SF-induced effects on cytokine mRNA expression levels in brain
There was a significant effect of acute SF on IL-1β and IL-6 expression levels in prefrontal cortex (IL-1β: F1,28 = 4.69, p = 0.039, IL-6: F1,28 = 8.07, p = 0.008, 2-way ANOVA), while acute SF induced effects on IL-1β expression levels in both prefrontal cortex and hippocampus were dependent on 6-OHDA treatment (prefrontal cortex: interaction- F1,28 = 7.81, p = 0.009, hippocampus: interaction - F1,26 = 6.52, p = 0.0169, 2-way ANOVA). Similarly, in hypothalamus, there was a significant effect of acute SF on IL-1β (F1,28 = 6.07, p = 0.020), and an interaction of acute SF and 6-OHDA on IL-6 (F1,28 = 16.21, p = 0.0004; 2-way ANOVA). TNFα levels were unaffected by the experimental treatment in brain tissues while TGFβ levels in all three brain regions were significantly affected by acute SF (prefrontal cortex: F1,28 = 8.62, p = 0.007; hippocampus: F1,28 = 14.29, p = 0.0008; hypothalamus: F1,28 = 15.07, p = 0.0006) and 6-OHDA treatment (except in prefrontal cortex; hippocampus: F1,28 = 14.77, p = 0.0006, hypothalamus: F1,28 = 10.55, p = 0.0030; 2-way ANOVA).
There was a significant effect of chronic SF on the expression levels of all genes assessed in prefrontal cortex (except TNFα; IL-1β: F1,30 = 4.29, p = 0.047; IL-6: F1,29 = 7.512, p = 0.0106; TGFβ: F1,29 = 6.510, p = 0.0163) along with a significant effect of 6-OHDA treatment and its interaction with chronic SF on TNFα and IL-1β, respectively (TNFα: 6-OHDA- F1,31 = 4.94, p = 0.034; IL-1β: interaction- F1,30 = 4.498, p = 0.042). Further, hippocampal TGFβ expression was significantly affected by 6-OHDA (F1,30 = 4.43, p = 0.044) and its interaction with chronic SF (F1,30 = 6.17, p = 0.019; 2-way ANOVA). Similarly, hypothalamic IL-1β expression was significantly affected by 6-OHDA treatment (F1,29 = 19.11, p = 0.0002), and hypothalamic TNFα levels were significantly affected by chronic SF (F1,29 = 8.27, p = 0.0076) and 6-OHDA (F1,29 = 28.16, p < 0.0001; 2-way ANOVA).
One week of recovery sleep significantly affected IL-1β expression in hippocampus (F1,28 = 5.97, p = 0.021) and hypothalamus (F1,28 = 5.13, p = 0.032), along with a significant effect of recovery and 6-OHDA treatment on TNFα expression in brain (hippocampus: recovery - F1,28 = 51.91, p < 0.0001, 6-OHDA- F1,28 = 6.49, p = 0.0166; hypothalamus: recovery - F1,28 = 4.36, p = 0.046, 6-OHDA- F1,28 = 4.84, p = 0.036). IL-6 expression levels in prefrontal cortex and hippocampus were significantly affected by recovery (prefrontal cortex: F1,28 = 14.38, p = 0.0007; hippocampus: F1,28 = 7.97, p = 0.0087) and 6-OHDA treatment (prefrontal cortex: F1,28 = 14.98, p = 0.0006; hippocampus: F1,28 = 23.54, p < 0.0001), the expression levels in hypothalamus were significantly affected by 6-OHDA (F1,28 = 42.39, p < 0.0001) and its interaction with recovery (F1,28 = 10.30, p = 0.0034). Neural TGFβ expression levels were significantly affected by recovery (hippocampus: F1,28 = 11.00, p = 0.003; hypothalamus: F1,28 = 11.37, p = 0.002), 6-OHDA (prefrontal cortex: F1,28 = 5.56, p = 0.026; hippocampus: F1,28 = 12.75, p = 0.001; hypothalamus: F1,28 = 14.48, p = 0.0007) and their interaction (prefrontal cortex: F1,28 = 12.68, p = 0.0013; hypothalamus: F1,28 = 13.81, p = 0.0009), except the effect of recovery and its interaction with 6-OHDA in prefrontal cortex and hippocampus, respectively (2-way ANOVA).
