Selektiv Udtømning af Mikroglia fra cerebellare Granule cellekulturer anvendelse af L-leucin-methylester
Summary
Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.
Abstract
Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique – described here – is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer’s or Parkinson’s disease.
Introduction
Den menneskelige hjerne består af en anslået 85 milliarder neuroner og en yderligere 85 milliarder ikke-neuronale celler, herunder glia 1. For størstedelen af de seneste 100 år neuroforskere har fokuseret overvejende på neuronal celle befolkning, at tro gliaceller være lidt mere end passive støtte celler, der er fastsat strukturstøtte til neuronerne – dermed den græske etymologi af 'glia "oversat til engelsk som "lim". Imidlertid for nylig er det blevet mere klart, at neuronale-glia interaktioner kan være langt mere grundlæggende til grundlæggende aspekter af neurobiologi, neurofysiologi og tilblivelsen og progression af mange neurodegenerative sygdomme. Cerebellumgranulaceller (CGC), den mest rigelige homogen neuronal population i den menneskelige hjerne, dominerer cerebellum og udgør mere end 90% af dets cellulære bestanddele. Derfor er disse celler blevet anvendt i udstrakt grad in vitro som et modelsystem til tHan studerer af neuronal udvikling, funktion og patologi 2-6.
Imidlertid CGC kulturer indeholder stadig mikroglia og andre glia i velsagtens signifikante proportioner. Som et resultat heraf kan CGC data putativt udviser direkte neuronale reaktioner på forskellige celletyper behandlinger faktisk opstår – delvis eller i alt – fra den indirekte sekundær reaktion af tilstødende glia i kulturen. For at vurdere dette, vi selektivt elimineret mikroglial fra CGC neuronale kulturer ved hjælp af L-leucinmethylester (LME). LME er en lysomotropic agent oprindeligt brugt til selektivt at ødelægge makrofager 7, og er siden blevet brugt til også selektivt nedbryder microglia fra neural, astrocyt, og blandede glial kulturer 8,9,10. LME internaliseres af makrofager og mikroglia, hvor det forårsager lysosomal forstyrrelser og efterfølgende apoptose 13,14. Makrofager og mikroglia er karakteristisk rig i lysosomer, får dem til at være particularly sårbar ved eksponering for LME-behandling. Denne protokol giver en kraftig, men alligevel enkel og nem måde at fastslå bidrag mikroglia i forsøg udnytte CGC og andre neuronale / gliale dyrkningssystemer.
Protocol
Representative Results
Discussion
De vigtigste skridt til at sikre en vellykket selektiv fjernelse af mikroglia fra CGC og / eller blandede kulturer er: 1) at opretholde en steril og sund CGC kultur; 2) filtersterilisering LME-holdige medium og returnere opløsningen til pH 7,4; 3) at holde de tilbageholdte CGC medier og LME-holdige medier ved 37 ° C for at undgå varme chok; og 4) at arbejde hurtigt for at reducere den tid, cellerne holdes uden for inkubatoren.
Vi brugte 25 mM LME at nedbryder mikroglia fra vo…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.
Materials
| Forceps | Sigma-Aldrich | F4142 | The curved end facilitates removal of the cerebellum |
| Micro-dissecting scissors | Sigma-Aldrich | S3146 | Straight, sharp point facilitates rodent P4-7 dissection |
| L-leucine methyl ester hydrochloride | Sigma-Aldrich | 7517-19-3 | |
| EBSS solution | Sigma-Aldrich | E7510-500 ml | |
| Poly-D-lysine | Sigma-Aldrich | 27964-99-4 | Coat coverslips 1 day before use |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A9418 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich | P4417 | |
| DNase | Sigma-Aldrich | D5025 | |
| Soybean trypsin inhibitor | Sigma-Aldrich | T6414 | |
| Mouse anti-ED1 antibody | Abcam | ab31630 |
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