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. 2024 Nov 11;12(12):e0203124.
doi: 10.1128/spectrum.02031-24. Online ahead of print.

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation

Sara A Dochnal  1 , Patryk A Krakowiak  1 , Abigail L Whitford  1 , Anna R Cliffe  1
Affiliations

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation

Sara A Dochnal et al. Microbiol Spectr. .

Abstract

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.IMPORTANCEEstablishing models to investigate host-pathogen interactions requires mimicking physiological conditions as closely as possible. One consideration is the oxygen concentration used for in vitro tissue culture experiments. Standard incubators do not regulate oxygen levels, exposing cells to oxygen concentrations of approximately 18%. However, cells within the body are exposed to much lower oxygen concentrations, with physiological oxygen concentrations in the brain being 0.55%-8% oxygen. Here, we describe a model for herpes simplex virus 1 (HSV-1) latent infection using neurons cultured in 5% oxygen. We show that culturing neurons in more physiological oxygen concentrations improves neuronal health to permit long-term studies of virus-cell interactions and the impact on reactivation.

Keywords: herpes simplex virus; in vitro culture; latency; oxygen; reactivation; sympathetic neurons.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Neuronal health in atmospheric versus physioxic incubation conditions. (A–E) Primary sympathetic neurons were incubated in atmospheric (approximately 18% oxygen) or physioxic (5% oxygen) conditions. (A) Neurons were latently infected with Stayput-GFP at an MOI of 10 PFU/cell in the presence of acyclovir (ACV; 50 µM) for 10 days and cultures were imaged using phase contrast to document soma morphology. (B and C) Neurons were incubated for up to 30 days post-plating. Scores representing cell body (B) and axon (C) health, as described in Tables 1 and 2, were recorded over time. Biological replicates from two independent dissections; statistical comparisons were made using two-way analysis of variance. (D and E) Neurons were plated in microfluidic chambers. Ten days post-plating, cultures were fixed and stained for neuronal marker Beta III tubulin (white) (D). Scale bar 100 µm. Axonal degeneration was analyzed using these images (E). Biological replicates from four independent experiments; statistical comparisons were made using unpaired non-normal t-test. Individual biological replicates along with the means and SEMs are represented. *P < 0.05; **P < 0.01.
Fig 2
Fig 2
Physioxic incubation conditions reduce HSV-1 reactivation. (A–F) Primary neurons were latently infected with Stayput-GFP at an MOI of 10 PFU/cell in the presence of acyclovir (ACV; 50 µM) for 6 days and then reactivated 2 days after the removal of acyclovir with LY294002 (20 µM). Quantification of relative latent viral DNA load (A) and LAT expression (B) at 8 days post-infection. (C) Quantification of the number of GFP-positive neurons at 48 hours post-stimulus. (D–F) Relative viral gene expression at 18 hours post-stimulus compared to latent samples quantified by reverse transcription-quantitative PCR (RT-qPCR) for immediate early viral gene ICP27 (D), early viral gene ICP8 (E), or late gene gC (F) normalized to cellular control mGAPDH. Biological replicates from four independent dissections; statistical comparisons were made using paired t-tests. Individual biological replicates along with the means and SEMs are represented. *P < 0.05; **P < 0.01.
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