Systemic inflammation following traumatic injury and its impact on neuroinflammatory gene expression in the rodent brain

Abstract

Background: Trauma can result in systemic inflammation that leads to organ dysfunction, but the impact on the brain, particularly following extracranial insults, has been largely overlooked.

Methods: Building upon our prior findings, we aimed to understand the impact of systemic inflammation on neuroinflammatory gene transcripts in eight brain regions in rats exposed to (1) blast overpressure exposure [BOP], (2) cutaneous thermal injury [BU], (3) complex extremity injury, 3 hours (h) of tourniquet-induced ischemia, and hind limb amputation [CEI+tI+HLA], (4) BOP+BU or (5) BOP+CEI and delayed HLA [BOP+CEI+dHLA] at 6, 24, and 168 h post-injury (hpi).

Results: Globally, the number and magnitude of differentially expressed genes (DEGs) correlated with injury severity, systemic inflammation markers, and end-organ damage, driven by several chemokines/cytokines (Csf3, Cxcr2, Il16, and Tgfb2), neurosteroids/prostaglandins (Cyp19a1, Ptger2, and Ptger3), and markers of neurodegeneration (Gfap, Grin2b, and Homer1). Regional neuroinflammatory activity was least impacted following BOP. Non-blast trauma (in the BU and CEI+tI+HLA groups) contributed to an earlier, robust and diverse neuroinflammatory response across brain regions (up to 2-50-fold greater than that in the BOP group), while combined trauma (in the BOP+CEI+dHLA group) significantly advanced neuroinflammation in all regions except for the cerebellum. In contrast, BOP+BU resulted in differential activity of several critical neuroinflammatory-neurodegenerative markers compared to BU. t-SNE plots of DEGs demonstrated that the onset, extent, and duration of the inflammatory response are brain region dependent. Regardless of injury type, the thalamus and hypothalamus, which are critical for maintaining homeostasis, had the most DEGs. Our results indicate that neuroinflammation in all groups progressively increased or remained at peak levels over the study duration, while markers of end-organ dysfunction decreased or otherwise resolved.

Conclusions: Collectively, these findings emphasize the brain's sensitivity to mediators of systemic inflammation and provide an example of immune-brain crosstalk. Follow-on molecular and behavioral investigations are warranted to understand the short- to long-term pathophysiological consequences on the brain, particularly the mechanism of blood-brain barrier breakdown, immune cell penetration-activation, and microglial activation.

Keywords: Blast; Differential gene expression; Neuroinflammation; Polysystem injury; Secondary brain injury.

Fig. 1

Injury models and experimental design schematic. Adult male Sprague-Dawley rats (400–500 g) either received (1) head-on whole-body blast overpressure exposure [120 kPa; BOP], (2) a partial-thickness cutaneous thermal burn [BU], (3) BOP+BU, (4) complex extremity trauma involving closed femoral fracture and soft-tissue crush injury, 3 h of prolonged tourniquet-induced limb ischemia and limb amputation through the zone of injury [CEI+tI+HLA], or (5) BOP+CEI plus delayed hind limb amputation [BOP+CEI+dHLA]. Age- and sex-matched naïve rats (n = 7) served as controls. Following injury, whole blood obtained from tail vein venipuncture was obtained from each cohort (1, 3, or 72 hpi) at the timepoint that preceded euthanasia. At 6, 24, and 168 h postinjury (hpi), cohorts of rats (n = 5–7 timepoint/injury paradigm) were euthanized, and eight anatomic regions of the brain were dissected and profiled for neuroinflammatory-neurodegeneration gene expression signatures using a custom low-density RT‒qPCR microarray. The molecular heterogeneity of the gene profiles of the trauma-induced changes in the brain over time was compared with that of the naïve steady state of control animals. This scientific illustration was created in the Biorender web interface

Fig. 2

Clinical indicators of systemic inflammation and remote organ dysfunction. a Changes in serum cytokine/chemokine levels were profiled in the naïve and trauma cohorts using a multianalyte MesoScale diagnostic profiling platform. b Serum chemical markers of end-organ damage were measured using a Heska Element DCX chemistry analyzer. Serum values from naïve animals (n = 7) were used to construct the 95% confidence intervals (gray shading). All the graphs show the mean values (n = 6–7 samples/timepoint/injury model) ± SEMs; significant differences (p < .05) in naïve animals are denoted with closed circles, and nonsignificant values are denoted with open circles at the indicated timepoints postinjury. The values were compared using one-way ANOVA and Tukey post-hoc analysis. Abbreviations: alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr)

