6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice

We used 8–12-week-old C57BL/6 mice. The mice were kept in a barrier facility under specific pathogen-free conditions. All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (animal protocols 84-02.04.2012.A373 and 84-02.04.2011.A377) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Unless otherwise stated, all reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany).

Systemic inflammation was induced by cecal ligation and puncture (CLP) [20]. Mice were anesthetized by intraperitoneal injection of ketamine (125 μg/g body weight; Pfizer, New York, NY, USA) and xylazine (12.5 μg/g body weight; Bayer, Leverkusen, Germany). Sufficient anesthesia for the analgesic tolerance of surgical procedures was ensured by monitoring the response to a toe-pinch. The animals were killed by exsanguination through transcutaneous cardiac puncture after the experiment. CLP was performed as described before [20]. Briefly, a midline laparotomy incision was made after skin disinfection. The cecum was ligated 14 mm distal to the ileocecal valve so that continuity was preserved, and then it was punctured twice with a 20-gauge needle. It was returned to the peritoneal cavity, and the wound was closed in two layers. Mice were then allowed to recover and had free access to food and water. Animals in the sham group underwent the identical procedure without CLP. One hour after the procedure, the animals were administered 20 ml/kg HES 130/0.4 (Volulyte®) or a control solution (Isolyte® balanced electrolyte solution) by intravenous injection into the tail vein through an intravascular catheter. Volulyte® and Isolyte® share the same electrolyte constituency (Na⁺ 137 mmol/L, K⁺ 4 mmol/L, Mg²⁺ 1.5 mmol/L, Cl⁻ 110 mmol/L, CH₃COO⁻ 34 mmol/L, osmolality 286.5 mmol/L). The animals were randomized to treatment groups, and the investigators were blinded to these groups. An animal group size of eight mice was analyzed for each condition. Pulmonary inflammation induced by inhalation of lipopolysaccharide (LPS) from Salmonella enteritidis was performed as described previously [21]. Evans blue (EB) extravasation as a measure of vascular permeability was analyzed as described previously [22]. EB is a dye that binds to serum albumin after intravenous injection [23]. To investigate the influence of HES 130/0.4 administration on vascular permeability, mice were subjected to a sham or CLP operation and received a dose of 20 ml/kg HES 130/0.4 or Isolyte® (as a control) 1 h after surgery. EB (dissolved in 0.9% saline) was injected at a dose of 20 mg/kg body weight 30 minutes before the animals were killed. After the animals were killed, blood was obtained and centrifuged, and plasma was preserved for EB measurements. Afterward, the animals were subjected to perfusion through the right ventricle with PBS. The organs were harvested and weighed, and the tissue was homogenized in PBS (organ weight in milligrams × 3.5 = PBS volume in microliters) by mechanical mincing using a tissue homogenizer and incubated with formamide (vol/vol 2:1 ratio). The mixture was incubated for 12–18 h at 60 °C. After the incubation, the tissue homogenate was centrifuged at 5000 rpm for 30 minutes. EB absorbance measurements of the supernatants were performed on a spectrophotometer at 620 nm and 740 nm. The maximum absorption of EB occurs at 620 nm. No absorption occurs at 740 nm; the absorption at this wavelength is indicative of the amount of contaminating heme pigments. The degree of leak of EB is represented as a ratio of the corrected absorbance of EB in the tissue to the corrected absorbance of EB in the plasma. The extravasation of EB into peripheral organs was analyzed 4, 8, and 24 h after surgery. All animals survived the 24-h observation period. Each animal was examined at only one time point (4 h, 8 h, or 24 h), and we investigated eight mice at each time point for each experimental group in both models.

