In the pathophysiology of sepsis, impairment of the intestinal microcirculation is a major complication that can lead to a collapse of the intestinal barrier (1, 2). This collapse has been shown to enable translocation of bacteria and toxins into the bloodstream and local lymph system, thereby aggravating multiorgan failure and increasing mortality (3, 4). Thus, growing effort is made to maintain and restore gastrointestinal microcirculation in the therapy of critical illness (4, 5).
In this context, secondary actions of selected drugs gained attraction in the therapy of sepsis. For example, it could be demonstrated that HMG-CoA-reductase inhibitors such as pravastatin show immunomodulatory actions (e.g., inhibition of nuclear factor kappa-B, downregulation of pro-inflammatory cytokines, and protection against endothelial cell apoptosis) independent of lipid lowering and may improve survival in sepsis (6–8). Furthermore, it is well known that statins can also ameliorate microcirculation, especially in patients with coronary heart disease (9, 10). Experimental and human observational studies suggest that statins might also have ameliorated microcirculatory function in sepsis (11–13). However, the benefit of statin therapy is a subject of discussion, especially in combination with higher levels of carbon dioxide. A recent meta-analysis has shown that in patients with acute respiratory distress syndrome (ARDS), where moderate hypercapnia is clinically intended, statin therapy might not be beneficial (14).
This is in contrast to recent experimental studies where moderate hypercapnia alone ameliorates the impaired gastrointestinal microcirculatory oxygenation in hemorrhagic shock and sepsis and restores gut permeability (15–18). Moderate hypercapnia, also known as permissive hypercapnia, generally is a tolerated side effect during lung-protective ventilation with low lung stretch and reduced mortality in critically ill patients with sepsis and in particular with ARDS (19, 20). Furthermore, besides its ameliorative effect on intestinal microcirculatory oxygenation, hypercapnia has also anti-inflammatory effects, suppresses the production of reactive oxygen species, and thereby seems to play an important role in preservation of tissue integrity during sepsis (19, 21).
Thus, the aim of this study was to clarify if pravastatin ameliorates the impaired intestinal microvascular oxygenation and flow during sepsis and if this effect is abolished through combination of pravastatin and moderate hypercapnia. The results might give new insights in statin therapy in sepsis.
MATERIALS AND METHODS
The study was performed in accordance with NIH guidelines for animal care and in accordance with the ARRIVE guidelines (see appendix, http://links.lww.com/SHK/A845) (22). After approval by the local Animal Care and Use Committee (Landesamt für Natur, Umwelt und Verbraucherschutz, Recklinghausen, Germany, Az. 87–51.04.2010.A361) 40 male Wistar rats (320 g–380 g body weight) were randomized into four groups (n = 10) (Fig. 1). The animals were derived from the breeding facility at the Central Animal Research Facility of the Heinrich-Heine-University Duesseldorf. Half of them received 0.2 mg • kg−1 pravastatin (pravastatin sodium salt hydrate, P4498, Sigma-Aldrich, Taufkirchen, Germany; injected volume 1 mL • kg−1) subcutaneously, the other half received the same volume NaCl 0.9% as vehicle subcutaneously. After 18 h colon ascendens stent peritonitis (CASP)-surgery was carried out in all animals to develop sepsis using an established protocol as described previously (16, 23). The experiments started at 8:00 AM in the research laboratory of the Heinrich-Heine-University Duesseldorf. In brief, the animals were anesthetized with sevoflurane (3.0%–3.2 % end-expiratory, FiO2 0.5) and received buprenorphine (0.05 mg • kg−1, s.c.). An approximately 2-cm-long median laparotomy was performed. After locating the colon, it was penetrated on its antimesenteric wall, approximately 1 cm distal to the ileocecal valve, with a 16-gauge peripheral venous catheter (PVC, Vasofix safety, B. Braun Melsungen AG, Melsungen, Germany). The inner needle was withdrawn, allowing constant fecal leakage into the abdominal cavity to develop abdominal sepsis, subsequently. The PVC was sewed on the colon wall (6–0 Prolene, non-resorbable, Ethicon Inc, Somerville, Mass) and shortened to a total length of ∼8 mm. The intestine was placed back in its former position and the abdominal wall was closed in two layers.
After CASP surgery, animals were kept individually in separate plastic cages at a 12-h light/dark cycle with free access to water and food under controlled temperature (24°C ± 2°C) and humidity (50 ± 5%). Furthermore, animals were monitored with respect to their welfare, as previously reported (16). Buprenorphine (0.05 mg • kg−1 s.c.) was applied 12 h after surgery. Within 24 h after induction of sepsis, a total of eight animals died. Therefore, CASP surgery was performed on a total of 48 animals to complete 40 successful experiments (four groups with n = 10 per group).
