Figure 2. Treatment with ritonavir reduces CA1 neuron death and hippocampal injury in acutely infected mice.

TUNEL staining of the hippocampal region (a,c,e) and histological examination of the CA1 neuron layer (b,d,f) in uninfected mice (a,b), 4 dpi TMEV-infected mice treated with vehicle (c,d), and 4 dpi TMEV-infected mice treated with ritonavir (e,f) showed reduced neuron cell death only in ritonavir-treated mice. (g) Quantitation of the number of TUNEL-positive cells revealed that ritonavir treatment significantly protected the hippocampus (P < 0.001 between all groups by one-way ANOVA on ranks; SNK pairwise: VEH vs RIT, P < 0.05; n = 10 mice per group). Area of the hippocampus analyzed was not different between the groups (P = 0.613 by one-way ANOVA). (h) Mouse hippocampal neurons were treated with the calcium ionophore A23187 (5 μM or 10 μM) for 24 hr in the presence of 10 μg/mL ritonavir or DMSO. Neuron cell death was assessed by LDH assay. Ritonavir significantly decreased cell death in response to the ionophore (P < 0.001 by one-way ANOVA; SNK pairwise analysis indicates P < 0.001 for all ritonavir vs DMSO pairs; n = 4 samples per condition). TUNEL and histology images are representative of more than 10 mice per condition. Scale bar in (e) is 500 μm and refers to (a,c). Scale bar in (f) is 50 μm and refers to (b,d). UNINF = uninfected; VEH = vehicle-treated; RIT = ritonavir-treated; DMSO = vehicle only; RIT = ritonavir only; 5 mM A = 5 mM A23187 + VEH; RIT + 5A = 5 mM A23187 + RIT; 10 mM A = 10 mM A23187 + VEH; RIT + 10A = 10 mM A23187 + RIT. Graphs show means ± 95% confidence intervals.