NS65819 (CDJ ، TO) ، NS049720 (CDJ) ، CA097257 (CDJ ، TO) ، CIRM DR1 - 01426 نود أن نشكر راكيل سانتوس لتقديم المساعدة التقنية.

<ol>
	<li>Bobo, R. H., Laske, D. W., Akbasak, A., Morrison, P. F., Dedrick, R. L., Oldfield, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+macromolecules+in+the+brain.">Convection-enhanced delivery of macromolecules in the brain.</a> Proc. Natl. Acad. Sci. 91, 2076-2080 (1994).</li><li>Degen, J. W., Walbridge, S., Vortmeyer, A. O., Oldfield, E. H., Lonser, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+convection-enhanced+delivery+of+gemcitabine+or+carboplatin+in+a+malignant+glioma+model+in+rats.">Safety and efficacy of convection-enhanced delivery of gemcitabine or carboplatin in a malignant glioma model in rats.</a> J Neurosurg. 99, 893-898 (2003).</li><li>Dickinson, P. J., LeCouteur, R. A., Higgins, R. J., Bringas, J. R., Roberts, B., Larson, R. F., Yamashita, Y., Krauze, M. T., Noble, C. O., Drummond, D. C., Kirpotin, D. B., Park, J. W., Berger, M. S., Bankiewicz, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+model+of+convection-enhanced+delivery+of+liposomes+containing+CPT-11+monitored+with+real-time+magnentic+resonance+imaging:+laboratory+investigation.">Canine model of convection-enhanced delivery of liposomes containing CPT-11 monitored with real-time magnentic resonance imaging: laboratory investigation.</a> J Neurosurg. 108, 989-998 (2008).</li><li>Gill, S. S., Patel, N. K., Hotton, G. R., O'Sullivan, K., McCarter, R., Bunnage, M., Brooks, D. J., Svendsen, C. N., Heywood, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+brain+infusion+of+glial+cell+line-derived+neurotrophic+factor+in+Parkinson+disease.">Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease.</a> Nature Medicine. 9, 589-595 (2003).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+novel+therapeutic+agents+in+tumors:+physiological+barriers+and+strategies.">Delivery of novel therapeutic agents in tumors: physiological barriers and strategies.</a> J Natl Cancer Inst. 81, 570-576 (1989).</li><li>Krauze, M. T., Saito, R., Noble, C. O., Tamas, M., Bringas, J., Park, J. W., Berger, M. S., Bankiewicz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflux-free+cannula+for+convection-enhanced+high-speed+delivery+of+therapeutic+agents.">Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.</a> J Neurosurg. 103, 923-929 (2005).</li><li>Krauze, M. T., Vandenberg, S. R., Yamashita, Y., Saito, R., Forsayeth, J., Noble, C. O., Park, J. W., Bankiewicz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+real-time+convection-enhanced+delivery+of+liposomes+to+primate+brain:+a+long-term+retrospective.">Safety of real-time convection-enhanced delivery of liposomes to primate brain: a long-term retrospective.</a> Exp Neurol. 210, 638-644 (2008).</li><li>Murad, G. J., Walbridge, S., Morrison, P. F., Garmestani, K., Degen, J. W., Brechbiel, M. W., Oldfield, E. H., Lonser, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time,+image-guided,+convection-enhanced+delivery+of+interleukin+13+bound+to+pseudomonas+exotoxin.">Real-time, image-guided, convection-enhanced delivery of interleukin 13 bound to pseudomonas exotoxin.</a> Clin Cancer Res. 12, 3145-3151 .</li><li>Ozawa, T., James, C. D., Van Meir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Brain+Tumor+Cell+and+Tumor+Tissue+Transplantation+Models.">Human Brain Tumor Cell and Tumor Tissue Transplantation Models.</a> CNS Cancer: Models, Markers, Prognostic Factors, Targets, and Therapeutic Approaches. , 147-162 (2009).</li><li>Szerlip, N. J., Walbridge, S., Yang, L., Morrison, P. F., Degen, J. W., Jarrell, S. T., Kouri, J., Kerr, P. B., Kotin, R., Oldfield, E. H., Lonser, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+convection-enhanced+delivery+of+viruses+and+virus-sized+particles.">Real-time imaging of convection-enhanced delivery of viruses and virus-sized particles.</a> J Neurosurg. 107, 560-567 (2007).</li><li>Yamashita, Y., Krauze, M. T., Kawaguchi, T., Noble, C. O., Drummond, D. C., Park, J. W., Bankiewicz, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+a+topoisomerase+I+inhibitor+(nanoliposomal+topotecan)+and+a+topoisomerase+II+inhibitor+(pegylated+liposomal+doxorubicin)+in+intracranial+brain+tumor+xenografts.">Convection-enhanced delivery of a topoisomerase I inhibitor (nanoliposomal topotecan) and a topoisomerase II inhibitor (pegylated liposomal doxorubicin) in intracranial brain tumor xenografts.</a> Neuro Oncol. 9, 20-28 (2007).</li></ol>