Twenty-four hours after induction of sepsis, the animals were anesthetized by pentobarbital-injection (60 mg • kg −1 i.p.), placed on a heating pad, tracheotomized and mechanically ventilated in a volume-controlled, pressure-limited mode (70 min−1, VT 1.8 mL–2.5 mL, PAW < 17 cm • H2O, FiO2 = 0.3, FiN2 = 0.7) to maintain normocapnia (pCO2 40 ± 6 mm Hg) (Inspira Advanced safety Ventilator, Harvard Apparatus GmbH, March-Hugstetten, Germany).
To ensure continuous anesthesia, an external jugular vein catheter was established with subsequent continuous pentobarbital-infusion (10 mg • kg−1 • h−1). Additionally, 2 mg pancuronium were injected every 2 h. Blood pressure was measured in the left arteria carotis communis, 1 mL arterial blood was extracted for further analysis and 120 μL blood were extracted intermittently for blood gas analysis (ABL 715, Radiometer, Copenhagen, Denmark) throughout the experiment. A continuous infusion of Ringer solution (4 mL • h−1) was applied via the arterial access for volume replacement and to prevent blood coagulations. The animals were relaparotomized and a flexible light guide probe (O2C LW 2222, Lea Medizintechnik GmbH, Gießen, Germany; penetration depth 0.7 mm, tissue catchment area 7 mm2, separation 1 mm) was placed on the tunica serosa of the colon ascendens, 1 cm distal to the stent.
Microcirculatory flow (μflow) and microcirculatory oxygenation (μHBO2) were measured as previously described via laser Doppler flowmetry and reflectance spectrophotometry, respectively (16). These techniques simultaneously assess microcirculatory oxygenation and flow through the whole colonic wall (penetration depth of the probe: 0.7 mm; wall thickness 0.45 mm). Briefly, white light (450 nm–1,000 nm) and laser light (820 nm, 30 mW) are transmitted to the colonic tissue via a microlightguide and the reflected light is analyzed. The wavelength-dependent and overall absorption of the applied white light can be used to calculate the percentage of oxygenated hemoglobin in the microcirculation (μHbO2). Due to the Doppler effect, magnitude, and frequency distribution of changes in wavelength are proportional to the number of blood cells multiplied by the measured mean velocity of these cells. This product is proportional to flow and is expressed in arbitrary perfusion units. Hence, this method allows for the assessment and comparison of oxygenation and perfusion of the same region at the same time (24). Changes of flow can be attributed either to a change in velocity or in the number of red blood cells, comparable to the information gained by intravital microscopy (25). Only the microcirculation is measured as light entering vessels bigger than 100 μm is completely absorbed. The biggest fraction of the blood volume is stored in venous vessels (85%), so mostly postcapillary oxygenation is measured, which represents the critical partial pressure of oxygen for hypoxia (26). The flexible light-guide probe was placed on the tunica serosa of the colon ascendens. Online evaluation of the signal quality throughout the experiments allows verification of the correct position of the probe tip. The μHbO2 and μflow values reported are the means of the last 5 min every 30 min.
To establish moderate hypercapnia (pCO2 72 ± 10 mm Hg), after 30 min (baseline) with normocapnic ventilation (pCO2 40 ± 6 mm Hg), exogenous CO2 was added to the inspiratory air (FiO2 0.3, FiCO2 0.1). In normocapnic groups, pCO2 was maintained at 40 ± 6 mm Hg for 120 min. Thus, four different groups were analyzed: vehicle-treated normocapnic septic animals (NC-V), vehicle-treated hypercapnic septic animals (HC-V), pravastatin-pretreated normocapnic septic animals (NC-PS), as well as pravastatin-preatreated hypercapnic animals (HC-PS) (Fig. 1).
At the end of the experiments, the animals were euthanized by exsanguination under deep anesthesia.
To calculate the appropriate sample size a priori power analysis (G*Power Version 3.1.7, University of Dusseldorf, Germany) was performed. With n = 10 animals per group at a given α ≤ 0.05 (two-tailed) and an expected mean difference in μHbO2 of at least 20% (percentage points) with an expected standard deviation of 10% to 15% (based on previous studies) a power of 84.5% resulted.
