Lipopolysaccharide Exposure Differentially Alters Plasma and Brain Inflammatory Markers in Adult Male and Female Rats - PubMed (original) (raw)
Lipopolysaccharide Exposure Differentially Alters Plasma and Brain Inflammatory Markers in Adult Male and Female Rats
Hannah A Nonoguchi et al. Brain Sci. 2022.
Abstract
Humans and rodents have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. Therefore, we examined whether inflammatory response to LPS and the associated BBB disruption differed in male and female adult rats. Rats were treated with saline or two injections of 1 mg/kg LPS and studied 24 h after the second LPS injection. Plasma isolated from trunk blood and brain tissue homogenates of the prefrontal cortex (PFC), dorsal striatum (DS), hippocampus, and cerebellum were analyzed for cytokines and chemokines using a 9-plex panel from Meso Scale Discovery. BBB disruption was analyzed with tight junction proteins claudin-5 and VE-cadherin via Western blotting and VEGF by ELISA. This allowed us to compare sex differences in the levels of individual cytokines as well as associations among cytokines and expression of tight junction proteins between the plasma and specific brain regions. Higher levels of interferon-γ, interleukin-10 (IL-10), IL-13, IL-4, CXCL-1, and VEGF in the plasma were revealed compared to the brain homogenates, and higher levels of TNFα, IL-1β, IL-6, and IL-5 in the PFC were seen compared with plasma and other brain regions in males. Females showed higher levels of plasma CXCL1 and VEGF compared to males, and males showed higher levels of PFC TNFα, IL-6, IL-4, and VEGF compared to females. LPS induced significant increases in plasma cytokines and VEGF in both sexes. LPS did not significantly alter cytokines in brain tissue homogenates, however, it increased chemokines in the PFC, DS, and hippocampus. In the PFC, LPS produced BBB disruption, which is evident as reduced expression of claudin-5 in males and reduced expression of VE-cadherin in both sexes. Taken together, our results reveal significant sex differences in pro-inflammatory cytokine and chemokine levels in plasma and brain that were associated with BBB disruption after LPS, and validate the use of multiplex assay for plasma and brain tissue samples.
Keywords: LPS; dorsal striatum; hippocampus; meso scale discovery; prefrontal cortex; sex differences.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Schematic of the time line of body weight measures, LPS injections, and euthanasia (a). Body weight data expressed as percent change in weight 2 from weight 1 (b). Sagittal view of the adult rat brain indicating the region of interest for dissections (c). Prefrontal cortex, PFC; dorsal striatum, DS; hippocampus, Hipp; cerebellum, CB. Data are represented as mean ± S.E.M. n = 4 control female, n = 5 LPS female, n = 3 control male, n = 4 LPS male. p < 0.05 vs. controls by post-hoc tests.
Figure 2
A heatmap was generated to display 10 different measured inflammatory markers in healthy control (saline treated) female (F) and male (M) rats in plasma and brain tissue homogenates. Mean values without standard error are indicated in the heatmap. However, statistical analysis was performed on mean ± S.E.M. values. Prefrontal cortex, PFC; dorsal striatum, DS; hippocampus, Hipp; cerebellum, CB. White to purple transitions (color key; pg/mL) indicate, respectively, low to high average cytokine or chemokine or VEGF concentration. Data are represented as mean. n = 4 control female, n = 2–3 control male. * p < 0.05 vs. all other samples within each sex, # p < 0.05 vs. the opposite sex by post-hoc tests.
Figure 3
Cytokines, chemokines, and VEGF were measured in the plasma of female and male rats that were exposed to LPS or saline injections. control female, n = 5 LPS female, n = 3 control male, n = 4 LPS male. Pro-inflammatory cytokines are indicated in panels (a–d). Anti-inflammatory cytokines are indicated in panels (e–g). Chemokine is indicated in panel (h) and angiogenesis marker is indicated in panel (i). Main effect of LPS (p < 0.05) is indicated by solid line with flat arrowheads and main effect of sex (p < 0.05) is indicated by dashed line with flat arrowhead. Differences between saline and LPS within each sex and between sexes (p < 0.05) are indicated by separate solid lines.
Figure 4
Cytokines, chemokines, and VEGF were measured in the prefrontal cortex (PFC) of female and male rats that were exposed to saline or LPS challenges. Pro-inflammatory cytokines are indicated in panels (a–d). Anti-inflammatory cytokines are indicated in panels (e–h). Chemokine is indicated in panel (i) and angiogenesis marker is indicated in panel (j). Data are represented as mean ± S.E.M. n = 4 control female, n = 5 LPS female, n = 2 control male, n = 4 LPS male. Main effect of LPS (p < 0.05) is indicated by solid line with flat arrowheads, and main effect of sex (p < 0.05) is indicated by dashed line with flat arrowhead. Differences between saline and LPS within each sex and between sexes (p < 0.05) are indicated by separate solid lines.
Figure 5
Cytokines, chemokines, and VEGF were measured in the dorsal striatum (DS) of female and male rats that were exposed to saline or LPS challenges. Pro-inflammatory cytokines are indicated in panels (a–d). Anti-inflammatory cytokines are indicated in panels (e–h). Chemokine is indicated in panel (i) and angiogenesis marker is indicated in panel (j). Data are represented as mean ± S.E.M. n = 4 control female, n = 5 LPS female, n = 2 control male, n = 4 LPS male. Main effect of LPS (p < 0.05) is indicated by solid line with flat arrowheads, and main effect of sex (p < 0.05) is indicated by dashed line with flat arrowhead.
Figure 6
Cytokines, chemokines, and VEGF were measured in the hippocampus (Hipp) of female and male rats that were exposed to saline or LPS challenges. Pro-inflammatory cytokines are indicated in panels (a–d). Anti-inflammatory cytokines are indicated in panels (e–h). Chemokine is indicated in panel (i) and angiogenesis marker is indicated in panel (j). Data are represented as mean ± S.E.M. n = 4 control female, n = 5 LPS female, n = 2 control male, n = 4 LPS male. Interaction (p < 0.05) is indicated by a solid straight line with arrowheads, main effect of LPS (p < 0.05) is indicated by solid line with flat arrowheads, and main effect of sex (p < 0.05) is indicated by dashed line with flat arrowhead. Differences between saline and LPS within each sex and between sexes (p < 0.05) are indicated by separate solid lines.
Figure 7
Cytokines, chemokines, and VEGF were measured in the cerebellum (CB) of female and male rats that were exposed to saline or LPS challenges. Pro-inflammatory cytokines are indicated in panels (a–d). Anti-inflammatory cytokines are indicated in panels (e–h). Chemokine is indicated in panel (i) and angiogenesis marker is indicated in panel (j). Data are represented as mean ± S.E.M. n = 4 control female, n = 5 LPS female, n = 2 control male, n = 4 LPS male. No significant differences were seen in any of the markers studied.
Figure 8
Western blotting analysis of tight junction proteins and neuroinflammatory markers in the prefrontal cortex (PFC) of female and male rats that were exposed to saline or LPS challenges. Representative immunoblots with corresponding Coomassie (Coom) blot is indicated in (a). Coom band 150 kDa was used for female samples and band 250/150 kDa was used for male samples. Claudin-5, Cld-5; VE-Cadherin, VE-Cad; phospho NF-kB, pNF-kB; total NF-kB, tNF-kB; control, con. (b) Quantitative data represented as mean ± S.E.M. of percent change in density of protein from respective controls (indicated as dashed line). n = 4 control female, n = 5 LPS female, n = 3 control male, n = 4 LPS male. * p ≤ 0.05 between saline and LPS within each sex.
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