High molecular weight hyaluronan – a potential adjuvant to fluid resuscitation in porcine abdominal sepsis

While fluid resuscitation is fundamental in the treatment of sepsis-induced tissue hypo-perfusion, a sustained positive fluid balance is associated with excess mortality. Crystalloids are the mainstay of fluid resuscitation and use of either synthetic colloids or albumin is controversial. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water, has not been tested as adjuvant in fluid resuscitation. We sought to evaluate the effects of hyaluronan as an adjuvant to fluid resuscitation in peritonitis induced sepsis. In a prospective, parallel-grouped, blinded model of porcine peritonitis-sepsis, we randomized animals to intervention with adjuvant hyaluronan (add-on to standard therapy) (n=8) or 0.9% saline (n=8). After the onset of hemodynamic instability the animals received an initial bolus of 0.1 % hyaluronan 1 mg/kg/10 min or placebo (saline) followed by a continuous infusion of 0.1% hyaluronan (1 mg/kg/h) or saline during the experiment. We hypothesized that the administration of hyaluronan would reduce the volume of fluid administered (aiming at stroke volume variation <13%) and/or attenuate the inflammatory reaction. Total volumes of intravenous fluids infused were 17.5 ± 11 ml/kg/h vs. 19.0 ± 7 ml/kg/h in intervention and control groups, respectively (p = 0.442). Plasma IL-6 increased to 2450 (1420 – 6890) pg/ml and 3700 (1410 – 11960) pg/ml (18 hours of resuscitation) in the intervention and control groups (NS). In a post-hoc analysis, modified shock index remained lower in intervention group (p = 0.011 - 0.037). In conclusion adjuvant hyaluronan did not reduce the volume needed for fluid administration or decrease the inflammatory reaction. Adjuvant hyaluronan was, however, associated with lower modified shock index. Bearing in mind that the experiment has a limited group-size we suggest that further studies on hyaluronan in sepsis are warranted.

the onset of circulatory instability (S0). S6, S12 and S18 refer to consecutive time points, 149 respectively (hours after S0). 150 In order to simulate an intensive care setting, the respiratory, circulatory and metabolic 151 maintenance treatments followed a predefined protocol to support vital parameters according 152 to typical invasive monitoring and repeated measurements and sampling. Both intervention and 153 control groups were subject to a protocolized resuscitation that was intitiated at onset of 154 circulatory instability (S0) with Ringer's Acetate 10 ml/kg/h. The resuscitation protocol was 155 equal to both groups and aimed at MAP > 60 mmHg, guiding fluid and norepinephrine 156 administration by changes in SVV and MAP respectively. If MAP < 60 mmHg and SVV > 15 tapered down to 5 ml/kg/h, and if the animal continued to be stable and SVV maintained < 13% 162 the infusion was stopped [42]. 163 164 Experimental design 165 This was a prospective, parallel-grouped, blinded study with animals randomized (block 166 randomization, sealed opaque envelope) after peritonitis induction into two treatment groups:  Analyses and physiologic parameters 179 We analyzed arterial blood gases at baseline, at the onset of circulatory instability and every 180 hour for the following eighteen hours duration of the experiment. At the same time points, 181 hemodynamic parameters (systemic arterial and pulmonary arterial pressures, CO, heart rate), 182 respiratory parameters (F I O 2 , SaO 2 , ETCO 2 , static peak pressure, dynamic and static compliance) and urine output were measured. We calculated modified shock index based on 184 MAP [43,44] and also combined the calculation with hgb and norepinephrine dose.

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Every three hours mixed venous blood gas analyses was performed, while plasma and urinary 186 samples were collected for analysis every six hours. Stroke volume variation (SVV) was 187 monitored continuously in order to guide fluid resuscitation.  parts of the section), 3, severe: (numerous leukocytes in many parts of the section), 2, moderate (moderate numbers of leukocytes diffusely or focally distributed), 1, mild (low number of 207 leukocytes diffusely or focally distributed or 0, lesions were not observed.