تمت الموافقة على جميع الإجراءات التي أثبتت هنا الحيوان UCSF الرعاية المؤسسية واللجنة الاستخدام.

يجب تقييمها قبل السريرية لفعالية أي وكيل علاجية تأخذ بعين الاعتبار الخصائص الدوائية وكيل والنسيج المستهدف من الفائدة. في حين أن الأساليب المنهجية في الإدارة العامة أكثر سطحية وأكثر تحملا من قبل المرضى ، والانتقائية في حاجز الدم في الدماغ غالبا ما يستلزم التسليم المحلي من العلاج لعلاج مرض الجهاز العصبي المركزي. هنا ، لقد أثبتنا أسلوب واحد من التسليم المباشر للدماغ : CED. الأشكال الأخرى للتسليم المحلي إلى الدماغ وتشمل الإدارة المباشرة للالسائل النخاعي ، intratumoral حقن البلعة ، والحقن البطيني. عن هذه الأساليب ، الإنتشارية العلاجية من المهم للغاية ، مع الحد من انتشار سوء منطقة التغطية لبضعة ملليمترات من موقع الحقن (جاين 1989 ، بوبو 1994). في المقابل ، CED يستخدم الضغط الايجابي لزيادة مجال توزيع المخدرات (بوبو 1994) ، في حين لا تزال مواتية لاستهداف المنشآت العلاجية تشريحي عصبي معين. في المختبر لدينا ، لقد نجحنا في تنفيذ CED في الدماغ وكذلك في putamen المذنبة في القوارض (الشكل 4). أصبحت القدرة على إجراء CED في نماذج القوارض متزايد الأهمية في تكوين الجمعيات مع الاهتمام المتزايد في تعظيم الكفاءة العلاجية عند علاج مختلف أنواع مرض الجهاز العصبي المركزي. ويمكن استخدام CED لتقديم مجموعة متنوعة من العوامل ، بما في ذلك تنقية البروتينات والعقاقير جزيء صغير ، والفيروسات (جيل 2003 ، ديجين 2003 ، Szerlip 2007). مجموعة من الأمراض التي يمكن علاجها مع CED يشمل سرطانات الجهاز العصبي المركزي (ياماشيتا 2007) وكذلك الأمراض العصبية مثل مرض باركنسون (جيل 2003). والبحوث مستمرة في توسيع دائرة الهندسة المدنية ، وضرورة التصوير في الجسم الحي من CED تدار العلاج هو زيادة مماثلة. في حين أظهر التصوير فلوري هنا سيكون من المحتمل أن لا تكون مرئية من خلال الجمجمة البشرية ، وتوظيف أساليب أخرى شاركت في ضخ عوامل التباين التصوير بالرنين المغناطيسي (ديكنسون 2008) وجذب الاهتمام لتقييم تطبيق لرصد infusates في المرضى الذين يعانون من أمراض الجهاز العصبي المركزي.

