Figure 3.

Myelin deficits and sustained cognitive impairments eight weeks following juvenile H1N1 exposure.

(A) Schematic illustration of experimental timeline. Mice were intratracheally inoculated at P14 with PBS or H1N1, and endpoint analyses were performed at P70, 8 weeks post-infection (WPI).

(B) Time spent in center of the open field over 10-minute testing period. Open field performed 8 weeks post-infection. n = 10 control, n = 12 H1N1 mice.

(C) Total distance moved in the open field over 10-minute testing period. Open field performed 8 weeks post-infection. n = 10 control, n = 12 H1N1 mice.

(D) Novel preference for NORT performed 8 weeks post-infection. n = 10 control, n = 12 H1N1 mice.

(E) Quantification of white matter reactive microglia (IBA1⁺CD68⁺) 8 weeks post-infection in the cingulum. n = 8 mice per group.

(F) Representative confocal micrographs of white matter reactive microglia (IBA1, white; CD68, red) in the cingulum 8 weeks post-infection.

(G) Quantification of white matter mature oligodendrocytes (ASPA⁺) 8 weeks post-infection in the cingulum. n = 8 mice per group.

(H) Representative confocal micrographs of white matter mature oligodendrocytes (ASPA, white) in the cingulum 8 weeks post-infection.

(I) Schematic depicting the cingulum region of the corpus callosum where transmission electron microcopy (TEM) was performed 8 weeks post-infection in mice infected during the juvenile period (J-M) and mice infected during adulthood (N-P).

(J) Representative TEM images of mice infected with H1N1 during the juvenile period 8 weeks post-infection at the level of the cingulum of the corpus callosum in cross-section. Myelinated axons visible as electron-dense sheaths encircling axons.

(K) Scatter plots of g-ratio as a function of axon diameter 8 weeks post-infection for control axons (black dots) or H1N1 axons (blue triangles). A single point indicates the g-ratio for a single axon. Approximately 250–400 axons were quantified for each animal. n = 6 mice per group.

(L) Quantification of myelinated axon density. n = 6 mice per group.

(M) G-ratio relative to small (<500 nm), medium (500–1000 nm), and large (>1000 nm) caliber axons for control (black points) or H1N1 (blue points) treated mice. n = 6 mice per group.

(N) Quantification of myelinated axon density. n = 7 mice per group.

(O) G-ratio relative to small (<500 nm), medium (500–1000 nm), and large (>1000 nm) caliber axons for control (black points) or H1N1 (orange points) treated mice. n = 7 mice per group.

(P) Representative TEM images of mice infected with H1N1 during adulthood 8 weeks post-infection at the level of the cingulum of the corpus callosum in cross-section. Myelinated axons visible as electron-dense sheaths encircling axons.

Data shown as mean ± SEM (B-E, G, L, and N); each dot represents an individual mouse. ns: p > 0.05, * p < 0.05, ** p < 0.01, analyzed via (B, C, E, G, L, and N) unpaired two-tailed t-test, (D) Mann-Whitney U test for datasets that failed the Shapiro-Wilk test, or (M and O) unpaired two-tailed t-test with correction with multiple comparisons. Scale bars, (F and H) 40 μm, (J and P) 1 μm.