The Authors acknowledge the economic support by FAIP (Federazione Associazioni Italiane Paraplegici), &#8220;Neurogel-en-Marche&#8221; Foundation (France), Fondazione &#8220;La Colonna&#8221;.

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الكتاب تعلن أي مصلحة مالية المتنافسة.

في هذه الورقة وصفنا طريقة للحصول على نموذج استنساخه من إصابات في النخاع الشوكي باستخدام المتصادم الأفق اللانهائي في قوة من 70 kdyne (شديد). باستخدام نموذج قوة أكبر (80 kdyne)، ونحن يمكن أن يسبب إصابة أكثر الشديدة التي للأسف يرتبط معدل وفيات الفئران أعلى. من أجل تجنب هذه المشك?...

إصابة الحبل الشوكي (النخاع الشوكي) هو الإصابة الأكثر شيوعا التي تسببها الصدمة ذات الطاقة العالية مثل الحوادث والسيارات، وتقع والرياضة والعنف 1. في SCI شديد، القوة إصابة بتدمير أو أضرار الأنسجة العصبية، مما تسبب في خسارة مفاجئة من وظيفة الجهاز العصبي. صدمة اصابات النخاع الشوكي غالبا ما يحدث لدى البالغين الذين تتراوح أعمارهم بين 10 و 40 سنة من العمر. أنه يؤثر بشكل كبير الحالة العقلية والجسدية للمريض ويسبب تأثير اقتصادي هائل للمجتمع 2. نهج العلاج في المرحلة الحادة غالبا ما يقتصر على جرعة عالية من الستيرويدات القشرية، وتحقيق الاستقرار الجراحي وإزالة الضغط لربما تخفيف مزيد من الضرر 3-4، ولكن أدوار هذه الأساليب على الانتعاش الحركي بعد اصابات النخاع الشوكي لا تزال مثيرة للجدل. بالإضافة إلى فقدان الأنسجة الحادة، والإصابات وتفعيل آليات الثانوية من انحطاط سبب إزالة الميالين والموت من أنواع الخلايا متعددة 5-6. درجة استعادة وظيفة يمكن للعشرة تكون مرتبطة إلى حد المادة البيضاء يدخر في مكان الاصابه 7. ويمكن استخدام النماذج الحيوانية من اصابات النخاع الشوكي على حد سواء للتحقيق في الاستجابات البيولوجية للأنسجة للإصابة واختبار العلاجات المحتملة. وعلاوة على ذلك، وهو نموذج حيواني مفيد لأمراض البشري لديه ليس فقط لإعادة إنتاج بعض جوانب هذا الشرط ولكن أيضا يجب أن توفر المزايا على الملاحظة السريرية المباشرة والتجربة. النماذج المستخدمة على نطاق واسع من إصابة الحبل الشوكي تنطوي كدمة أو ضغط إصابة تسليمها إلى الحبل الشوكي يتعرض جراحيا 8. تطوير اصابة في الوزن الإفلات كدمة تسيطر تمثل معلما هاما في تاريخ البحوث اصابات النخاع الشوكي. مركز أبحاث النخاع الشوكي جامعة ولاية أوهايو انتهجت التحدي التكنولوجي للجهاز والتي يمكن استخدامها للحث على ضغط معين من الحبل الشوكي مع المعلمات من تأثير يسيطر عليها جهاز كمبيوتر 9. وقد تم تصميم هذا أصلا للاستخدام وايالفئران عشر. في وقت لاحق تم تعديله لتطبيق نحو 10 الفئران. مزايا هذا النوع من النهج هي أن الميكانيكا الحيوية الإصابة يمكن دراستها أكثر في العمق ويمكن تعريف المعلمات الإصابة بطريقة أكثر اكتمالا من أجل الحصول على نموذج تجريبي استنساخه، وبالتالي السماح للتقييم أكثر دقة للآثار العلاجات اختبارها على عملية الانتعاش وظيفية. وتقييم العديد من الدراسات الآثار زرع مجموعة متنوعة من الخلايا الجذعية في النماذج SCI 11. لقد عزل مؤخرا الكبار الخلايا الجذعية العصبية من المنطقة الفرعية البطيني (SVZ) بعد عدة ساعات من وفاة المتبرع الماوس 12-13. يوفر هذا الإجراء السكان من الخلايا الجذعية العصبية، ودعا تشريح الجثة السلائف العصبية (PM-الشخصيات)، والتي يبدو أن من المفيد في نهج الطب التجديدي لعلاج اصابات النخاع الشوكي. في هذه الورقة سوف نظهر: ط) بروتوكول لوصفها خلية مع التتبع PKH26 الحيوي، والثاني) في سورغالإجراء كال لأداء على الصدمة اصابات النخاع الشوكي، والثالث) في الوريد (IV) إدارة الخلايا المسمى. وعلاوة على ذلك، في هذا العمل نظهر أن الخلايا المزروعة تهاجر إلى العمود الفقري مواقع الآفة الحبل وتفرق معظمهم في أنيبيب المرتبطة بروتين (MAP) 2 خلايا إيجابية. وعلاوة على ذلك، ويرافق التفريق من قبل تعزيز انتعاش مستقر في وظيفة هند أطرافهم.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">PKH26GL-1KT&#160;</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:119px;">091M0973</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Infinite horizon (IH) Impactor device&#160;</td>
			<td style="width:115px;">Precision Systems and Instrumentation, LLC</td>