In comparison to controls, IL-1β expression under both SF regimes was significantly increased in brain regions (except hippocampus of chronic SF exp; Bonferroni posthoc test, p < 0.05, Fig. 2a, e, i, 3a, i). Acute SF caused a significant reduction in expression levels of TGFβ in all three brain tissues, and in IL-6 in prefrontal cortex and hypothalamus (Bonferroni posthoc test, p < 0.05, Fig. 2b, d, f, h, j, l), as opposed to chronic SF which lead to a significant increase in IL-6 expression in prefrontal cortex, TNFα in hypothalamus and TGFβ in hippocampus (Bonferroni posthoc test, p < 0.05, Fig. 3b, h, k).
A recovery period of 1 week after chronic SF was enough to negate the effects of chronic SF (except IL-6 in prefrontal cortex). In fact, IL-6, TNFα and TGFβ expression in hippocampus and hypothalamus and IL-1β expression in hypothalamus was significantly lower in recovery mice compared with controls (Bonferroni posthoc comparison, p < 0.05, Fig 4f-l). In addition, lack of significant differences between 6-OHDA administered controls and experimental groups in all experiments (except hypothalamic TNFα of chronic SF+R experiment), along with significant reduction in hypothalamic IL-1β and TNFα in 6-OHDA-treated chronic SF mice, and in TGFβ in hippocampus and cortex of 6-OHDA treated acute and chronic SF mice, indicated that chemical sympathectomy significantly attenuated SF-induced neuroinflammatory responses (Bonferroni posthoc test, p < 0.05, fig. 2-4).
SF-induced effects on microglia activity
Iba-ir cell number was significantly affected by acute SF (cortex: F1,16 = 15.60, p = 0.001; hippocampus: F1,16 = 14.79, p = 0.001; pre-optic area: F1,16 = 6.03, p = 0.026), 6-OHDA treatment (cortex: F1,16 = 15.60, p = 0.001; hippocampus: F1,16 = 16.33, p = 0.001), and their interaction (cortex: F1,16 = 26.49, p < 0.0001; hippocampus: F1,16 = 10.45, p = 0.005; pre-optic area: F1,16 = 5.56, p = 0.031) in all three brain regions, except an effect of 6-OHDA in pre-optic area, as determined by 2-way ANOVA. In all three brain regions, acute SF significantly increased Iba-ir, which was significantly decreased with 6-OHDA treatment (Bonferroni posthoc test, p < 0.05, Fig. 5a, e, i). Similarly, Iba-ir soma area was significantly affected by acute SF in pre-optic area (F1,96 = 6.46, p = 0.012), by 6-OHDA treatment in cortex (F1,96 = 12.04, p = 0.0008), and by their interaction in hippocampus (F1,96 = 4.87, p = 0.029) and pre-optic area (F1,96 = 4.07, p = 0.047; 2-way ANOVA), with significantly greater –ir soma area in pre-optic area of mice subjected to acute SF compared with controls (Bonferroni posthoc test, p < 0.05, Fig. 5b, f, j). Iba-ir soma area in cortex of acute SF mice was significantly greater than their counterparts pre-treated with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig. 5b).
In the second experiment, there was a significant effect of SF on Iba-ir cell number in cortex (F1,23 = 26.24, p < 0.0001) and hippocampus (F1,23 = 4.43, p = 0.023), of 6-OHDA treatment and its interaction with SF in cortex (6-OHDA: F1,23 = 8.10, p = 0.009; SF x 6-OHDA: F2,23 = 19.30, p < 0.0001; 2-way ANOVA). Interestingly, Iba-ir cell number in cortex and hippocampus of mice subjected to chronic SF > recovery > controls (Bonferroni posthoc test, p < 0.05 Fig. 6a, e). Furthermore, 6-OHDA treatment significantly reduced Iba-ir in mice subjected to chronic SF (Bonferroni posthoc test, p < 0.05; Fig. 6a, e, i). There was a significant effect of SF on Iba-ir soma area in cortex (F2,139 = 6.49, p = 0.002) and hippocampus (F2,139 = 4.68, p = 0.011), and of 6-OHDA treatment (F1,139 = 12.51, p = 0.0006) and its interaction with SF (F2,139 = 6.73, p = 0.002) on Iba-ir soma area in pre-optic area (2-way ANOVA). Iba-ir soma area was significantly increased in chronic SF mice compared with recovery and controls (except in hippocampus; Bonferroni posthoc test, p < 0.05, Fig. 6b, f, j). Further, chronic SF mice had significantly lower Iba-ir soma area in pre-optic area when treated with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig. 6j).
