Septic Shock reduces overall Intestinal Microcirculation and specifically Anastomotic Perfusion: Insights from a Porcine Model Using Laser Speckle Contrast Imaging

Septic Shock reduces overall Intestinal Microcirculation and specifically Anastomotic Perfusion: Insights from a Porcine Model Using Laser Speckle Contrast Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Septic Shock reduces overall Intestinal Microcirculation and specifically Anastomotic Perfusion: Insights from a Porcine Model Using Laser Speckle Contrast Imaging Rupan Paramasivam, Claudia Jaensch, Nickolai Malte Kristensen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6567985/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Septic shock can severely compromise intestinal perfusion, increasing the risk of poor anastomotic healing and surgical complications. This study investigated the effects of septic shock on intestinal microcirculation and anastomotic perfusion in a porcine model using Laser Speckle Contrast Imaging. Ten pigs underwent laparotomy with creation of four anastomoses— one hand-sewn and one stapled in both small intestine and colon. Microcirculatory measurements were taken before and after anastomosis formation, and repeatedly during the development of septic shock induced by intravenous Escherichia coli infusion. Septic shock led to a significant reduction in microcirculation across both untouched bowel and anastomoses. Hand-sewn anastomoses maintained higher perfusion than stapled anastomoses throughout all time points. This study shows that septic shock significantly impairs intestinal microcirculation indicating a risk of intestinal ischemia if bacteriaemia and subsequent septic shock is untreated. Due to diminish blood flow following septic shock, the anastomotic healing may be compromised leading to increased risk of anastomotic leakage. Conclusively, this study provides a foundation for optimizing surgical strategies and improving patient outcomes in this high-risk population. Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer Health sciences/Pathogenesis/Infection Health sciences/Risk factors Sepsis intestinal anastomoses microcirculation Anastomotic perfusion porcine model Laser Speckle Contrast Imaging (LSCI) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Sepsis is a complex and life-threatening condition that disrupts multiple organ systems, including the microcirculation[ 1 ]. The effects of sepsis on mesenteric blood flow and its underlying pathophysiology have been extensively studied[ 2 ]. Sepsis, characterized by systemic inflammation and the release of toxins, can cause profound alterations in the splanchnic circulation, including reduced mesenteric perfusion and intestinal ischemia[ 3 – 5 ]. Nevertheless, our understanding of how intestinal microcirculation and healing is affected during sepsis remains limited[ 6 ]. Some studies have demonstrated higher rates of complications, such as anastomotic leakage (AL) and delayed wound healing, likely due to compromised tissue perfusion[ 7 , 8 ]. However, other studies have suggested that performing an anastomosis in emergency colorectal surgery is safe, with no increased overall risk of AL[ 9 ]. Nonetheless, these studies also report a significantly higher risk of AL if the patient remains septic and requires vasopressor therapy during secondary anastomoses. Subsequently, the management of patients with sepsis, whether preexisting or developing postoperatively, remains without clear evidence-based guidelines. Experimental studies using advanced imaging techniques, such as Sidestream Dark Field (SDF) imaging[ 10 ] and Laser Doppler Flowmetry (LDF)[ 11 , 12 ], have demonstrated significant reductions in intestinal microcirculation during sepsis. These findings strongly support the hypothesis that ischemic injury to the gut plays a pivotal role in sepsis-related surgical complications. However, these studies also have notable limitations. In the LDF study[ 11 ], the sepsis model was based on fecal peritonitis, which itself is a known risk factor for postoperative complications, potentially confounding the results and limiting the applicability of the findings in other sepsis etiologies. Similarly, the SDF study[ 10 ] measured intraluminal microcirculation in the small intestine via a stoma and in the colon only through the rectum. This restricted sampling approach may not provide a comprehensive understanding of the microvascular changes throughout the entire intestinal tract. Thus, critical questions are still unanswered about the safety and outcomes of surgical interventions in septic patients. To date, no studies have directly evaluated the effects of sepsis on intestinal anastomoses or their healing potential. This study aims to bridge this gap by using Laser Speckle Contrast Imaging (LSCI) to investigate the direct effects of septic shock on the microcirculation of both the colon and small intestine, as well as the impact on hand-sewn and stapled anastomoses. As a novel imaging modality, LSCI offers significant advantages by enabling repeated, quantitative measurements of microcirculation under dynamic conditions, providing a robust and detailed assessment that may help inform clinical decision-making[ 13 – 15 ]. By exploring how sepsis influences intestinal microcirculation, this study seeks to provide insights that could inform clinical practice; especially concerning acute intestinal surgery. Materials and methods The impact of septic shock on the microcirculation of the small intestine and colon, as well as hand-sewn and stapled anastomoses in both small intestine and the colon, was assessed using an experimental setup in a porcine model. Porcine model The anesthesia protocol for the porcine model has been described in detail previously[ 15 ]. Briefly, ten female Danish Landrace/Yorkshire/Duroc pigs, weighing an average of 43.35 ± 2.5 kg and aged 14–16 weeks, were sedated, and anesthetized. Propofol and fentanyl infusions maintained anesthesia throughout the experiment. The animals were intubated and mechanically ventilated, receiving Lactated Ringer's solution at a rate of 5 ml/kg/h. The septic response was defined by characteristic alterations in systemic physiology, including a progressive decline in hemodynamic parameters (mean arterial pressure (MAP) and systolic blood pressure), accompanied by rising lactate levels and decreasing arterial pH. These variables were continuously monitored throughout the experiment to confirm the onset and progression of septic shock. Surgical procedure Following induction of general anesthesia, a midline laparotomy was performed to expose the small intestine and colon. In each pig, two types of anastomoses were created: a single-layer, hand-sewn seromuscular end-to-end anastomosis[ 16 ] and a stapled anti-mesenteric side-to-side anastomosis[ 17 ] on both the small intestine and colon, resulting in a total of four anastomoses per pig. LSCI measurements were systematically performed on all four anastomoses, as well as on predefined untouched segments of the small intestine and colon, following a standardized protocol (Fig. 1 ). Baseline measurements (T B ) were made prior to anastomosis formation, with additional measurements immediately after the anastomoses were completed (T A ). The intestines were then allowed to rest for one hour without further surgical manipulation, after which the T 0 measurements were obtained. Sepsis was induced by intravenously injection of live Escherichia coli ( E.coli ) (ATCC 25922). The initial infusion rate was set at 16 ml/hour, and the rate was doubled every 10 minutes until approximately 240 ml of the E.coli solution had been administered. LSCI measurements and arterial blood gas analyses were performed at 30-minute intervals, starting at T 30 . This continued until T 150 , after which the pigs were euthanized with intravenous phenobarbital while they were still fully anesthetized. Laser Speckle Contrast Imaging, LSCI Microcirculation was evaluated using LSCI at a wavelength of 785 nm (MoorFLPI-2, Moor Instruments, Axminster, UK). This method enabled assessment of microcirculation to a depth of approximately 1 mm. The LSCI device was positioned 25 cm above the intestinal surface, ensuring coverage of at least 5 cm on either side of the region of interest (ROI). Measurements were captured over a 30-second period with a sampling rate of 25 frames per second[ 18 ]. Results were presented both as a picture heatmap and in arbitrary Laser Speckle Perfusion Units (LSPU). E.coli infusate The E.coli sepsis model used in this study has been described previously[ 19 ]. In summary, cultures of E.coli (strain ATCC 25922, Department of Medical Microbiology, Aarhus University Hospital) were grown on 5% sheep blood agar plates (Statens Serum Institut, Copenhagen, Denmark) for 24 hours at 37°C. During the log phase of growth, colonies were harvested from the agar surface, suspended in normal saline and then adjusted to OD 620 = 1.2 (approximately 1 × 10^9 CFU/ml). The following day, the bacterial suspension was serially diluted in triplicates and plated on to sheep blood agar plates and incubated for 24h at 37°C followed by CFU enumeration on the agar plates, to verify the concentration and ensure accuracy. Prior to administration, the suspension was stored at 4°C to prevent bacterial replication under low-oxygen conditions of the infusate. Histopathology Tissue samples from the small intestine and colon were collected at baseline and at the end of the experiment. These samples were sent for histological analysis to evaluate any changes associated with E.coli sepsis. Briefly, the biopsies were fixated in 10% neutral buffered formalin and processed through graded concentrations of alcohol and embedded in paraffin wax. Tissue sections were cut (4 µm) and stained with hematoxylin and eosin (HE). HE stained sections were evaluated patho-morphologically with special focus on identifying (yes/no) an acute inflammatory response. The following parameters were evaluated in mucosa and submucosae, respectively; epithelial