Virus Infections on Prion Diseased Mice Exacerbate Inflammatory Microglial Response

Abstract

We investigated possible interaction between an arbovirus infection and the ME7 induced mice prion disease. C57BL/6, females, 6-week-old, were submitted to a bilateral intrahippocampal injection of ME7 prion strain (ME7) or normal brain homogenate (NBH). After injections, animals were organized into two groups: NBH (n = 26) and ME7 (n = 29). At 15th week after injections (wpi), animals were challenged intranasally with a suspension of Piry arbovirus 0.001% or with NBH. Behavioral changes in ME7 animals appeared in burrowing activity at 14 wpi. Hyperactivity on open field test, errors on rod bridge, and time reduction in inverted screen were detected at 15th, 19th, and 20th wpi respectively. Burrowing was more sensitive to earlier hippocampus dysfunction. However, Piry-infection did not significantly affect the already ongoing burrowing decline in the ME7-treated mice. After behavioral tests, brains were processed for IBA1, protease-resistant form of PrP, and Piry virus antigens. Although virus infection in isolation did not change the number of microglia in CA1, virus infection in prion diseased mice (at 17th wpi) induced changes in number and morphology of microglia in a laminar-dependent way. We suggest that virus infection exacerbates microglial inflammatory response to a greater degree in prion-infected mice, and this is not necessarily correlated with hippocampal-dependent behavioral deficits.

Figure 1

Influences of virus infection on prion disease progression. Burrowing activity was the most sensitive test to detect earlier associated behavioral changes occurring in both ME7 infected group (ME7) and virus infected prion diseased animals (ME7+Py). All other tests showed significant changes only at later stages of the disease. ∗ indicates significant differences between ME7 groups and respective controls (NBH versus ME7; NBH+Py versus ME7+Py; Bonferroni posttest p < 0.01). # indicates significant differences between NBH+Py and NBH groups (Bonferroni posttest p < 0.05).

Figure 2

Photomicrographs from immunolabeled sections to detect Piry virus antigens in the brain of NBH and ME7 virus infected groups. Virus antigens were indirectly detected as immunostained cell somas and neuronal primary dendrites in the olfactory pathways in both NBH+Py (a) and ME7+Py (b) groups. Note the absence of virus antigens selective immunolabeling in NBH control group (c). We also did not observe any labeling after immunoreaction without anti-Piry polyclonal primary antibody (not illustrated). Scale bar = 25 μm.

Figure 3

Photomicrographs to illustrate protease resistant PrPc immunolabeled hippocampal sections from control (a and c) and ME7 infected (b and d) subjects, counterstained with cresyl violet. Low power (a and b); scale bar = 50 μm; high power (c and d) = 25 μm.

Figure 4

Photomicrographs of CA1 microglial cells taken from sections of NBH (a, e, and i); NBH+Py (b, f, and j); ME7 (c, g, and k); ME7+Py (d, h, and l). From left to right the 1st, 2nd, 3rd, and 4th columns correspond, respectively, to subjects intracerebrally (i.c.) injected with normal brain homogenate, NBH, with NBH and nasal instilled with Piry virus suspension, NBH+Py, i.c., injected with ME7 infected brain homogenate, ME7, i.c., injected with ME7 and nasal instilled with Piry virus suspension, ME7+Py. First row corresponds to pyramidal cell layer (Pyr) and stratum radiatum (Rad). Second row corresponds to lacunosum molecular layer (Lac-Mol), hippocampal fissure (Fiss), and small portion of the molecular layer of dentate gyrus below hippocampal fissure. The third row corresponds to high power pictures to illustrate different stages of microglial morphological activation. Note that microglia from ME7+Py group is closer to the amoeboid format (last stage of morphological change of activated microglia). Scale bars (a)–(h) = 50 μm; (i)–(l) = 25 μm.

Figure 5

Stereological estimate of total microglia (CA1 total) and on CA1 lacunosum molecular (Lac-Mol) and radiatum and pyramidal layers. Note that as compared to all other groups, Piry (Py) virus infections in combination with intracerebral injection of ME7 infected brain homogenate (ME7+Py), significantly increased the number of microglia in all layers. Normal brain homogenate intracerebrally (i.c.) injection (NBH); NBH i.c. injection and virus suspension instilled intranasally (NBH+Py); ME7 infected brain homogenate (ME7). ∗ indicates significant differences between ME7+Py and all other groups and layers (one-way ANOVA, p < 0.01), and # indicates p < 0.05. Two-way ANOVA revealed that virus infection interacts with prion disease and exacerbates microglial response (p = 0.01).