Introduction: Healing is essential for successful colorectal surgery. Optimal microcirculation is needed to ensure this; however, this is only subjectively assessed by the surgeon. Laser speckle contrast imaging (LSCI) is an objective noncontact, image-based method to quantify microcirculation in bowel ends. This study aimed to evaluate the application of LSCI in an open surgery porcine model, determine differences between normal and impaired microcirculation, and test the LSCI applicability to repeated measurements. Method: A midline laparotomy was made in ten healthy female pigs to expose the colon and small intestine. Subsequently, baseline measurements were conducted. A local arteria supplying the colonic or small intestine mesentery was clamped for 5 min and LSCI measures were made again. After an hour’s rest, LSCI measurements were done in two unaffected areas on the colon and the small intestine, and baseline values were recorded. Hypotension was induced with rapid bleeding and LSCI measurements were done. After the mean arterial blood pressure (MAP) dropped to 50–60 mm Hg, norepinephrine infusion was started. At a stable MAP of 85–100 mm Hg, LSCI measurements were repeated at 0 min and 30 min during continuous norepinephrine infusion. Results: Cross-clamping caused LSCI levels to drop equally in both the colon and small intestine by 60% in the entire clamped zone. Compared to baseline, the microcirculation measured by LSCI in the unclamped adjacent transition zone was diminished by 33% and 40% in the colon and small intestines, respectively. During hypotension due to bleeding, LSCI decreased as expected. When MAP was stabilized by norepinephrine infusion, LSCI values dropped further: compared to baseline, measurements decreased with 24% and 20% in the colon and small intestines, respectively. Conclusion: LSCI can be used as a quantitative, real-time, non-contact method to detect changes in the microcirculation during open intestinal surgery with large changes in microcirculation due to, e.g., hypovolemic and norepinephrine infusion. It is simple to use and in contrast to the existing intraoperative microcirculation assessment techniques, LSCI stands out primarily for its elimination of the requirement for a dye. As our study has shown, this feature allows us to perform time-independent measurements and repeat them indefinitely in nearby regions without compromising the effectiveness of the method.

Colorectal surgery is performed for both malignant and benign cases [1]. Optimal microcirculation is an essential aspect of colorectal surgery to ensure healing and prevent anastomotic leakage, minimizing postoperative morbidity and mortality [2, 3]. Currently, the microcirculation in intestinal surgery is assessed subjectively by the surgeon by evaluating, e.g., sufficient pulsation in a terminal artery, pulse, tissue color, and bleeding from surfaces. However, the ideal estimation of the microcirculation should be objectively quantifiable, reproducible, minimally invasive, and manageable in a clinical setting [4].

Laser speckle contrast imaging (LSCI) is a dye-free, noncontact, noninvasive image-based method, capable of quantitative estimation of the local intestinal perfusion. Further, it allows for time-independent repeated measurements. LSCI employs coherent laser light with a specific wavelength. When a tissue is uniformly illuminated with laser light, it creates a laser speckle where the scattered light creates a random interference effect. This is in contrast to normal, incoherent light, where interference does occur but the interference pattern fluctuates randomly and therefore forms no speckle pattern. When an object is in motion or contains moving particles, such as blood cells in circulation, this leads to phase variations and the creation of a dynamic speckle pattern. Based on this principle, LSCI can be used to assess the blood flow within the tissue (microcirculation) [5]. Thus, LSCI cannot measure absolute flow, but rather, contrast changes and results are expressed as flux in arbitrary laser speckle perfusion units (LSPUs), where higher LSPU values correspond to higher flow [5, 6]. LSCI is validated and used in small animal and human studies of the gastrointestinal tract, but has not been thoroughly tested in a true clinical setting [7‒9].

To prepare for the translation of LSCI into a clinical setting, this study aimed to (1) evaluate if LSCI is applicable to open intestinal surgery, (2) investigate if LSCI measurements differ between tissue with normal and severely impaired microcirculation, (3) describe the applicability of the method for repeated measurements, and (4) describe the effect of norepinephrine on the microcirculation during perioperative hypovolemia.

Study design: To evaluate whether LSCI was applicable in an open surgery setting and could reliably measure the intestinal microcirculation during standard colorectal surgery, two experimental protocols were designed on a porcine model (Fig. 1). For this, ten healthy female pigs of Danish Landrace with a weight of 41.75 ± 1.58 kg (mean ± SD) and age 14–16 weeks were used.

