Name: neural stem cell transplantation in experimental contusive model of spinal cord injury
Uploaded: 2014-12-17T13:00:00
Description: Watch this Scientific Journal Video about Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury at JoVE.com

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

Bemærk: Alle procedurer blev godkendt af Review Committee for universitetet i Milano og mødte de italienske retningslinjer for forsøgsdyr i overensstemmelse med Europæiske Fællesskabets direktiv fra november 1986 (86/609 / EØF). 1. Fremstilling af celler til transplantation BEMÆRK: Brug neurale stamceller mellem 5. og 9. passage i kultur i disse forsøg; teste kulturer til proliferation og differentiering evne før at blive stemplet til transplantation. Best...

<ol>
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I dette papir beskrev vi en metode til at opnå en reproducerbar model af traumatisk rygmarvsskade ved hjælp af en Infinite Horizon Impactor ved en kraft på 70 Kdyne (alvorlig). Ved hjælp af en større kraft paradigme (80 Kdyne), kan vi give en mere alvorlig skade, som desværre er forbundet med højere mus dødelighed. For at undgå dette problem, vi almindeligvis vælge en moderat kraft paradigme (70 Kdyne), der er knyttet til en gentagelig læsion med en gradvis genopretning af funktion og lavere dødelighed. For ...

En rygmarvsskade (SCI) er den mest almindelige skade forårsaget af højenergi traumer lignende motorkøretøjer ulykker, fald, sport og vold 1. Ved svær SCI, skaden kraft ødelægger eller skader nervevæv, der forårsager pludselige tab af neurologiske funktion. Traumatisk SCI ofte opstår hos unge voksne mellem 10 og 40 år. Det i høj grad påvirker patientens psykiske og fysiske tilstand og forårsager enorme økonomiske konsekvenser for samfundet 2. Behandlingen tilgang i den akutte fase er ofte begrænset til en høj dosis af kortikosteroid, kirurgisk stabilisering og dekompression til eventuelt dæmpe yderligere skader 3-4, men rollerne for disse metoder på bevægeapparatet opsving efter SCI er stadig kontroversiel. Ud over akut tab af væv, traumatiske skader og aktiveringen af sekundære mekanismer degeneration årsag demyelinering og død af flere celletyper 5-6. Graden af ​​genvinding af funktionen kan afti korreleres til udstrækningen af sparet hvide substans ved læsionsstedet 7. Dyremodeller af SCI kan anvendes både til at undersøge de biologiske reaktioner i vævet skade og til at teste potentielle terapier. Desuden er en nyttig dyremodel for en menneskelig patologi ikke blot at gengive nogle aspekter af denne betingelse, men også skal tilbyde fordele i forhold direkte klinisk observation og eksperiment. De mest udbredte modeller af rygmarvsskade involverer kontusion eller kompression skade leveres til kirurgisk udsatte rygmarven 8. Udviklingen af ​​en kontrolleret vægt-drop kontusion skade udgør en vigtig milepæl i historien om SCI forskning. Den Ohio State University rygmarv forskningscenter udøvet den teknologiske udfordring af en anordning, der kan bruges til at fremkalde en bestemt kompression af rygmarven med parametre for effekt styres af en computer 9. Dette blev oprindeligt designet til brug with rotter; senere blev det ændret til at anvende over for mus 10. Fordelene ved denne form for tilgang er, at biomekanik skaden kan studeres mere i dybden og parametrene for skaden kan defineres på en mere fuldstændig måde for at opnå en reproducerbar eksperimentel model, derfor tillader mere præcis vurdering af virkningerne af testede behandlinger på den funktionelle helbredelsesprocessen. Mange undersøgelser har evalueret transplantation virkninger af en bred vifte af stamceller i SCI-modeller 11. Vi har voksne neurale stamceller fra Sub-ventrikulær zone (SVZ) flere timer nylig isoleret efter død mus donor 12-13. Denne procedure giver en population af neurale stamceller, kaldet post mortem neurale forstadier (PM-NPCs), som synes at være fordelagtigt i en regenerativ medicin fremgangsmåde til hærdning SCI. I dette papir vil vi demonstrere: i) protokollen for celle mærkning med den vitale sporstof PKH26, ii) Surgical procedure at udføre på traumatisk SCI, og iii) intravenøs (IV) administration af mærkede celler. Desuden i dette arbejde viser vi, at transplanterede celler migrerer til rygmarv læsioner sites og differentiere det meste i mikrotubuli protein (MAP) 2-positive celler. Endvidere er differentieringen ledsaget af fremme en stabil genvinding af bagbenet funktion.