damage, hemorrhage, hyperemia, edema, neutrophil infiltration, and necrosis. Computational and statistical analysis Four regions of interest (ROIs) were selected for analysis (Fig. 2 ) for each anastomosis. ROI 1 was centered directly on the anastomosis, ROI 2 was positioned 5 mm away from the anastomosis on either side, ROI 3 was located 10 mm from the anastomosis on either side, and the final ROI was placed on a predefined untouched segment of the small intestine/colon. Each ROI measured 5 mm in width and spanned the entire intestinal diameter, with ROI 1–3 arranged adjacently. Median microcirculatory values in LSPU were calculated for each ROI. Data was analyzed using linear mixed models to account for repeated measurements within the same animal. Results were presented as LSPU changes relative to baseline, which was defined as the average LSPU across all ten animals in the study at T B . Statistical significance was defined as p < 0.05. Statistical analyses were performed using STATA version 18 (StataCorp LLC, College Station, TX, USA), while microcirculation flux data were processed using MoorFLPI2 Research software v2.x (Moor Instruments, Axminster, UK). Ethics The trial was conducted under the supervision of veterinary personnel at the Department of Animal Science, Aarhus University, AU Foulum, and the Principles of Laboratory Animal Care[ 20 ] were followed. The trial was approved by the Danish Animal Experiments Inspectorate (No. 2022-15-0201-01331). All methods were carried out in accordance with relevant guidelines and regulations. Pigs were purchased from Department of Animal Science, Aarhus University, AU Foulum. All methods are reported in accordance with the ARRIVE guidelines. Results Sepsis model All ten pigs underwent surgery without complications or adverse events. To induce septic shock, an average of 235.5 ml of E.coli suspension was administered (range: 210–240 ml), with a mean bacterial concentration of 1.11 × 10 9 CFU/ml (range: 8 × 10 8 – 1.4 × 10 9 CFU/ml). The hemodynamic data (Fig. 3 ) during sepsis show a significant decrease in systolic blood pressure (p < 0.001) and MAP (p < 0.001), accompanied by a significant increase in pulse (p < 0.001). Additionally, there was a significant reduction in pH levels (p < 0.001) and a significant increase in lactate levels (p < 0.001). LSCI measurements were successfully performed at all predefined time points in all pigs. Representative LSCI images illustrating microcirculation around the anastomoses are shown in Fig. 2 (colon) and Supplementary Fig. 1 (small intestine). Relative changes in LSPU compared to baseline are presented in Fig. 4, while absolute LSPU values are provided in Supplementary Table 1. Microcirculation in Untouched Colon and Small Intestine In the untouched colon, no statistically significant difference in microcirculation was seen during anastomosis formation or after 1 hour of rest. After E.coli infusion, a decline was seen: perfusion continued to drop over time, reaching statistically significant reductions at T 60 (91%, 95%CI: 80–99%); T 90 (81%, 95%CI: 80–86%); and T 150 (69%, 95%CI: 60–78%). In the untouched small intestine, a significant reduction in perfusion was observed already during anastomose formation, with a drop to 84% (95%CI: 80–88%. After one hour of rest, perfusion increased slightly to 90% (95%CI: 85–95%), though still significantly lower than baseline. After E.coli infusion, perfusion declined further to 76% of baseline (95%CI: 70–82%) at T 30 ; 69% (95%CI: 60–74%) at T 60 ; and 66% (95%CI: 60–71%) at T 150 (Fig. 4). All measurements following E.coli infusion were statistical significant lower compared to baseline. Colonic Microcirculation at Hand-Sewn and Stapled Anastomoses ROI 1, Anastomosis The perfusion in hand-sewn anastomoses at T A was 55% of baseline (95%CI: 43–67%), which was significantly higher than the perfusion observed in stapled anastomoses of 42% (95%CI: 35–49) (p = 0.011). This finding persisted throughout the study: After 1 hour rest (T 0 ), perfusion increased in both groups and was significantly higher in hand-sewn anastomoses; After E.coli infusion, the perfusion levels decreased and at T 150 , perfusion in hand-sewn anastomoses dropped to 51% of baseline (95%CI: 40–63%), whereas the stapled anastomoses’ perfusion dropped to 34% (95%CI: 30–43%) which was significantly lower than the hand-sewn anastomoses (p = 0.002). ROI 2,±5 mm Adjacent to the Anastomosis Perfusion within 5 mm of the anastomosis was higher than at ROI 1 for hand-sewn and stapled anastomoses after the anastomosis formation (92% vs. 88%). This finding persisted at T 0 through to T 150 , where perfusion adjacent to hand-sewn anastomoses decreased to 64% (95%CI: 50–75%) compared to 55% (95%CI: 50–65%) in stapled anastomoses. ROI 3, ± 10 mm Adjacent to the Anastomosis At ± 10 mm from the anastomosis, no significant differences were observed between hand-sewn and stapled anastomoses across all time points compared to baseline. After E.coli infusion, the impact on perfusion was similar to that seen in untouched intestine, with microcirculation at T 150 reduced to 68% of baseline (95%CI: 60–80%) for hand-sewn anastomoses and 61% of baseline (95%CI: 50–69%) for stapled anastomoses. In summary, microcirculation across all ROIs during sepsis was significantly reduced compared to baseline. Hand-sewn anastomoses consistently demonstrated higher perfusion than stapled anastomoses at all time points, with statistically significant differences observed at the anastomotic site itself. In contrast, differences between hand-sewn and stapled techniques were less pronounced at ± 5 mm and ± 10 mm from the anastomosis, where a more gradual decline in perfusion was observed—mirroring the pattern seen in the untouched colon. Small intestinal Microcirculation in Hand-Sewn and Stapled Anastomoses ROI 1, Anastomosis Region In the anastomosis region, perfusion after anastomosis formation was similar between hand-sewn and stapled anastomoses, with values of 37% (95%CI: 30–43%) and 37% (95%CI: 32–42%) of baseline, respectively (p = 0.952). However, significant differences emerged during subsequent time points. At T 0 , perfusion in hand-sewn anastomoses increased to 53% (95%CI: 49–57%), compared to 41% (95%CI: 36–47) in stapled anastomoses (p = 0.010). During sepsis, perfusion in hand-sewn anastomoses continued to decrease significantly less relative to stapled anastomoses; at T 150 , the perfusion in hand-sewn anastomoses was 45% (95%CI: 40–54%), and in stapled anastomoses 36% of baseline (95%CI: 30–42%, p = 0.014), respectively. ROI 2, ± 5 mm Adjacent to the Anastomosis At ROI 2, microcirculation was generally higher than at the anastomosis site. At T A no significant difference was found in stapled anastomoses (71%, 95%CI: 67–76%) compared to hand-sewn anastomoses (64%, 95%CI: 59–69%, p = 0.071). At T 0 , perfusion was significantly higher in hand-sewn anastomoses (81%) compared to stapled anastomoses (70%, p = 0.004). Similar findings were observed at T 30 , T 90 , and T 150 , where perfusion decreased to 62% (95%CI: 60–68%) for hand-sewn anastomoses and 57% (95%CI: 50–62%) for stapled anastomoses (p = 0.005). ROI 3, ± 10 mm Adjacent to the Anastomosis At ± 10 mm from the anastomosis, perfusion was similar between hand-sewn and stapled anastomoses after anastomosis formation (78% vs. 82%, p = 0.303). At T 0 , hand-sewn anastomoses had significantly higher perfusion (88%, 95%CI: 83–92%) than stapled anastomoses (78%, 95%CI: 74–82%, p = 0.031). Similar differences were found at T 90 , T 120 , and T 150 , where perfusion declined to 67% (95%CI: 60–74%) in hand-sewn anastomoses and 62% (95%CI: 60–68%) in stapled anastomoses (p = 0.026). Recapitulating, hand-sewn anastomoses had consistently higher perfusion compared to stapled anastomoses, particularly within the anastomosis region and at ± 5 mm from the site. Differences at ± 10 mm were less pronounced, but still statistically significant one hour after E.coli sepsis was induced. Pathology Histological results are presented in Supplementary table 2 and revealed no significant abnormalities in the small intestine or colon. The most common observation was post-operative hyperemia in the colon, characterized by an increased presence of red blood cells within the vessels compared to baseline. In a few samples, an elevated number of neutrophils were noted on the epithelial surface, particularly between villi and within crypts. However, no infiltration was observed in the mucosal or submucosal layers. Discussion This study provides novel insights into the effects of septic shock on intestinal microcirculation, comparing untouched segments of the small intestines and colon to hand-sewn and stapled anastomoses: We demonstrated a significant and progressive decline in microcirculation in both untouched bowel and especially anastomotic regions during septic shock. This may indicate that septic shock impairs intestinal perfusion in general and especially around surgical resection sites, which we hypothesize could impair intestinal healing and increase e.g. anastomotic leak rates. Thus, our findings contribute to our understanding of microcirculatory changes during sepsis and raise important considerations for surgical decision-making in septic patients. Our sepsis model effectively induced a state of septic shock, as demonstrated by significant reductions in systemic parameters, including MAP and pH, alongside elevated lactate levels. These findings are consistent with established septic shock models. Unlike other studies, such as Krejci et. al[ 11 ], who utilized fecal peritonitis as the sepsis model, our approach avoided localized peritoneal contamination. Despite this, the intestines still exhibited pronounced microcirculatory impairments, emphasizing the systemic effects of sepsis on intestinal perfusion, independent of localized contamination. Furthermore, unlike the SDF study[ 10 ], which assessed intraluminal microcirculation, our use of LSCI allowed for direct, repeated measurements on both the small and large intestine, including