نشكر حنا وارنر للمساهمة بيانات الشكل (3) والأستاذ بيرجكويست جوناس للمساهمة قيمة. السويدية للمجلس البحوث (جرانت 522-2006-6416 (MA)، 521-2007-5407 (MA)، ومؤسسة Wiberg آك ل(ماجستير، JH)، والأكاديمية الملكية السويدية للعلوم (MA، JH)، والمواد الكيميائية السويدية والتقدير والإمتنان المجتمع (JH) للحصول على الدعم المالي.

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هناك العديد من المزايا المتمثلة في استخدام الطيف الكتلي MALDI التصوير في دراسة نيوروببتيد. ويمكن تحليل غير متحيز للبيانات MS تكشف عن أن نواة الدماغ معينة فقط، أو كما في النتائج المقدمة هنا حيث يرتبط فقط على جزء من المخطط ظهراني جانبي مع الحالة المرضية في جسم المريض معينة. عن طريق الإبقاء على المعلومات المكانية ومن ثم من الممكن لإعادة تحديد المناطق ذات الأهمية للتحليل احصائى مع حساسية أعلى، وانخفاض التقلبات مقارنة مع تحليل لأقسام الدماغ كله أو باستخدام التقليدية peptidomics دراسات على مقتطفات الببتيد. وبالإضافة إلى ذلك، من المهم أن ندرك MALDI IMS بسهولة يمكن الكشف لم تكن معروفة سابقا في مرحلة ما بعد التعديلات متعدية، ولكن التحليلات الهيكلية يجب أن تتبع لتحديد المواقع بدقة من الأحماض الأمينية التي يتم تعديلها. المخاطر المشتركة في وضع الرؤية وIMS البيانات MALDI تشمل رسم خريطة كثافة الذروة القصوى لنطاق بصري خطي من BLACK (0٪) إلى لون (100٪) عن كل قسم في سلسلة التجارب (الشكل 3)، بدلا من رسم خرائط لجميع الفروع على نطاق ومطلق مشترك بنسبة 100٪ حيث هو كثافة الذروة القصوى من جميع أقسام (الشكل 5) . الأسلوب الأخير يسمح بمقارنة البيانات المجموعة وتصور من الاختلافات بين مجموعات العلاج. يشكل عقبة رئيسية في تحليل IMS MALDI هو مهمة من الببتيدات إلى قمم كتلة محددة. على النسيج جنبا إلى جنب، قياس الطيف الكتلي الممكن في بعض الأحيان، ولكن غالبا ما يكون صعبا للغاية 13،14. فإننا نجد أنه يمكن استخدام نهج أكثر التقليدية بما في ذلك تجزئة إعدادي قوي على الكاتيون تبادل اللوني، تليها مرحلة LC-MS/MS معكوسة إلى نيوروببتيد تسلسل بنجاح العديد من الببتيدات الأفيونية خاصة. فإنه لا يزال من غير المألوف للحصول على نوعية جيدة MS / MS الأطياف التي لا تتطابق مع اي مداخلة على قاعدة البيانات باستخدام محركات البحث الشائعة مثل تعويذة. في هذه الحالات من جديد، من جهة تسلسل هو فقط. tواي خيار. يمكن الحصول على الدليل القاطع على هوية الذروة من قبل IMS MALDI من المقاطع النسيجية من الماوس خروج المغلوب المناسبة، ولكن هذه ليست دائما متاحة أو ممكنة. وبديل ذلك هو للتحقق من صحة النتائج وفقا لطريقة مختلفة تماما، على سبيل المثال بواسطة immunoblotting الغربي أو المناعية. وهذا يمكن أن تشمل في كثير من الأحيان رفع الأجسام المضادة، وقدرا كبيرا من العمل التحقق من الأجسام المضادة الجديدة. تم تحسين الاستراتيجية العامة المقدمة في هذا البروتوكول على نطاق واسع للتجارب neuropeptide IMS MALDI بما في ذلك عدة أقسام والظروف التجريبية. وقد تم تحسين البروتوكول وخاصة بالنسبة للالببتيدات الأفيونية، وسيكون له أثر كبير في الدراسات المستقبلية، كما يعملون في مجالات متنوعة من الأبحاث بما في ذلك الآليات الكامنة وراء الألم والاستجابة الذاتية للعقاقير الإدمان.

