Name: a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice
Uploaded: 2022-05-11T14:00:00
Description: Watch this Scientific Journal Video about A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (Tilskud: 82071380, 81873743).

Alle dyreprocedurer blev godkendt af Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, Kina. Voksne C57BL/6 han- og hunmus (vildtype, WT; 20-25 g; 8-10 uger gamle) blev anvendt til denne undersøgelse. Musene blev fremstillet fra kommercielle kilder (se Materialetabel). Mus blev opstaldet i et specifikt patogenfrit (SPF) dyreanlæg med vand og mad leveret ad libitum. De blev holdt i en vekslende 12 timers periode med lys og mørk cyklus ...

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MS, en kronisk demyeliniserende sygdom i CNS, er en af de mest almindelige årsager til neurologisk dysfunktion hos unge voksne20. Klinisk oplever ca. 60%-80% af MS-patienter cyklussen af tilbagefald og remissioner, før de udvikler en sekundær progressiv MS21,22, og det fører i sidste ende til kumulative bevægelsesforstyrrelser og kognitive underskud over tid23. I øjeblikket dækker ingen enkelt eksperimentel m...

Receptormedieret lysophospholipid signalering involverer forskellige fysiologiske processer i næsten alle organsystemer1. I centralnervesystemet (CNS) spiller denne signalering en kritisk rolle i patogenerne af autoimmune neurologiske sygdomme såsom multipel sklerose (MS). Multipel sklerose er en kronisk immunmedieret lidelse karakteriseret ved patologisk demyelinering og inflammatorisk respons, der forårsager neurologisk dysfunktion og kognitiv svækkelse 2,3. Efter kontinuerlig tilbagefald og remittering under den tidlige sygdom udvikler de fleste patienter sig til sidst til det sekundære progressive stadium, hvilket kan forårsage irreversibel skade på hjernen og deraf følgende handicap4. Det antages, at det patologiske kendetegn ved det sekundære progressive kursus er demyeliniserende plaques forårsaget af inflammatoriske læsioner5. Eksisterende behandlinger for MS kan reducere risikoen for tilbagefald betydeligt. Der er dog stadig ingen effektiv terapi til langvarig demyeliniserende skade forårsaget af progressiv MS6. Således kræves en stabilt etableret og let reproducerbar model for at studere prækliniske terapier, der fokuserer på degeneration af hvidt stof.
Demyelinering og remyelinering er to store patologiske processer i udviklingen af multipel sklerose. Demyelinisering er tabet af myelinskede omkring axoner induceret af microglia med proinflammatoriske fænotyper7, og det fører til langsom ledning af nerveimpulser og resulterer i tab af neuroner og neurologiske lidelser. Remyelinering er et endogent reparationsrespons medieret af oligodendrocytter, hvor lidelser kan føre til neurodegeneration og kognitiv svækkelse8. Det inflammatoriske respons er afgørende for hele processen og påvirker både graden af myelinskader og reparation.
Derfor er en stabil dyremodel med vedvarende inflammatorisk demyelinering meningsfuld for yderligere udforskning af terapeutiske strategier for MS. På grund af kompleksiteten af MS er der etableret forskellige typer dyremodeller til at efterligne demyeliniserende læsioner in vivo, herunder eksperimentel autoimmun encephalomyelitis (EAE), toksisk-demyeliniserende modeller, cuprizon (CPZ) og lysophosphatidylcholin (LPC)9 . LPC er et endogent lysophospholipid forbundet med inflammation, og det kan fremkalde hurtig skade med toksicitet for myelinlipider, hvilket fører til fokal demyelinisering. Baseret på tidligere rapporter og forskning 10,11 leveres en detaljeret protokol med topunktsinjektion med nogle ændringer. Generelt producerer den klassiske etpunkts LPC-injektionsmodel kun lokal demyelinering på injektionsstedet og ledsages ofte af spontan remyelinering12,13. Imidlertid kan topunktsinjektions-LPC-modellen demonstrere, at LPC direkte kan inducere demyelinisering i musens corpus callosum og forårsage mere holdbar demyelinisering med lidt myelinregenerering.