Fig. 1.

Experimental timeline. Overview of the experiment detailing all steps and time points for LSCI measurements. MAP, mean arterial pressure; NE, norepinephrine.

Fig. 1.

Experimental timeline. Overview of the experiment detailing all steps and time points for LSCI measurements. MAP, mean arterial pressure; NE, norepinephrine.

Close modal

Experimental Protocol 1

To investigate aim 1 of whether LSCI could measure microcirculation in open intestinal surgery, baseline measures were made in healthy large and small intestines in the porcine model 30 min after laparotomy. To address aim 2, the arterial blood supply for a part of the intestines was cross-clamped 40 min after laparotomy. Since our aim was to investigate severely impaired microcirculation, we waited 4–5 min to ensure ischemia, and then we conducted LSCI measurements. We divided the measured areas into five regions of interest (ROIs): the ROIs were placed with a 5-mm gap between them. This minimized the risk of one intestinal ROI moving into the adjacent ROI due to the slight peristaltic movements of the intestine in the LSCI images. ROI 3 was placed in the transition zone which we defined as “the transition from clamped to non-clamped tissue,” equal to the end of the clamping forceps in the experiment. As a result, ROIs 1–2 were placed toward the cross-clamp-affected intestine and ROIs 4–5 were placed toward the normal intestine at a distance of ±5 and 10 mm from the transition zone, respectively.

Experimental Protocol 2

To evaluate aims 3 and 4, baseline measurements were conducted again on untouched intestine 2 h after laparotomy. To investigate microcirculatory changes in open intestinal surgery, we introduced hypovolemic hypotension in the porcine model and then corrected the blood pressure with norepinephrine. Throughout the experiment, repeated LSCI measurements (specified in Fig. 1) were made in four predefined ROIs: 1 m and 1.5 m distal to the terminal ileum (colon) and 2 m and 2.5 m proximal to the terminal ileum (small intestines). Hypotension (MAP of 50–60 mm Hg) was induced by rapid bleeding done by aspirating blood from an arterial catheter placed in the femoral artery. Norepinephrine infusion was initiated to normalize the blood pressure until the desired MAP of 85–100 mm Hg was reached. Norepinephrine infusion remained constant and secured a stable MAP of 85–100 mm Hg throughout the remaining experiment.

Anesthesia

All pigs were sedated according to the standard procedure with intramuscular Zoletil 50 vet (25 mg/kg), xylazine, ketamine, and butorphanol. To ensure stress reduction in the pigs, they were moved to the stable next to the operating room 24 h before the operation to acclimatize. Anesthesia was induced and maintained with propofol (15 mg/kg/h) and fentanyl (5 μg/kg/h). After endotracheal intubation, the pigs were ventilated mechanically with positive pressure ventilation using 40% oxygen and a respiratory rate of 14–18/min [5]. Lactated Ringer’s solution was administrated continuously at a rate of 5 mL/kg/h and a urinary catheter was installed to account for urine production. 8F arterial catheters were placed in both femoral arteries for continuously arterial pressure monitoring and blood samples. Arterial blood gas was analyzed at each LSCI measurement (ABL 90; Radiometer Medical ApS) in order to monitor the ventilation and the general condition of the pig. The pigs were euthanized at the end of the experiment with intravenous phenobarbital while they were still fully anesthetized.

Microcirculation

The microcirculation was assessed with LSCI using a wavelength of 785 nm (MoorFLPI-2, Moor Instruments, Axminster, UK). The LSCI was placed perpendicular to the surface and fixed at 25 cm high. It was set to cover a minimum of 5 cm intestine from the area of interest on each side. Each measurement was recorded over 30 s, with a sampling rate of 25 frame/s.

Computational and Statistical Analysis

The initial analysis of flow data was performed using software installed on a standard desktop (MoorFLPI2 Research software v2.x). Each ROI was 5 mm wide and covered the entire diameter of the intestine. Flow data from each 30-s measurement were acquired and median values were extracted through the software [10], and statistical analysis was performed in STATA version 17 (StataCorp LLC, College Station, TX). Data were tested for normal distribution and repeated measurement was analyzed within pigs by linear mixed models. Two-sided p values <0.05 were considered statically significant. The data were presented as LSPU percentage differences relative to the mean baseline LSPU value established across all ten animals in the study.