1.1 جمع العينات وإعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Isoflurane (Apoteksbolaget AB، السويد) </li><li style=";text-align:right;direction:rtl"> الجراحية الأدوات؛ اثنين من أزواج من مقص وقراضة، ملعقة </li><li style=";text-align:right;direction:rtl"> الثلج الجاف سحق ناعما </li><li style=";text-align:right;direction:rtl"> رقائق الألومنيوم وأكياس بلاستيكية للتخزين </li><li style=";text-align:right;direction:rtl"> ناظم البرد (Microm، هايدلبرغ، ألمانيا) </li><li style=";text-align:right;direction:rtl"> الإنديوم والقصدير أكسيد (ITO) المغلفة الشرائح MALDI (بروكر Daltonics، ألمانيا) </li><li style=";text-align:right;direction:rtl"> درجة الحرارة المثلى متوسطة القطع (أكتوبر) (ساكورا Finetek أوروبا، هولندا) </li><li style=";text-align:right;direction:rtl"> فراغ dessicator </li></ol> 1.2 مصفوفة التطبيق <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأسيتونيتريل (ACN)، والميثانول (MeOH)، والإيثانول (EtOH) المؤيدة للتحليل الصف (ميرك، ألمانيا). </li><li style=";text-align:right;direction:rtl"> Trifluoroacetic حامض (TFA) (سيغما الدريتش، وسانت لويس، MO). </li><li style=";text-align:right;direction:rtl"> تمت تنقية المياه مع نظام تنقية ملي-Q (ميليبور، بيدفورد، ماجستير). </li><li style=";text-align:right;direction:rtl"> رقاقة 1000 (شيمادزو، اليابان) أو صورة 630 نصاب (LabCyte، ​​سانيفيل كاليفورنيا) </li></ol> 1.3 قياس الطيف الكتلي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ULtraflex الثاني (بروكر Daltonics) </li><li style=";text-align:right;direction:rtl"> FlexImaging البرمجيات (V 2.0، بروكر Daltonics) </li><li style=";text-align:right;direction:rtl"> الببتيد أو البروتين المعايرة القياسي (بروكر Daltonics) </li></ol> 1.4 معالجة البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأصل v.8.1 (OriginLab كورب، Norhampton، ماجستير) </li><li style=";text-align:right;direction:rtl"> MATLAB الإصدار 7.9.0.529 (برنامج Mathworks، ناتيك، ماجستير) </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 تحديد Neuropeptide <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموجات فوق الصوتية sonicator </li><li style=";text-align:right;direction:rtl"> 10 كيلو دالتون الوزن الجزيئي قطع فلتر (ميليبور، بيدفورد، ماجستير) </li><li style=";text-align:right;direction:rtl"> SP Sephadex C 25 هلام (سيغما، ألمانيا) </li><li style=";text-align:right;direction:rtl"> Hypersil العمود (ThermoFisher) </li><li style=";text-align:right;direction:rtl"> بيريدين، حمض الفورميك (FA)، الأسيتونيتريل (ACN)، والميثانول (MeOH)، والإيثانول (EtOH) المؤيدة للتحليل الصف (ميرك، ألمانيا). </li><li style=";text-align:right;direction:rtl"> Trifluoroacetic حامض (TFA) سيغما الدريخ (سانت لويس، MO، الولايات المتحدة الأمريكية). </li><li style=";text-align:right;direction:rtl"> اجيلنت 1100 nanoflow LC، microWPS، العمود C18 (0.075x150mm) </li><li style=";text-align:right;direction:rtl"> اجيلنت 1100 الصغرى، fractioncollector، الهدف anchorchip Prespotted لوحة (بروكر Daltonics) </li><li style=";text-align:right;direction:rtl"> BioTools البرمجيات (V 3.1 SR2، بروكر Daltonics) </li><li style=";text-align:right;direction:rtl"> يعمل البرنامج الحيوي (v.3.3، الحرارية فيشر؛ <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ) </li><li style=";text-align:right;direction:rtl"> برنامج التميمة (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 قاعدة البيانات ( <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 تصوير الطيف الكتلي (IMS) هو نهج القوية التي تسهل التحليل المكاني للأنواع الجزيئية في عينات الأنسجة البيولوجية 2 (Fig.1). ويغطي القسم A 12 ميكرون رقيقة الأنسجة مع مصفوفة MALDI، مما يسهل والامتزاز التأين من الببتيدات سليمة والبروتينات التي يمكن الكشف عنها مع محلل الشامل، وذلك باستخدام عادة MALDI TOF / TOF مطياف الكتلة. عموما يمكن أن تقيم مئات من القمم في مقطع واحد الفئران أنسجة المخ. وعلى النقيض من تقنيات التصوير المستخدمة، وهذا النهج لا يتطلب معرفة مسبقة من الجزيئات من الفائدة ويسمح للتحليل غير خاضعة للرقابة وشاملة من الأنواع الجزيئية متعددة مع الحفاظ على خصوصية الجزيئية العالية وحساسية 2. نحن هنا وصف نهج IMS MALDI مقرها لتوضيح ملامح التوزيع الخاصة بكل منطقة من نيوروببتيد في الدماغ الفئران من مرض حيوان نموذج الشلل الرعاش (PD). PD هو مرض شائع الاعصاب مع انتشار قدرها 1٪ بالنسبة للأشخاص فوق سن 65 من العمر 3،4. ويستند العلاج الأكثر شيوعا أعراض على استبدال الدوبامين باستخدام لام دوبا 5. لكن يرافق ذلك من خلال آثار جانبية خطيرة بما في ذلك تحركات غير طبيعية غير الطوعي، ووصف لام دوبا الناجم عن خلل الحركة (غطاء) 1،3،6. واحد من التغيير الجزيئي الأبرز في LID هو upregulation من مرنا السلائف prodynorphin الأفيونية 7. والببتيدات dynorphin تعدل العصبي في مناطق الدماغ التي تشارك أساسا في مراقبة الحركة 7،8. لكن، حتى الآن على وجه الدقة الببتيدات الأفيونية التي تنشأ من تجهيز السلائف neuropeptide لم يتميز. ولذلك، تستخدم نحن MALDI IMS في نموذج حيواني من مرض باركنسون والتجريبية لام دوبا خلل الحركة التي يسببها. أثبتت MALDI التصوير قياس الطيف الكتلي ليكون من المفيد وخاصة فيما يتعلق characteriza neuropeptideنشوئها، منذ النهج الأجسام المضادة المستخدمة القائم يستهدف سلاسل الببتيد معروف سابقا، ولاحظ التعديلات بعد متعدية. على النقيض من ذلك يمكن أن MALDI IMS كشف رواية تجهيز المنتجات الببتيد، وبالتالي تكشف عن الآليات الجزيئية الجديدة من تعديل neuropeptide من انتقال الخلايا العصبية. في حين لا يمكن أن المبلغ المطلق من نيوروببتيد يحددها IMS MALDI، أن يرسم الوفرة النسبية للايونات الببتيد من أطياف الشامل، وإعطاء الأفكار حول تغيير مستويات في الصحة والمرض. في الأمثلة المعروضة هنا، تم العثور على شدة ذروة B dynorphin، ألفا neoendorphin وف المادة المراد زيادة كبيرة في ظهراني جانبي، ولكن ليس ظهراني إنسي، المخطط للحيوانات مع خلل الحركة الشديدة التي تنطوي على الوجه والجذع وعضلات orolingual (الشكل 5). وعلاوة على ذلك، كشفت MALDI IMS وجود علاقة بين شدة خلل الحركة ومستويات DES-التيروزين neoendorphin ألفا، وهو ما يمثل آلية لم تكن معروفة سابقا من inacti وظيفيvation من dynorphins في المخطط مثل إزالة N-محطة التيروزين يقلل من dynorphin في مستقبلات المواد الأفيونية قدرة ملزم 9. هذه هي أول دراسة على توصيف neuropeptide في LID باستخدام MALDI IMS، وإبراز النتائج المحتملة لهذه التقنية لتطبيقها في جميع مجالات البحوث الطبية الحيوية.