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			<td >L-&#945;-Lysophosphatidylcholine from egg yolk</td>
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			<td >high speed skull drill</td>
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			<td >drill</td>
			<td >Hager &#38; Meisinger, Germany&#160;</td>
			<td >REF 500 104 001 001 005</td>
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			<td >Matrx Animal Aneathesia Ventilator</td>
			<td >MIDMARK</td>
			<td >VMR</td>
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			<td >Portable Stereotaxic Instrument for Mouse</td>
			<td >Reward</td>
			<td >68507</td>
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			<td >Micro syringe</td>
			<td >Reward</td>
			<td >KDS LEGATO 130</td>
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			<td >Isoflurane&#160;</td>
			<td >VETEASY</td>
			<td ></td>
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			<td >Paraformaldehyde</td>
			<td >Servicebio</td>
			<td >G1101</td>
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			<td >Phosphate buffer</td>
			<td >BOSTER</td>
			<td >PYG0021</td>
			<td></td>
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			<td >LuxoL fast bLue</td>
			<td >Servicebio</td>
			<td >G1030-100ML</td>
			<td></td>
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			<td >Suture</td>
			<td >FUSUNPHARMA</td>
			<td >20152021225</td>
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			<td >Brain mold</td>
			<td >Reward</td>
			<td >68707</td>
			<td></td>
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			<td >Electron microscope fixative</td>
			<td >Servicebio</td>
			<td >G1102-100ML</td>
			<td></td>
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			<td >Neutral red (C.I. 50040), for microscopy Certistain</td>
			<td >Sigma-Aldrich</td>
			<td >1.01376</td>
			<td></td>
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		<tr >
			<td >Anti-Myelin Basic Protein Antibody&#160;</td>
			<td >Millipore</td>
			<td >#AB5864</td>
			<td></td>
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			<td >Anti-GST-P pAb</td>
			<td >MBL</td>
			<td >#311</td>
			<td></td>
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			<td >Ki-67 Monoclonal Antibody (SolA15)</td>
			<td >Thermo Fisher&#160;Scientific</td>
			<td >14-5698-95</td>
			<td></td>
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			<td >Beta Actin Monoclonal Antibody</td>
			<td >Proteintech</td>
			<td >66009-1-Ig&#160;</td>
			<td></td>
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			<td >Myelin Basic Protein Polyclonal Antibody</td>
			<td >Proteintech</td>
			<td >10458-1-AP</td>
			<td></td>
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			<td >OLIG2 Polyclonal Antibody</td>
			<td >Proteintech</td>
			<td >13999-1-AP</td>
			<td></td>
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			<td >Alexa Fluor 488&#160;AffiniPure&#160;Donkey anti-Rabbit&#160;IgG (H+L)</td>
			<td >YEASEN</td>
			<td >34206ES60</td>
			<td></td>
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			<td >Alexa Fluor 594 AffiniPure Donkey Anti-Rat IgG (H+L)&#160;</td>
			<td >YEASEN</td>
			<td >34412ES60</td>
			<td></td>
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			<td >Alexa Fluor 594&#160;AffiniPure Donkey Anti-Rabbit IgG (H+L)&#160;</td>
			<td >YEASEN</td>
			<td >34212ES60</td>
			<td></td>
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			<td >HRP Goat Anti-Rabbit IgG (H+L)</td>
			<td >abclonal</td>
			<td >AS014</td>
			<td></td>
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			<td >HRP Goat Anti-Mouse IgG (H+L)&#160;</td>
			<td >abclonal</td>
			<td >AS003</td>
			<td></td>
		</tr>
		<tr >
			<td >Adult C57BL/6 male and female mice</td>
			<td >Hunan SJA Laboratory Animal Co. Ltd</td>
			<td ></td>
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a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice

Receptormedieret lysophospholipid signalering bidrager til patofysiologien af forskellige neurologiske sygdomme, især multipel sklerose (MS). Lysophosphatidylcholin (LPC) er et endogent lysophospholipid forbundet med inflammation, og det kan fremkalde hurtig skade med toksicitet for myelinlipider, hvilket fører til fokal demyelinination. Her præsenteres en detaljeret protokol for stereotaktisk topunkts LPC-injektion, der direkte kan forårsage alvorlig demyelinering og replikere den eksperimentelle demyelineringsskade hurtigt og stabilt hos mus ved kirurgisk procedure. Denne model er således yderst relevant for demyelineringssygdomme, især MS, og den kan bidrage til den relaterede fremadskridende klinisk relevante forskning. Immunofluorescens og Luxol hurtige blå farvningsmetoder blev også brugt til at skildre tidsforløbet for demyelinisering i corpus callosum hos mus injiceret med LPC. Derudover blev adfærdsmetoden brugt til at evaluere musens kognitive funktion efter modellering. Samlet set er topunktsinjektionen af lysophosphatidylcholin via en stereotaxisk ramme en stabil og reproducerbar metode til at generere en demyelineringsmodel hos mus til yderligere undersøgelse.

Topunktsinjektion af LPC resulterede i en mere holdbar demyelinering LPC fører hovedsageligt til hurtig skade med toksicitet for myelin og spaltning af axonintegriteten15. Injektionsdagen blev betragtet som dag 0. Mus blev opbevaret i en periode på 10-28 dage (10 dpi og 28 dpi). Luxol fast blue (LFB) farvning10 blev brugt til at evaluere området for demyelinisering hos mus på disse tidspunkter. I topunktsinjektionsmodellen var der signifikant demyeli...

Watch this Scientific Journal Video about A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice at JoVE.com

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

Den nuværende protokol beskriver en topunktsinjektion af lysophosphatidylcholin via en stereotaksisk ramme for at generere en stabil og reproducerbar demyeliniseringsmodel hos mus.