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Wet-to-dry ratio was measured in samples from the above mentioned locations. Samples were 209 weighed, and dried in an oven, at 50° C, until the weight did not differ between two consecutive 210 measurements.

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Bacterial investigations 213 Every third hour 0.5 mL arterial blood was collected from a sterile arterial catheter for 214 quantitative blood cultures. Therefrom a 100 µl was cultured on three separate cysteine lactose 215 electrolyte deficient (CLED) agar plates, then cultured at 37C overnight and colony forming 216 units (CFU) quantified with viable count technique the following day. CFU on only one CLED 217 plate from a time point was interpreted as a contamination otherwise the median of counted 218 CFU/mL was calculated. More than 1 CFU/mL were considered a positive blood culture.

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Colonies were sent to specification to a MALDI Biotyper (tof-user@FLEX-PC).

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Samples from lung, spleen and liver were also collected for tissue culture after spraying the 221 organ (surface) with 99% ethanol. App. 1 gram of each tissue was placed in a sterile mortar and 222 mashed in 3 mL saline 0.9%, from where 200 µl was cultured on CLED plates and quantified 223 as described above. To determine sample size we used data from a previous peritonitis protocol where the fluid 227 balance of the control group had a standard deviation of ± 4 ml/kg/h. Aiming at detecting a 228 difference of 6 ml/kg/h between groups in fluid balance, a power of 0.8 and a significance level of < 0.05 yielded a sample size of eight animals in each group. We tested data for normality by 230 applying the Shapiro-Wilk's test.

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To describe each group separately from baseline to onset of circulatory instability (S0) we used 232 the Student's t-test, whereas the one-way ANOVA was used to describe the groups separately 233 throughout the experiment. The two-tailed Student´s t-test, the Mann-Whitney U test and the 234 two-way ANOVA were used to compare the two groups, pending distribution of data. Multiple 235 imputation was used in order to replace missing data due to early deaths.

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The data are expressed as mean ± SD or median (IQR) as appropriate. We conducted the 237 statistical analyses using SPSS v. 27.0.0 software (SPSS, Inc., Chicago, IL, USA). A p-value 238 of < 0.05 was considered to be statistically significant. Bonferroni correction was not used.

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The results are presented as n = 8 per group at the baseline, at the onset of circulatory instability 240 (S0), at six (S6) and twelve (S12) hours after onset of circulatory instability, as well as at the 241 end (last observation, prior to imminent death or at 18 hours (S18)). Hourly recordings of

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Values reported as mean ± SD. Significance level as range after performing multiple imputation (p range). * Baseline vs S0,

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Lactate increased in intervention group during the same period, but not in the control group 289 (Table 3).

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All hemodynamic parameters as well as arterial blood lactate changed comparably in the two 291 groups as a function of time over the length of the resuscitation period (Tables 2 and 3). Neither 292 did the groups differ in regard to wedge pressure, nor CVP as a function of time (Tables 2 and   293 3).

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Onset of circulatory instability was accompanied by comparable decrease of SaO 2 and P/F ratio 303 from baseline in the two groups. There was a gradual decrease in both dynamic and static 304 compliance in both intervention and control groups respectively, throughout the protocol (

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Finally, there were no differences between groups in urine creatinine, urea, sodium, or 345 potassium concentrations between groups as a function of time (Table 5).

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One animal died before finishing the protocol in both groups, last sample before imminent death is included in the analyses 355 of the time point after death, that is S12 for the animal that died in group 1 (204 at S8), and S18 for the animal that died in 356 group 2 (207 at S14), before finishing the protocol. Values as median (95% CI). * Baseline vs S0, p < 0.05, p -value from 357 paired t-test.

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Wet-to-dry ratio 360 Wet-to-dry ratios at the end of the experiment were comparable in the two groups (Additional 361 file 28).  The main finding of the present study was that, contrary to our hypothesis, high molecular

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In conclusion, the current study does not support the hypothesis that HMW-HA reduces the