The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration

Abstract

Aims To develop human organotypic retinal cultures (HORCs) to study retinal ganglion cell (RGC) death in response to ischaemic and excitotoxic insults, both known to cause loss of RGCs and proposed as mechanisms involved in glaucomatous retinal neurodegeneration. Methods Human donor eyes were obtained within 24 h post mortem. The retina was isolated and explants cultured using two techniques. THY-1 mRNA (assessed by real-time quantitative PCR) and neuronal nuclei (NeuN) (assessed by immunohistochemistry) were used as markers of RGCs. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Results The distribution of THY-1 mRNA and NeuN-labelling within the human retina was consistent with the expected distribution of RGCs. Gross morphology and retinal architecture remained stable over a 96 h culture period. THY-1 mRNA and NeuN-labelled RGC layer cells decreased over the culture period, and there was an increase in TUNEL-labelling with time, but HORCs cultured in serum-free DMEM/HamF12 medium were useful for up to 48 h in culture. N-methyl-d-aspartate (10 µM) caused a reduction in THY-1 mRNA by 24 h and decreased the numbers of NeuN-labelled RGC layer neurons by 48 h, suggesting that the loss of THY-1 mRNA was a marker of RGC stress prior to death. Simulated ischaemia (60 min oxygen/glucose deprivation) caused a reduction at 24 h in both THY-1 mRNA and the numbers of NeuN-labelled neurons of HORCs. Conclusion HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes

P2X7Receptor Activation Mediates Retinal Ganglion Cell Death in a Human Retina Model of Ischemic Neurodegeneration

“…Note that there is no loss of NeuN staining over the 24 hour culture period. 27 P2X 7 Receptor. Macular samples were fixed with 4% formaldehyde in PBS (24 hours; 48C), then dehydrated through graded ethanol (30%, 50%, 70%, 90%, and 100% EtOH: water; 30 minutes each; 48C), followed by 50:50 xylene: EtOH, then 100% xylene for 30 minutes at room temperature (RT).…”

“…Note that there is a loss of THY-1 signal over the 24 hour culture period. 27 Retinal Sections. Following dissection from the globe, a 4-mm diameter sample was taken from the macular region of each retina.…”

“…Total RNA, measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), was reverse transcribed to synthesize cDNA, as previously described. 27 The ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK) was used for QRT-PCR. cDNA was mixed with Human THY-1 probe (Hs00174816_m1; Applied Biosystems), or Human P2X7R probe (Hs0017521_m1; Applied Biosystems), and TaqMan PCR Master Mix (Applied Biosystems).…”

“…Immunohistochemistry for NeuN on HORC sections has been previously described. 27 The primary monoclonal antibody for NeuN (Millipore, Watford, UK) was used at 1:200 and was incubated at 48C overnight. The secondary antibody was Alexa Fluor 488-conjugated (Invitrogen) used at 1:1000, and incubation was for 2 hours at room temperature.…”

“…We have developed a human organotypic retinal culture (HORC) system to investigate RGC death, 27 which was used in the present study to assess a potential role for the P2X 7 R in loss of RGCs within the human retina. We demonstrated for the first time that simulated ischemia (oxygen/glucose deprivation [OGD]) resulted in loss of RGCs via a mechanism that involved P2X 7 R activation.…”