Normal distribution of data was assessed and confirmed in Q-Q-plots (IBM SPSS Statistics, International Business Machine Corp, Armonk, New York). Data were analyzed with a two-way ANOVA for repeated measures, followed by Dunnett post-hoc test for differences versus baseline, and Tukey post-hoc test for differences between groups (GraphPad software v 6.0, Int, La Jolla, Calif).
Data are presented as means ± SD, P < 0.05 was considered significant.
Wherever delta values are presented, the absolute baseline value was subtracted from the absolute value at the respective observation points to individualize the data to each rat's baseline. Therefore, the data are provided as absolute percentage points with regard to baseline values and not as relative changes.
Table 1, Figures 2 and 3 summarize the effects of hypercapnia and pravastatin on systemic hemodynamics as well as on intestinal microcirculatory oxygenation and flow in sepsis. 24 h after CASP surgery, baseline values did not differ significantly between the groups (data not shown).
The effect of normocapnia
In normocapnic septic animals (NC-V) μHBO2 decreased significantly over time (90 min: −6.6 ± 6.4%; 120 min: −8.4 ± 8.7%; P < 0.05 vs. baseline) (Fig. 2). μflow did not change over time (Fig. 3). There were also no significant changes in mean arterial blood pressure (MAP) and heart rate (HR) (Table 1).
The effect of hypercapnia
In contrast to normocapnia, μHBO2 remained constant over time in septic animals with hypercapnic ventilation (HC-V) (90 min: 3.1 ± 5.7%; 120 min: 0.9 ± 8.5% vs. baseline) and was significantly higher than in normocapnic animals (NC-V) after 60, 90, 120 min (Fig. 2). Like in normocapnic animals, μflow (Fig. 3), MAP and HR (Table 1) remained unchanged.
The exogenous CO2 addition to establish hypercapnic ventilation led to a significant rise in pCO2 and a significant decrease in pH (Table 1).
The effect of pravastatin
In animals pretreated with pravastatin (NC-PS), μHBO2 did not change in the course of time (90 min: + 2.0 ± 5.9%; 120 min: −1.9 ± 5.7% vs. baseline) (Fig. 2). Furthermore, after 90 min μHBO2 was significantly higher compared with vehicle-treated animals (NC-V). Pravastatin had no effect on μflow (Fig. 3), MAP, and HR (Table 1).
The effect of pravastatin and hypercapnia
μHBO2 declined in hypercapnic pravastatin pretreated animals (HC-PS) significantly over time (90 min: −6.6 ± 6.7%; 120 min: −8.9 ± 11.8%; P < 0.05 vs. baseline). With combination of pravastatin and hypercapnia the effect of hypercapnia alone and pravastatin alone was abolished, μHBO2 of HC-PS-treated animals was significantly lower compared with HC-V (90, 120 min) as well as to NC-PS (90 min) (Fig. 2). μflow did not change over time in hypercapnic septic animals with previous pravastatin-injection (Fig. 3) and there were also no significant changes in MAP and HR (Table 1).
The exogenous CO2 addition to establish hypercapnic ventilation led also in this group to a significant rise in pCO2 and a significant decrease in pH (Table 1).
This study was carried out to evaluate the role of pravastatin pretreatment with and without additional moderate hypercapnia on the intestinal microvascular oxygenation and flow under septic conditions. The main results are:
- Hypercapnia prevents the deterioration of the intestinal microcirculatory oxygenation during sepsis.
- Pretreatment with pravastatin ameliorates the intestinal microcirculatory oxygenation during sepsis.
- Hypercapnia abolishes the effect of pravastatin pretreatment on the microcirculatory oxygenation of the colon during sepsis.
- Intestinal microcirculatory oxygenation is improved without changes in intestinal microvascular flow.
We used the well-established CASP model of sepsis to simulate closely the pathophysiology of human abdominal sepsis (23). This model led to a continuously increasing systemic inflammation and provided us with the opportunity to study microcirculatory alterations of the intestine seen in the development of sepsis (23, 27). Thereby, in the context of replacement, refinement or reduction of animal experiments (the 3Rs), it is important to note that sepsis could only be investigated rationally in an animal model because a cell model could not reflect the systemic influence of sepsis on microcirculation, immune system and sympathetic nervous system. However, the observed mortality rate of 17% is comparable to previous observations and reflects the moderate severity of our sepsis model to avoid scarification of experimental animals (16, 23). There was no difference in mortality between animals receiving pravastatin pretreatment or vehicle. However, this study was not powered to detect any mortality differences. It is important to note that our animals do not suffer hypotension as expected in septic shock. Instead, our therapeutic approach starts before development of septic shock and aims to prevent this. Nevertheless, we have clear signs of infection: visible peritonitis, mortality rate of 17%, and high cytokine plasma levels (as shown in previous studies with the same sepsis model (16)).