anastomoses. This comprehensive approach provides a more detailed and accurate representation of intestinal microcirculation under septic conditions, enhancing the translational relevance of our findings. First, our results demonstrate a consistent and significant decline in microcirculation in both the untouched colon and small intestine during septic shock. A notable finding was the difference in microcirculatory responses between the untouched small intestine and colon; The small intestine exhibited a higher baseline perfusion, which may explain the more pronounced reduction observed during sepsis. In contrast, the colon demonstrated a more gradual decline in perfusion. The general decline in both colonic and small intestinal perfusion aligns with the existing understanding of the splanchnic circulatory response to sepsis, where mesenteric blood flow is redistributed to other critical organs (lung, heart, kidney) at the expense of the bowel[ 12 ]. The observed decrease in perfusion could predispose intestinal tissue to ischemia, which raises concerns in cases with both sepsis and surgical treatment including anastomosis formation. To evaluate the effect of surgery on anastomoses and whether normal pathological processes were disturbed during sepsis, tissue was sampled for histological analysis. However, these revealed no significant pathological changes: The primary histological finding was pronounced hyperemia and vascular stasis, consistent with the early vascular phase of the acute inflammatory response[ 21 ]. It is important to note that cellular signs of inflammation, such as neutrophil infiltration, are typically not evident histologically until approximately two hours after the onset of injury, with infiltration peaking around 24 hours. These findings reflect the relatively short duration of the experiment (2.5 hours). If histopathological changes are to accurately reflect impaired healing following sepsis, tissue sampling should be performed 4–7 days postoperatively[ 22 ], as studies have shown this to be the typical timeframe for the development of anastomotic leakage. However, such a setup was beyond the ethical permission given for this porcine model, but could be integrated in a future research model. Second, our study indicates that in cases where anastomoses are made during sepsis, hand-sewn anastomoses present consistently higher perfusion compared to stapled anastomoses across all time points. This suggests that hand-sewn techniques may preserve microcirculatory integrity better than stapled approaches during sepsis and increase the healing potential. However, both types of anastomoses experienced significant declines in perfusion during sepsis, which underlines that cautious surgical planning in septic patients is necessary; salvage surgery without primary anastomosis could be preferable until the septic condition is treated to reduce the risk of anastomotic leakage from impaired microcirculation. Previous research has suggested that the lower threshold for safe anastomotic perfusion is a 30% reduction in microcirculation[ 23 ]; In our study, we demonstrate that sepsis reduced the intestinal microcirculation at the untouched intestine with 33% for the small intestine and 30% for the colon, and even more at the anastomotic sites especially at ROI 1 and ROI 2, which is much more than the suggested 30%. Moreover, the clinical use of vasopressors to stabilize systemic blood pressure during sepsis, such as norepinephrine, has been shown to further compromise intestinal perfusion, aggravating the impaired healing in intestinal surgery[ 15 ]. Limitations and Future Perspectives While this study provides valuable insights into the effects of septic shock on intestinal microcirculation in general and around anastomoses, several limitations must be addressed. First, the study period of 150 minutes of E.coli infusion may not capture the long-term effects of sepsis on anastomotic healing. Future studies with extended experimental durations are necessary to better understand the progression of microcirculatory impairment and its impact on surgical outcomes. Another limitation is the use of a porcine model. While pigs are a translationally relevant model for studying human intestinal microcirculation, their sepsis pathophysiology may not fully replicate the complex clinical scenarios encountered in humans. This highlights the need for careful interpretation of the findings and underscores the importance of validating results in clinical settings. The sample size of ten pigs also represents a limitation, as a larger cohort could reduce the risk of type II errors and potentially yield more statistically significant results. However, ethical considerations place constraints on animal research, and future studies must continue to balance the need for statistical power with ethical responsibility. Beyond addressing these limitations, future research should investigate potential therapeutic interventions, such as targeted vasodilators, to preserve intestinal microcirculation during sepsis. These approaches may provide novel strategies to improve surgical outcomes and reduce complications, such as anastomotic leakage. Moreover, the potential clinical applications of LSCI warrant further exploration. LSCI offers a unique capability for real-time, quantitative assessment of microcirculation, making it a promising tool for intraoperative decision-making in septic patients. For instance, its use could help surgeons evaluate the viability of intestinal segments before performing an anastomosis, minimizing the risk of ischemia-related complications. By integrating advanced imaging modalities like LSCI into acute surgical settings, clinicians may be able to enhance decision-making and improve patient outcomes in high-risk scenarios. Conclusion This study highlights the impact of septic shock in intestinal microcirculation, revealing differences between untouched and anastomotic regions, as well as between small and large intestine. Also, hand-sewn anastomoses preserve microcirculation better than stapled ones, but both show a drastic decline, underscoring the significant risk of anastomotic leakage in septic patients. These findings provide a basis for optimizing surgical strategies and improving outcomes in a state of sepsis. Declarations Acknowledgments AU Foulum is acknowledged for its invaluable aid and supervision during experiments. Special gratitude is extended to animal caretaker Birgitte Frydkjær for her assistance during the operations. We extend our sincere gratitude to the Department of Medical Microbiology, Aarhus University Hospital, for their invaluable support in the preparation and handling of E.coli . Biorender.com was used for the design, as shown in Figure 1. Author Contributions R.P.: Formal analysis, investigation, resources, data curation, writing – original draft, visualization, and funding acquisition. N.M.K., S.P. and H.W.: Investigation and writing, review, and editing. L.K.J., C. J. and A.H.M.: Conceptualization, methodology, supervision, writing, review, and editing. M.W.Ø.: Conceptualization, methodology, supervision, writing – original draft, and writing – review and editing. Data Availability Statement 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 authors. Conflict of Interest Statement The authors declare no conflicts of interest . Funding Sources No specific funding was received for the study. Rupan Paramasivam is supported by grants from the NEYE Foundation, Dagmar Marshalls Foundation, and NIDO Denmark. The funders did not play any role in the design of the study. Statement of Ethics The trial was conducted under the supervision of veterinary personnel at the Department of Animal Science, Aarhus University, AU Foulum, and the Principles of Laboratory Animal Care[20] were followed. The trial was approved by the Danish Animal Experiments Inspectorate (No. 2022-15-0201-01331). References Shepherd, A.P., et al., Effects of vasoconstrictors on intestinal vascular resistance and oxygen extraction. Am J Physiol, 1976. 230 (2): p. 298-305. Reilly, P.M., et al., The mesenteric hemodynamic response to circulatory shock: an overview. Shock, 2001. 15 (5): p. 329-43. Deitch, E.A., et al., A study of the relationship among survival, gut-origin sepsis, and bacterial translocation in a model of systemic inflammation. J Trauma, 1992. 32 (2): p. 141-7. Ruokonen, E., et al., Regional blood flow and oxygen transport in septic shock. Crit Care Med, 1993. 21 (9): p. 1296-303. Fink, M.P., Adequacy of gut oxygenation in endotoxemia and sepsis. Crit Care Med, 1993. 21 (2 Suppl): p. S4-8. Wang, X. and D. Liu, Hemodynamic Influences on Mesenteric Blood Flow in Shock Conditions. Am J Med Sci, 2021. 362 (3): p. 243-251. Patel, S. and A. Duncan, Anaesthesia and intestinal anastomosis. BJA Educ, 2021. 21 (11): p. 433-443. Huisman, D.E., et al., Fluid management and vasopressor use during colorectal surgery: the search for the optimal balance. Surg Endosc, 2023. 37 (8): p. 6062-6070. Fischer, P.E., et al., Vasopressor use after initial damage control laparotomy increases risk for anastomotic disruption in the management of destructive colon injuries. Am J Surg, 2013. 206 (6): p. 900-3. Pranskunas, A., et al., Early course of microcirculatory perfusion in eye and digestive tract during hypodynamic sepsis. Crit Care, 2012. 16 (3): p. R83. Krejci, V., L.B. Hiltebrand, and G.H. Sigurdsson, Effects of epinephrine, norepinephrine, and phenylephrine on microcirculatory blood flow in the gastrointestinal tract in sepsis. Crit Care Med, 2006. 34 (5): p. 1456-63. Hiltebrand, L.B., et al., Redistribution of microcirculatory blood flow within the intestinal wall during sepsis and general anesthesia. Anesthesiology, 2003. 98 (3): p. 658-69. Heeman, W., et al., Application of laser speckle contrast imaging in laparoscopic surgery. Biomed Opt Express, 2019. 10 (4): p. 2010-2019. Heeman, W., et al., Clinical applications of laser speckle contrast imaging: a review. J Biomed Opt, 2019. 