Inhaled Nitric Oxide with Intravenous Hydrocortisone Does Not Improve Microvascular Perfusion in Septic Pigs.

Background Capillary ow restoration in sepsis may prevent organ dysfunction caused by a prolonged impairment of microvascular ow. The main aim of the study was to investigate the microcirculatory effect of inhaled nitric oxide (iNO) combined with intravenous hydrocortisone in a porcine model of sepsis. The second aim was to evaluate the inuence of hemodynamic resuscitation with noradrenaline and crystalloids on capillary ow. In we and We found no effect of iNO with hydrocortisone on microcirculation in septic piglets. Fluid and vasopressor treatment postponed microvascular ow impairment and led to a higher microcirculatory ow index, greater proportion of perfused vessels, and perfused vessel density.


Introduction
Every year, 31 million people are hospitalized because of sepsis, and over 5 million die with this diagnosis [1]. For this reason, early detection and treatment of sepsis have been of great interest to intensivists.
Sepsis and its complications including multiorgan failure seem to result from changes in capillary perfusion, which may precede more severe symptoms [2,3]. It has been proven that persistent microcirculatory derangement is strongly related to poor outcome and can be observed in patients with multiorgan dysfunction even after shock resolution [4,5]. Therefore, capillary ow monitoring appears to be essential for early and successful management of sepsis.
There are multiple studies concerning septic patients and septic animal models that provide reproducible information about microcirculation changes occurring in sepsis [6-8]. Various processes activated by in ammation lead to the cessation of ow in some capillaries and augmentation of ow in others [9]. An increased distance between perfused capillaries impedes oxygen diffusion and its delivery to cells, creating microcirculatory shunt [10]. It leads to oxygen de cit in tissues, increased lactate production, shock, and, nally, organ dysfunction [2,11,12]. Microvascular monitoring is crucial not only for early detection of pathologies but it has also been used to assess the effects of treatment with multiple drugs e.g. diuretics, hydrocortisone, phenylephrine, nitroglycerin, or red blood cell transfusion [13][14][15][16].
Therapies tested in sepsis (e.g. corticosteroids) modulate the in ammatory response. The use of hydrocortisone in septic shock seems bene cial not only due to its anti-in ammatory properties, since glucocorticoids inhibit pro-in ammatory gene expression and activate anti-in ammatory protein production [17]. It also increases vessel responsiveness to catecholamines [18]. Moreover, moderate doses of hydrocortisone administered in early stages of septic shock improve capillary perfusion [13].
The role of corticosteroids in the management of septic shock is still controversial. Nevertheless, low doses of corticosteroids may restore circulation and reduce mortality caused by septic shock [13,19,20].
As far as sepsis treatment is concerned, much attention has also been paid to nitric oxide (NO) with multiple trials targeting both NO production inhibition and external NO delivery [21]. Generally, the release of nitric oxide is increased in septic shock. However, in some areas NO production may be impaired, which is one of the factors leading to heterogeneity and hypoperfusion of the microvascular bed [12,21,22]. The delivery of exogenous NO, a very potent vasodilator, might potentially resuscitate microcirculation and restore tissue perfusion [21,23]. Inhaled nitric oxide (iNO) is one of the donors tested not only because of its pulmonary activity, but also because of recognized peripheral effects [24].
Experimental models of sepsis revealed that inhaled nitric oxide up-regulates glucocorticoid receptors and blunts the in ammatory reaction [25].
For the above reasons, our objective was to assess the effect of iNO and hydrocortisone on microcirculatory ow in a porcine septic model. The aim of the study was to verify the theory that iNO and hydrocortisone administered in sepsis preserve microvascular ow. We also wished to test whether uid resuscitation combined with vasopressors resuscitates microcirculation. For microvascular ow assessment, we applied sidestream dark eld imaging described elsewhere [26].

