CD40-CD40 Ligand Pathway is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis

Abstract

Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40-CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40-CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40-CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40-CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis.

Figure 1

(A) Western blotting analysis on activated microglial culture with LPS and treated with antiCD40 and CD40L. (B) Expression CD40 and CD40L for Western blotting 24 h in the hippocampus of rats submitted for sham and CLP or CLP and CLP plus minocycline. Levels of CD40 and CD40L (C) 12 h (D) 24 h and (E) 48 h by Western blotting. Data are expressed as protein levels/β-actin. For five independent experiments performed in duplicate (mean ± SD). Representative bands. *Different from sham (p < 0.05); ^#different from CLP plus control (p < 0.05).

Figure 2

Cytokine levels (A) TNF-α, (B) IL-1β and (C) IL-6 in microglial culture activated with LPS and treated or not treated with anti-CD40; cytokines (D) TNFα, (E) IL-1β and (F) IL-6 in the hippocampus of rats submitted to sepsis by CLP treated or not with anti-CD40 (1, 10 or 100 μg/kg) and MPO activity in the hippocampus of rats submitted to sepsis by CLP treated or not treated with anti-CD40 (1, 10 or 100 μg/kg). Data are expressed as pg/mg protein and nmol/mg protein, for independent experiments performed in duplicate (mean ± SD). *Different from sham (p < 0.05); ^#different from CLP plus saline (p < 0.05); ^&different from CLP plus anti-CD401 μg/kg (p < 0.05).

Figure 3

Nitrate concentration (A) thiobarbituric acid reactive substances (TBARS) (B) and permeability of blood brain barrier (BBB) (C) in the hippocampus of rats submitted to sepsis by CLP treated or not treated with anti-CD40 (1, 10 or 100 μg/kg). Data are expressed as nmol/mg protein and ng/mL, for independent experiments performed in duplicate (mean ± SD). *Different from sham (p < 0.05); ^#different from CLP plus saline (p < 0.05); ^&different from CLP plus anti-CD401 μg/kg (p < 0.05).

Figure 4

Kaplan-Meier curve of survival times in sham, CLP and CLP plus anti-CD40 (100 μg/kg) groups. *Different from sham (p < 0.05). Data are analyzed to log-rank.

Figure 5

Habituation on (A) the open field and (B) inhibitory avoidance. Animals were submitted to sham-operated and CLP treated or not treated with anti-CD40 (100 μg/kg). Data are presented as mean ± SEM to open field and as median and interquartile ranges to inhibitory avoidance, n = 10 rats per group. *Different training (p < 0.05). sec, Seconds.