Brain tight junction protein expression in sepsis in an autopsy series

The study was approved by the ethics committee of the Oulu University Hospital and the Northern Ostrobothnia Hospital District (224/2013). The use of brain tissue samples obtained in the autopsy was approved by the National Institute for Health and Welfare (BB_2017_1003). This retrospective observational cohort study was conducted at the Oulu University Hospital, Oulu, Finland, an academic tertiary level referral hospital. Adult patients deceased with sepsis who underwent postmortem examination during the years 2007–2015 and had brain tissue specimens available were included in the study. Medical non-forensic autopsies are performed if antemortem data or clinical examination does not provide the clinician enough information to issue a death certificate and to better understand the disease course, or in cases in which the next of kin request it. The final decision is made at the discretion of the treating physician. All patients were treated by a multidisciplinary team of intensivists and infectious disease specialists in our 26 beds, closed adult intensive care unit. Sepsis was defined according to the American College of Chest Physicians/Society of Critical care medicine criteria [14]. The intensive care treatment was performed according to normal intensive care unit (ICU) protocols and sepsis guidelines.

Clinical parameters were gathered from the ICU clinical data management system database (Centricity Critical Care, Clinisoft; GE Healthcare, Helsinki, Finland). On admission, the severity of illness was determined by the Acute Physiology and Chronic Health (APACHE II) score [15], and daily total and individual organ group sequential organ failure assessment score (SOFA) was used as a measure of organ dysfunction [16]. The Glasgow Coma Score (GCS) was employed for assessing the impairment of patients’ level of consciousness [17]. The presence of multi-organ failure (MOF) during the ICU treatment was defined as more than two organs failing based on grade 3 or 4 sequential organ failure assessment [16]. Data regarding age, sex, time to death, focus of infection, and blood culture positivity were collected. Procalcitonin (PCT) on admission and its highest value, as well as white blood cell count were retrieved. Clinical laboratory samples were analyzed by commercially available laboratory methods in the hospital accredited central laboratory (NordLab, Oulu University Hospital, Oulu, Finland).

The median time from the death to autopsy was 5 (3–6) days. At the autopsy, brain specimens were routinely collected, usually from a least two anatomical locations. A systematic neuropathological examination was performed when the pathologist in charge considered it necessary or by request of the clinician, for example, in cases when the patient had neurological symptoms. For each deceased subject, brain tissue specimens were categorized according to anatomical location (cerebrum, cerebellum), and one representative tissue block from each location was selected for the study. The exact location of the specimens had not been registered in all cases, but according to the suggested sampling routine of our hospital, likely followed in most cases, cerebral samples represented the precentral gyrus, and the cerebellar samples the lateral aspect of either hemisphere. The immunohistochemical stainings were performed in accordance with the manufacturer’s recommendation, and a set of samples was used to optimize the dilution of primary antibodies. We used the following antibodies and conditions: (1) human occludin (rabbit polyclonal, catalog no: 711500, Invitrogen, Frederick, MD, USA), with pretreatment using pronase, at a dilution of 1:800, incubation at room temperature (RT) 60 min; (2) ZO-1 (rabbit polyclonal; Zymed, cat no: 61-7300, Carlsbad CA, USA), pretreatment with 15 min boiling in a TRIS-EDTA buffer, dilution at 1:400, incubation for 60 min at RT); and (3) claudin-5 (mouse monoclonal, clone 4C3C2, Zymed), pretreatment with TRIS-EDTA, dilution at 1:50, incubation for 60 min at RT. For all antibodies, the detection was performed with a polymer-based kit (Envision, Dako, Copenhagen, Denmark). Diaminobenzidine (Dako basic DAB-kit, Dako) was used as a chromogen. All stainings were performed with the Dako Autostainer (Dako, Copenhagen, Denmark). Validation of our immunohistochemical analysis was performed through a series of negative controls by omitting the primary antibody.

Stained sections were digitized (Leica-Aperio AT2; Leica Biosystems, Nussloch, Germany) and assessed using the Aperio ImageScope program independently by two researchers (KE and ME) supervised by an experienced neuropathologist (HT), all blinded for the clinical and outcome data. Endothelial cells of the capillaries were analyzed for each anatomic location. Expression was considered positive if 50% or more showed a positive reaction of any degree and were negative in the absence of any staining or if less than 50% showed a positive reaction.

Occludin is the key tight junction (TJ) protein in cerebral endothelial cells which modulates blood-brain barrier (BBB) functions and maintains TJ stability, and accordingly, patients were categorized as having BBB damage if there was no expression of occludin in the endothelium of the cerebral microvessels [18, 19].

The associations of an absence of occludin to the severity of illness were evaluated by comparison with the maximum SOFA score recorded during the ICU stay, an increase of PCT to > 10 μg/L and CRP to > 100 mg/L. The cut-off point for CRP as a marker of inflammation was chosen according to Falk et al. [20]. The cutoff value for PCT > 10 μg/L was used as a marker of organ dysfunction in sepsis [21].

Statistical analyses were performed with SPSS for Windows (2017 release, Version 25; IBM Corporation, Armonk, NY, USA). Data are expressed as the percentage of stained cells as median (25–75th percentile) in continuous variables. Categorical data were analyzed with Fisher’s exact test. Mann-Whitney U test was applied to distributions across the two groups. Spearman’s correlation coefficient (rho) was calculated. Two-sided p values are presented and a p < 0.05 was considered statistically significant. However, the level of statistical significance should be treated with caution, given the large number of statistical tests performed.

Our study stresses the importance of BBB damage in sepsis. The results of the present study support those reported in experimental studies, but also new findings of clinical associations were found. Our findings suggest that analyzing of blood occludin concentration might serve as a biomarker in human sepsis for the detection of BBB damage, as already shown in a rat study with cerebral arterial occlusion [30]. We have earlier shown that delirium in septic shock was associated with an elevation in protein S-100β expression [31]. Modulating BBB function may also offer new treatment possibilities for preventing brain dysfunction. For example, an in vitro model of human BBB composed of brain microvascular endothelial cells showed that anti-TNF-α antibody improved the upregulation of tight junction protein expressions following oxidative stress [32].

The collection of multiple brain samples was not standardized as the autopsies were performed when judged to be clinically pertinent. It is possible that the autopsies were only performed in the deceased with whom there were diagnostic difficulties during their ICU stay. Moreover, at the time of their death, the patients were at different stages of inflammation. Due to the retrospective study design, we were not able to include focused data on the brain function during the ICU stay including EEG or MRI of the brain. Further studies with comparison to non-septic patients are needed to explore the link between sepsis and TJ proteins.