Results
All eleven animals developed SIRS (Systemic In ammatory Response Syndrome) ( Table 1). There were no differences between the three studied groups as far as initial microcirculatory parameters are concerned (Fig. 2). The 30-hour analysis showed no in uence of iNO and hydrocortisone on microvascular bed perfusion. In both groups, microvascular ow index (MFI), small vessel microvascular ow index (sMFI), perfused vessel density (PVD), perfused small vessel density (sPVD), proportion of perfused vessels (PPV), and proportion of perfused small vessels (sPPV) signi cantly dropped. There were no marked intergroup differences in perfusion impairment (Table 2). Table 2. Table demonstrating changes of the TVD, sVD, and de Backer score over time. At all time points, there were no signi cant differences in the above variables between the studied groups (p>0.05). The TVD, sVD, and de Backer score did not change over time. The data are presented as medians (25th-75th percentiles).
The statistical analysis of HI (mean ± SD) was not possible due to the small size of the analyzed groups. In Group 3, perfusion deteriorated to a much greater extent than in other groups at 20 hours of observation (MFI, sMFI, PVD, sPVD, PPV and sPPV). There were no signi cant intergroup differences in the total vessel density (TVD) and small vessel density (sVD) and de Backer score (Table 2).
Throughout the study, the only consistent correlations between microvascular ow and systemic ow were observed in Group 3 ( Table 3). Impairment of microvascular ow (expressed by changes in the PPV, sPPV, PVD, sPVD, MFI, sMFI) were related to the heart rate (HR), mean arterial pressure (MAP),diastolic arterial pressure (DAP), systemic vascular resistance (SVRI) with p < 0.05 in all cases. None of these parameters depended on the cardiac output (CO), cardiac index (CI), or pulmonary capillary wedge pressure (PCWP). Nevertheless, they were inversely related to pulmonary pressures -mean pulmonary artery pressure (MPAP), systolic pulmonary artery pressure (SPAP), diastolic artery pressure (DPAP). In terms of the laboratory test results, capillary perfusion deterioration was related to organ dysfunction with a signi cant decrease of pH and BE, diuresis reduction, increase in creatinine and urea only in Group 3 (Table 3).

