J Vet Sci.  2018 Nov;19(6):750-758. 10.4142/jvs.2018.19.6.750.

Neonatal influenza virus infection affects myelination in influenza-recovered mouse brain

• Kim JH
• Yu JE
• Chang BJ
• Nahm SS

• KMID: 2427023
• DOI: http://doi.org/10.4142/jvs.2018.19.6.750

Abstract

Influenza virus infection is a zoonosis that has great socioeconomic effects worldwide. Influenza infection induces respiratory symptoms, while the influenza virus can infect brain and leave central nervous system sequelae. As children are more vulnerable to infection, they are at risk of long-term neurological effects once their brains are infected. We previously demonstrated that functional changes in hippocampal neurons were observed in mice recovered from neonatal influenza infection. In this study, we investigated changes in myelination properties that could affect neural dysfunction. Mice were infected with the influenza virus on postnatal day 5. Tissues were harvested from recovered mice 21-days post-infection. The expression levels for myelin basic protein (MBP) were determined, and immunohistochemical staining and transmission electron microscopy were performed. Real-time polymerase chain reaction and Western blot analyses showed that mRNA and protein expressions increased in the hippocampus and cerebellum of recovered mice. Increased MBP-staining signal was observed in the recovered mouse brain. By calculating the relative thickness of myelin sheath in relation to nerve fiber diameter (G-ratio) from electron photomicrographs, an increased G-ratio was observed in both the hippocampus and cerebellum of recovered mice. Influenza infection in oligodendrocyte-enriched primary brain cell cultures showed that proinflammatory cytokines may induce MBP upregulation. These results suggested that increased MBP expression could be a compensatory change related to hypomyelination, which may underlie neural dysfunction in recovered mice. In summary, the present results demonstrate that influenza infection during the neonatal period affects myelination and further induces functional changes in influenza-recovered mouse brain.

Keyword

cerebellum; hippocampus; influenza; myelin; oligodendrocyte

MeSH Terms

Animals
Blotting, Western
Brain*
Cell Culture Techniques
Central Nervous System
Cerebellum
Child
Cytokines
Hippocampus
Humans
Influenza, Human*
Mice*
Microscopy, Electron, Transmission
Myelin Basic Protein
Myelin Sheath*
Nerve Fibers
Neurons
Oligodendroglia
Orthomyxoviridae*
Real-Time Polymerase Chain Reaction
RNA, Messenger
Up-Regulation
Cytokines
Myelin Basic Protein
RNA, Messenger

Figure

• 

  Fig. 1 Real-time polymerase chain reaction analysis of myelin basic protein (MBP) expression in the hippocampus and cerebellum of control and influenza-recovered mice. MBP expression in recovered mice was significantly increased both in the hippocampus and cerebellum (n = 5; *p < 0.05).

• 

  Fig. 2 Representative images (A) and quantification data from Western blot analyses for myelin basic protein (MBP) expression in the hippocampus (HP) and cerebellum (CB) of control and recovered mice (B). MBP expression in recovered mice was significantly increased both in the hippocampus and cerebellum (n = 4; *p < 0.05, **p < 0.01).

• 

  Fig. 3 Representative photomicrographs of myelin basic protein (MBP) immunohistochemical staining in the hippocampus and cerebellum of control (A and C) and recovered (B, D–F) mice. Positive staining for MBP expression was detected in the molecular layer (ML) of the hippocampus and in the white matter (WM) of the cerebellum. High magnification images of the indicated fields in the hippocampus (B) and the cerebellum (D) are shown in panels E and F in Fig. 3, respectively. The optical density measured from the stained areas was significantly higher in the cerebellar WM in recovered mice (G; *p < 0.05). Scale bars = 200 µm (A and B), 50 µm (C–E), 100 µm (F).

• 

  Fig. 4 Representative transmission electron microscopic photographs of the myelin sheath in control (A and C) and recovered (B and D) mice. Cross sections of myelinated axons are visible. The appearance and thickness of the myelin sheaths are comparable between the control and recovered mouse brain. Scale bars = 5 µm (A–D).

• 

  Fig. 5 A stepwise image conversion process to calculate the G-ratio. (A) An original image was converted to a black and white image by applying a threshold that would only reveal myelin sheaths. Scale bar = 5 µm. (B) Image analysis software was then used to obtain the area of the myelin sheath. (C) The areas that were occupied by axons were filled manually. The entire area occupied by the myelin sheaths and axons was then obtained. (D) Schematic representation (of an axon and myelin sheath) of how the G-ratio is obtained. By deducting the area occupied by the myelin sheaths from the entire area occupied by both the myelin sheaths and axons, the radius of the axons (r) and the thickness of the myelin sheaths (R – r) were obtained. The G-ratio was obtained by using the following formula: G = r/R. (E) A graph showing the G-ratio for the hippocampus and cerebellum. *p < 0.05.

• 

  Fig. 6 (A) The viability of primary brain cell cultures enriched with oligodendrocyte precursor cells. Real-time polymerase chain reaction analysis of myelin basic protein (MBP; B), interleukin-1β (IL-1β; C), interleukin-6 (IL-6; D), and tumor necrosis factor-α (TNF-α; E) expressions in control and influenza-infected cells (n = 6; *p < 0.05, **p < 0.01).

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