Rat model of sepsis-associated encephalopathy
All experiments were performed according to local ethics committee (University College London) and Home Office (UK) guidelines under the 1986 Scientific Procedures Act. Adult male Wistar rats (approximate body weight 300 g, 12–14 weeks old) were used to generate a 3-day in vivo sepsis model of fecal peritonitis. Sepsis induction and experimental procedures were performed as described in detail before . Four naive (noninstrumented) rats, eleven sham-operated, and fourteen septic rats were included. All rats were instrumented under brief anesthesia with tunneled carotid arterial (left side) and jugular venous (right side) catheters to monitor hemodynamics, sample blood, and infuse fluids. The catheters were mounted onto a swivel-tether system, allowing the rat, on recovery from anesthesia, to have free movement in its cage and ad libitum access food and water. Sepsis was induced 24 h later by an intraperitoneal injection of fecal slurry (0.63 mg/100 g body weight, prepared from bowel contents of rats from the same batch). From 2 h postinsult, fluid resuscitation using a 1:1 solution of colloid and 5% glucose was administered at a rate of 10 ml/h for the first 24 h and then halved on successive days to ensure normovolemia and normoglycemia. All septic animals showed clinical signs of sepsis by decreased movements, decreased alertness, hunched posture, and piloerection from about 12 h postinjection of fecal slurry. Rats were killed by cervical dislocation under deep isoflurane anesthesia either 24, 48, or 72 h after sepsis induction. After craniectomy, brains were removed within a few minutes of death. The brain was dissected through the midline into halves. One half was snap-frozen in liquid nitrogen; the other half was placed in formalin.
Histology, immunohistochemistry, and protein extraction from septic rat and human brain
Neuropathologists (MG, FS) were masked to the condition of rat and human brain samples. Human and rat brain tissue from frontal lobe areas and the cerebellum were processed in paraffin wax using a standard 7-day dehydration and paraffin-embedding protocol on an automated tissue processor. Five-micrometer paraffin sections were cut, mounted onto SuperfrostTM glass slides and dried overnight at 37 °C. For the general histologic examination, sections were dewaxed, rehydrated, and stained with H&E using a standard protocol. For the immunohistochemical detection of neurofilaments (Nf) and glial fibrillary acidic protein (GFAP), the sections were dewaxed and rehydrated before being placed in 600 ml of 0.1 M citrate buffer (pH 6.0) and microwaved at full power for 15 minutes in a 850-W microwave oven. The four sections were allowed to cool before being rinsed in 0.05 M PBS, pH 7.4, and incubated overnight at room temperature in either a mouse monoclonal antibody directed against an epitope common to the 70 and 200 kDa Nf proteins (clone 2 F11 diluted 1:20; MP Biomedicals Inc., Santa Ana, CA, USA), β-tubulin (1:200; Sigma-Aldrich, Gillingham, UK), β-APP (1:500; Dako, Ely, UK), or a rabbit polyclonal antibody directed against GFAP (diluted 1:1500; Dako). After being washed in PBS, the sections were incubated in a biotinylated secondary antibody for 1 h (Dako), followed by a washing step and incubation in peroxidase-conjugated streptavidin for 1 h (diluted 1:300; Sigma-Aldrich) All dilutions were in PBS with 0.1% Triton-X. Antibody localization was visualized by incubating the sections for 10 minutes at room temperature in 0.05% diaminobenzidine with 0.04% NiCl2 and 0.01% hydrogen peroxide added. The sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted.
The dry weight of the snap-frozen rat brain tissue was 0.56 to 1.94 g. Barbitone ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.6) containing a protease inhibitor cocktail (P8340; Sigma-Aldrich) was added to 1:2 wt/vol. The samples were homogenized on ice using an ULTRA-TURRAX T 25 instrument (IKA-Werke GmbH & Co., Staufen, Germany) for 1 minute, followed by sonication on ice for 1 minute. Samples were refrozen at −70 °C and then thawed at 30 °C, and 2 ml of sample was added to 5 ml of diisopropyl ether and 2 ml of barbitone EDTA buffer. After a mixing step, the samples were spun at 25,000 × g and 4 °C for 30 minutes, and the protein soluble fraction was collected. Tissue levels of Nf heavy chain (NfHSMI35) and GFAP were measured by enzyme-linked immunosorbent assay (ELISA), and total protein was measured using the Lowry method [26, 27].