The dosage of pravastatin used in this study (0.2 mg • kg−1 s.c.) is adapted from the literature (11, 28). In our study, the animals received their statin dose before sepsis induction, analog to other studies (12, 28). The question if only pretreatment with statins might be beneficial in sepsis or if de novo therapy after sepsis-induction could also be advantageous is an ongoing discussion. In a recent multicenter trial Kruger et al. (29) found that only the prior statin users rather than de novo users had a lower mortality risk compared with placebo. Ou et al. (30) came to the same conclusion in their analysis. However, in a murine model of sepsis also the statin therapy after onset of sepsis is beneficial (11). Furthermore, a single-center study on 100 critically ill patients also found that acute administration of statins may prevent sepsis progression (31). In this study, we focused on the effects of pravastatin on the septic intestinal microvascular oxygenation and flow over a short observation period and therefore used pravastatin pretreatment.
We analyzed microcirculatory alterations in the colon via reflectance spectrophotometry and laser Doppler flowmetry, as in our previous studies (15, 16, 23). The combination of these two techniques allows a reliable detection of microcirculatory oxygen supply, especially of the postcapillary area (16, 26). This technique has been validated in various tissues and used in several clinical settings (32–35). The monitoring method is in principle suitable to measure parallel alteration between microcirculatory oxygen and flow as we have proved in a mild hemorrhagic shock model in dogs (loss of 20% of the estimated total blood volume). This mild hemorrhagic shock induces a combined and significant reduction in microcirculatory flow and oxygenation in the oral and gastric mucosa, demonstrating that flow induced ischemia is accompanied by decreases in tissue oxygenation (36). However, as we discuss below in the current study intestinal microcirculatory oxygenation is improved without changes in intestinal microvascular flow. Furthermore, it is important to note that the analyzed postcapillary area is mainly affected by septic derangements and that microcirculatory oxygen alterations in this area correlate with outcome of septic patients (37, 38). Besides, Yeh et al. (39) demonstrated in their animal study that ameliorated intestinal oxygenation in septic rats augment intestinal barrier function and reduce mucosal cell death as indirect parameters for improved outcome.
However, the used microcirculatory imaging technique of this study is limited in assessment of capillary density or heterogeneity of blood flow as it averages the whole catchment area. Future studies should aim to combine the measurement with other imaging techniques like sidestream dark field (SDF) imaging to further strengthen the validity of data on microcirculatory alterations (40). Furthermore, it might be a limitation of our current study that we have not taken any histology to examine if changes in microcirculatory oxygenation lead on to tissue damage. However, we expected that only tissue function, i.e., barrier function, is altered without (irreversible) damage to the tissue. Recent data suggest that even mild changes in regional oxygenation, which most likely do not lead to irreversible damage as shown in a canine model, have impact on barrier function (41). In this setting, i.e., in sepsis before visible tissue damage occurs, we have an opportunity for therapy in the clinical setting, whereas once the tissue is obviously damaged, interventional therapy might be too late. Nevertheless, it is a limitation of our study that the used microcirculatory imaging technique averages the whole colonic wall and so we do not only measured the microvascular oxygenation and flow of the particular interesting intestinal mucosa (penetration depth of the probe: 0.7 mm; wall thickness 0.45 mm). However, to limit surgical stress due to opening the intestine and subsequently impair intestinal microcirculatory oxygenation and flow per se, we have placed the flexible light guide probe atraumatic on the tunica serosa of the colon.
In this study, we focused on the examination of microcirculatory intestinal microvascular oxygenation and blood flow. Restoration of macrohemodynamic parameters, as for example cardiac output, in sepsis does not seem to be the crucial point, but rather the improvement of microcirculatory circulation seems to be vital for treatment of critically ill patients and the strongest predictor of outcome (37). We have recently demonstrated that hypercapnic ventilation restored the aggravated gastrointestinal microcirculatory oxygenation during sepsis (16, 23). The hypercapnia-induced amelioration was confirmed in the present study. It is important to note that we could demonstrate in a previous study that this amelioration seems to be independent of pH. Despite normal pH, due to buffering the hypercapnic acidosis, the hypercapnic effect was maintained (16).