			<td style="width:119px;">Model 0400 Serial 0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Gentamycin 10mg/ml</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ECM0011B</td>
			<td>1mg/ml in sterile saline solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoflurane-Vet 250ml</td>
			<td style="width:115px;">Merial</td>
			<td style="width:119px;">B142J12A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Blefarolin POM OFT 10g</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Slide Warmer</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">HB101-sm-402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Scalpel, size 10</td>
			<td style="width:115px;">Lance Paragon</td>
			<td style="width:119px;">26920</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Small Graefe Forceps</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">11023-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rongeur</td>
			<td style="width:115px;">Medicon Instruments</td>
			<td style="width:119px;">07 60 07</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Micro scissors</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Absorbable sutures (4/0)</td>
			<td style="width:115px;">Safil Quick</td>
			<td style="width:119px;">C0046203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hemostat</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">13014-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Reflex 7 wound clip applicator</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">12031-07</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">7mm Reflex wound clips</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">12032-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">NGS</td>
			<td style="width:115px;">Euroclone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Triton X 100</td>
			<td style="width:115px;">Merck Millipore</td>
			<td style="width:119px;">1086431000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Anti Microtubule Assocoated Protein&#160; (MAP) 2</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">AB5622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:119px;">A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluorSave Reagent&#160;</td>
			<td style="width:115px;">Calbiochem</td>
			<td style="width:119px;">345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Neural stem cells medium</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td>DMEM-F12 medium (Euroclone) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma-Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 &#956;g/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/mL; Life Technologies) and EGF (human recombinant, 20 ng/mL; Life Technologies)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM-F12</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ASM5002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">l-glutamine</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">glucose</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">putrescine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5780-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">progesterone</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6149-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium-selenite</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9133-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">T 5391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">I1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Heparin sodium-salt</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0200000</td>
			<td></td>
		</tr>
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neural stem cell transplantation in experimental contusive model of spinal cord injury