The trial was approved by the Danish Animal Experiments Inspectorate (No. 2020-15-0201-00695) and conducted under the supervision of veterinarian personal at AU Foulum, Department of Animal Science, Aarhus University, Denmark.

A total of ten pigs underwent surgical procedures without complications or adverse events. During the rapid bleeding, mean aspiration of 752 mL (min;max 350;1400 mL) blood induced the desired hemodynamic changes. Hemodynamics are presented in Figure 2.

Fig. 2.

Hemodynamic variables (mean ± SD) as they changed during the experiment. Time points on the x-axis and mm Hg or beats per minute (bpm) on the y-axis. BP Sys, systolic blood pressure; MAP, mean arterial pressure; NE, norepinephrine.

Fig. 2.

Hemodynamic variables (mean ± SD) as they changed during the experiment. Time points on the x-axis and mm Hg or beats per minute (bpm) on the y-axis. BP Sys, systolic blood pressure; MAP, mean arterial pressure; NE, norepinephrine.

Close modal

Experimental Protocol 1

The results are presented in Figure 3 and Table S1 (for all online suppl. material, see https://doi.org/10.1159/000535525). Baseline microcirculation values differed significantly between the small and large intestines (p < 0.001). Colon LSPU values averaged 850 (95% confidence interval (95% CI): 688–1011), while the small intestine exhibited higher values at 1750 (95% CI: 1585–1915). During arterial cross-clamping, LSCI measurements decreased by 60% compared to baseline for both intestines. Microcirculation was lowest within the cross-clamped area (ROI 1) but improved toward the transition zone (ROI 2). At ROI 3, LSCI values were decreased 33% and 40% for the large and small intestines, respectively. At ROI 4, microcirculation levels reached 90% of baseline, while at ROI 5, LSCI measurements resembled the baseline. ROI 3 to ROI 1 showed significant differences from baseline for both large and small intestines, while ROI 4 and ROI 5 did not.

Fig. 3.

Experimental protocol 1: cross-clamped blood supply to the small intestine. Regions of interest (ROIs) are placed 5 mm apart. ROI 3 is placed in the transition zone, placing ROIs 1–2 toward the cross-clamp-affected intestine and ROIs 4–5 toward the normal intestine at a distance of ±5 and 10 mm from the transition zone, respectively. The arrows mark the transitional zone where a forceps is seen cross-clamping the intestinal loop arterial blood supply. Graphical representation; mean LSCI measurements from ROI 1 to ROI 5 in the large and small intestines. Dots represent mean flow, whiskers 95% CI.

Fig. 3.

Experimental protocol 1: cross-clamped blood supply to the small intestine. Regions of interest (ROIs) are placed 5 mm apart. ROI 3 is placed in the transition zone, placing ROIs 1–2 toward the cross-clamp-affected intestine and ROIs 4–5 toward the normal intestine at a distance of ±5 and 10 mm from the transition zone, respectively. The arrows mark the transitional zone where a forceps is seen cross-clamping the intestinal loop arterial blood supply. Graphical representation; mean LSCI measurements from ROI 1 to ROI 5 in the large and small intestines. Dots represent mean flow, whiskers 95% CI.

Close modal

Experimental Protocol 2

There was no statistically significant microcirculatory difference between the colon 1 m and 1.5 m distal to neither the terminal ileum nor for the small intestines 2 m and 2.5 m proximal to the terminal ileum (online suppl. Table S2). Therefore, data from the two ROIs were pooled in the following analyses. The results from the experiment are shown in Figure 4.

Fig. 4.

Experimental protocol 2: LSCI measurements prior and during hypovolemic hypotension. A Colon. B Small intestine. C Graphical representation of the relative change from baseline in microcirculation during the experiment for both colon and small intestine, measured by LSCI, whiskers represent 95% CI. Time points: baseline (a); hypotension (MAP of 50–60 mm Hg) (b); norepinephrine at 0 min (c); norepinephrine after 30 min (d).

Fig. 4.

Experimental protocol 2: LSCI measurements prior and during hypovolemic hypotension. A Colon. B Small intestine. C Graphical representation of the relative change from baseline in microcirculation during the experiment for both colon and small intestine, measured by LSCI, whiskers represent 95% CI. Time points: baseline (a); hypotension (MAP of 50–60 mm Hg) (b); norepinephrine at 0 min (c); norepinephrine after 30 min (d).