Fig. 3

Heatmap depicting the expression of 74 neuroinflammatory genes in eight brain regions according to the type of trauma. The values reported were calculated based on the 2^−ΔΔC(t) method and normalized to the geometric mean of three specific, constitutively active, and stable housekeeping genes (B2m, Gapdh, Hprt1) and regional brain biopsy samples from naïve uninjured animals. Blue shading represents a decrease in expression compared to that in the naïve group, red shading represents an increase in expression compared to that in the naïve group, and white shading represents nonsignificant changes (− 2 to 2 fold-change). The color intensity correlates with the magnitude of gene expression relative to that in naïve, uninjured rats

Fig. 4

The magnitude and neuroinflammatory gene expression response to trauma are brain region dependent, and the mode of injury is independent. t-distributed stochastic neighbor embedding (tSNE) data visualization and analysis were used to plot the 2^−ΔΔC(t) profiles of 74 genes for each regional brain biopsy sample, and the results demonstrated that the gene expression profiles of individual samples tended to cluster by region as opposed to timepoint or injury model. The dotted area around the clusters represents the circle of best fit at an 85% confidence interval for gene expression

Fig. 5

Brain region-specific changes in neuroinflammatory gene transcript levels over time correlate with injury severity. Stacked bar charts are used to illustrate the number of differentially expressed genes (DEGs; up- or downregulated) in eight anatomical brain regions for each injury pattern. The data quantitatively reflect the number of genes with significant expression changes compared to those in naïve, uninjured control animals. The brain regions are displayed in ascending order based on the aggregate number of significantly altered genes (DEG counts irrespective of injury pattern and timepoint: cerebellum: 87, hippocampus: 96, amygdala: 98, striatum: 106, prefrontal cortex: 116, neocortex: 138, thalamus: 170, and hypothalamus: 180)

Fig. 6

Neuroinflammatory gene signatures remain elevated at 168 hpi following various forms of trauma. The mean relative expression of individual genes across brain regions at 168 hpi, depicted by injury mode, was normalized to the geometric mean of three specific, constitutively active, and stable housekeeping genes (B2m, Gapdh, Hprt1) and regional brain biopsies from naïve uninjured animals. Two-way ANOVA was used to assess the main effect of injury on each of the brain regions. Tukey‒Kramer post hoc analyses were used to assess differences between injured and naïve expression changes. Asterisks (*) indicate significant differences from naïve uninjured controls. *p < .05, **p < .01, and ***p < .001

Fig. 7

Protein–protein interaction networks of the differentially expressed genes in each trauma model. STRING protein–protein interaction (PPI) software was used to construct the gene networks of the differentially expressed genes (DEGs) determined in different brain regions for each injury group to highlight gene–gene interactions. Each color represents a different gene cluster

Fig. 8

Systemic inflammation elicited persistent acute neuroinflammation following non-blast or blast-associated trauma. The mean levels of serum cytokines (IL-1β, IL-6, CXCL1, TNF-α, MCP-1, and IFN-γ) and clinical chemistry markers of end organ damage (BUN:Cr ratio, AST, ALT, and Albumin) normalized to those of naïve uninjured controls of individual animals were used to compute the relative intensity of (a) systemic inflammation and (b) level of end organ damage following three types of injury: cranial trauma (as represented by BOP), noncranial trauma (as represented by BU and CEI+tI+HLA), or combined cranial/noncranial trauma (as represented by BOP+BU and BOP+CEI+dHLA) over time. The number of significantly differentially expressed genes was utilized to compute the (c) relative intensity of trauma-induced neuroinflammation gene expression profile for each type of trauma over time. Serum systemic inflammation and circulating clinical biomarkers of end-organ damage peak at 6 h postinjury (hpi) and wane or return to baseline/naïve levels by 168 h postinjury, whereas the neuroinflammatory gene expression profiles in the context of various forms of trauma remain elevated by 168 hpi compared to those in the naïve steady state