24 (8): p. 1-11. Paramasivam, R., et al., Laser speckle contrast imaging for intraoperative assessment of intestinal microcirculation in normo- and hypovolemic circulation in a porcine model. Eur Surg Res, 2023. Saeger, H.D., et al., [Course system of the Working Group for Gastro-intestinal Surgery Davos]. Chirurg, 2010. 81 (1): p. 25-30. Russell, K.W., et al., The Barcelona Technique for Ileostomy Reversal. J Gastrointest Surg, 2015. 19 (12): p. 2269-72. Ambrus, R., et al., Laser Speckle Contrast Imaging for Monitoring Changes in Microvascular Blood Flow. Eur Surg Res, 2016. 56 (3-4): p. 87-96. Dehring, D.J., et al., Comparison of live bacteria infusions in a porcine model of acute respiratory failure. J Surg Res, 1983. 34 (2): p. 151-8. American Society of Laboratory Animal Practitioners Position Statement: Animal Care Principles. J Am Assoc Lab Anim Sci, 2024. 63 (5): p. 470-1. Jarczak, D., S. Kluge, and A. Nierhaus, Sepsis-Pathophysiology and Therapeutic Concepts. Front Med (Lausanne), 2021. 8 : p. 628302. Tsai, Y.Y. and W.T. Chen, Management of anastomotic leakage after rectal surgery: a review article. J Gastrointest Oncol, 2019. 10 (6): p. 1229-1237. Kashiwagi, H., The lower limit of tissue blood flow for safe colonic anastomosis: an experimental study using laser Doppler velocimetry. Surg Today, 1993. 23 (5): p. 430-8. Additional Declarations No competing interests reported. Supplementary Files S1.tiff Suppl.Table12.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6567985","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":465400659,"identity":"0af90d11-cb48-4948-a84a-e3294dae27ee","order_by":0,"name":"Rupan Paramasivam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBAC+wYwZQMijInTYnAATKWRruUwKVpu5B578HPP+XzdBubNBkRpsZ+Rl27Y8+y25bYDbMUJRGmxk8gxk+A5cNvA7ACP8QGitBgDtUj+OXCOBC2GM3LMpHkOHABrIc5hBmfepRvLHEg2MDvMVkyc9w2O5x57+OaAnYHZ8ebNEkRpYWDgYYPQzESqR9IyCkbBKBgFowAXAADsqS+FHhN3uAAAAABJRU5ErkJggg==","orcid":"","institution":"Godstrup Hospital","correspondingAuthor":true,"prefix":"","firstName":"Rupan","middleName":"","lastName":"Paramasivam","suffix":""},{"id":465400660,"identity":"162cd98e-ea35-4a28-8cb1-fb7ca18522ac","order_by":1,"name":"Claudia Jaensch","email":"","orcid":"","institution":"Godstrup Hospital","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Jaensch","suffix":""},{"id":465400665,"identity":"d63a8f74-dac5-4179-9d48-8e861f7e2e41","order_by":2,"name":"Nickolai Malte Kristensen","email":"","orcid":"","institution":"Godstrup Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nickolai","middleName":"Malte","lastName":"Kristensen","suffix":""},{"id":465400668,"identity":"78a44871-7c5d-41e1-a10f-eec681549082","order_by":3,"name":"Susie Paramasivam","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Susie","middleName":"","lastName":"Paramasivam","suffix":""},{"id":465400669,"identity":"5e7bbc35-cfd7-4129-aacc-e5d0ad81c053","order_by":4,"name":"Helen Woldeselassie","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"","lastName":"Woldeselassie","suffix":""},{"id":465400670,"identity":"b85d011b-de6e-4583-982b-0fb525737998","order_by":5,"name":"Louise Kruse Jensen","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"Kruse","lastName":"Jensen","suffix":""},{"id":465400671,"identity":"f9e5af53-5b67-4543-9791-d5553ba7cc92","order_by":6,"name":"Anders Husted Madsen","email":"","orcid":"","institution":"Godstrup Hospital","correspondingAuthor":false,"prefix":"","firstName":"Anders","middleName":"Husted","lastName":"Madsen","suffix":""},{"id":465400672,"identity":"d63ce4b1-706e-48da-a33c-a8d2232f0af8","order_by":7,"name":"Mai-Britt Worm Ørntoft","email":"","orcid":"","institution":"Godstrup Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mai-Britt","middleName":"Worm","lastName":"Ørntoft","suffix":""}],"badges":[],"createdAt":"2025-04-30 21:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6567985/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6567985/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83918862,"identity":"4367a06e-8ac4-4794-ad79-055506d4cce9","added_by":"auto","created_at":"2025-06-04 13:14:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":616414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimeline for the porcine sepsis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverview of the study set-up and LSCI measurement time points. TB=Baseline measurements; TA= Immediately after anastomosis formation; T0 = After one hour of rest, start of\u0026nbsp;\u003cem\u003eE.coli (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)\u003c/em\u003e\u0026nbsp;infusion; T30, T60, T90, T120, T150 = Measurements taken at different time points, in minutes, during\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion/sepsis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/4b3768e126c0eda82080eadb.png"},{"id":83917817,"identity":"2b370233-c18e-46ac-8d89-22a0d36f8e0d","added_by":"auto","created_at":"2025-06-04 13:06:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLSCI measurements of the anastomose for colon\u003c/strong\u003e \u003cstrong\u003eat specific time points: A, Colon Hand-sewn; B, Colon Stapled\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/em\u003eThe placement of regions of interest (ROIs) is indicated on all white light images using bold markings: ROIs were placed adjacent to each other, with ROI 1 centered on the anastomosis, ROI 2 ±5 mm on either side, and ROI 3 ±10 mm on either side of the anastomosis. A color scale ranging from red to blue indicates good to poor perfusion, respectively. T\u003csub\u003eA \u003c/sub\u003e= Immediately after anastomosis formation; T\u003csub\u003e0\u003c/sub\u003e = One hour of rest/start of\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion; T\u003csub\u003e60\u003c/sub\u003e, \u003csub\u003eT120,\u003c/sub\u003e T\u003csub\u003e150\u003c/sub\u003e = Measurements taken during\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion/sepsis, number indicates minutes from start of \u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/4de954b6e7ac6448ab84a1b9.png"},{"id":83917822,"identity":"bbda2d0b-2426-4158-ad0e-0b32bbf748fe","added_by":"auto","created_at":"2025-06-04 13:06:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHemodynamic changes and sepsis variables in the porcine model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraph A shows the hemodynamic parameters measured throughout the experiment: Systolic blood pressure, Mean arterial blood pressure, and Pulse from start (right after anesthesia induction prior to surgery) to T\u003csub\u003e150\u003c/sub\u003e. Following the initiation of the \u003cem\u003eE.coli\u003c/em\u003e infusion (at T0), hemodynamic parameters were recorded every 10 minutes. The y-axis represents values in mmHg (for blood pressure and MAP), beats per minute (for pulse). Whiskers represent SD.\u003cbr\u003e\nGraph B illustrates the progression of pH and lactate levels throughout the duration of the experiment. The graph shows the mean value ± SD (whiskers). X-axis represents time points: Start=After induced anesthesia and prior to surgery; T\u003csub\u003eB\u003c/sub\u003e=Baseline measurements; T\u003csub\u003eA\u003c/sub\u003e= Immediately after anastomosis formation; T\u003csub\u003e30\u003c/sub\u003e, T\u003csub\u003e60\u003c/sub\u003e, T\u003csub\u003e90\u003c/sub\u003e, T\u003csub\u003e120\u003c/sub\u003e, T\u003csub\u003e150\u003c/sub\u003e = Measurements taken during\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion/sepsis, number indicates minutes from start of \u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion. The y-axis represents lactate levels in mmol/L, or pH values.\u003c/p\u003e\n\u003cp\u003eAbbreviations: SysBP = systolic blood pressure, MAP = mean arterial pressure, \u003cem\u003eE.coli = \u003c/em\u003e\u003cstrong\u003eEscherichia coli, SD=\u003c/strong\u003estandard deviation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/bc61363ff8efd2bb65f6a296.png"},{"id":83917819,"identity":"7e38b4b5-a304-4bf0-9c6f-31643f2a7bbc","added_by":"auto","created_at":"2025-06-04 13:06:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of the relative change in microcirculation over time compared to baseline: A, Colon Hand-sewn; B, Colon Stapled; C, Small Intestine Hand-sewn; D, Small Intestine Stapled\u003c/strong\u003e\u003cbr\u003e\nThe y-axis represents the relative change compared to baseline values. X-axis represents time points: T\u003csub\u003eB\u003c/sub\u003e=Baseline measurements; T\u003csub\u003eA\u003c/sub\u003e= Immediately after anastomosis formation; T\u003csub\u003e0\u003c/sub\u003e = After One hour of rest, start of\u0026nbsp;\u003cem\u003eE.coli \u003c/em\u003einfusion; T\u003csub\u003e30\u003c/sub\u003e, T\u003csub\u003e60\u003c/sub\u003e, T\u003csub\u003e90\u003c/sub\u003e, T\u003csub\u003e120\u003c/sub\u003e, T\u003csub\u003e150\u003c/sub\u003e = Measurements taken during\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion/sepsis, number indicates minutes from start of \u003cem\u003eE.coli\u003c/em\u003e\u0026nbsp;infusion.\u003cem\u003e\u003cbr\u003e\nE.coli = \u003c/em\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/2a52fb250b25c38d9ded1280.png"},{"id":86386699,"identity":"433f9491-fe58-4b41-aa02-367165130fee","added_by":"auto","created_at":"2025-07-10 06:01:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1777810,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/30ac0b05-ce5b-42b8-a5ad-b6af28b34e86.pdf"},{"id":83917825,"identity":"10d8c73c-0519-469b-8dbb-4ee59e9f7d77","added_by":"auto","created_at":"2025-06-04 13:06:56","extension":"tiff","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":34790902,"visible":true,"origin":"","legend":"","description":"","filename":"S1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/2dc33b504e77acca78b954b8.tiff"},{"id":83917824,"identity":"d2400169-13de-4ac6-ae77-4b7d9367135c","added_by":"auto","created_at":"2025-06-04 13:06:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":156027,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table12.