Close modal

After 2 h rest, no change in microcirculation was observed at the colon (p = 0.356) or at the small intestine (p = 0.952) compared to baseline (1 h, experimental protocol 1). After induced hypovolemic hypotension, a statistically significant drop in the microcirculation was measured in both the colon (14%, p = 0.003) and the small intestine (13%, p < 0.001). The norepinephrine infusion resulted in a further microcirculatory reduction of 16% in both the colon and small intestine. After 30 min of continuous norepinephrine infusion, at a stable MAP, the colonic and small intestinal microcirculation was decreased to 24% (p < 0.001) and 20% (p < 0.001) of baseline, respectively.

In this study, we used LSCI to measure the microcirculation in open surgery on porcine intestines and to detect changes in the microcirculation during hypovolemia due to bleeding. LSCI was able to quantitatively detect real-time microcirculatory changes, with repeated measurement regardless of time, without contact to the tissue, and the method proved easy and simple to use.

In protocol 1, LSCI distinguished microcirculatory changes in adjacent ROIs only 5 mm apart with good performance and great reproducibility. This ability to distinguish microcirculatory changes is in line with previous findings in both human and porcine models [11, 12]. This high discriminatory ability is an important feature, if LCSI is to be implemented in a clinical setting as a supporting tool for perioperative perfusion assessment to, e.g., decrease anastomotic leakage rates in colorectal surgery [13]. In this study, we show that within the ischemic zone (ROI 1), the microcirculation is the poorest, but as we approach the transition zone, it increases. At ROI 5, 10 mm from the transition zone, the LSPU values seem to plateau, indicating that microcirculation is nearly re-established. However, it would be interesting to include more ROIs further away from the transition zone to establish a distance at which the microcirculation restored. The experiment shows that even though the arterial supply for ROI 4 was not cross-clamped, the microcirculation was still impaired, and only at ROI 5, were LSPU values approximating baseline values. These measurements across ROI 4–5 indicate that even though there is in principle blood supply to the tissue adjacent to an ischemic area, the microcirculation is impaired at least 10 mm into the tissue adjacent to the transition zone. These findings suggest that even if the surgeon observes active bleeding from the marginal arteria adjacent to the transition zone (where the anastomotic resection incision is planned), the microcirculation is likely to be impaired in the remaining area, which will be unrecognized. Currently, the ability to distinguish between good and suboptimal perfusion is reliant on subjective evaluation alone. Our experiment illustrates the low precision of this method and may underline why no subjective methods have yet been able to show a consistent effect in decreasing the anastomose leakage rate [4, 14‒19].

However, one innate challenge of using LSCI for microcirculation assessment is that measurements are given in “LSPU,” an arbitrary laser unit. LSPU values vary inter-individually, as indicated by the broad confidence intervals in online supplementary Tables S1 and S2. Furthermore, LSPU values differ between tissues intra-individually: the absolute mean LSPU value of the small intestine was approximately 1,500 but only 850 for the large intestine, and during cross-clamping, the absolute LSPU values for ischemic small intestines were higher than the baseline LSPU values for the well-perfused colon. In online supplementary Table S2, we observe wide 95% CIs for both the colon and small intestine. At baseline, this interval ranges from 836 to 1219 LSPU. Similarly, when examining the decrease in microcirculation during continuous norepinephrine infusion in the colon, a relative change of 24% was found, and the 95% CI ranges up to an LSPU value of 994. This illustrates that cautious interpretation of absolute LSPU values is warranted and analysis of relative changes in LSPU as previously suggested is more meaningful [20]. Thus, an absolute threshold value of “ischemia,” applicable to all tissue, will probably be indeterminable. Instead, perfusion change over time or perfusion change in comparison to adjacent, unaffected tissue may be alternative ways to investigate whether a threshold value for “poor perfusion” can be established; such a threshold value holds clinical perspective in the evaluation of appropriate timing for, e.g., resections of tissue with irreversible ischemic damage, anastomoses, and wound closure.

