Astrocytes Protect against Isoflurane Neurotoxicity by Buffering pro-brain-derived Neurotrophic Factor.
BACKGROUND
Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain-derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75). Astrocytes express p75, but their role in neuronal p75-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75 neurotoxicity.


METHODS
Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75 in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times.


RESULTS
Exposure to isoflurane significantly (P < 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75 inhibition. Astrocyte-targeted p75 knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold).


CONCLUSIONS
The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis.
From the Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University School of Medicine, Stanford, California.
Creed M
CM
Stary
Xiaoyun
X
Sun
Rona G
RG
Giffard
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D019208:Brain-Derived Neurotrophic Factor / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D002478:Cells, Cultured; D018920:Coculture Techniques; D007530:Isoflurane / Q000633:toxicity*; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000096:biosynthesis*
D019208:Brain-Derived Neurotrophic Factor; D011498:Protein Precursors; C426674:brain-derived neurotrophic factor precursor; D007530:Isoflurane