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6567985/v1/84008ecff9dffa7eed02718e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Septic Shock reduces overall Intestinal Microcirculation and specifically Anastomotic Perfusion: Insights from a Porcine Model Using Laser Speckle Contrast Imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a complex and life-threatening condition that disrupts multiple organ systems, including the microcirculation[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The effects of sepsis on mesenteric blood flow and its underlying pathophysiology have been extensively studied[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Sepsis, characterized by systemic inflammation and the release of toxins, can cause profound alterations in the splanchnic circulation, including reduced mesenteric perfusion and intestinal ischemia[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, our understanding of how intestinal microcirculation and healing is affected during sepsis remains limited[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Some studies have demonstrated higher rates of complications, such as anastomotic leakage (AL) and delayed wound healing, likely due to compromised tissue perfusion[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, other studies have suggested that performing an anastomosis in emergency colorectal surgery is safe, with no increased overall risk of AL[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nonetheless, these studies also report a significantly higher risk of AL if the patient remains septic and requires vasopressor therapy during secondary anastomoses. Subsequently, the management of patients with sepsis, whether preexisting or developing postoperatively, remains without clear evidence-based guidelines.\u003c/p\u003e \u003cp\u003eExperimental studies using advanced imaging techniques, such as Sidestream Dark Field (SDF) imaging[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and Laser Doppler Flowmetry (LDF)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], have demonstrated significant reductions in intestinal microcirculation during sepsis. These findings strongly support the hypothesis that ischemic injury to the gut plays a pivotal role in sepsis-related surgical complications. However, these studies also have notable limitations. In the LDF study[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], the sepsis model was based on fecal peritonitis, which itself is a known risk factor for postoperative complications, potentially confounding the results and limiting the applicability of the findings in other sepsis etiologies. Similarly, the SDF study[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] measured intraluminal microcirculation in the small intestine via a stoma and in the colon only through the rectum. This restricted sampling approach may not provide a comprehensive understanding of the microvascular changes throughout the entire intestinal tract. Thus, critical questions are still unanswered about the safety and outcomes of surgical interventions in septic patients.\u003c/p\u003e \u003cp\u003eTo date, no studies have directly evaluated the effects of sepsis on intestinal anastomoses or their healing potential. This study aims to bridge this gap by using Laser Speckle Contrast Imaging (LSCI) to investigate the direct effects of septic shock on the microcirculation of both the colon and small intestine, as well as the impact on hand-sewn and stapled anastomoses. As a novel imaging modality, LSCI offers significant advantages by enabling repeated, quantitative measurements of microcirculation under dynamic conditions, providing a robust and detailed assessment that may help inform clinical decision-making[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By exploring how sepsis influences intestinal microcirculation, this study seeks to provide insights that could inform clinical practice; especially concerning acute intestinal surgery.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe impact of septic shock on the microcirculation of the small intestine and colon, as well as hand-sewn and stapled anastomoses in both small intestine and the colon, was assessed using an experimental setup in a porcine model.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePorcine model\u003c/h2\u003e \u003cp\u003eThe anesthesia protocol for the porcine model has been described in detail previously[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, ten female Danish Landrace/Yorkshire/Duroc pigs, weighing an average of 43.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 kg and aged 14\u0026ndash;16 weeks, were sedated, and anesthetized. Propofol and fentanyl infusions maintained anesthesia throughout the experiment. The animals were intubated and mechanically ventilated, receiving Lactated Ringer's solution at a rate of 5 ml/kg/h. The septic response was defined by characteristic alterations in systemic physiology, including a progressive decline in hemodynamic parameters (mean arterial pressure (MAP) and systolic blood pressure), accompanied by rising lactate levels and decreasing arterial pH. These variables were continuously monitored throughout the experiment to confirm the onset and progression of septic shock.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurgical procedure\u003c/h3\u003e\n\u003cp\u003eFollowing induction of general anesthesia, a midline laparotomy was performed to expose the small intestine and colon. In each pig, two types of anastomoses were created: a single-layer, hand-sewn seromuscular end-to-end anastomosis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and a stapled anti-mesenteric side-to-side anastomosis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] on both the small intestine and colon, resulting in a total of four anastomoses per pig. LSCI measurements were systematically performed on all four anastomoses, as well as on predefined untouched segments of the small intestine and colon, following a standardized protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Baseline measurements (T\u003csub\u003eB\u003c/sub\u003e) were made prior to anastomosis formation, with additional measurements immediately after the anastomoses were completed (T\u003csub\u003eA\u003c/sub\u003e). The intestines were then allowed to rest for one hour without further surgical manipulation, after which the T\u003csub\u003e0\u003c/sub\u003e measurements were obtained. Sepsis was induced by intravenously injection of live \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE.coli\u003c/em\u003e) (ATCC 25922). The initial infusion rate was set at 16 ml/hour, and the rate was doubled every 10 minutes until approximately 240 ml of the \u003cem\u003eE.coli\u003c/em\u003e solution had been administered. LSCI measurements and arterial blood gas analyses were performed at 30-minute intervals, starting at T\u003csub\u003e30\u003c/sub\u003e. This continued until T\u003csub\u003e150\u003c/sub\u003e, after which the pigs were euthanized with intravenous phenobarbital while they were still fully anesthetized.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLaser Speckle Contrast Imaging, LSCI\u003c/h3\u003e\n\u003cp\u003eMicrocirculation was evaluated using LSCI at a wavelength of 785 nm (MoorFLPI-2, Moor Instruments, Axminster, UK). This method enabled assessment of microcirculation to a depth of approximately 1 mm. The LSCI device was positioned 25 cm above the intestinal surface, ensuring coverage of at least 5 cm on either side of the region of interest (ROI). Measurements were captured over a 30-second period with a sampling rate of 25 frames per second[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Results were presented both as a picture heatmap and in arbitrary Laser Speckle Perfusion Units (LSPU).\u003c/p\u003e \u003cp\u003e \u003cb\u003eE.coli\u003c/b\u003e \u003cb\u003einfusate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE.coli\u003c/em\u003e sepsis model used in this study has been described previously[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In summary, cultures of \u003cem\u003eE.coli\u003c/em\u003e (strain ATCC 25922, Department of Medical Microbiology, Aarhus University Hospital) were grown on 5% sheep blood agar plates (Statens Serum Institut, Copenhagen, Denmark) for 24 hours at 37\u0026deg;C. During the log phase of growth, colonies were harvested from the agar surface, suspended in normal saline and then adjusted to OD\u003csub\u003e620\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.2 (approximately 1 \u0026times; 10^9 CFU/ml). The following day, the bacterial suspension was serially diluted in triplicates and plated on to sheep blood agar plates and incubated for 24h at 37\u0026deg;C followed by CFU enumeration on the agar plates, to verify the concentration and ensure accuracy. Prior to administration, the suspension was stored at 4\u0026deg;C to prevent bacterial replication under low-oxygen conditions of the infusate.\u003c/p\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eTissue samples from the small intestine and colon were collected at baseline and at the end of the experiment. These samples were sent for histological analysis to evaluate any changes associated with \u003cem\u003eE.coli\u003c/em\u003e sepsis. Briefly, the biopsies were fixated in 10% neutral buffered formalin and processed through graded concentrations of alcohol and embedded in paraffin wax. Tissue sections were cut (4 \u0026micro;m) and stained with hematoxylin and eosin (HE). HE stained sections were evaluated patho-morphologically with special focus on identifying (yes/no) an acute inflammatory response. The following parameters were evaluated in mucosa and submucosae, respectively; epithelial damage, hemorrhage, hyperemia, edema, neutrophil infiltration, and necrosis.