Translational evidence for two distinct patterns of neuroaxonal injury in sepsis: a longitudinal, prospective translational study

Abstract

Background: Brain homeostasis deteriorates in sepsis, giving rise to a mostly reversible sepsis-associated encephalopathy (SAE). Some survivors experience chronic cognitive dysfunction thought to be caused by permanent brain injury. In this study, we investigated neuroaxonal pathology in sepsis.

Methods: We conducted a longitudinal, prospective translational study involving (1) experimental sepsis in an animal model; (2) postmortem studies of brain from patients with sepsis; and (3) a prospective, longitudinal human sepsis cohort study at university laboratory and intensive care units (ICUs). Thirteen ICU patients with septic shock, five ICU patients who died as a result of sepsis, fourteen fluid-resuscitated Wistar rats with fecal peritonitis, eleven sham-operated rats, and three human and four rat control subjects were included. Immunohistologic and protein biomarker analysis were performed on rat brain tissue at baseline and 24, 48, and 72 h after sepsis induction and in sham-treated rats. Immunohistochemistry was performed on human brain tissue from sepsis nonsurvivors and in control patients without sepsis. The clinical diagnostics of SAE comprised longitudinal clinical data collection and magnetic resonance imaging (MRI) and electroencephalographic assessments. Statistical analyses were performed using SAS software (version 9.4; SAS Institute, Inc., Cary, NC, USA). Because of non-Gaussian distribution, the nonparametric Wilcoxon test general linear models and the Spearman correlation coefficient were used.

Results: In postmortem rat and human brain samples, neurofilament phosphoform, β-amyloid precursor protein, β-tubulin, and H&E stains distinguished scattered ischemic lesions from diffuse neuroaxonal injury in septic animals, which were absent in controls. These two patterns of neuroaxonal damage were consistently found in septic but not control human postmortem brains. In experimental sepsis, the time from sepsis onset correlated with tissue neurofilament levels (R = 0.53, p = 0.045) but not glial fibrillary acidic protein. Of 13 patients with sepsis who had clinical features of SAE, MRI detected diffuse axonal injury in 9 and ischemia in 3 patients.

Conclusions: Ischemic and diffuse neuroaxonal injury to the brain in experimental sepsis, human postmortem brains, and in vivo MRI suggest these two distinct lesion types to be relevant. Future studies should be focused on body fluid biomarkers to detect and monitor brain injury in sepsis. The relationship of neurofilament levels with time from sepsis onset may be of prognostic value.

Trial registration: ClinicalTrials.gov, NCT02442986 . Registered on May 13, 2015.

Keywords: Animal models; Biomarkers; Encephalopathy; Intermediate filaments; Rats; SAE; Sepsis; Sepsis-associated encephalopathy.

Conflict of interest statement

Authors’ information

Not applicable.

Ethics approval and consent to participate

All experiments were performed according to the University College London Ethics Committee and Home Office (UK) guidelines under the 1986 Scientific Procedures Act. The study was approved by the local ethics board at Rostock University (A 2012-0058) and by the Comité Consultatif de Protection des Personnes se Prêtant à la Recherche Biomédicale de Saint Germain en Laye, France. All patients or their legal representatives signed written informed consent forms before study inclusion.

Consent for publication

Not applicable.

Competing interests

AP is supported by the Moorfields Biomedical Research Centre and the Dutch MS Research Foundation and received honoraria from Novartis for quality control reading (PASSOS study). The other authors declare that they have no competing interests.

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Figures

Fig. 1

Brain lesions seen in rat sepsis model. a Central brain white matter immunohistochemistry in sham-treated animals (controls) shows characteristic neuronal soma with restricted staining for β-amyloid precursor protein (βAPP) (arrows). b In septic animals, brain tissue from the same locations showed abnormal and more widespread axonal staining extending from the axonal hillock to the proximal axon (arrows). c Abnormal axonal βAPP staining follows white matter tracts (arrows). d There are also pockets of inflammatory and ischemic brain lesions seen in the rat sepsis model (H&E stain; arrow). e Staining of such lesions shows intense neuronal and axonal staining for βAPP (arrows). f Staining of lesions of sham-treated animals for β-tubulin in the magnification field is crisp and shows integrity of the neuroaxonal compartment (overview, inset; original magnification × 10). g Likewise, the integrity of white matter tracts in sham-treated animals can be seen (×10; inset, original magnification × 40). h There is severe structural disorganization of the β-tubulin network in white matter tracts of the septic animals. i The level of structural β-tubulin disorganization in the septic rat brain is best observed at greater magnification (original magnification × 40) of the neuroaxonal compartment from the same location as that taken from the sham model shown in (f)

Fig. 2

Brain lesions seen in postmortem human tissue. a Patient 1 (control): Diffuse staining of axons (arrows), extending from the axonal hillock (β-amyloid precursor protein [βAPP]). b Extensive diffuse axonal injury is shown in patient 2 (sepsis). Staining for βAPP is not restricted to the axonal hillock but is seen throughout the white matter tracts. Multiple axonal endbulbs can also be seen (small arrowheads). c Disruption of the deep white matter axons and the presence of axonal end bulbs are widespread based on dephosphorylated neurofilament heavy chain (SMI32). d Patient 3: Small areas of ischemic lesions can be seen throughout the brain (βAPP). e Patient 4: One type of lesion not observed in the animal model is shown. Amyloid plaques (arrow) are present and scattered throughout the brain tissue (βAPP). f In this patient, diffuse deep white matter axonal damage (arrows) is the most severe of this series (βAPP)

Fig. 3

Brain magnetic resonance imaging of three patients during septic shock. The images are fluid-attenuated inversion recovery (FLAIR; a, c, and e) and echo planar imaging diffusion-weighted imaging (DWI; b, d, and f) scans. a and b An 81-year-old male patient with urosepsis. a Axial FLAIR image obtained on day 5 after the onset of septic shock shows punctiform and confluent white matter hyperintensities (WMH) in both paraventricular and paramedian regions (grade 2 leukoencephalopathy). b DWI study shows subacute ischemic lesion in the left occipital paramedian region. c and d An 80-year-old female patient with urosepsis. (c) Axial FLAIR image obtained 9 days after the onset of septic shock shows confluent WMH in the left periventricular region (grade 2 leukoencephalopathy). d DWI study shows bilateral ischemic lesions in the frontal region. e and f An 80-year-old female patient with urosepsis. e Axial FLAIR performed 8 days after onset of septic shock revealing a single punctiform WMH in the left periventricular region (grade 1 leukoencephalopathy). f DWI study shows punctiform ischemic lesions in the left occipital and parietal (inset) regions