En stabilt etableret topunktsinjektion af lysophosphatidylcholin-induceret fokal demyeliniseringsmodel hos mus

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

Vores model er yderst relevant for demyeliniseringssygdomme, især multipel sklerose. Og det kan bidrage til den relaterede fremme klinisk relevant forskning. Det er en detaljeret protokol, der direkte kan forårsage alvorlig demyelinisering hurtigt og stabilt hos mus ved kirurgisk procedure.Mens du prøver denne teknik for første gang, skal du være tålmodig og sikre dig, at koordinaterne er nøjagtige. Begynd med at forbinde en 32 gauge nål til en 5 mikroliter sprøjte. Træk 5 mikroliter LPC-opløsning til fremstilling af injektionen.Bekræft derefter dybden af musens anæstesi ved manglen på tåspidsrefleks, mens vejrtrækningen er glat. Placer musen i den stereotaxiske ramme med rygsiden opad, og fastgør hovedet med en næsekegle og tandklemme. Fastgør musen til det stereotaxiske apparat med bilaterale ørestænger.Sørg for, at ørestængerne er plane, og hovedet er vandret og stabilt. Desinficer hovedhuden ved at tørre med iodophor. Brug derefter skalpel til at skære et lille snit til ca. 1,5 centimeter langs hovedbundens midterlinje for at udsætte kraniet.Tør færdigheden af med en vatpind dyppet i 1% hydrogenperoxid, indtil bregma, lambda og bageste fontanelle er udsat. Placer sprøjten på det stereotaxiske apparat. Sørg for vandret positionering af dyrets hoved, og find corpus callosum.Når injektionsstedet er bestemt, skal du registrere koordinaterne og lave et mærke med en markør på kraniet. Bor forsigtigt det markerede sted med en kranieboremaskine. Pas på at undgå blødning.Flyt langsomt nålen til de givne koordinater. For at inducere corpus callosum demyelinisering injiceres 2 mikroliter LPC-opløsning på hvert injektionssted med en hastighed på 0,4 mikroliter pr. Minut. Efter injektion skal du holde nålen på hvert sted i yderligere 10 minutter.Til sidst sutureres huden med en 4-0 sutur, og vent på, at dyret vågner op inden for 10 minutter. Det repræsentative billede viser påvisning af de hvide stoflegioner via Luxol hurtig blå farvning. Fra resultaterne kan stabil demyelinisering af corpus callosum ses 10 og 28 dage efter injektion.Kvantificeringen af demyeliniseringsområdet på forskellige tidspunkter er vist her. Gennem immunfluorescensfarvning af nedbrudt myelinbasisk protein blev der observeret en bemærkelsesværdig stigning i dMBP i den LPC-injicerede gruppe 10 dage efter injektion, hvilket repræsenterer myelintab i corpus callosum. Western blot-analyse af myelinbasisk protein eller MBP-ekspression viste et signifikant tab af MBP efter injektion af LPC.Beta-actin blev brugt som indlæsningskontrol til at måle basislinjeudtrykket. Elektronmikroskopibilleder bekræfter den myeliniserede ultrastruktur i corpus callosum af sham-gruppe og LPC-injiceret gruppe. GST-pi er markøren for oligodendrocytprækursorcelle eller OPC, differentieringsmodning.Efter 10 dages LPC-injektion faldt GST-pi sammenlignet med skingruppen. Samtidig kan oligodendrocytternes proliferationsevne afspejles af forholdet mellem oligodendrocytter proliferere og de samlede oligodendrocyt-afstamningsceller. Det højere forhold repræsenterer mere spredning af oligodendrocytter efter 10 dage efter injektion.De repræsentative billeder viser musenes svømmebane i hver gruppe, i platformens tilstedeværelse og efter fjernelse af platformen. Resultaterne tyder på, at den rumlige hukommelse hos demyeliniserede mus er signifikant svækket i topunkts LPC-injektionsmodellen. Mens du forsøger denne procedure, skal du sørge for, at injektionshastigheden er nøjagtig, og holde nålen på hvert sted i yderligere 10 minutter.

Research

JoVE Journal

Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitorisk synapsedannelse i en Co-kultur model, som indeholder GABAerge Medium Langustre neuroner og HEK293-celler stabilt udtrykker GABA<sub&gt; A</sub&gt; Receptorer

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Eksperimentel Demyelinering og remyelinering af murine Spinal Cord ved Focal Injektion af Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Visuelt fremkaldt Potentiel optagelse i en rotte model for Eksperimentel Optic Nerve Demyelinisering

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Delvis synsnerven Transection i rotter: en standardformular, som udarbejdes med en ny operativ tilgang til at vurdere sekundære Degeneration af Retinal Ganglion celler

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Rottemodel af udbredt cerebral kortikal demyelinering induceret af en intracerebral injektion af proinflammatoriske cytokiner

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Direkte Intratekal injektion af rekombinant Adeno-associeret virus i voksen mus

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Dobbelt direkte injektion af blod i Cisterna Magna som en model af Subarachnoid Blødning

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

A Rat Model of EcoHIV Brain Infection

En rottemodel af EcoHIV-hjerneinfektion

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...