وأيد هذا العمل من قبل المعاهد الوطنية للصحة / NINDS المنح وNS26380 NS55976 ، وكريستوفر ودانا ريف مؤسسة وصندوق للبحوث هيومان دانيال الحبل الشوكي. جامعة دريكسيل كلية الطب الحبل الشوكي مركز أبحاث يوفر الدعم للمرافق الأساسية المستخدمة لإكمال هذا العمل.

<ol>
	<li>Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+the+potential+for+chronically+injured+neurons+to+regenerate+axons+into+intraspinal+peripheral+nerve+grafts.">Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts.</a> Exp. Neurol. 113, 1-9 (1991).</li><li>Ye, J. H., Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+the+chronically+injured+spinal+cord+with+neurotrophic+factors+can+promote+axonal+regeneration+from+supraspinal+neurons.">Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons.</a> Exp. Neurol. 143, 70-81 (1997).</li><li>Houle, J. D., Tom, V., Mayes, D., Wagoner, G., Phillips, N., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+an+autologous+peripheral+nerve+bridge+and+matrix+modification+by+chondroitinase+allows+robust+functional+regeneration+across+a+hemisection+lesion+of+the+adult+rat+spinal+cord.">Combining an autologous peripheral nerve bridge and matrix modification by chondroitinase allows robust functional regeneration across a hemisection lesion of the adult rat spinal cord.</a> J. Neurosci. 26, 7405-7415 (2006).</li><li>Tom, V., Houle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspinal+microinjection+of+chondroitinase+ABC+following+injury+promotes+axonal+regeneration+out+of+a+peripheral+nerve+graft.">Intraspinal microinjection of chondroitinase ABC following injury promotes axonal regeneration out of a peripheral nerve graft.</a> Exp. Neurol. 211, 315-319 (2008).</li></ol>

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لاستنساخ التجربة أنه من المهم أن مستوى إصابة الحبل الشوكي تكون متسقة من الحيوان الى الحيوان. ولذلك فمن الأهمية بمكان أن يتم تحديد العملية المناسبة الفقري لالثقب. لأننا باستخدام نموذج transection تنصيف أو الآفة لهذه الدراسة لا يوجد أي غموض حول حجم الآفة أو ما ?...

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<td>10-0 silk suture</td>
<td>&nbsp;</td>
<td>ArosSurgical</td>
<td>T5A10N10</td>
<td>&nbsp;</td>
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<td>6-0 silk suture</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>2693</td>
<td>&nbsp;</td>
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<td>Ampicillin</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>483549</td>
<td>&nbsp;</td>
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<td>Antibody to cFos</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F7799</td>
<td>&nbsp;</td>
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<td>Biotinylated dextran Amine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D7135</td>
<td>&nbsp;</td>
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<td>Buprenorphin (.3mg/ml)</td>
<td>&nbsp;</td>
<td>McKesson</td>
<td>12496075701</td>
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<td>Associates of Cape Cod</td>
<td>100332-1A</td>
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<td>Euthasol</td>
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<td>Webster Veterinary</td>
<td>07-805-9296</td>
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<td>209-1966</td>
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<td>Fine Science Tools</td>
<td>12040-02</td>
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<td>Neurotrace Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>N7167</td>
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<td>True Blue</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T5891</td>
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<td>Clever System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Infinite Horizon Impact Device</td>
<td>&nbsp;</td>
<td>Precision Systems and Instrumentation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Magnetic Stimulation Device</td>
<td>&nbsp;</td>
<td>MagStim Inc.</td>
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combining peripheral nerve grafting and matrix modulation to repair the injured rat spinal cord

