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. 2019 Feb 28;20(5):1058.
doi: 10.3390/ijms20051058.

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection

Sarah Schwarz  1   2 , Ingo Spitzbarth  3   4 , Wolfgang Baumgärtner  5   6 , Annika Lehmbecker  7   8
Affiliations

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection

Sarah Schwarz et al. Int J Mol Sci. .

Abstract

Canine dorsal root ganglion (DRG) neurons, isolated post mortem from adult dogs, could provide a promising tool to study neuropathogenesis of neurotropic virus infections with a non-rodent host spectrum. However, access to canine DRG is limited due to lack of donor tissue and the cryopreservation of DRG neurons would greatly facilitate experiments. The present study aimed (i) to establish canine DRG neurons as an in vitro model for canine distemper virus (CDV) infection; and (ii) to determine whether DRG neurons are cryopreservable and remain infectable with CDV. Neurons were characterized morphologically and phenotypically by light microscopy, immunofluorescence, and functionally, by studying their neurite outgrowth and infectability with CDV. Cryopreserved canine DRG neurons remained in culture for at least 12 days. Furthermore, both non-cryopreserved and cryopreserved DRG neurons were susceptible to infection with two different strains of CDV, albeit only one of the two strains (CDV R252) provided sufficient absolute numbers of infected neurons. However, cryopreserved DRG neurons showed reduced cell yield, neurite outgrowth, neurite branching, and soma size and reduced susceptibility to CDV infection. In conclusion, canine primary DRG neurons represent a suitable tool for investigations upon the pathogenesis of neuronal CDV infection. Moreover, despite certain limitations, cryopreserved canine DRG neurons generally provide a useful and practicable alternative to address questions regarding virus tropism and neuropathogenesis.

Keywords: canine; canine distemper virus (CDV); cryopreservation; dog; dorsal root ganglion (DRG) neurons; neuroinfection; paramyxovirus.

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Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

