Anesthesia-induced developmental neurotoxicity in the setting of systemic inflammation: the role of microglia

Abstract

Although it is well documented in animal research that an early exposure to general anesthetics during critical stages of synaptogenesis disturbs normal brain development ultimately leading to cognitive and affective impairments, it is less clear whether and how surgical interventions and/or underlying systemic inflammation impact the detrimental effects of general anesthetics. Some emerging evidence suggests that aseptic systemic inflammation preceding exposure to the commonly used general anesthetics worsens anesthesia-induced neuroapoptosis and activates inflammasome pathways while resulting in impaired cognitive-affective behaviors. To improve our understanding of the underlying mechanisms, here we focused on multicellular interactions between damaged neurons and microglia since microglia is the resident macrophages within the brain that respond to stress. Using infant rats (post-natal day 7) and most commonly used inhaled anesthetic, sevoflurane, we examine microglia role in sevoflurane-induced inflammation-propagated developmental neurotoxicity. We show that sevoflurane exposure leads to a significant neuroapoptosis in young rat pup hippocampal subiculum, a neuroapoptosis that is worsened in the setting of systemic inflammation caused by either lipopolysaccharide (LPS) injection or trauma (tibial fracture). The worsening is not only shown in terms of the intensity of neuroapoptosis but in its duration and onset. We further report that sevoflurane-induced neuroapoptosis triggers activation of microglia, which in turn releases proinflammatory cytokine MCP-1 and upregulates endothelial cell adhesion molecule, ICAM-1. This leads to T-lymphocyte infiltration in the hippocampal subiculum, an event that further perpetuates microglia activation in an attempt to control neuroapoptosis which is suggested by the fact that microglia depletion leads to a significant worsening of sevoflurane-induced developmental neuroapoptosis. Our work gets us a step closer to making our animal work more relevant to the clinical setting and hence more translational. This is vitally important considering that exposure to anesthesia is exceedingly rare in the absence of any kind of a pathological process.

Keywords: developmental neurotoxicity; lipopolysaccharide; microglia; neonatal rats; neuroinflammation; sevoflurane; tibial fracture.

FIGURE 1

Preceding tibial fracture worsens sevoflurane-induced neuroapoptosis in the hippocampal subiculum of PND7 rat pups. Histomorphological analyses of neuronal apoptosis in the hippocampal subiculum are shown in the representative images of activated caspase-3 (AC-3⁺) staining in the hippocampal subiculum in control (A), trauma (B), sevoflurane (C) and trauma + sevoflurane (D) groups. The number of AC-3⁺ cells was increased in trauma + sevoflurane group compared with controls. ×10 magnification, scale bar is 100 μm. Bar graphs show the quantification analysis of AC-3⁺ positive cells per square millimeter in the hippocampal subiculum. Although trauma alone was indistinguishable from the control group, 3 hours of sevoflurane exposure significantly increased AC-3+cells compared with the control or trauma group. However, sevoflurane exposure in the setting of trauma (trauma + sevo group) the number of AC-3⁺ cells were greater compared with either treatment alone. Because there were no gender differences in AC-3⁺ cells densities, the results from both sexes were combined. For the ease of side-by-side comparisons, we include shadow bar graph in the upper right corner that shows previously published effects of lipopolysaccharide (LPS) on sevo-(sevoflurane) induced developmental neuroapoptosis. [12]. One-way ANOVA with Tukey’s post hoc. ns - non-significant, *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 2

Short (1 hour) sevoflurane exposure in the setting of systemic inflammation induces significant neuroapoptosis in the hippocampal subiculum of PND7 rat pups. Quantitative analyses of AC-3⁺ positive cells per square millimeter in the hippocampal subiculum showed that a short exposure to sevoflurane alone is similar to controls, whereas the AC3⁺ level in sevoflurane group in the setting of LPS-induced systemic inflammation (LPS + sevo group) is significantly upregulated when compared to sham controls or the other two experimental groups. Because there were no gender difference in AC-3⁺ cells densities, the results from both sexes are combined. One-way ANOVA with Tukey’s post hoc. **p < 0.01, ***p < 0.001.