\u003c/p\u003e\n\u003ch3\u003eComputational and statistical analysis\u003c/h3\u003e\n\u003cp\u003eFour regions of interest (ROIs) were selected for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) for each anastomosis. ROI 1 was centered directly on the anastomosis, ROI 2 was positioned 5 mm away from the anastomosis on either side, ROI 3 was located 10 mm from the anastomosis on either side, and the final ROI was placed on a predefined untouched segment of the small intestine/colon. Each ROI measured 5 mm in width and spanned the entire intestinal diameter, with ROI 1\u0026ndash;3 arranged adjacently. Median microcirculatory values in LSPU were calculated for each ROI. Data was analyzed using linear mixed models to account for repeated measurements within the same animal. Results were presented as LSPU changes relative to baseline, which was defined as the average LSPU across all ten animals in the study at T\u003csub\u003eB\u003c/sub\u003e. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using STATA version 18 (StataCorp LLC, College Station, TX, USA), while microcirculation flux data were processed using MoorFLPI2 Research software v2.x (Moor Instruments, Axminster, UK).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003eThe trial was conducted under the supervision of veterinary personnel at the Department of Animal Science, Aarhus University, AU Foulum, and the Principles of Laboratory Animal Care[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were followed. The trial was approved by the Danish Animal Experiments Inspectorate (No. 2022-15-0201-01331). All methods were carried out in accordance with relevant guidelines and regulations. Pigs were purchased from Department of Animal Science, Aarhus University, AU Foulum. All methods are reported in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSepsis model\u003c/h2\u003e \u003cp\u003eAll ten pigs underwent surgery without complications or adverse events. To induce septic shock, an average of 235.5 ml of \u003cem\u003eE.coli\u003c/em\u003e suspension was administered (range: 210\u0026ndash;240 ml), with a mean bacterial concentration of 1.11 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/ml (range: 8 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e \u0026ndash; 1.4 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCFU/ml).\u003c/p\u003e \u003cp\u003eThe hemodynamic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) during sepsis show a significant decrease in systolic blood pressure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and MAP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), accompanied by a significant increase in pulse (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, there was a significant reduction in pH levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a significant increase in lactate levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLSCI measurements were successfully performed at all predefined time points in all pigs. Representative LSCI images illustrating microcirculation around the anastomoses are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (colon) and Supplementary Fig.\u0026nbsp;1 (small intestine).\u003c/p\u003e \u003cp\u003eRelative changes in LSPU compared to baseline are presented in Fig.\u0026nbsp;4, while absolute LSPU values are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicrocirculation in Untouched Colon and Small Intestine\u003c/h2\u003e \u003cp\u003eIn the untouched colon, no statistically significant difference in microcirculation was seen during anastomosis formation or after 1 hour of rest. After \u003cem\u003eE.coli\u003c/em\u003e infusion, a decline was seen: perfusion continued to drop over time, reaching statistically significant reductions at T\u003csub\u003e60\u003c/sub\u003e (91%, 95%CI: 80\u0026ndash;99%); T\u003csub\u003e90\u003c/sub\u003e (81%, 95%CI: 80\u0026ndash;86%); and T\u003csub\u003e150\u003c/sub\u003e (69%, 95%CI: 60\u0026ndash;78%).\u003c/p\u003e \u003cp\u003eIn the untouched small intestine, a significant reduction in perfusion was observed already during anastomose formation, with a drop to 84% (95%CI: 80\u0026ndash;88%. After one hour of rest, perfusion increased slightly to 90% (95%CI: 85\u0026ndash;95%), though still significantly lower than baseline. After \u003cem\u003eE.coli\u003c/em\u003e infusion, perfusion declined further to 76% of baseline (95%CI: 70\u0026ndash;82%) at T\u003csub\u003e30\u003c/sub\u003e; 69% (95%CI: 60\u0026ndash;74%) at T\u003csub\u003e60\u003c/sub\u003e; and 66% (95%CI: 60\u0026ndash;71%) at T\u003csub\u003e150\u003c/sub\u003e (Fig.\u0026nbsp;4). All measurements following \u003cem\u003eE.coli\u003c/em\u003e infusion were statistical significant lower compared to baseline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eColonic Microcirculation at Hand-Sewn and Stapled Anastomoses\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eROI 1, Anastomosis\u003c/h2\u003e \u003cp\u003eThe perfusion in hand-sewn anastomoses at T\u003csub\u003eA\u003c/sub\u003e was 55% of baseline (95%CI: 43\u0026ndash;67%), which was significantly higher than the perfusion observed in stapled anastomoses of 42% (95%CI: 35\u0026ndash;49) (p\u0026thinsp;=\u0026thinsp;0.011). This finding persisted throughout the study: After 1 hour rest (T\u003csub\u003e0\u003c/sub\u003e), perfusion increased in both groups and was significantly higher in hand-sewn anastomoses; After \u003cem\u003eE.coli\u003c/em\u003e infusion, the perfusion levels decreased and at T\u003csub\u003e150\u003c/sub\u003e, perfusion in hand-sewn anastomoses dropped to 51% of baseline (95%CI: 40\u0026ndash;63%), whereas the stapled anastomoses\u0026rsquo; perfusion dropped to 34% (95%CI: 30\u0026ndash;43%) which was significantly lower than the hand-sewn anastomoses (p\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eROI 2,\u0026plusmn;5 mm Adjacent to the Anastomosis\u003c/h2\u003e \u003cp\u003ePerfusion within 5 mm of the anastomosis was higher than at ROI 1 for hand-sewn and stapled anastomoses after the anastomosis formation (92% vs. 88%). This finding persisted at T\u003csub\u003e0\u003c/sub\u003e through to T\u003csub\u003e150\u003c/sub\u003e, where perfusion adjacent to hand-sewn anastomoses decreased to 64% (95%CI: 50\u0026ndash;75%) compared to 55% (95%CI: 50\u0026ndash;65%) in stapled anastomoses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eROI 3, \u0026plusmn;\u0026thinsp;10 mm Adjacent to the Anastomosis\u003c/h2\u003e \u003cp\u003eAt \u003cem\u003e\u0026plusmn;\u003c/em\u003e\u0026thinsp;10 mm from the anastomosis, no significant differences were observed between hand-sewn and stapled anastomoses across all time points compared to baseline. After \u003cem\u003eE.coli\u003c/em\u003e infusion, the impact on perfusion was similar to that seen in untouched intestine, with microcirculation at T\u003csub\u003e150\u003c/sub\u003e reduced to 68% of baseline (95%CI: 60\u0026ndash;80%) for hand-sewn anastomoses and 61% of baseline (95%CI: 50\u0026ndash;69%) for stapled anastomoses.\u003c/p\u003e \u003cp\u003eIn summary, microcirculation across all ROIs during sepsis was significantly reduced compared to baseline. Hand-sewn anastomoses consistently demonstrated higher perfusion than stapled anastomoses at all time points, with statistically significant differences observed at the anastomotic site itself. In contrast, differences between hand-sewn and stapled techniques were less pronounced at \u0026plusmn;\u0026thinsp;5 mm and \u0026plusmn;\u0026thinsp;10 mm from the anastomosis, where a more gradual decline in perfusion was observed\u0026mdash;mirroring the pattern seen in the untouched colon.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSmall intestinal Microcirculation in Hand-Sewn and Stapled Anastomoses\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eROI 1, Anastomosis Region\u003c/h2\u003e \u003cp\u003eIn the anastomosis region, perfusion after anastomosis formation was similar between hand-sewn and stapled anastomoses, with values of 37% (95%CI: 30\u0026ndash;43%) and 37% (95%CI: 32\u0026ndash;42%) of baseline, respectively (p\u0026thinsp;=\u0026thinsp;0.952). However, significant differences emerged during subsequent time points. At T\u003csub\u003e0\u003c/sub\u003e, perfusion in hand-sewn anastomoses increased to 53% (95%CI: 49\u0026ndash;57%), compared to 41% (95%CI: 36\u0026ndash;47) in stapled anastomoses (p\u0026thinsp;=\u0026thinsp;0.010). During sepsis, perfusion in hand-sewn anastomoses continued to decrease significantly less relative to stapled anastomoses; at T\u003csub\u003e150\u003c/sub\u003e, the perfusion in hand-sewn anastomoses was 45% (95%CI: 40\u0026ndash;54%), and in stapled anastomoses 36% of baseline (95%CI: 30\u0026ndash;42%, p\u0026thinsp;=\u0026thinsp;0.014), respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eROI 2, \u0026plusmn;\u0026thinsp;5 mm Adjacent to the Anastomosis\u003c/h2\u003e \u003cp\u003eAt ROI 2, microcirculation was generally higher than at the anastomosis site. At T\u003csub\u003eA\u003c/sub\u003e no significant difference was found in stapled anastomoses (71%, 95%CI: 67\u0026ndash;76%) compared to hand-sewn anastomoses (64%, 95%CI: 59\u0026ndash;69%, p\u0026thinsp;=\u0026thinsp;0.071). At T\u003csub\u003e0\u003c/sub\u003e, perfusion was significantly higher in hand-sewn anastomoses (81%) compared to stapled anastomoses (70%, p\u0026thinsp;=\u0026thinsp;0.004). Similar findings were observed at T\u003csub\u003e30\u003c/sub\u003e, T\u003csub\u003e90\u003c/sub\u003e, and T\u003csub\u003e150\u003c/sub\u003e, where perfusion decreased to 62% (95%CI: 60\u0026ndash;68%) for hand-sewn anastomoses and 57% (95%CI: 50\u0026ndash;62%) for stapled anastomoses (p\u0026thinsp;=\u0026thinsp;0.005).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eROI 3, \u0026plusmn;\u0026thinsp;10 mm Adjacent to the Anastomosis\u003c/h2\u003e \u003cp\u003eAt \u0026plusmn;\u0026thinsp;10 mm from the anastomosis, perfusion was similar between hand-sewn and stapled anastomoses after anastomosis formation (78% vs. 82%, p\u0026thinsp;=\u0026thinsp;0.303). At T\u003csub\u003e0\u003c/sub\u003e, hand-sewn anastomoses had significantly higher perfusion (88%, 95%CI: 83\u0026ndash;92%) than stapled anastomoses (78%, 95%CI: 74\u0026ndash;82%, p\u0026thinsp;=\u0026thinsp;0.031). Similar differences were found at T\u003csub\u003e90\u003c/sub\u003e, T\u003csub\u003e120\u003c/sub\u003e, and T\u003csub\u003e150\u003c/sub\u003e, where perfusion declined to 67% (95%CI: 60\u0026ndash;74%) in hand-sewn anastomoses and 62% (95%CI: 60\u0026ndash;68%) in stapled anastomoses (p\u0026thinsp;=\u0026thinsp;0.026).