Figures

Figure 1
Figure 1
Overlay of phase-contrast and immunofluorescence-images of a non-infected, non-cryopreserved canine DRG neuron at 6 DiV: green = pan-NF; blue = bisBenzimide nuclear stain. The neuron has a large, granular cell body with a phase-bright halo (arrow), is adherent to the substrate, and shows outgrowth of branched neurites (arrowhead). It is surrounded by spindeloid, bi- to multipolar glial cells with delicate processes (blue nuclei) lacking neurofilament staining. Pan-NF = pan-neurofilament, DiV = days in vitro, DRG = dorsal root ganglion.
Figure 2
Figure 2
A: Y axis: neuronal cell yield [number of neurons] in cell suspensions after isolation/thawing, respectively. Cryo cultures show reduced neuronal cell yield when compared to non-cryo cultures. Non-cryo and cryo: n = 8 dogs. B: Y axis: neuronal viability [%] at 6 DiV. Cryo neurons show reduced viability when compared to non-cryo neurons. Non-cryo and cryo: n = 24 wells. C: Y axis: area of neuronal soma [µm2] at 6 DiV. The cell bodies of cryo neurons are generally smaller than the ones of non-cryo neurons. Non-cryo: n = 98 neurons, cryo: n = 70 neurons. D: Y axis: neurons expressing cleaved Caspase 3 [%]. A higher percentage of non-cryo neurons express the apoptotic marker cleaved Caspase 3 when compared to cryo neurons. Non-cryo and cryo: n = 12 wells. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Employed statistical test: Mann-Whitney U-test. DiV = days in vitro, non-cryo = non-cryopreserved, cryo = cryopreserved. Graphs show median value, quartils, minimum and maximum values.
Figure 3
Figure 3
Comparative morphometric analysis of non-infected non-cryo (6 DiV) and cryo (6 and 12 DiV) neurons. A: Y axis: neurons showing neurite outgrowth [%]. Non-cryo cultures contain a higher percentage of neurons showing neurite outgrowth compared to cryo cultures. Non-cryo 6 DiV, cryo 6 DiV, and cryo 12 DiV: n = 24 wells. B: Y axis: number of primary neurites per neuron. Among the neurons showing neurite outgrowth, non-cryo neurons possess a higher number of primary neurites per neuron. Non-cryo: n = 72 neurons, cryo 6 DiV: n = 24 neurons, cryo 12 DiV: n = 37 neurons. C: Y axis: number of neurite branching points per neuron. Non-cryo neurons form a higher number of neurite branching points per neuron than cryo neurons. Non-cryo: n = 72 neurons, cryo 6 DiV: n = 24 neurons, cryo 12 DiV: n = 37 neurons. D: Y axis: length of longest neurite [µm] per neuron. Non-cryo and cryo neurons show no significant difference in the mean length of their longest neurite. Non-cryo: n = 72 neurons, cryo 6 DiV: n = 24 neurons, cryo 12 DiV: n = 37 neurons. E–H: Immunofluorescence-images of non-cryo (E,G) and cryo neurons (F,H) at 6 DiV; green = pan-NF, blue = bisBenzimide nuclear stain. More neurons without neurites (F: arrows) can be found in cryo cultures. Cryo neurons generally show fewer and less branched neurites and smaller cell bodies (F,H) compared to non-cryo neurons (E,G). ** = p < 0.01, *** = p < 0.001. Employed statistical test: Mann–Whitney U-test. Pan-NF = pan-neurofilament, DiV = days in vitro, non-cryo = non-cryopreserved, cryo = cryopreserved. Graphs show median value, quartils, minimum and maximum values.
Figure 4
Figure 4
Y axis: neurons infected with different CDV strains [%] at 6 dpi. Cryo neurons show reduced susceptibility to infection with both CDV-strains (CDV R252 and CDV-5804 PeGFP) when compared to non-cryo neurons. Generally CDV R252 infects a higher percentage of neurons than CDV-5804 PeGFP. Non-cryo and cryo: n = 24 wells. *** = p < 0.001. Employed statistical test: Mann–Whitney U-test., dpi = days post infection, non-cryo = non-cryopreserved, cryo = cryopreserved, CDV = canine distemper virus, eGFP = enhanced green fluorescent protein. Graphs show median value, quartils, minimum and maximum values.
Figure 5
Figure 5
Immunofluorescence images of non-cryo neurons infected with CDV-5804 PeGFP (A–C) and CDV R252 (D–F) at 6 dpi. A: NF (red) indicates neuron with several branched neurites (arrow). B: eGFP-expression (green) indicating infection by CDV -5804 PeGFP in a neuron (arrow) and several glial cells. C: Merged image of A and B including bisBenzimide nuclear stain (blue) shows expression of eGFP indicating infection with CDV-5804 PeGFP in the cell body of the neuron (arrow) and in the cell bodies and processes of glial cells. D: NF (green) indicating neuron (arrow) with short, branched neurites. E: Red signal (CDV-nucleoprotein) is visible in the cell-body as well as the neurites of a non-cryopreserved neuron (arrow). F: Merged image of D and E including bisBenzimide nuclear stain (blue) indicating a neuron infected with CDV R242 (arrow) with CDV nucleoprotein present in the cell body and neurites. A–C: Bar = 200 µm. D–F: Bar = 100 µm. Non-cryo = non-cryopreserved, CDV = canine distemper virus, eGFP = enhanced green fluorescent protein, dpi = days post-infection, NF = neurofilament.
Figure 6
Figure 6
Experimental design: while non-cryo neurons were directly seeded after isolation, dissociation and counting, cryo-neurons were cryopreserved an thawed after 3 months. The day the neurons were seeded represents day 0 of the experiment. Part of the cultures were fixed for comparative immunofluorescence and morphometric analysis at 6 DiV for non-cryo and cryo neurons and at an additional time-point (12 DiV) for cryo neurons. The remaining DRG neuronal cultures were infected with 2 different strains of CDV (CDV-5804 PeGFP, CDV R252) at 6 DiV and kept in culture until 12 DiV or 6 dpi, respectively, when they were processed for immunostaining and further analysis. Non-cryo = non-cryopreserved neurons, cryo = cryopreserved neurons, DiV = days in vitro, dpi = days post infection, CDV = canine distemper virus, eGFP = enhanced green fluorescent protein.
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