To investigate the influence of HES 130/0.4 administration on glycocalyx integrity during systemic inflammation in vivo, we performed intravital microscopy (IVM) of the cremaster muscle and lung as described previously [24‐27]. Following induction of general anesthesia, the cremaster muscle was exteriorized and prepared for imaging on a custom-built stage. IVM was carried out on an upright microscope (Axioskop; Carl Zeiss, Göttingen, Germany) with × 40 magnification and a 0.75 numerical aperture saline immersion objective. For IVM of the lung, a right lateral thoracotomy was performed, and the lung was positioned under the window of a custom-built fixation device. A mild vacuum was applied to hold the lung in position during microscopy. Images were captured with a charge-coupled device camera (Sensicam; PCO, Kehlheim, Germany). Fluorescein isothiocyanate (FITC)-dextran (150 kDa) was used to assess the glycocalyx width in these experiments as previously described (see also the scheme in Fig. 3a) [28‐30]. HES 130/0.4 or Isolyte® (20 ml/kg) was administered 1 h after CLP or sham surgery. IVM was performed after 4, 8, or 24 h. A different animal was used at each time point (4, 8, or 24 h) for IVM because it was not possible to continuously monitor a single animal over the whole observation period. Analysis of the recorded data files was performed by blinding the type of fluid used before evaluation.

Plasma levels of syndecan-1, heparanase, hyaluronic acid, and hyaluronidase were quantified using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturers’ instructions (syndecan-1 and heparanase, Cusabio Biotech, College Park, MD, USA; hyaluronic acid, BlueGene, Shanghai, China; hyaluronidase, USCN Life Science, Wuhan, China). Hyaluronidase activity was analyzed using an ELISA-based assay (Echelon Biosciences, Salt Lake City, UT, USA).

Statistical analysis was performed using IBM SPSS version 22.0 software (IBM, Armonk, NY, USA) with the Wilcoxon test or the t test as appropriate. More than two groups were compared using two-way analysis of variance followed by the Bonferroni test. Data distribution was assessed using the Kolmogorov-Smirnov test or the Shapiro-Wilk test. All data are presented as mean ± SEM. A p value < 0.05 was considered statistically significant.

To investigate the integrity of the glycocalyx under different inflammatory conditions, we determined the plasma levels of syndecan-1, hyaluronic acid, and heparanase. After induction of systemic inflammation, syndecan-1, heparanase, and hyaluronic acid levels in the plasma increased over the first 24 h (Fig. 1a–c). Four and eight hours following CLP, the plasma levels of syndecan-1 and heparanase were significantly reduced in HES-treated mice compared with Isolyte®-treated mice (Fig. 1a and b). Mice in the HES-treated groups also had significantly reduced hyaluronic acid plasma levels 8 and 24 h following CLP compared with the Isolyte® control groups (Fig. 1c). In contrast, hyaluronidase plasma levels showed a trend toward higher levels in the HES 130/0.4 group at 8 and 24 h after CLP, which did not reach statistical significance (Additional file 1: Figure S1). To specifically address the higher, although statistically not significant, hyaluronidase levels in CLP animals following HES administration, we performed an additional hyaluronidase enzyme activity assay. Interestingly, we could detect a statistically significant reduction in hyaluronidase activity in blood samples from CLP animals that had received HES compared with Isolyte®-treated animals (Fig. 1d). This could possibly explain why HES administration reduces circulating hyaluronic acid concentrations, whereas hyaluronidase serum concentrations appear to be increased, although this did not reach statistical significance in the CLP model. We also analyzed the plasma levels of syndecan-1, hyaluronic acid, hyaluronidase, and heparanase in mice after LPS-induced pulmonary inflammation (Fig. 2). HES 130/0.4 significantly decreased the plasma levels of syndecan-1 after 8 h, but not after 24 h, compared with the Isolyte® control group (Fig. 2a). Furthermore, HES 130/0.4 significantly decreased the plasma levels of heparanase and hyaluronic acid plasma levels 8 and 24 h after LPS exposure (Fig. 2b and c).

