Early course of microcirculatory perfusion in eye and digestive tract during hypodynamic sepsis

Lithuanian White pigs were fasted 12 hours before the experiment but were allowed free access to water. All pigs were premedicated with intramuscular ketamine hydrochloride (20 mg/kg) and xylazine (2 mg/kg), followed by cannulation of an ear vein. Orotracheal intubation (6.5- to 7.5-mm tracheal tubes) was performed after intravenous induction of general anesthesia with 6 mg/kg of thiopental sodium. The animals were ventilated with a volume-controlled mode with a positive end-expiratory pressure of 5 cm H₂O (Drager, Lubeck, Germany). Tidal volume was kept at 10 to 12 ml/kg, and the respiratory rate was adjusted (14 to 16 breaths/min) to maintain end-tidal carbon dioxide tension between 35 and 45 mm Hg (4.7 to 6.0 kPa). General anesthesia and muscle relaxation were maintained with a continuous intravenous infusion of fentanyl, 15 μg/kg/h, midazolam (0.2 mg/kg/h), and pipecuronium bromide IV boluses as needed. The stomach was emptied with an orogastric tube.

Both femoral veins and the right femoral artery were surgically dissected and catheterized. An arterial catheter was placed into the right femoral artery to measure invasive arterial blood pressure and to obtain blood gases. A standard thermodilution 7F Swan-Ganz catheter (TD-I; B. Braun Medical, Bethlehem, PA, USA) was advanced into the pulmonary artery through an introducer in the right femoral vein. A central vein catheter was introduced into the left femoral vein for delivery of medications and fluids. A Foley catheter was inserted into the urinary bladder through a small suprapubic incision.

A proximal-loop jejunostomy was constructed for microcirculatory assessment through a 5- to 7-cm incision on the right side of the abdominal wall. The jejunostomy was covered with moisturized gauze with local hourly administration of saline solution to maintain mucosal integrity.

Ringer lactate solution was infused at rate of 5 ml/kg/h during the surgical procedures. After surgical preparations, the animals were allowed to stabilize for 1 hour before recording the baseline measurements.

After basal measurements, the pigs were assigned randomly to either the septic or the sham group. Sepsis was induced by an infusion of live Escherichia coli (E. coli, 1.8 × 10⁹/kg colony-forming units) over a 1-hour period. E. coli was isolated from blood cultures from a patient who died of septic shock, according to a previous published model [10]. During and after the infusion of E. coli, Ringer lactate was continuously titrated to maintain CVP and PAOP at baseline levels. In addition to this, mean arterial pressure (MAP) was maintained > 70 mm Hg by providing standardized fluid boluses of 250 ml in 15 minutes until the increase in cardiac index (CI) did not exceed 10%.

Images of the sublingual, conjunctival, jejunal, and rectal microcirculation were obtained with SDF videomicroscopy (Microscan; Microvision Medical, Amsterdam, The Netherlands) [11]. After gentle removal of saliva and other secretions with isotonic saline-drenched gauze, the device was applied to the sublingual region, avoiding pressure artifacts by establishing a threshold image. Before evaluation of conjunctiva, the eye was washed with 0.9% saline solution. An SDF objective with a disposable sterile lens cap was gently applied to the perilimbal surface of the bulbar conjunctiva, preventing the collapse and decrease of flow of large vessels. Movement artifacts were prevented by attaching the SDF objective to a finger of the other hand placed on the periocular bones and by additional fixation of the eyelid. An additional person was often needed to optimize the brightness and sharpness of the image. The eye conjunctiva was regularly rinsed with 0.9% saline solution to prevent local inflammation and was further protected by wet gauze on the eye between measurements. Measurements were performed on the same eye during all experiments.

The microcirculation of the small intestine was investigated through a jejunostomy by insertion of an SDF objective to the depth of 5 to 7 cm from the edge of the stoma and slight angulation.

The microcirculation of rectum was investigated through the anus by insertion of an SDF objective to the depth of 6 to 10 cm from the edge.