\u003c/p\u003e \u003cp\u003eRecapitulating, hand-sewn anastomoses had consistently higher perfusion compared to stapled anastomoses, particularly within the anastomosis region and at \u003cem\u003e\u0026plusmn;\u003c/em\u003e\u0026thinsp;5 mm from the site. Differences at \u003cem\u003e\u0026plusmn;\u003c/em\u003e\u0026thinsp;10 mm were less pronounced, but still statistically significant one hour after \u003cem\u003eE.coli\u003c/em\u003e sepsis was induced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePathology\u003c/h2\u003e \u003cp\u003eHistological results are presented in Supplementary table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and revealed no significant abnormalities in the small intestine or colon. The most common observation was post-operative hyperemia in the colon, characterized by an increased presence of red blood cells within the vessels compared to baseline. In a few samples, an elevated number of neutrophils were noted on the epithelial surface, particularly between villi and within crypts. However, no infiltration was observed in the mucosal or submucosal layers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides novel insights into the effects of septic shock on intestinal microcirculation, comparing untouched segments of the small intestines and colon to hand-sewn and stapled anastomoses: We demonstrated a significant and progressive decline in microcirculation in both untouched bowel and especially anastomotic regions during septic shock. This may indicate that septic shock impairs intestinal perfusion in general and especially around surgical resection sites, which we hypothesize could impair intestinal healing and increase e.g. anastomotic leak rates. Thus, our findings contribute to our understanding of microcirculatory changes during sepsis and raise important considerations for surgical decision-making in septic patients.\u003c/p\u003e \u003cp\u003eOur sepsis model effectively induced a state of septic shock, as demonstrated by significant reductions in systemic parameters, including MAP and pH, alongside elevated lactate levels. These findings are consistent with established septic shock models. Unlike other studies, such as Krejci et. al[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], who utilized fecal peritonitis as the sepsis model, our approach avoided localized peritoneal contamination. Despite this, the intestines still exhibited pronounced microcirculatory impairments, emphasizing the systemic effects of sepsis on intestinal perfusion, independent of localized contamination.\u003c/p\u003e \u003cp\u003eFurthermore, unlike the SDF study[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which assessed intraluminal microcirculation, our use of LSCI allowed for direct, repeated measurements on both the small and large intestine, including anastomoses. This comprehensive approach provides a more detailed and accurate representation of intestinal microcirculation under septic conditions, enhancing the translational relevance of our findings.\u003c/p\u003e \u003cp\u003eFirst, our results demonstrate a consistent and significant decline in microcirculation in both the untouched colon and small intestine during septic shock. A notable finding was the difference in microcirculatory responses between the untouched small intestine and colon; The small intestine exhibited a higher baseline perfusion, which may explain the more pronounced reduction observed during sepsis. In contrast, the colon demonstrated a more gradual decline in perfusion. The general decline in both colonic and small intestinal perfusion aligns with the existing understanding of the splanchnic circulatory response to sepsis, where mesenteric blood flow is redistributed to other critical organs (lung, heart, kidney) at the expense of the bowel[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The observed decrease in perfusion could predispose intestinal tissue to ischemia, which raises concerns in cases with both sepsis and surgical treatment including anastomosis formation. To evaluate the effect of surgery on anastomoses and whether normal pathological processes were disturbed during sepsis, tissue was sampled for histological analysis. However, these revealed no significant pathological changes: The primary histological finding was pronounced hyperemia and vascular stasis, consistent with the early vascular phase of the acute inflammatory response[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is important to note that cellular signs of inflammation, such as neutrophil infiltration, are typically not evident histologically until approximately two hours after the onset of injury, with infiltration peaking around 24 hours. These findings reflect the relatively short duration of the experiment (2.5 hours). If histopathological changes are to accurately reflect impaired healing following sepsis, tissue sampling should be performed 4\u0026ndash;7 days postoperatively[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], as studies have shown this to be the typical timeframe for the development of anastomotic leakage. However, such a setup was beyond the ethical permission given for this porcine model, but could be integrated in a future research model.\u003c/p\u003e \u003cp\u003eSecond, our study indicates that in cases where anastomoses are made during sepsis, hand-sewn anastomoses present consistently higher perfusion compared to stapled anastomoses across all time points. This suggests that hand-sewn techniques may preserve microcirculatory integrity better than stapled approaches during sepsis and increase the healing potential. However, both types of anastomoses experienced significant declines in perfusion during sepsis, which underlines that cautious surgical planning in septic patients is necessary; salvage surgery without primary anastomosis could be preferable until the septic condition is treated to reduce the risk of anastomotic leakage from impaired microcirculation. Previous research has suggested that the lower threshold for safe anastomotic perfusion is a 30% reduction in microcirculation[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; In our study, we demonstrate that sepsis reduced the intestinal microcirculation at the untouched intestine with 33% for the small intestine and 30% for the colon, and even more at the anastomotic sites especially at ROI 1 and ROI 2, which is much more than the suggested 30%. Moreover, the clinical use of vasopressors to stabilize systemic blood pressure during sepsis, such as norepinephrine, has been shown to further compromise intestinal perfusion, aggravating the impaired healing in intestinal surgery[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and Future Perspectives\u003c/h2\u003e \u003cp\u003eWhile this study provides valuable insights into the effects of septic shock on intestinal microcirculation in general and around anastomoses, several limitations must be addressed. First, the study period of 150 minutes of \u003cem\u003eE.coli\u003c/em\u003e infusion may not capture the long-term effects of sepsis on anastomotic healing. Future studies with extended experimental durations are necessary to better understand the progression of microcirculatory impairment and its impact on surgical outcomes.\u003c/p\u003e \u003cp\u003eAnother limitation is the use of a porcine model. While pigs are a translationally relevant model for studying human intestinal microcirculation, their sepsis pathophysiology may not fully replicate the complex clinical scenarios encountered in humans. This highlights the need for careful interpretation of the findings and underscores the importance of validating results in clinical settings.\u003c/p\u003e \u003cp\u003eThe sample size of ten pigs also represents a limitation, as a larger cohort could reduce the risk of type II errors and potentially yield more statistically significant results. However, ethical considerations place constraints on animal research, and future studies must continue to balance the need for statistical power with ethical responsibility.\u003c/p\u003e \u003cp\u003eBeyond addressing these limitations, future research should investigate potential therapeutic interventions, such as targeted vasodilators, to preserve intestinal microcirculation during sepsis. These approaches may provide novel strategies to improve surgical outcomes and reduce complications, such as anastomotic leakage.\u003c/p\u003e \u003cp\u003eMoreover, the potential clinical applications of LSCI warrant further exploration. LSCI offers a unique capability for real-time, quantitative assessment of microcirculation, making it a promising tool for intraoperative decision-making in septic patients. For instance, its use could help surgeons evaluate the viability of intestinal segments before performing an anastomosis, minimizing the risk of ischemia-related complications. By integrating advanced imaging modalities like LSCI into acute surgical settings, clinicians may be able to enhance decision-making and improve patient outcomes in high-risk scenarios.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the impact of septic shock in intestinal microcirculation, revealing differences between untouched and anastomotic regions, as well as between small and large intestine. Also, hand-sewn anastomoses preserve microcirculation better than stapled ones, but both show a drastic decline, underscoring the significant risk of anastomotic leakage in septic patients. These findings provide a basis for optimizing surgical strategies and improving outcomes in a state of sepsis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAU Foulum is acknowledged for its invaluable aid and supervision during experiments. Special gratitude is extended to animal caretaker Birgitte Frydkj\u0026aelig;r for her assistance during the operations. We extend our sincere gratitude to the Department of Medical Microbiology, Aarhus University Hospital, for their invaluable support in the preparation and handling of\u0026nbsp;\u003cem\u003eE.coli\u003c/em\u003e. Biorender.com was used for the design, as shown in Figure 1.