Long-Term Kinetics of Immunologic Components and Neurological Deficits in Rats Following Repetitive Mild Traumatic Brain Injury

Abstract

BACKGROUND Despite growing awareness of repetitive mild traumatic brain injury (rmTBI), understanding of the involvement of long-term kinetics of immunologic components in the central and peripheral immune system took part remains incomplete. The present study aimed to provide a quantitative assay for certain immune system parameters in rmTBI rats. MATERIAL AND METHODS Neurological functions were assessed by modified Neurological Severity Score (mNSS) and Morris Water Maze (MWM), immunologic components from brain and peripheral blood were analyzed by flow cytometry (FCM), and concentrations of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were measure by enzyme-linked immunosorbent assay (ELISA). RESULTS Neurological functions of rmTBI rats were seriously impaired. In the brain, T cells were up-regulated and peaked at week 1. The percentage of CD4+ T cells decreased from week 1 to week 4, while CD8+ T cells notably decreased at week 1, then increased until week 4. The infiltration proportion of Treg cells was reduced at week 1 and peaked at week 2. CD86+/CD11b+ M1 peaked at week 4 and CD206+/CD11b+ M2 rose at week 1. IL-6/IL-10 showed a similar pattern, whose rise corresponded to the decrease in TNF-α at week 2 after rmTBI. FCM demonstrated peripheral immune dysfunction after rmTBI. CONCLUSIONS mNSS and MWM demonstrated neuronal deficits in rmTBI rats, and central and peripheral immune systems were implicated in the pathophysiological processes of rmTBI. Long-term immune response may play dual roles in injury and repair of rmTBI.

Figure 1

The impact of rmTBI on long-term neurological function. Long-term neurological function was evaluated by mNSS (A) and MWM (n=10), which includes spatial acquisition trial (B, C), probe trial (D), swimming velocity (E), and heading angle (F). ^# indicates P<0.05, ^## indicates P<0.01, ^### indicates P<0.001 when comparing mNSS and MWM between rmTBI groups and the sham group.

Figure 2

Analysis of T cells in the brain during 4 weeks post-injury. Dot plots of isolated live immune cells in the brain during 4 weeks post-injury (A). Representative FCM for CD3⁺ cells in injured brain at the indicated weeks (B). Statistical data for CD3⁺ cells (C). ^# P<0.05, CD3⁺ cells at 2 and 4 weeks compared with sham, ^## P<0.01, CD3⁺ cells at 1 week compared with sham.

Figure 3

Changes in T cell subsets in the brain during 4 weeks post-injury. Representative FCM for CD3⁺CD4⁺ cells (A) and CD3⁺CD8⁺ cells (B) in injured brain after rmTBI. Statistical data for CD3⁺CD4⁺ cells (C) and CD3⁺CD8⁺ cells (3D). ^# indicates P<0.05, ^## indicates P<0.01 when compared CD3⁺CD4⁺ cells or CD3⁺CD8⁺ cells with cells in the sham group.

Figure 4

Infiltration proportion of Treg cell in the brain during 4 weeks post-injury. Dot plots of isolated live immune cells in the brain (A). Representative FCM for Treg cells (CD4⁺, CD25⁺ and Foxp3⁺) in injured brain (B). Quantitative data for accumulated Treg cells (C). ^# P<0.05 at 1 week, ^## P<0.01 at 2 and 4 weeks compared with sham.

Figure 5

Changes in specific subsets of microglia in the brain during 4 weeks post-injury. Dot plots of isolated specific subsets of microglia in the brain (A). Representative FCM for CD86⁺/CD11b⁺ M1-like microglia (B). Representative FCM for CD206⁺/CD11b⁺ M2-like microglia (C). Quantitative data for CD86⁺/CD11b⁺ cells (D). Statistical data for CD206⁺/CD11b⁺ cells (E). ^# P<0.05, rmTBI group vs. sham group.

Figure 6

Inflammatory cytokine levels during 4 weeks post-injury. Expression levels of inflammatory cytokines IL-6 (A), TNF-α (B), and IL-10 (C) during 4 weeks post-injury. ^# P<0.05, ^## P<0.01, ^### P<0.001, rmTBI group vs. sham group.

Figure 7

T cell number in the peripheral blood during 4 weeks post-injury. Dot plots of isolated immune cells in the peripheral blood (A). Representative FCM for CD3⁺ cells in the peripheral blood (B). Statistical data for accumulated T cells in the peripheral blood (C). ^# P<0.05 at 1 week, ^## P<0.01 at 2 and 4 weeks compared with sham.

Figure 8

T cell subsets in the peripheral blood during 4 weeks post-injury. Representative FCM for CD3⁺CD4⁺ cells in the peripheral blood (A). Representative FCM data for CD3⁺CD8⁺ cells in the peripheral blood (B). Quantitative data for the percentages of T cell subsets (C, D). ^# P<0.05, ^## P<0.01 compared with sham.

Figure 9

Treg cells in the peripheral blood during 4 weeks post-injury. Dot plots of isolated immune cells in the peripheral blood (A). Representative FCM for Treg cells (CD4⁺, CD25⁺, and Foxp3⁺) in the peripheral blood (B). Quantitative data for accumulated Treg cells in the peripheral blood (C). ^# P<0.05 at 4 weeks compared with no injury.