Lethal versus surviving sepsis phenotypes displayed a partly differential regional expression of neurotransmitters and inflammation and did not modify the blood-brain barrier permeability in female CLP mice

Abstract

Background: Septic encephalopathy is frequent but its pathophysiology is enigmatic. We studied expression of neurotransmitters, inflammation and integrity of the blood-brain barrier (BBB) in several brain regions during abdominal sepsis. We compared mice with either lethal or surviving phenotype in the first 4 sepsis days. Mature CD-1 females underwent cecal ligation and puncture (CLP). Body temperature (BT) was measured daily and predicted-to-die (within 24 h) mice (for P-DIE; BT < 28 °C) were sacrificed together (1:1 ratio) with mice predicted-to-survive (P-SUR; BT > 35 °C), and healthy controls (CON). Brains were dissected into neocortex, cerebellum, midbrain, medulla, striatum, hypothalamus and hippocampus.

Results: CLP mice showed an up to threefold rise of serotonin in the hippocampus, 5-hydroxyindoleacetic and homovanillic acid (HVA) in nearly all regions vs. CON. Compared to P-SUR, P-DIE mice showed a 1.7 to twofold rise of HVA (386 ng/g of tissue), dopamine (265 ng/g) and 3,4-Dihydroxyphenylacetic acid (DOPAC; 140 ng/g) in the hippocampus, hypothalamus and medulla (174, 156, 82 ng/g of tissue, respectively). CLP increased expression of TNFα, IL-1β and IL-6 mRNA by several folds in the midbrain, cerebellum and hippocampus versus CON. The same cytokines were further elevated in P-DIE vs P-SUR in the midbrain and cerebellum. Activation of astrocytes and microglia was robust across regions but remained typically phenotype independent. There was a similar influx of sodium fluorescein across the BBB in both P-DIE and P-SUR mice.

Conclusions: Compared to survivors, the lethal phenotype induced a stronger deregulation of amine metabolism and cytokine expression in selected brain regions, but the BBB permeability remained similar regardless of the predicted outcome.

Keywords: Abdominal sepsis; Brain amines; Cytokines; Outcome prediction; Phenotyping.

Fig. 1

Regional concentrations of neurotransmitters and their selected metabolites (A–F) in the brains of septic (CLP) and healthy control mice. Septic mouse data were pooled regardless of the predicted outcome. Mice were sacrificed within days 2–4 post-CLP. CLP mice: n = 15–16 in all regions except neocortex (n = 10) and striatum (n = 7–10). Control mice: n = 7 in all regions except neocortex (n = 4) and striatum (n = 3). Data as mean ± SD. *P < 0.05. HVA homovanillic acid, HIAA hydroxyindoleacetic acid, DOPAC dihydroxyphenylacetic acid

Fig. 2

Regional concentrations of neurotransmitters and their selected metabolites (A–F) in the brains of septic (CLP) mice predicted-to-die (P-DIE), predicted-to-survive (P-SUR) and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. P-DIE: n = 7–8 in all regions except neocortex (n = 5) and striatum (n = 3). P-SUR: n = 7–8 in all regions except neocortex (n = 5) and striatum (n = 4) Control mice: n = 7 in all regions except neocortex (n = 4) and striatum (n = 3). Data as mean ± SD. *P < 0.05 between P-DIE and P-SUR. HVA homovanillic acid, HIAA hydroxyindoleacetic acid; DOPAC: dihydroxyphenylacetic acid

Fig. 3

Comparison of cytokine gene expression in the midbrain of septic (CLP) mice predicted-to-die (P-DIE), predicted-to-survive (P-SUR) and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. Insets depict comparison of all pooled septic mice (regardless of the predicted outcome) to healthy controls. k = 1000 ×

Fig. 4

Comparison of cytokine gene expression in the cerebellum of septic (CLP) mice predicted-to-die (P-DIE), predicted-to-survive (P-SUR) and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. Insets depict comparison of all pooled septic mice (regardless of the predicted outcome) to healthy controls. k = 1000 ×

Fig. 5

Comparison of cytokine gene expression in the hypothalamus of septic (CLP) mice predicted-to-die (P-DIE), predicted-to-survive (P-SUR) and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. Insets depict comparison of all pooled septic mice (regardless of the predicted outcome) to healthy controls. k = 1000 ×

Fig. 6

Comparison of cytokine gene expression in the hippocampus of septic (CLP) mice predicted-to-die (P-DIE), predicted-to-survive (P-SUR) and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. Insets depict comparison of all pooled septic mice (regardless of the predicted outcome) to healthy controls. k = 1000 ×

Fig. 7

Low and high-resolution images of immunohistochemical staining with GFAP- (A–C) and IBA-1 (E, F) antibodies in the medulla oblongata of septic P-DIE mice (A, D), P-SUR mice (B, E) and CON mice (C, F). bar = 160 µm for low resolution images; bar = 40 µm for high resolution images; P-DIE predicted-to-die, P-SUR predicted-to-survive, IBA-1 ionized calcium-binding protein (microglia/macrophage-specific), GFAP glial fibrillary acidic protein (astrocyte-specific)

Fig. 8

Comparison of blood–brain barrier (BBB) permeability with sodium-fluorescein (NaFl) in the midbrain (A), neocortex (B) and cerebellum (C) of septic P-DIE mice, P-SUR mice and healthy control mice. P-DIE mice were sacrificed within days 2–4 post-CLP and matched with P-SUR mice. P-DIE predicted-to-die, P-SUR predicted-to-survive. Insets depict comparison of all pooled septic mice (regardless of the predicted outcome) to healthy controls

Fig. 9

Representative images of the thalamus of septic P-DIE (A), P-SUR (B) and healthy control (C) mice in transmission electron microscopy (TEM). No lesions in cellular ultrastructure were detectable in any of the evaluated animals regardless of group assignment. The blood–brain barrier (BBB) was intact even in animals of the P-DIE group. The insert shows the magnification of an intact tight junction (arrowheads) annotated by a rectangle (A). Note mild perivascular edema (E) present in all groups. Bars = 2500 nm, bar in insert = 500 nm; E perivascular edema, L capillary lumen, N endotheliocyte nucleus, P-DIE predicted-to-die, P-SUR predicted-to-survive