Discussion
Flow impairment and heterogeneity are the main pathologies accounting for microcirculatory derangement in sepsis. Endothelium dysregulation, arteriolar constriction, increased leukocyte and platelet adhesion, and heterogeneous expression of inducible nitric oxide synthase cease the ow in some capillaries, reducing functional capillary density [9]. Areas without ow are adjacent to hyperperfused regions. The off-load time for oxygen exceeds the time of erythrocyte passage through capillaries with hyperdynamic ow, which results in the inability of oxygen to detach from hemoglobin and diffuse [11]. It seems that ow heterogeneity has a worse impact on tissue oxygenation than equally impaired perfusion [2,27,28]. The restoration of ow in closed capillaries by NO, a strong vasodilator, might improve tissue oxygenation and elimination of anaerobic metabolism products. Total nitric oxide production in sepsis is increased but its distribution is very heterogenous mainly because of uneven expression of inducible NO synthase [12,22,23,29,30]. This creates areas with NO de ciency that might bene t from exogenous NO delivery [21,23]. The effect of iNO on microcirculation remains unclear [23,31]. In our study, iNO potentiated by intravenous hydrocortisone had no effect on the proportion of perfused vessels, perfused capillary density, and the microcirculatory ow index. Unfortunately, our study had certain limitations. The studied groups were not numerous, which made statistical analysis impossible for some variables including the heterogeneity index of microvascular perfusion, hemodynamic and laboratory data. Moreover, for technical reasons, lactates were not monitored during the study. Even though functional capillary density was not affected by iNO with hydrocortisone, we are unable to evaluate changes in the heterogeneity of perfusion, lactate washing, or organ function.
Microcirculatory variables changed more profoundly in piglets not resuscitated with uids and vasopressors, with a statistically greater decrease of the MFI, sMFI, PPV, sPPV, PVD and sPVD. Only in this group was capillary ow strongly related to systemic ow. In groups receiving uids and noradrenaline, hemodynamic variables were stabilized but these changes did not lead to microcirculatory ow normalization. This observation supports the theory that resuscitation of systemic ow does not resuscitate microcirculation to the same degree as systemic circulation. Capillary ow impairment still remains, but it proceeds more slowly. The discrepancy between hemodynamics and microcirculatory variables has already been described by other authors [3,5,32]. According to De Backer et al., microcirculatory derangement may precede systemic hemodynamic compromise and the relation between hemodynamic and microcirculatory indices may be loose [5]. Still, changes in cardiac output and mean arterial pressure may in uence capillary perfusion [5]. In a single-center prospective observational study, de Backer proved that when sepsis develops at early stages of resuscitation, hemodynamic measurements are related to microvascular ow indices, and this correlation disappears at later stages [3]. In the human subject research, our observations are limited to septic and resuscitated groups, and are usually compared to healthy control groups. For obvious reasons, a control non-resuscitated group cannot be permitted. It is possible that for this reason no association between systemic hemodynamic and capillary ow indices was observed at advanced stages of sepsis. There is no doubt that hemodynamic resuscitation in sepsis is bene cial [33]. In recent years, we have been seeking correlations between macro-and microcirculatory indices in sepsis. This correlation exists when sepsis proceeds undisturbed. Perhaps the search for a direct relationship between the indicators of micro-and microcirculation and the expectation of microcirculation normalization as soon as global hemodynamics improvement is reached are some erroneous assumptions at an early stage of the therapeutic process and treatment optimization. The fact that microcirculation and systemic circulation are not coherent in resuscitated patients does not mean that hemodynamic resuscitation is not effective in terms of capillary perfusion resuscitation. As shown above, microcirculation deteriorated in resuscitated groups, but more slowly. This suggests that resuscitation may buy time necessary for the immune system and antibiotics to overcome the infection. This observation suggests that the management of sepsis should rst concentrate on the monitoring and stabilization of systemic circulation and -at later stages -on the evaluation and resuscitation of microcirculation [5].
Moreover, in the non-resuscitated group, microcirculatory parameters were signi cantly related to the progress of acidosis and renal failure. This correlation was not observed in resuscitated groups, where acidosis and organ dysfunction did not proceed as fast in the non-treated group. In Group 3, the combination of capillary perfusion deterioration and related metabolic acidosis supports the theory of microcirculatory pathogenesis of anaerobic metabolism.
In our study, the TVD, sVD and de Backer score did not follow any certain pattern and their changes over time were not statistically signi cant. This observation does not support the results presented by Massey from the ProCESS trial [34]. In the above study, the TVD and de Backer score appeared to be strongly associated with outcome in septic patients. However, their prognostic value appeared to be signi cant after 72 hours of observation. The observed difference in the TVD and de Backer score value might result from a shorter time of observation and a smaller group of animals in our study.
Our study had certain limitations. The number of animal subjects in the study groups was limited and the statistical analysis of some parameters was not possible.
Finally, our observations of microcirculatory alterations were limited to the sublingual area. The sublingual mucosa and the digestive mucosa have the same embryologic origin, and changes in their capnometry correlate quite well, showing similar alterations [21,35,36]. Capillary perfusion in the sublingual area seems to re ect the ow in the splanchnic mucosa and is easily accessible. Nevertheless sidestream dark eld imaging does not allow direct in vivo observation of microcirculation in vital organs.

Ethical Issues
The study was approved by the Animal Research Ethics Committee of the Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland (permission number 7/05) and reported in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Care provided to animals was compliant with the European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scienti c Purposes [37].
Throughout the research, care was taken to guarantee maximum comfort to animals, which were generally anesthetized, and the depth of anesthesia was adjusted to the level of painful stimulation. Those that survived the study period were euthanized with pentobarbital.