Furthermore, we have demonstrated that pretreatment with pravastatin could also maintain intestinal microcirculatory oxygenation in sepsis. HMG-CoA-reductase inhibitors induce protective effects on microcirculation in different tissues: La Mura et al. (12) observed that prophylactic simvastatin prior to LPS administration prevents the development of microvascular dysfunction in the liver. Another study demonstrated vessel relaxation after pravastatin pretreatment in the mesentery during LPS administration (28). Possible explanations for the described statin effects are restoration of the impaired parasympathetic microvascular innervation during sepsis and the increased expression of endothelial nitric oxide with consecutive microcirculatory vasodilatation (28, 42). In our study, we did not observe a difference in μflow as indicator for perfusion changes between pravastatin-treated and vehicle-treated septic animals. However, pravastatin pretreatment prevented the decline of microcirculatory oxygenation seen in septic animals. This demonstrates on the one hand that there is no strict connection between microvascular oxygenation and flow and that overall flow might be maintained, when an increase of vessel diameter reduces velocity. On the other hand, it is important to note that the oxygenation seems to be the crucial parameter for evaluating the microcirculation. This is supported by our recently published results showing that gastrointestinal barrier function rather depends on adequate oxygenation than on perfusion (41). A possible explanation for the increased intestinal microcirculatory oxygenation might be that pravastatin optimizes mitochondrial function as observed in cardiac cells (43). Another explanation could be an increased shunting of oxygen to the venular compartment with a change from aerobic to anerobic energy extraction (37), which we cannot further address since lactate values were not measured in this study. Nevertheless, it is important to note that several studies indicate that a preserved mixed venous oxygen saturation as well as preserved microcirculatory oxygenations per se are markers for improved outcome (38, 44). However, it is a limitation of our study that we have not measured changes in cell metabolism.
Therefore, further studies have to be executed to study the pravastatin effect on mitochondrial function in the gut. Furthermore, it is worth to investigate if an improvement of diffusion distances or an optimization of heterogeneous perfusion might provide an explanation for the increased microcirculatory oxygenation, for example using other imaging techniques, like SDF imaging. Especially, because our imaging technique averages the whole catchment area and therefore existing differences in μflow due to heterogeneity of blood flow could not be detected.
Nevertheless, optimized intestinal microcirculatory oxygenation with improved gastrointestinal barrier function might be a possible explanation for improved survival seen in other septic studies with statin pretreatment, besides other beneficial mechanisms like preservation of cardiac function and reduced inflammatory response (8, 11, 30, 41). This survival benefit due to statin pretreatment seems to persist after sepsis is survived (30).
However, the effect of statin therapy is subject of discussion, especially in patients with elevated levels of carbon dioxide. Two recently published meta-analyses do not provide evidence for a clinical benefit of statin therapy in septic patients with ARDS (14, 45). In these patients, permissive hypercapnia is clinically intended (19, 20). The reasons for these conflicting results can be various. For example, the included studies showed a significant heterogeneity with regard to statin pretreatment versus de novo statin therapy and in ARDS severity. Thus, it remains unclear which specific groups of patients might benefit from statins.
We have demonstrated, in the present study, that hypercapnia abolishes the ameliorative effect of pravastatin pretreatment on the intestinal microvascular oxygenation under septic conditions. These results might provide an explanation for the lacking clinical benefit described in statin-treated patients with severe ARDS and associated hypercapnia. The potentially insufficient intestinal microvascular oxygenation with consecutive insufficient gastrointestinal barrier function could worsen patient's outcome. A possible explanation for the abolished ameliorative effect of statin therapy under hypercapnic conditions might be that hypercapnia as well as HMG-CoA reductase inhibitors have an influence on vasopressin (15, 46, 47). Vasopressin, however, seems to be crucial for the intestinal microcirculatory oxygenation under septic conditions (15, 48). Nevertheless, further studies need to clarify if there are counteracting effects of hypercapnia and statins on the intestine in patients with severe sepsis and ARDS.
Here, we studied the role of statins in a rodent model of sepsis and not in human sepsis, so these data and their clinical impact should be interpreted with care. Nevertheless, our CASP model is one of the closest experimental models to imitate anastomosis failure with consecutive abdominal sepsis. Therefore, our results highlight the potential of statin pretreatment in sepsis to ameliorate intestinal microcirculatory oxygenation and thus preventing the vicious circle with intestinal hypoxia, translocating bacteria (and toxins), and aggravation of sepsis (3).
In conclusion, our study showed that pravastatin pretreatment ameliorates the intestinal microvascular oxygenation in sepsis without changes in microcirculatory flow. In addition, we demonstrated that additional hypercapnia abolishes this ameliorative effect of pravastatin on the septic intestinal microcirculatory oxygenation, indicating why septic ARDS patients might not benefit from pravastatin therapy. Further studies are needed to explore these experimental results in septic patients.
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