إصابة الحبل الشوكي هو شرط السريرية مدمرة، وتتميز مجموعة معقدة من الاختلالات العصبية. نماذج حيوانية من إصابة الحبل الشوكي يمكن استخدام كلا للتحقيق في الاستجابات البيولوجية للإصابة واختبار العلاجات المحتملة. كدمة أو ضغط إصابة تسليمها إلى الحبل الشوكي يتعرض جراحيا هي النماذج الأكثر استخداما على نطاق واسع من الأمراض. في هذا التقرير يتم تنفيذ كدمة التجريبية باستخدام اللانهائي الأفق (IH) جهاز المتصادم، والذي يسمح بإنشاء نموذج حيواني إصابة استنساخه من خلال تعريف المعلمات إصابة محددة. يعتبر زرع الخلايا الجذعية عادة استراتيجية يمكن أن تكون مفيدة لعلاج هذا الشرط المنهكة. وتقييم دراسات عديدة آثار زراعة مجموعة متنوعة من الخلايا الجذعية. نحن هنا لشرح طريقة تكييفها لإصابة الحبل الشوكي يليه الذيل الوريد حقن الخلايا في الفئران CD1. باختصار، نحن نقدم إجراءات ل: ط) وضع العلامات خلية وايعشر التتبع الحيوي، والثاني) الرعاية ما قبل المنطوق من الفئران، الثالث) تنفيذا لإصابة الحبل الشوكي contusive، والرابع) الوريد من بعد الوفاة السلائف العصبية. ويمكن استخدام هذا النموذج كدمة لتقييم فعالية وسلامة زرع الخلايا الجذعية في نهج الطب التجديدي.

العدد الكلي للخلايا المزروعة هو 1 × 10 6 خلايا وقسمت إلى ثلاث حقن متتالية في الوريد الذيل. نحن تدار 3.3 × 10 5 خلايا في 50 ميكرولتر من حل العازلة الفوسفات (PBS). تم إجراء الحقن الأولى في غضون 30 دقيقة بعد الإصابة، في وقت لاحق 6 ساعة الثانية والساعة ال 18 الماضية بعد الآ?...

Watch this Scientific Journal Video about Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury at JoVE.com

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

إصابة الحبل الشوكي هي حالة الصدمة التي تسبب الاعتلال الشديد وارتفاع معدل الوفيات. في هذا العمل ونحن تصف بالتفصيل نموذج كدمة من إصابة الحبل الشوكي في الفئران تليها زرع الخلايا الجذعية العصبية.

العصبية زرع الخلايا الجذعية في التجريبية Contusive نموذج من إصابة الحبل الشوكي

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

neural-stem-cell-transplantation-experimental-contusive-model-spinal

Research

JoVE Journal

Medicine

12.8K Views.  University of Milan.  إصابة الحبل الشوكي هي حالة الصدمة التي تسبب الاعتلال الشديد وارتفاع معدل الوفيات. في هذا العمل ونحن تصف بالتفصيل نموذج كدمة من إصابة الحبل الشوكي في الفئران تليها زرع الخلايا الجذعية العصبية. 

Video: Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

داخل النخاع زرع الخلايا لاستهداف القرن بطني عنق الرحم في التصلب الجانبي وصدمة إصابة النخاع الشوكي

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

الحادة والمزمنة الاختبارات الحسية اللمس بعد إصابة الحبل الشوكي في الجرذان

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

نموذج من جانب واحد راض عنق الرحم لاصابة النخاع الشوكي باستخدام المتصادم الأفق اللانهائي

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

زرع الخلايا الجذعية في في المختبر مقلد نقص التروية / إعادة إشباع الخلايا النموذجي

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

نموذج كدمة شديدة اصابات الحبل الشوكي في الجرذان

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

رواية الفقري طريقة لتحقيق الاستقرار لإنتاج Contusive إصابة الحبل الشوكي

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

تسيطر الموديل أثر القشرية لإصابات الدماغ الرضية

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

ونظام القياس عن بعد راديو لمراقبة وظيفة القلب والأوعية الدموية في الحبل الشوكي مع الفئران Transection والعصبية الجذعية الجنينية الطعوم خلية

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

استخدام التآزر من الخلايا العصبية السلائف والببتيدات الذاتي تجميع في التجريبية عنق الرحم إصابة الحبل الشوكي

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

معايرة الملقط نموذج من الحبل الشوكي ضغط الإصابات

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...