imaging mass spectrometry of neuropeptides in parkinson's disease

MALDI הדמיה ספקטרומטריית מסה (IMS) היא גישה רבת עוצמה המאפשרת ניתוח מרחבי של מינים מולקולריים דגימות רקמה ביולוגית 2 (תמונה 1). סעיף 12 דק מיקרומטר רקמות מכוסה MALDI מטריקס, המאפשר desorption ו יינון של פפטידים וחלבונים שלמים כי ניתן לאתר עם נתח המונית, בדרך כלל באמצעות TOF MALDI / ספקטרומטר TOF המוני. בדרך כלל מאות פסגות ניתן להעריך סעיף חולדה אחת רקמות המוח. לעומת שיטות הדמיה נפוצים, גישה זו אינה דורשת ידע מוקדם של מולקולות של עניין ומאפשר ניתוח ללא השגחה ומקיף של מינים מולקולרית מרובים תוך שמירה על סגוליות גבוהה ורגישות מולקולרית 2. כאן נתאר את גישת IMS MALDI מבוסס על הבהרת האזור הספציפי פרופילים ההפצה של נוירופפטידים במוח החולדה למחלות בעלי חיים דגם הפרקינסון (PD). פ&quot;ד היא מחלה ניוונית נפוצה עם שכיחות של 1% עבור אנשים מעל גיל 65 לגיל 3,4. טיפול סימפטומטי הנפוץ ביותר מבוסס על החלפת דופמין באמצעות L-DOPA 5. אולם זה מלווה תופעות לוואי חמורות, כולל תנועות לא רצוניות חריגות, המכונות L-DOPA-Induced dyskinesias (מכסה) 1,3,6. אחד שינוי מולקולרי הבולט ביותר הוא מכסה upregulation של mRNA prodynorphin אופיואידים מבשר 7. פפטידים dynorphin לווסת עצבית באזורים במוח המעורבים למעשה בשליטה התנועה 7,8. עם זאת, עד כה פפטידים אופיואידים מדויקים שמקורם עיבוד מקדים neuropeptide לא אופיינו. לכן, השתמשנו MALDI IMS במודל חיה של מחלת פרקינסון ניסיוני ו-L-DOPA dyskinesia המושרה. MALDI הדמיה ספקטרומטריית מסה התגלה יתרון במיוחד ביחס neuropeptide characterization, שכן הגישות הנפוצות מבוססות נוגדנים מטרות רצף הפפטיד ידועים שנצפה קודם לכן לאחר שינויים של תרגום. לעומת זאת MALDI IMS יכול לפענח הרומן עיבוד מוצרים פפטיד ובכך לחשוף את המנגנונים המולקולריים החדשים של neuropeptide אפנון של שידור עצבי. בעוד הסכום המוחלט של נוירופפטידים לא ניתן לקבוע על ידי MALDI IMS, השפע היחסי של יוני פפטיד ניתן התחום של הספקטרום המונית, נותן תובנות אודות שינוי רמות בריאות ומחלות. בדוגמאות המוצגות כאן, לעוצמות השיא של dynorphin B, אלפא neoendorphin וחומר P נמצאו גדל באופן משמעותי דורסולטרלי, אבל לא הסטריאטום dorsomedial, בעלי חיים עם dyskinesia חמורה מעורבים המטען הפנים, שרירי orolingual (איור 5). יתר על כן, גילה MALDI IMS מתאם בין חומרת dyskinesia ורמת neoendorphin אלפא des-tyrosine, המייצג מנגנון ידוע עד כה inacti תפקודיתvation של dynorphins בסטריאטום כמו הסרת טירוזין N-מסוף מפחית את הקולטן אופיואידים, של dynorphin קיבולת מחייב 9. זהו המחקר הראשון על אפיון neuropeptide בלוד באמצעות MALDI IMS ואת התוצאות מדגישות את הפוטנציאל של הטכניקה ליישום בכל תחומי מחקר ביו.

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

טיפול תרופתי חלופי באמצעות דופמין L-DOPA הוא טיפול סימפטומטי הנפוץ ביותר של מחלת פרקינסון, אך מלווה תופעות לוואי, כולל תנועות לא רצוניות חריגות, dyskinesia כינה<sup&gt; 1</sup&gt;. כאן, פרוטוקול עבור MALDI הדמיה ספקטרומטריית מסה מוצג המאתר שינויים במוח חולדה רמות ונוירופפטידים הקשורים dyskinesia.

MALDI הדמיה ספקטרומטריית מסה של נוירופפטידים אצל חולי פרקינסון

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

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MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

25.8K Views.  Uppsala University. טיפול תרופתי חלופי באמצעות דופמין L-DOPA הוא טיפול סימפטומטי הנפוץ ביותר של מחלת פרקינסון, אך מלווה תופעות לוואי, כולל תנועות לא רצוניות חריגות, dyskinesia כינה 1. כאן, פרוטוקול עבור MALDI הדמיה ספקטרומטריית מסה מוצג המאתר שינויים במוח חולדה רמות ונוירופפטידים הק...

Video: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

פיתוח מודל חד צדדי, lesioned מאוס 6-OHDA של מחלת פרקינסון

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

תרבות explant עור אגרוף ביופסיה לגזירה של fibroblasts אדם היסודי

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

השימוש בפיברובלסטים אנושיים ראשוניים לניטור פנוטיפים מיטוכונדריאלי בתחום מחלת פרקינסון

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

מודלים לאינטראקציה בין סביבת גנים כדי לחשוף מנגנוני רגישות במחלת פרקינסון

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

שליטה מחלת פרקינסון עם מסתגל עמוק גירוי המוח

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

דימות תהודה מגנטית כמותי של מחלת שריר שלד

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Mass כמותי spectrometric פרופיל של Proteomes סרטן התאים נגזרים מנוזלים וגידולים מוצקים

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

פיתוח של מודל עכברוש מבוסס אלפא-synuclein עבור מחלת פרקינסון באמצעות הזרקת stereotactic של וקטור ויראלי הקשורים מוקטורים

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

מתכות הדמיה ברקמת המוח על ידי אבלציה לייזר - פלזמה מצמידים אינדוקטיבי - ספקטרומטריית מסה (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

מוח-כל פילוח וניתוח נקודת השינוי האנטומי במוח MRI – היישום ב- Premanifest של מחלת הנטינגטון

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...