In protocol 2, we applied the principle of relative LSPU changes for assessment of microcirculation: Hypovolemic hypotension was induced, blood pressure decreased, and in parallel, LSPU values decreased with 14%. When blood pressure was normalized by continuous norepinephrine infusion for 30 min, the microcirculation was further reduced to 24% and 20% of baseline in the large and small intestines, respectively. This indicates that the large intestines are more susceptible to microcirculatory changes than the small intestines. We hypothesize that this susceptibility is reflected in the higher anastomosis leakage rates of large intestinal anastomosis compared to small intestinal anastomosis [21], where a decrease of 25% in the microcirculation has previously been identified as the lower limit for acceptable perfusion [22]. Regarding norepinephrine, it is a widely used vasopressor substance but has been shown to negatively affect intestinal microcirculation by 23% measured with laser doppler flow [23]. We confirm this effect by real-time LSCI in our porcine model. These findings underline that a normal blood pressure postoperatively is not necessarily a marker of restored intestinal microcirculation if norepinephrine has been given. Alternatives to norepinephrine should be considered to promote tissue healing.

An alternative method for evaluation of the microcirculation, which has gained interest in recent years, is the use of indocyanine green fluorescence angiography (ICG-FA), as it is currently the only commercially available assessment method that can be combined with minimally invasive endoscopic surgery. Nevertheless, the predictive value of ICG-FA to prevent anastomosis leakage has shown potential but is not consistent [16‒19, 24]. The method is impaired by three major limitations: The administration of the ICG dye makes it time-dependent; it limits repeated measurements [25]; and the interpretation of the fluorescence intensity, as a proxy for increased circulation at the anastomosis site, is still dependent on clinical evaluation [26]. In contrast, LSCI distinguishes itself particularly in one aspect by eliminating the need for a dye. As demonstrated in our study, this has enabled us to conduct time-independent measurements and repeat them infinitely in adjacent areas without compromising the method’s performance. One of the major limitations of LSCI is its susceptibility to motion artifacts. While LSCI is highly sensitive to the minute movements of red blood cells, it tends to capture unwanted motion artifacts within the measured signal, and effectively separating artifacts from the true data proves to be a challenging task [27]. Nevertheless, promising results have emerged regarding the adaptation of LSCI for minimally invasive surgery [28]. This advancement would render the technique significantly more attractive, given that it is currently only commercially available for open surgery.

The present study has some limitations in extrapolating results to a clinical setting. We use a porcine model, as it is impossible to perform LCSI measurements in similar, standardized circumstances in humans. Thus, results may not be directly transferable to humans, though porcine models are thought to provide the best translation of experimental results to human intestinal physiology [29]. Another limitation is the small number of porcine experiments. However, we met our objectives and demonstrated a consistent effect with consideration to the ethical aspects of animal model research [30]. In protocol 2, we normalized blood pressure solely using norepinephrine to isolate its specific effect; in a clinical scenario with acute bleeding, the use of vasopressors would be combined with crystalloids and blood transfusions as well.

The presented LSCI porcine model provides means to investigate the impact of microcirculation on open gastrointestinal surgery in various scenarios. Moving forward, we aim to investigate how different types of anastomoses and sepsis affect microcirculation, as well as examine the differential effects of inotropic drugs on the intestines. Further research is necessary to determine whether LSCI can be used as a reliable predictor for critical perianastomotic perfusion values in a clinical setting.

AU Foulum is acknowledged for their invaluable aid and supervision during the experiments. Special gratitude is extended to animal caretaker Emilie Sommerlund for her assistance during the operations. Biorender.com was used for design of Figure 1.

This study protocol was reviewed and approved by Danish Animal Experiments Inspectorate (No. 2020-15-0201-00695) and conducted under the supervision of veterinarian personal at AU Foulum, Department of Animal Science, Aarhus University, Denmark.

The authors have no conflicts of interest to declare.

No specific funding was obtained for this study. Rupan Paramasivam is supported by grants from the NEYE Foundation, the Dagmar Marshalls Foundation, and NIDO Denmark. The funders did not play a role in the study’s design.

R.P.: formal analysis, investigation, resources, data curation, writing – original draft, visualization, and funding acquisition. N.M.K. and M.S.: investigation and writing – review and editing. R.A. and A.H.M.: conceptualization, methodology, supervision, and writing – review and editing. M.W.Ø.: conceptualization, methodology, supervision, writing – original draft, and writing – review and editing.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.

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