FIGURE 3

Sevoflurane exposure in the setting of LPS-induced systemic inflammation causes prolonged neuroapoptosis in the hippocampal subiculum of PND7 rat pups. Quantitative analyses of AC-3⁺ positive cells per square millimeter in hippocampal subiculum showed that the AC3⁺ levels in the LPS and sevoflurane alone groups at 8 h post-exposure are back to the control levels whereas the AC3⁺ levels in sevo-treated groups in the setting of systemic inflammation (LPS + sevo group) remain significantly upregulated when compared to either control, LPS or sevoflurane alone groups. The bar to the far right shows that the AC3⁺ levels in LPS + sevo group are of the similar magnitude at 8 h post-sevoflurane exposure as the one detected at 2 h post-sevoflurane exposure. Because there were no gender difference in AC-3⁺ cells densities, the results from both sexes are combined. One-way ANOVA with Tukey’s post hoc. ns - non-significant, ****p < 0.0001.

FIGURE 4

Sevoflurane exposure causes microglia activation in the hippocampal subiculum of PND7 rat pups that is worsen in the setting of trauma- and LPS-induced systemic inflammation. (A) Resting microglia (left panel) are ramified with processes (arrows) that probe the extracellular environment and become amoeboid (right panel) in activated state (arrowheads). ×60 magnification, scale bar 20 μm. (B) At 2 h post-sevoflurane exposure we detect significant (∼2-fold) increase in microglia activation in LPS or sevoflurane alone groups in the subiculum compared to their respective controls. When sevoflurane was administered following LPS, we report a further increase in microglial activation (∼3-fold above controls). (C) At 2 h post-sevoflurane exposure we detect significant (∼2-fold) increase in microglia activation in trauma (tibia fracture) or sevoflurane alone groups in the subiculum compared to their respective controls. When sevoflurane was administered following trauma, we report a further increase in microglial activation (∼4.5-fold above controls). Because there were no gender difference in microglia activation densities, the results from both sexes are combined. One-way ANOVA with Tukey’s post hoc. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Microglia inhibition with minocycline results in worsening of sevoflurane-induced neuroapoptosis in the setting of LPS-induced systemic inflammation. When PND7 rat pups were treated with minocycline (Mino) at the time of LPS administration and again 12 h later at the time of sevoflurane (sevo) exposure we found that the density of AC3⁺ cells evaluated 2 hours post-sevoflurane was significantly increased in neuroapoptosis compared to those without minocycline. The difference between minocycline-pretreated groups and other animal groups (controls, LPS alone, sevoflurane alone) was not significant. Because there were no gender difference in microglia activation densities, the results from both sexes are combined. Two-way ANOVA with Sidak’s post hoc. ****p < 0.0001.

FIGURE 6

Microglia depletion with PLX5622 results in worsening of sevoflurane-induced neuroapoptosis in the setting of LPS-induced systemic inflammation. When PND7 rat pups were treated with a more selective microglia inhibitor, PLX5622 at the time of LPS administration and again 12 h later at the time of sevoflurane exposure, we found that the density of AC3⁺ cells evaluated 2 hours post-sevoflurane was significantly increased compared to vehicle-treated counterparts. The difference between PLX5622-pretreated animals and corresponding vehicle-treated animals within groups (controls, LPS alone, sevoflurane alone) was not significant. Because there were no gender difference in microglia activation densities, the results from both sexes are combined. ×10 magnification, scale bar 150 μm. Two-way ANOVA with Sidak’s post hoc, **p < 0.01.

FIGURE 7

Sevoflurane exposure in the setting of LPS-induced systemic inflammation causes a significant increase in microglia-secreted cytokine, MCP-1 and cell adhesion molecule, ICAM-1 resulting in T-lymphocyte infiltration in hippocampal subiculum of PND7 rat pups. (A) There is a significant upregulation MCP-1 mRNA in LPS + sevo group when compared to controls and sevo-alone groups. (B) Similarly, we found that, when compared to controls and sevo alone groups, there is a significant upregulation of ICAM-1 mRNA expression in LPS + sevo group. (C) There is a significant increase in the proportion of T-lymphocytes in the hippocampus of LPS + sevo rat pups compared to controls. (D) Representative flow scatter plots in control group. (E) Representative flow scatter plots in sevoflurane group in the setting of systemic inflammation. Because there were no gender difference, the results from both sexes are combined. One-way ANOVA with Tukey’s post hoc. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 8

Schematic diagram of the proposed pathway in the setting of sevoflurane-induced systemic inflammation-propagated developmental neuroapoptosis in the hippocampal subiculum of PND7 rat pups. Sevoflurane-induced neuroapoptosis triggers activation of neuroprotective microglia, which in turn releases proinflammatory cytokine, MCP-1 and activates endothelial cell adhesion molecule, ICAM-1. This leads to T-lymphocyte infiltration in the subiculum, an event that further propagates neuroprotective microglia activation in an attempt to control neuroapoptosis, clear the debris and restore the neuropil.