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<td>2% Chlorhexidine</td>
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<td>Stereotaxic Frame</td>
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<td>51725</td>
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<td>Upchurch Scientific</td>
<td>1520</td>
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<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
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systemic and local drug delivery for treating diseases of the central nervous system in rodent models

اختبار دقيق قبل السريرية للنظام العصبي المركزي (CNS) المداواة يتضمن النظر في طرق الإدارة وكيل biodistribution في تقييم فعالية العلاج. بين التصنيفين الرئيسية للإدارة ، ويفضل كثير من الأحيان المحلية مقابل النظامية ، ونهج التسليم النظامية نظرا لسهولة الإدارة. ومع ذلك ، قد يؤدي إلى تسليم النظامية تركيز الدواء الأمثل يتحقق في الجهاز العصبي المركزي ، وتؤدي إلى استنتاجات خاطئة بشأن فعالية عامل. الطرق المحلية تسليم المخدرات هي أكثر الغازية ، ولكن قد يكون ضروريا لتحقيق مستويات الدواء العلاجية الجهاز العصبي المركزي. هنا ، علينا أن نظهر الأسلوب السليم لمدة ثلاث طرق لتقديم الأدوية الجهازية : الحقن في الوريد ، وحقن داخل الصفاق ، وبالتزقيم عن طريق الفم. بالإضافة إلى ذلك ، نعرض طريقة لتسليم المحلي إلى الدماغ : الحمل محسنة التسليم (CED). يتم تضمين استخدام المركبات التي تحمل علامات fluorescently عن التصوير في الجسم الحي ، والتحقق من الدواء المناسب. وتعرض الأساليب باستخدام نماذج الفئران ، ولكن يمكن بسهولة أن تتكيف لاستخدامها في الفئران.

Watch this Scientific Journal Video about Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models at JoVE.com

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

اختبار دقيق قبل السريرية من الأدوية التي تعمل في الجهاز العصبي المركزي غالبا ما ينطوي على تقييم ومقارنة biodistribution المخدرات بالتعاون مع الادارة وتحديد طرق محددة. هنا ، أظهرت ثلاث طرق شائعة الاستخدام المنهجي للتسليم (في الوريد ، داخل الصفاق ، والشفوي) ، فضلا عن طريقة لتسليم المحلي (الحراري محسنة التسليم) في الفئران.

النظامية والمحلية تسليم المخدرات لعلاج أمراض الجهاز العصبي المركزي في المجموعات القوارض

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

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30.2K Views.  University of California, San Francisco - UCSF. اختبار دقيق قبل السريرية من الأدوية التي تعمل في الجهاز العصبي المركزي غالبا ما ينطوي على تقييم ومقارنة biodistribution المخدرات بالتعاون مع الادارة وتحديد طرق محددة. هنا ، أظهرت ثلاث طرق شائعة الاستخدام المنهجي للتسليم (في الوريد ، داخل الصفاق ، والشفوي) ، فضلا عن طري?...

Video: Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

التصوير المتعدد الوسائط من زرع الخلايا الجذعية في الجهاز العصبي المركزي من الفئران

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

انتاج ناقلات Lentiviral للخلايا Transducing من الجهاز العصبي المركزي

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي<em&gt; ذبابة الفاكهة الدروسوفيلا</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

مباشرة داخل البطين تسليم المخدرات الى القوارض الجهاز العصبي المركزي

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

تحليل المناعى في الجهاز العصبي المركزي والمحيطي الجرذ العقدة الليمفاوية أقسام الأنسجة

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

في الجسم الحي Optogenetic تحفيز الجهاز العصبي المركزي القوارض

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

المقاصة البصري للنظام العصبي المركزي الماوس عن طريق وضوح السلبي

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug.
The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors.
Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders.
A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.

A validated computerized battery of neuropsychological and neurophysiological tests is used to study pharmacodynamic effects on the central nervous system of newly developed drugs in early phase development. To demonstrate the test battery, the acute effects of mecamylamine and the reversal of these effects by two agonist drugs are described.

بطارية اختبار محوسبة لدراسة تأثيرات ميكروبيولوجية على الجهاز العصبي المركزي المخدرات اتروبين في المرحلة المبكرة من تطوير العقاقير

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

بيولوجيا الخلية كمية من نيوروديجينيريشن في المورفولوجية من خلال تحليل غير منحازة للبروتينات فلوريسسينتلي المعلمة باستخدام إيماجيج

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

توصيل البراز المضاد للندرة في الجهاز العصبي المركزي للفئران

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...