Systemic inflammation, as induced, for example, by CLP, causes degradation of the vascular glycocalyx [28]. The width of the vascular glycocalyx, analyzed using a FITC-dextran exclusion technique (Fig. 3a), remained unchanged in animals that received Isolyte® or HES 130/0.4 after sham surgery at all time points (Fig. 3b). The induction of systemic inflammation following CLP caused a significant decrease in glycocalyx thickness in animals that received Isolyte® compared with sham animals after 8 and 24 h (Fig. 3b). HES 130/0.4 administration significantly attenuated glycocalyx degradation after 8 h (0.70 ± 0.12 μm vs. 1.06 ± 0.11 μm) and 24 h (0.78 ± 0.09 μm vs. 1.39 ± 0.10 μm) compared with Isolyte®-treated mice, with no significant differences within the HES-treated mice (Fig. 3b). To investigate whether HES 130/0.4 also affects glycocalyx degradation during inflammatory processes in the lung, mice were exposed to nebulized LPS or saline, and HES 130/0.4 or Isolyte® (20 ml/kg) was administered after 1 h. Saline inhalation did not cause glycocalyx degradation, and control mice that received Isolyte® or HES 130/0.4 after saline inhalation did not show significant differences in glycocalyx thickness (Fig. 3c). The induction of pulmonary inflammation by LPS inhalation caused a significant decrease in glycocalyx thickness after 8 and 24 h in animals that received Isolyte® compared with control animals (Fig. 3c). HES 130/0.4 administration significantly attenuated glycocalyx degradation after 8 h (0.98 ± 0.09 μm vs. 1.50 ± 0.20 μm) and 24 h (0.81 ± 0.09 μm vs. 1.49 ± 0.12 μm) (Fig. 3c).

CLP causes a systemic inflammation response in mice [20]. Increased plasma extravasation caused by increased vascular permeability is one hallmark of this systemic inflammation. Vascular permeability increased in all organs 8 and 24 h following CLP (Fig. 4a–d). Compared with Isolyte®-treated mice, HES 130/0.4 administration caused significantly decreased vascular permeability in the lung after 8 and 24 h (Fig. 4a). In the kidney (Fig. 4b), the liver (Fig. 4c), and brain tissue (Fig. 4d), HES 130/0.4 decreased the vascular permeability significantly after 24 h following CLP compared with Isolyte® treatment. To specifically address the contribution of glycocalyx degradation on the observed HES effect on vascular permeability changes, we performed additional experiments with the heparanase inhibitor N-desulfated/re-N-acetylated heparin (NAH). NAH coadministration together with HES abolished the vascular permeability decrease exerted by HES alone by 57–86%, depending on the specific organ (Fig. 4e). Interestingly, the administration of NAH alone did not modulate vascular permeability in our CLP model (Additional file 2: Figure S2). These data indicate that glycocalyx degradation does not mediate vascular permeability changes alone, but rather in addition to direct effects on the vascular endothelial cells. During LPS-induced pulmonary inflammation, vascular permeability increased in all organs 8 and 24 h after LPS inhalation, whereas the vascular permeability in saline-treated control animals did not change (Fig. 5a–d). HES 130/0.4 significantly decreased vascular permeability in the lung, liver, kidney, and brain 24 h after LPS inhalation compared with Isolyte®-treated mice (Fig. 5a–d). To investigate whether the observed protective effect of HES is compound-specific or a common property of colloids in general, we performed additional experiments with albumin. Whereas HES significantly reduced vascular permeability in the lung, liver, kidney, and brain at 24 h after CLP induction compared with Isolyte® treatment, this effect was observed in the brain only with albumin (Additional file 3: Figure S3A–D). To further characterize the severity grade of our CLP model, we analyzed different serum markers for organ damage 24 h after CLP induction. CLP induction did not readily increase serum creatinine (Additional file 3: Figure S3E), whereas it induced a modest increase in serum urea (Additional file 3: Figure S3F). Serum glutamate-pyruvate transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) as markers of hepatic cell injury were also modestly increased 24 h after CLP induction. Serum lactate levels in control animals 24 h after the CLP procedure averaged 2.8 mmol/L, indicating a modest degree of circulatory disorder. All mice survived the 24-h observation period; thus, our CLP was targeted at a rather modest severity level. Neither HES 130/0.4 nor albumin significantly altered the increase in serum urea, GPT, or GOT (Additional file 3: Figure S3F–H).