\u003cbr\u003e \u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.P.: Formal analysis, investigation, resources, data curation, writing \u0026ndash; original draft, visualization, and funding acquisition. N.M.K., S.P. and H.W.: Investigation and writing, review, and editing. L.K.J., C. J. and A.H.M.: Conceptualization, methodology, supervision, writing, review, and editing. M.W.\u0026Oslash;.: Conceptualization, methodology, supervision, writing \u0026ndash; original draft, and writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Data Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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 authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Conflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest\u003cstrong\u003e.\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;Funding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was received for the study. Rupan Paramasivam is supported by grants from the NEYE Foundation, Dagmar Marshalls Foundation, and NIDO Denmark. The funders did not play any role in the design of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Statement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial was conducted under the supervision of veterinary personnel at the Department of Animal Science, Aarhus University, AU Foulum, and the Principles of Laboratory Animal Care[20] were followed. The trial was approved by the Danish Animal Experiments Inspectorate (No. 2022-15-0201-01331).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShepherd, A.P., et al., \u003cem\u003eEffects of vasoconstrictors on intestinal vascular resistance and oxygen extraction.\u003c/em\u003e Am J Physiol, 1976. \u003cstrong\u003e230\u003c/strong\u003e(2): p. 298-305.\u003c/li\u003e\n\u003cli\u003eReilly, P.M., et al., \u003cem\u003eThe mesenteric hemodynamic response to circulatory shock: an overview.\u003c/em\u003e Shock, 2001. \u003cstrong\u003e15\u003c/strong\u003e(5): p. 329-43.\u003c/li\u003e\n\u003cli\u003eDeitch, E.A., et al., \u003cem\u003eA study of the relationship among survival, gut-origin sepsis, and bacterial translocation in a model of systemic inflammation.\u003c/em\u003e J Trauma, 1992. \u003cstrong\u003e32\u003c/strong\u003e(2): p. 141-7.\u003c/li\u003e\n\u003cli\u003eRuokonen, E., et al., \u003cem\u003eRegional blood flow and oxygen transport in septic shock.\u003c/em\u003e Crit Care Med, 1993. \u003cstrong\u003e21\u003c/strong\u003e(9): p. 1296-303.\u003c/li\u003e\n\u003cli\u003eFink, M.P., \u003cem\u003eAdequacy of gut oxygenation in endotoxemia and sepsis.\u003c/em\u003e Crit Care Med, 1993. \u003cstrong\u003e21\u003c/strong\u003e(2 Suppl): p. S4-8.\u003c/li\u003e\n\u003cli\u003eWang, X. and D. Liu, \u003cem\u003eHemodynamic Influences on Mesenteric Blood Flow in Shock Conditions.\u003c/em\u003e Am J Med Sci, 2021. \u003cstrong\u003e362\u003c/strong\u003e(3): p. 243-251.\u003c/li\u003e\n\u003cli\u003ePatel, S. and A. Duncan, \u003cem\u003eAnaesthesia and intestinal anastomosis.\u003c/em\u003e BJA Educ, 2021. \u003cstrong\u003e21\u003c/strong\u003e(11): p. 433-443.\u003c/li\u003e\n\u003cli\u003eHuisman, D.E., et al., \u003cem\u003eFluid management and vasopressor use during colorectal surgery: the search for the optimal balance.\u003c/em\u003e Surg Endosc, 2023. \u003cstrong\u003e37\u003c/strong\u003e(8): p. 6062-6070.\u003c/li\u003e\n\u003cli\u003eFischer, P.E., et al., \u003cem\u003eVasopressor use after initial damage control laparotomy increases risk for anastomotic disruption in the management of destructive colon injuries.\u003c/em\u003e Am J Surg, 2013. \u003cstrong\u003e206\u003c/strong\u003e(6): p. 900-3.\u003c/li\u003e\n\u003cli\u003ePranskunas, A., et al., \u003cem\u003eEarly course of microcirculatory perfusion in eye and digestive tract during hypodynamic sepsis.\u003c/em\u003e Crit Care, 2012. \u003cstrong\u003e16\u003c/strong\u003e(3): p. R83.\u003c/li\u003e\n\u003cli\u003eKrejci, V., L.B. Hiltebrand, and G.H. Sigurdsson, \u003cem\u003eEffects of epinephrine, norepinephrine, and phenylephrine on microcirculatory blood flow in the gastrointestinal tract in sepsis.\u003c/em\u003e Crit Care Med, 2006. \u003cstrong\u003e34\u003c/strong\u003e(5): p. 1456-63.\u003c/li\u003e\n\u003cli\u003eHiltebrand, L.B., et al., \u003cem\u003eRedistribution of microcirculatory blood flow within the intestinal wall during sepsis and general anesthesia.\u003c/em\u003e Anesthesiology, 2003. \u003cstrong\u003e98\u003c/strong\u003e(3): p. 658-69.\u003c/li\u003e\n\u003cli\u003eHeeman, W., et al., \u003cem\u003eApplication of laser speckle contrast imaging in laparoscopic surgery.\u003c/em\u003e Biomed Opt Express, 2019. \u003cstrong\u003e10\u003c/strong\u003e(4): p. 2010-2019.\u003c/li\u003e\n\u003cli\u003eHeeman, W., et al., \u003cem\u003eClinical applications of laser speckle contrast imaging: a review.\u003c/em\u003e J Biomed Opt, 2019. \u003cstrong\u003e24\u003c/strong\u003e(8): p. 1-11.\u003c/li\u003e\n\u003cli\u003eParamasivam, R., et al., \u003cem\u003eLaser speckle contrast imaging for intraoperative assessment of intestinal microcirculation in normo- and hypovolemic circulation in a porcine model.\u003c/em\u003e Eur Surg Res, 2023.\u003c/li\u003e\n\u003cli\u003eSaeger, H.D., et al., \u003cem\u003e[Course system of the Working Group for Gastro-intestinal Surgery Davos].\u003c/em\u003e Chirurg, 2010. \u003cstrong\u003e81\u003c/strong\u003e(1): p. 25-30.\u003c/li\u003e\n\u003cli\u003eRussell, K.W., et al., \u003cem\u003eThe Barcelona Technique for Ileostomy Reversal.\u003c/em\u003e J Gastrointest Surg, 2015. \u003cstrong\u003e19\u003c/strong\u003e(12): p. 2269-72.\u003c/li\u003e\n\u003cli\u003eAmbrus, R., et al., \u003cem\u003eLaser Speckle Contrast Imaging for Monitoring Changes in Microvascular Blood Flow.\u003c/em\u003e Eur Surg Res, 2016. \u003cstrong\u003e56\u003c/strong\u003e(3-4): p. 87-96.\u003c/li\u003e\n\u003cli\u003eDehring, D.J., et al., \u003cem\u003eComparison of live bacteria infusions in a porcine model of acute respiratory failure.\u003c/em\u003e J Surg Res, 1983. \u003cstrong\u003e34\u003c/strong\u003e(2): p. 151-8.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eAmerican Society of Laboratory Animal Practitioners Position Statement: Animal Care Principles.\u003c/em\u003e J Am Assoc Lab Anim Sci, 2024. \u003cstrong\u003e63\u003c/strong\u003e(5): p. 470-1.\u003c/li\u003e\n\u003cli\u003eJarczak, D., S. Kluge, and A. Nierhaus, \u003cem\u003eSepsis-Pathophysiology and Therapeutic Concepts.\u003c/em\u003e Front Med (Lausanne), 2021. \u003cstrong\u003e8\u003c/strong\u003e: p. 628302.\u003c/li\u003e\n\u003cli\u003eTsai, Y.Y. and W.T. Chen, \u003cem\u003eManagement of anastomotic leakage after rectal surgery: a review article.\u003c/em\u003e J Gastrointest Oncol, 2019. \u003cstrong\u003e10\u003c/strong\u003e(6): p. 1229-1237.\u003c/li\u003e\n\u003cli\u003eKashiwagi, H., \u003cem\u003eThe lower limit of tissue blood flow for safe colonic anastomosis: an experimental study using laser Doppler velocimetry.\u003c/em\u003e Surg Today, 1993. \u003cstrong\u003e23\u003c/strong\u003e(5): p. 430-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sepsis, intestinal anastomoses, microcirculation, Anastomotic perfusion, porcine model, Laser Speckle Contrast Imaging (LSCI)","lastPublishedDoi":"10.21203/rs.3.rs-6567985/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6567985/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeptic shock can severely compromise intestinal perfusion, increasing the risk of poor anastomotic healing and surgical complications. This study investigated the effects of septic shock on intestinal microcirculation and anastomotic perfusion in a porcine model using Laser Speckle Contrast Imaging. Ten pigs underwent laparotomy with creation of four anastomoses— one hand-sewn and one stapled in both small intestine and colon. Microcirculatory measurements were taken before and after anastomosis formation, and repeatedly during the development of septic shock induced by intravenous Escherichia coli infusion. Septic shock led to a significant reduction in microcirculation across both untouched bowel and anastomoses. Hand-sewn anastomoses maintained higher perfusion than stapled anastomoses throughout all time points. This study shows that septic shock significantly impairs intestinal microcirculation indicating a risk of intestinal ischemia if bacteriaemia and subsequent septic shock is untreated. Due to diminish blood flow following septic shock, the anastomotic healing may be compromised leading to increased risk of anastomotic leakage. Conclusively, this study provides a foundation for optimizing surgical strategies and improving patient outcomes in this high-risk population.\u003c/p\u003e","manuscriptTitle":"Septic Shock reduces overall Intestinal Microcirculation and specifically Anastomotic Perfusion: Insights from a Porcine Model Using Laser Speckle Contrast Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 13:06:50","doi":"10.21203/rs.3.rs-6567985/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"db8d20ff-1449-44bb-83d6-d3c8cf7286a3","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49406451,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer"},{"id":49406452,"name":"Health sciences/Pathogenesis/Infection"},{"id":49406453,"name":"Health sciences/Risk factors"}],"tags":[{"value":"featured","date":"2025-06-04 13:56:18"}],"updatedAt":"2025-07-10T05:53:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 13:06:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6567985","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6567985","identity":"rs-6567985","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}