Inhibition of complement C5a prevents breakdown of the blood-brain barrier and pituitary dysfunction in experimental sepsis

Abstract

Introduction: Septic encephalopathy secondary to a breakdown of the blood-brain barrier (BBB) is a known complication of sepsis. However, its pathophysiology remains unclear. The present study investigated the effect of complement C5a blockade in preventing BBB damage and pituitary dysfunction during experimental sepsis.

Methods: Using the standardised caecal ligation and puncture (CLP) model, Sprague-Dawley rats were treated with either neutralising anti-C5a antibody or pre-immune immunoglobulin (Ig) G as a placebo. Sham-operated animals served as internal controls.

Results: Placebo-treated septic rats showed severe BBB dysfunction within 24 hours, accompanied by a significant upregulation of pituitary C5a receptor and pro-inflammatory cytokine expression, although gene levels of growth hormone were significantly attenuated. The pathophysiological changes in placebo-treated septic rats were restored by administration of neutralising anti-C5a antibody to the normal levels of BBB and pituitary function seen in the sham-operated group.

Conclusions: Collectively, the neutralisation of C5a greatly ameliorated pathophysiological changes associated with septic encephalopathy, implying a further rationale for the concept of pharmacological C5a inhibition in sepsis.

Figure 1

Anti-C5a ameliorates impairment of the blood-brain barrier after caecal ligation and puncture (CLP). (a-e) Brains were surgically removed, snap-frozen and tissue sections (10 μm) were obtained. Cerebral extravasation of rat albumin was assessed by immunohistochemistry 24 hours after CLP or sham procedure, three samples per experimental condition. Stains displayed are representative of three independent experiments. (f, g) Comparison of Evans Blue extravasation into the cerebellum and pituitary in IgG-treated or anti-C5a treated rats 24 hours post CLP. Displayed depictions are representative of four animals. (h, i) Quantification of Evans Blue extravasation into the brain or pituitary by determination of mg Evans Blue/mg tissue ratio in different groups at indicated time-points, four for each experimental condition. # p < 0.05 between sham and 24 hours CLP animals; * p < 0.05 between IgG-treated and anti-C5a-treated rats.

Figure 2

Pituitary expression of C5a receptor (R) during experimental sepsis in IgG or anti-C5a IgG treated sham animals and septic littermates 24 hours after caecal ligation and puncture (CLP) procedure. (a) Following total RNA isolation from pituitary tissue, pituitary C5aR mRNA expression was assessed by real-time PCR. For each bar, sample size was five to seven. (b) Five pituitary tissue samples were removed at indicated time-points, homogenised and C5aR protein expression was analysed by Western blotting. For sham groups, two samples were taken; for CLP groups, three samples were taken. Blot is representative for three independent experiments. GAPDH = glyceraldehyde 3-phosphate dehydrogenase. # p < 0.05 between sham and 24 hours CLP animals; * p < 0.05 between IgG-treated and anti-C5a-treated rats.

Figure 3

Expression of inflammatory mediators in the pituitary. Pituitary tissue samples were surgically removed, snap-frozen, homogenised and total RNA was extracted. Samples were then analysed by quantitative real-time PCR analysis. Expression of (a) TNF and (b) IL-6 mRNA in the pituitary 24 hours after sham procedure or caecal ligation and puncture (CLP). Five to seven samples were taken per experimental condition. GAPDH = glyceraldehyde 3-phosphate dehydrogenase. # p < 0.05 between sham and 24 hours CLP animals; * p < 0.05 between IgG-treated and anti-C5a-treated rats.

Figure 4

Evaluation of pituitary function after caecal ligation and puncture (CLP). Pituitary tissue samples were removed from four to five mice, snap-frozen, homogenised in Trizol and total RNA was extracted. Assessment of mRNA expression of (a) proopiomelanocortin (POMC) and (b) growth hormone (GH) 24 hours after CLP or sham operation by real-time PCR. Whole blood samples were drawn at given time-points by puncture of the inferior vena cava, plasma was obtained by centrifugation and subjected to ELISA analysis. Samples were assessed for (c) GH or (d) corticosterone under identical conditions. For all graphs, there were five to seven samples per experimental condition. GAPDH = glyceraldehyde 3-phosphate dehydrogenase. # p < 0.05 between sham and 24 hours CLP animals; * p < 0.05 between IgG-treated and anti-C5a-treated rats.