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			<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>
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			<td height="21" style="height:21px;width:287px;">PKH26GL-1KT&#160;</td>
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			<td style="width:119px;">091M0973</td>
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			<td height="84" style="height:84px;">Infinite horizon (IH) Impactor device&#160;</td>
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			<td height="42" style="height:42px;width:287px;">Slide Warmer</td>
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			<td style="width:119px;">HB101-sm-402</td>
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			<td height="42" style="height:42px;">Small Graefe Forceps</td>
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			<td style="width:119px;">11023-14</td>
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			<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>
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			<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>
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			<td height="42" style="height:42px;">Hemostat</td>
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			<td style="width:119px;">13014-14</td>
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			<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>
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		<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>
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			<td>ECS0200D</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:287px;">Neural stem cells medium</td>
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			<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>
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			<td height="21" style="height:21px;width:287px;">DMEM-F12</td>
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			<td style="width:119px;">ASM5002</td>
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			<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>
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			<td height="21" style="height:21px;width:287px;">glucose</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-100G</td>
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			<td height="21" style="height:21px;width:287px;">putrescine</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">I1882</td>
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			<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>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">bFGF</td>
			<td style="width:115px;">Life Technology</td>
			<td style="width:119px;">PHG0024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">h-EGF</td>
			<td style="width:115px;">Life Technology</td>
			<td style="width:119px;">PHG6045</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:287px;">Syringe 0.33cc 29G</td>
			<td style="width:115px;">Terumo</td>
			<td style="width:119px;">MYJECTOR&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:287px;">buprenorphine</td>
			<td style="width:115px;">Schering Plough SpA</td>
			<td style="width:119px;">TEMGESIC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">eye gel</td>
			<td style="width:115px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">LIPOSIC</td>
			<td></td>
		</tr>
	</tbody>
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neural stem cell transplantation in experimental contusive model of spinal cord injury

Rygmarvsskade er en ødelæggende klinisk tilstand, kendetegnet ved et kompleks af neurologiske dysfunktioner. Dyremodeller for rygmarvsskade kan bruges både til at undersøge de biologiske reaktioner af skader og til at teste potentielle behandlinger. Kontusion eller kompression skade leveres til kirurgisk eksponeret rygmarven er de mest anvendte modeller af patologi. I denne rapport den eksperimentelle kontusion udføres ved hjælp af Infinite Horizon (IH) Impactor anordning, som gør det muligt at oprette en reproducerbar skade dyremodel gennem fastlæggelse af specifikke skade parametre. Stamcelletransplantation er almindeligt anset for en potentielt nyttig strategi for at kurere denne invaliderende tilstand. Talrige undersøgelser har evalueret virkningen af ​​transplantere en række stamceller. Her vise vi en tilpasset fremgangsmåde til rygmarvsskade efterfulgt af haleveneinjektion af celler i CD1 mus. Kort sagt, vi leverer procedurer for: i) cellemærkning with en vital sporstof, ii) præoperativ pleje af mus, iii) gennemførelse af en contusive rygmarvsskade, og iv) intravenøs administration af post mortem neurale forstadier. Denne kontusion model kan anvendes til at evaluere effekten og sikkerheden af ​​stamcelletransplantation i en regenerativ medicin tilgang.

Det samlede antal transplanterede celler er 1 x 10 6 celler og blev opdelt i tre på hinanden følgende injektioner i halevenen. Vi administreret 3,3 x 10 5 celler i 50 pi phosphatpufferopløsning (PBS). Den første injektion blev udført inden for 30 min efter skade, den anden 6 timer senere, og de sidste 18 timer efter læsionen. Valget af en frist på 18 timer efter SCI til administration PM-NPC&#39;ere blev bestemt ved den optimale permeabilitet af blod-hjerne barrieren på dette tidspunkt <sup...

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

Rygmarvsskade er en traumatisk tilstand, der forårsager alvorlig sygelighed og høj dødelighed. I dette arbejde har vi beskriver i detaljer en kontusion model af rygmarvsskade i mus, efterfulgt af en transplantation af neurale stamceller.

Neural stamcelletransplantation i Eksperimentel Contusive Model for Rygmarvsskader

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

Research

JoVE Journal

Medicine

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) ...

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

Akut og kronisk Tactile sensorisk analyse efter Rygmarvsskader i rotter

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

En Contusive Model af Unilateral Cervikal Rygmarvsskader Brug af Uendelige 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 ...

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

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

Stamcelletransplantation i en In vitro Simuleret Iskæmi / reperfusion Model

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

En Kontusion Model of Svær Rygmarvsskader i rotter

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

A Novel Vertebral Stabilization fremgangsmåde til fremstilling Contusive Rygmarvsskader

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

Kontrolleret Cortical Impact Model for Traumatisk Hjerneskade

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

En radio-telemetri system til overvågning Kardiovaskulær funktion i rotter med Spinal Cord transection og embryonale neural stamcelle transplantater

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

Fordelagtig samordning af neurale precursorceller og selvsamlende peptider i Experimental Cervikal Rygmarvsskader

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

Kalibreret pincet Model af rygmarvskompression Skade

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