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

الدوبامين استبدال العلاج الدوائي باستخدام لام دوبا هو العلاج الأكثر شيوعا من أعراض مرض باركنسون، لكن يرافقه من الآثار الجانبية بما في ذلك تحركات غير طبيعية غير الطوعي، وخلل الحركة ووصف<sup&gt; 1</sup&gt;. هنا، يتم تقديم بروتوكول للكتلة MALDI التصوير الطيفي أن يكشف تغيرات في مستويات الفئران neuropeptide الدماغ المرتبطة خلل الحركة.

MALDI الطيف الكتلي التصوير من نيوروببتيد في مرض باركنسون

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. الدوبامين استبدال العلاج الدوائي باستخدام لام دوبا هو العلاج الأكثر شيوعا من أعراض مرض باركنسون، لكن يرافقه من الآثار الجانبية بما في ذلك تحركات غير طبيعية غير الطوعي، وخلل الحركة ووصف 1. هنا، يتم تقديم بروتوكول للكتلة MALDI التصوير الطيفي أن يكشف تغيرات...

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.

تطوير نموذج من جانب واحد lesioned الفأر 6 OHDA من مرض باركنسون

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.

الجلد لكمة خزعة الثقافة ازدراع لاشتقاق الخلايا الليفية الإنسان الابتدائية

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.

استخدام الخلايا الليفية الإنسان الأساسية لرصد نمط ظاهري الميتوكوندريا في مجال مرض باركنسون

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.

نماذج التفاعل بين الجينات والبيئة لكشف آليات الحساسية في مرض باركنسون

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.

السيطرة على مرض باركنسون مع التكيف تحفيز الدماغ العميق

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.

الكمي التصوير بالرنين المغناطيسي من مرض العضلات والهيكل العظمي

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.

الكمي قداس الطيفية من التنميط Proteomes خلايا السرطان المستمدة من السائل والأورام الصلبة

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.

تطوير وبناء الجرذ نموذج ألفا synuclein لمرض باركنسون عن طريق حقن المجسم من النواقل الفيروسية المؤتلف المرتبطة الغدة

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.

المعادن التصوير في أنسجة المخ عن طريق الاستئصال بالليزر - إلى جانب بالحث البلازما - الطيف الكتلي (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.

الدماغ كل تجزئة وتحليل التغيير-نقطة من الدماغ التشريحية التصوير بالرنين المغناطيسي – التطبيق في مرض هنتنغتون بريمانيفيست

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