Three-factor univariate general linear models indicated a significant effect of acute SF and 6-OHDA treatment on microglial ramification in (F1,1824 = 19.66, p < 0.0001) and pre-optic area (F1,1824 = 43.31, p < 0.0001). Microglia ramification significantly varied with distance from soma (cortex: F18,1824 = 38.38, p < 0.0001; hippocampus: F18,1824 = 20.97, p < 0.0001; pre-optic area: F18,1824 = 10.02, p < 0.0001) and acute SF induced effects were dependent on 6-OHDA treatment in all three regions (acute SF x 6-OHDA interaction - cortex: F1,1824 = 6.124, p = 0.013; hippocampus: F1,1824 = 25.76, p < 0.0001; pre-optic area: F1,1824 = 15.99, p < 0.0001). There was a significant three-way interaction only in pre-optic area (acute SF x 6-OHDA x distance from soma: F18,1824 = 3.38, p < 0.0001, univariate GLM). In cortex, acute SF significantly increased the ramification at 6.5 µm from the centre of soma when compared with controls (Bonferroni posthoc test, p < 0.05, Fig 5c). 6-OHDA significantly affected the microglia ramification in controls, with higher ramification in cortex (between 9 to 16 µm from the soma centre), hippocampus (23 µm from the soma centre) and pre-optic area (at 26 and 29 µm from soma centre; Bonferroni posthoc test, p < 0.05, Fig 5c, d, g, h, k, l). Similarly, acute SF mice when injected with 6-OHDA, showed higher ramification in hippocampus at a distance of 29-30.5 µm from the centre of soma (Bonferroni posthoc test, p < 0.05, Fig 5g, h).
Chronic SF had more profound effects on microglia morphology with a significant effect of (cortex: F2,2754 = 108.79, p < 0.0001; hippocampus: F2,2754 = 24.59, p < 0.0001; pre-optic area: F2,2754 = 72.93, p < 0.0001), 6-OHDA treatment (cortex: F1,2754 = 5.14, p = 0.023; hippocampus: F1,2754 = 16.51, p < 0.0001; pre-optic area: F1,2754 = 113.17, p < 0.0001), distance from soma (cortex: F18,2754 = 40.82, p < 0.0001; hippocampus: F18,2754 = 37.55, p < 0.0001; pre-optic area: F18,2754 = 29.74, p < 0.0001), and SF x 6-OHDA interaction (cortex: F2,2754 = 52.79, p < 0.0001; hippocampus: F2,2754 = 37.78, p < 0.0001; pre-optic area: F2,2754 = 28.68, p < 0.0001) in all three brain regions, as determined by univariate GLM. In addition, there was a significant interaction effect of distance from soma with SF (F36,2754 = 1.83, p = 0.001) and 6-OHDA treatment (F18,2754 = 2.65, p < 0.0001) in cortex and pre-optic area, respectively. There was a significant three-way interaction in hippocampus (F36,2754 = 1.91, p = 0.001) and pre-optic area (F36,2754 = 2.16, p < 0.0001), but not in cortex. In comparison with controls, chronic SF caused microglia hyper-ramification in all three brain regions (distance from centre of soma: 14 to 32 µm in cortex, 17 to 26 µm in hippocampus, and 20 to 35 µm in pre-optic area). A significantly higher ramification in cortex at 11 to 32 µm, in hippocampus at 26 to 27.5 and 32 to 33.5 µm, and in pre-optic area at 20 to 35 µm from centre of soma was observed in chronic SF mice compared with recovery mice (Bonferroni posthoc test, p < 0.05, Fig 6c, d, g, h, k, l).
Hippocampal microglial response was still observed after 1 week of recovery, as ramification was significantly higher in mice subjected to recovery at 12.5 to 14 µm distance from centre of soma (Bonferroni posthoc test, p < 0.05, Fig 6g, h). Further, 6-OHDA administration diminished the neuroinflammatory response from chronic SF, as evidenced by significantly lower microglia ramification in cortex (at 23 to 33.5 µm distance from centre of soma), hippocampus (at 17 to 23 µm, 26 and 30.5 µm distance from centre of soma) and pre-optic area (at 14, and 20 to 30.5 µm distance from centre of soma) in mice subjected to 6-OHDA treatment following chronic SF, relative to their counterparts injected with vehicle (Bonferroni posthoc test, p < 0.05, Fig 6c, d, g, h, k, l). The pre-optic area of recovery mice injected with 6-OHDA had lower microglia ramification at 11 to 14 µm distance from centre of soma, compared with recovery mice not injected with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig 6k, l).