In vivo neurologic assessment of patients with sepsis
The study was approved by the local ethics board at Rostock University (A 2012-0058) and registered as a clinical trial (ClinicalTrials.gov, NCT02442986). The patient recruitment period was from November 2012 to May 2015. All patients or their legal representatives signed written informed consent forms before study inclusion. Inclusion criteria for participants were aged ≥ 18 years and the presence of severe sepsis or septic shock according to the criteria used at the time . Exclusion criteria were preexisting cerebrovascular diseases, including dementia, preexisting neuromuscular disease, high-dose glucocorticoid administration (>300 mg hydrocortisone or equivalent per day), preexisting renal replacement therapy, coagulopathy with active bleeding, and frequent administration of neuromuscular blocking agents (more than three times per week). Twenty patients with septic shock were included prospectively in the study. Seven participants without magnetic resonance imaging (MRI) examinations were excluded for the following reasons: death before MRI performed (n = 1), only cranial computed tomographic scan available owing to contraindication for MRI (n = 2), disclaimer for MRI from patient/legal representative after study inclusion (n = 2), and repeated surgery/unstable patient (n = 2). In total, 13 patients were enrolled prospectively in this single-center, longitudinal, observational study.
Clinical assessment protocol
All patients were clinically assessed by an interdisciplinary team consisting of intensivists and neurologists experienced in critical and neurocritical care using a validated scales for severity of disease: Acute Physiology and Chronic Health Evaluation II at ICU admission and the Sepsis-related Organ Failure Assessment score [29, 30]. All patients received standardized management according to guideline recommendations [2, 28]. After study inclusion, patients were longitudinally evaluated (study days 1, 3, 7, and 28) for their level of consciousness and for signs of SAE, such as confusion, agitation, hallucinations, or acute changes in mental status using the Glasgow Coma Scale, the Richmond Agitation-Sedation Scale, and the Confusion Assessment Method in the Intensive Care Unit (CAM-ICU) [31,32,33,34]. A medical history was taken from all patients, if obtainable, or from their next of kin. A standardized neurologic examination was performed on all patients by an experienced neurologist (MW). This comprised a detailed status of the level of consciousness; brainstem reflexes and function; deep tendon reflexes; and sensory and motor function, including muscular strength testing using the Medical Research Council dyspnea scale score [35, 36].
In addition to clinical assessment, all patients underwent electroencephalography (EEG) within the first 72 h after sepsis was diagnosed. The international 10–20 system was used for standard electrode placements with impedance level < 5 kΩ on a mobile EEG unit (ED 14; Madaus Schwarzer, Munich, Germany). All EEG recordings were done over 30 minutes. Patients were stimulated by verbal command. If no response to verbal stimulation could be obtained, sternal rub or nail bed compression were performed. The EEG recordings were assessed by an experienced accredited reader (MW) according to the method described by Young et al. . Patients with analgosedation (standard regimen with continuous infusion of propofol and sufentanil) had a sedation holiday of 30 minutes before EEG recording.
Magnetic resonance imaging
A 1.5-T magnet system (MAGNETOM Avanto; Siemens Healthcare, Erlangen, Germany) was used in seven patients, and a 3.0-T magnet system (MAGNETOM Verio; Siemens Healthcare) was used in six patients. A standardized MRI protocol was used, and all MRI findings were analyzed by an experienced neuroradiologist (AG). MRI examinations included coronal T1-weighted images (with and without contrast medium), sagittal and axial T2-weighted sequences, axial fluid-attenuated inversion recovery (FLAIR), and axial T2*-weighted gradient recalled echo sequences. Additionally, axial echo planar imaging diffusion-weighted imaging (DWI) sequences, apparent diffusion coefficient maps, and time-of-flight magnetic resonance angiography were performed. The extent of white matter hyperintensities (WMH) as an imaging marker of brain injury was graded on a previously validated scale. WMH were scored according to their number and size from grade 0 (no lesions) through grade 1 (punctiform), grade 2 (patchy or confluent), and grade 3 (diffuse) [21, 38]. An MRI examination was performed as soon as the patient was clinically stable for in-house transfer using continuous patient monitoring (Expression MR400 monitor; Phillips Healthcare Deutschland GmbH, Hamburg, Germany).
All 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 was used for comparing two independent variables. Two-way unbalanced analysis of variance (general linear model) was used for comparing more than two independent variables, followed post hoc analysis if significance was achieved. The F values providing the degrees of freedom and the number of samples included in each particular analysis are shown. The linear correlation between continuous variables was evaluated using the Spearman correlation coefficient. Linear regression analysis was performed using the least squares method. A p value < 0.05 was considered significant.