إصابة مؤلمة للنخاع الشوكي (SCI) يسبب موت الخلايا العصبية ، تعطل المحرك والألياف العصبية الحسية (محور عصبي) الممرات وتعطل الاتصالات مع الدماغ. واحد من أهداف بحثنا في تعزيز التجدد محوار لاستعادة الاتصال عبر موقع الآفة. لإنجاز هذا طورنا الأعصاب الطرفية (PN) تطعيم تقنية حيث يتم وضعها إما شرائح العصب الوركي مباشرة بين طرفي التالفة من الحبل الشوكي أو تستخدم لتشكيل جسر عبر الآفة. هناك مزايا عديدة لهذا النهج بالمقارنة مع زراعة الأنسجة العصبية الأخرى ، ويمكن أن توجه نحو تجديد محاور منطقة مستهدفة محددة ، فإن من السهل التحقق من عدد ومصدر للتجديد المحاور عن طريق تتبع تقنيات ، يمكن استخدام والكسب غير المشروع لتجارب لقياس كهربية انتعاش الفنية المرتبطة المحاوير في الكسب غير المشروع ، وأنه من الممكن لاستخدام عصب ذاتي لتقليل احتمال رفض الكسب غير المشروع. في المختبر لدينا كل من أجرينا ذاتي (المانحة والمتلقية هي نفس الحيوان) ، ومغايرة (المانحة والمتلقية هي حيوانات مختلفة) ترقيع مع نتائج مماثلة. وقد استخدم هذا النهج بنجاح في حالات الاصابة الحادة والمزمنة. المحاوير المجددة التي تصل إلى نهاية البعيدة للطعم PN غالبا ما تفشل لتمديد مرة أخرى في الحبل الشوكي ، ولذلك نحن نستخدم microinjections chondroitinase أن تتحلل من الجزيئات المثبطة المرتبطة ندبا المحيطة مجال اصابات النخاع الشوكي. في نفس الوقت وجدنا أن توفير النمو الخارجية والجزيئات الغذائية تشجع إعادة نمو محواري أطول مسافة في الحبل الشوكي. بعد عدة أشهر من الزرع ونحن أداء مجموعة متنوعة من الاختبارات التشريحية والسلوكية والكهربية لتقييم استعادة وظيفة في حيواناتنا النخاع الشوكي المصاب. وقد استخدم هذا المنهج التجريبي بنجاح في العديد من نماذج اصابات النخاع الشوكي ، على مستويات مختلفة من الإصابة بأنواع مختلفة و(الجرذان والفئران والقطط). الأهم ، فإن النهج الأعصاب الطرفية تطعيم فعال في تعزيز التجدد بواسطة الخلايا العصبية الحادة وجرح مزمن.

Watch this Scientific Journal Video about Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord at JoVE.com

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

صدمة إصابة في النخاع الشوكي يعطل التواصل مع الدماغ. لاستعادة الاتصال فقدنا الاستفادة من الكسب غير المشروع الأعصاب الطرفية لتوفير التحتية للألياف تجديد بالاقتران مع عوامل عصبية والانزيمات مصفوفة تحوير لإزالة الجزيئات المثبطة لتعزيز النمو طويل المسافة.

الجمع بين الأعصاب الطرفية التطعيم والتحوير ماتريكس لإصلاح الحبل الشوكي المصاب الجرذ

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

combining-peripheral-nerve-grafting-matrix-modulation-to-repair

Research

JoVE Journal

Biology

16.3K Views.  Drexel University College of Medicine. صدمة إصابة في النخاع الشوكي يعطل التواصل مع الدماغ. لاستعادة الاتصال فقدنا الاستفادة من الكسب غير المشروع الأعصاب الطرفية لتوفير التحتية للألياف تجديد بالاقتران مع عوامل عصبية والانزيمات مصفوفة تحوير لإزالة الجزيئات المثبطة لتعزيز النمو طويل المسافة.

Video: Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

الحبل الشوكي الكهربية

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

باستخدام optokinetic استجابة لدراسة وظيفة البصرية من الزرد

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

في فيفو سيرنا ترنسفكأيشن والجينات ضربة قاضية في الحبل الشوكي عن طريق السريع موسع قطني داخل القراب الحقن في الفئران

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

الجمع بين التلاعب جزيء واحدة والتصوير لدراسة التفاعلات-DNA البروتين

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

المجهر الإلكتروني من الأنسجة متآكلة إلى تصور مصفوفة خارج الخلية

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

قياسات العضلة العاصرة الاحليل سيستوميتريك والخارجية في الفئران مستيقظا مع القسطرة مزروع واقطاب السماح للقياسات المتكررة

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

قياس ميتوفاجي الحساسة بالتدفق الخلوي استخدام تعتمد على درجة الحموضة "مراسل الفلورسنت" طن متري-كيما

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

أديبو--واضحة: منديل مسح طريقة لتصوير ثلاثي الأبعاد للأنسجة الدهنية

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

تقييم استجابه نغمه الاوعيه الدموية باستخدام الشرايين المساريقيه المعزولة مع التركيز علي التحوير بواسطة الانسجه الدهنية الوعائية

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

نهج تفكك خلية واحدة للتحليل الجزيئي للمثانة البولية في الماوس بعد إصابة الحبل الشوكي

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...