Animal Preparation
A detailed description of the instrumentation was presented elsewhere earlier [38]. This study is part of a large project covering a sequence of studies on iNO delivery to septic piglets.
The experiment was conducted at the Department of Surgery at the Wroclaw University of Environmental and Life Sciences, Poland. Originally, the study group consisted of 12 piglets of Polish breed. One of them was excluded as it developed severe hypotonia unresponsive to treatment during instrumentation and died. All animals were of the same age (2 months) and their median body weight was 18.5 kg (range 17-22 kg).
All piglets fasted during nocturnal hours before the experiment; however, they were allowed to drink water at will. To induce anesthesia, we administered zolazepam/tiletamine 4 mg×kg − 1 dissolved in medetomidine 0.08 mg×kg − 1 intramuscularly. All animals were successfully intubated and mechanical

Study Protocol
The timeline of interventions is presented in Fig. 1. After a one-hour rest, all piglets underwent a surgical midline laparotomy. The descending colon was visualized, a 3-cm incision was performed, and 1.5 g/kg of fecal content were removed, mixed with blood and then deposited close to the diaphragm, in order to cause a septic-like condition. Then, the laparotomy was closed.
Next, the piglets were randomly allocated to one of three groups: 1. Group 1 -piglets receiving iNO (Pulmonox-Messer Griesheim 800 ppm iNO in 9000 nitrogen) at a concentration of 30 ppm and hydrocortisone (75 mg intravenously every 7 hrs). Hypotensive piglets (MAP < 60 mmHg for more than 3 minutes) were treated with a bolus of Ringer's lactate (300 ml) and norepinephrine infusion (if unresponsive to uids) to maintain MAP > = 65.
2. Group 2 -piglets not receiving iNO or hydrocortisone but treated for hypotension with uids and noradrenaline infusion as in the case of Group 1; 3. Group 3 -piglets not receiving iNO and hydrocortisone, not resuscitated with uids or noradrenaline infusion

Microcirculation Assessment
The study evaluated microcirculation with the MicroScan™ device (MicroVisionMedical, Amsterdam, the Netherlands) based on sidestream dark eld imaging (SDF). We assessed the microvascular bed in the sublingual region of the oral cavity in four different sequences at every time point. Every record was stored by a random number code and the analyzing researcher was blinded to all clinical data. To optimize image quality and avoid artefacts, we ensured maximum camera stability, removed saliva from the sublingual area using sterile gauze, and minimized pressure on the mucosa.
For the microcirculation evaluation, we used AVA 3.0, which automatically identi es vessels. It calculates total vessel density, the number of vessels crossing a grid of six lines (dividing the screen image into 16 equal areas), and the de De Backer score (total number of vessel crossings per grid length). Further images were assessed in two semi-quantitative methods described elsewhere by Boerma and de Backer [3,6]. The analysis provided information about the proportion of perfused vessels (PPV), perfused vessel density (PVD = PPVxTVD), and microvascular ow index (MFI). All the above variables were separately calculated for small vessels whose diameter was smaller than 20 µm (small vessel MFI-sMFI, small vessel density-sVD, proportion of perfused small vessels-sPPV, perfused small vessel density-sPVD). We also calculated the heterogeneity index (HI), which was proposed by Trzeciak et al. for microvascular evaluation [3].

Statistical Analysis
The data were analyzed using STATISTICA ver. 12. A one-way ANOVA was performed for a comparative analysis of microcirculatory variables obtained for the studied groups at each time point. Pearson's coe cients were used to assess the correlation between microcirculatory indices and hemodynamic variables, and between microcirculatory parameters and laboratory data. A p-value of < 0.05 was considered signi cant. All values are reported as medians, unless otherwise stated.

DATA AVAILABILITY
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

AUTHOR CONTRIBUTIONS
MG performed the experiments, acquisited, analyzed and interpreted the data, wrote the manuscript.
WG designed the study, supervised this study, performed the experiments, analyzed and interpreted the data.
PH performed the experiments, acquisited and analyzed the data.