Sequences of 20 seconds from at least three areas within one vascular bed were recorded on a hard disk of a personal computer and AVA v3.0 software (Microvision Medical, Amsterdam, The Netherlands). Video clips were blindly analyzed offline by two investigators in random order to prevent coupling. Assessment of microcirculatory parameters of convective oxygen transport (microvascular flow index (MFI) and proportion of perfused vessels (PPVs)), and diffusion distance (perfused vessel density (PVD) and total vessel density (TVD)) was done according to published recommendations [12]. Vessels were separated into large (mostly venules) and small (mostly capillaries) by using a diameter cutoff value of 20 μm. The final MFI score is the average value of all quadrants in three different areas of one vascular bed. The heterogeneity index was calculated as the difference between the highest and lowest MFI, divided by the mean MFI of all sites at a single time point [5]. Microvascular beds of gut villi (small intestine) and crypts (colon) consist only of small vessels. In addition to MFI, we also calculated PPV as the percentage of perfused villi or crypts divided by the total number of villi or crypts times 100%, in accordance with definitions in the sublingual region [12].

The primary end point was the MFI of small vessels in all investigated locations. The secondary end point was the correlation between sublingual and others locations of the microcirculation. The Statistical Package for Social Sciences (SPSS 15.1 for Windows, Chicago, IL, USA) was used for statistical analysis. Data were assessed for normality by using the Shapiro-Wilk test and presented as mean ± SD. Repeated-measures analysis of variance (ANOVA) for time comparison was used with Bonferroni correction for post hoc analysis. Differences between sepsis and control animals were analyzed by using the Mann-Whitney U test. Correlation between the sublingual microcirculation and that of other locations was assessed with the Spearman rho. A P value < 0.05 was considered statistically significant.

The kappa coefficient for interrater variability was 0.86 (P < 0.001), and for intrarater variability was 0.79 (P < 0.001). The percentage of disagreement for the MFI in the various vascular beds was as follows: eye, 5.6%; sublingual, 5.2%; intestinal stoma, 8.6%; and rectal, 9.0%. Initial microcirculatory parameters at the starting point were similar in sepsis and control groups (Table 3). The distribution of vessels according to internal diameter and length in sublingual and eye conjunctival locations at baseline are shown in Figure 1. In the conjunctiva, 77% of the vessel length was < 8 μm in internal diameter, and 92% was < 20 μm. In the sublingual mucosa, 81% of the vessel length was < 8 μm in internal diameter, and 90% was < 20 μm.

A significant decrease of microcirculatory parameters of convective oxygen transport (MFI and PPV of small vessels) was observed in all locations (sublingual, jejunal, rectal mucosa, and ocular conjunctiva) in the sepsis group (Table 3). In contrast, we observed a significant decrease in only one parameter of diffusion distance (PVD of small vessels) in the conjunctival but not in the other locations. TVD remained unaltered in both the sublingual and conjunctival microcirculatory beds (Table 3). Statistical analysis of MFIs in different microcirculatory locations of the sepsis animals revealed that the lowest MFIs could be detected in the sublingual region 3 hours and 5 hours after the induction of sepsis with infusion of E. coli (Figure 2).

We observed no correlation between MFI and CI, MAP, or lactate in almost all locations. Only sublingual MFI significantly correlated with lactate (r _s = 0.9; P = 0.037).

A statistically significant correlation could be demonstrated between the sublingual and conjunctival (r _s = 0.80; P = 0.036), sublingual and jejunal (r _s = 0.80; P = 0.044), and sublingual and rectal (r _s = 0.79; P = 0.030) MFI measurements 3 hours after the start of E. coli infusion. However, this correlation between the sublingual and conjunctival, and the jejunal and rectal regions disappeared 5 hours after the start of E. coli infusion (Figure 3). Correlations of MFI between others areas except the sublingual are shown in Additional file 1. Correlations between beds at different times for PPV, PVD, and TVD of small vessels are shown in Additional file 2.

We observed a tendency toward an increased heterogeneity index level at 3 hours after the start of E. coli infusion (0.11 ± 0.07 versus 0.25 ± 0.15; P = 0.068), and a significant difference at 5 hours after the start of E. coli infusion (0.11 ± 0.07 versus 0.59 ± 0.20; P = 0.025) in comparison with baseline. Digital microphotographs of microcirculation in the sepsis group are presented in Figure 4.