Prevention of ischemia-reperfusion injury on the porcine model of supra-renal aortic clamp by sulodexide *

Abstract

Background

The ischemia-reperfusion injury (IRI) is unavoidable in vascular surgery. Damage to the microcirculation and endothelial glycocalyx might set up a shock with loss of circulatory coherence and organ failure. Sulodexide may help to protect endothelial glycocalyx and alleviate the ischemia-reperfusion injury.

Methods

Twenty female piglets underwent surgery with a 30-min-long suprarenal aortic clamp, followed by two hours of reperfusion. Ten piglets received sulodexide before the clamp, and 10 received normal saline. Blood and urine samples were taken at baseline and in 20-min intervals until the 120^th minute to analyze the serum syndecan-1, E-selectin, and thrombomodulin. Albumin and glycosaminoglycans were examined in the urine. The kidney biopsies before and after the protocol were examined by light microscopy with hematoxylin-eosin staining. The sublingual microcirculation was recorded by side-stream dark field imaging at the time as blood and urine.

Results

Based on the 2-way ANOVA testing, there was no statistically significant difference in the parameters of sublingual microcirculation. Serum markers of endothelial cell activation and damage (E-selectin and thrombomodulin) did not show any statistically significant difference either. Syndecan-1, a marker of glycocalyx damage, showed statistically significantly higher values based on the 2-way ANOVA testing (p < 0.0001) with the highest difference in the 80^th minute: 7.8 (3.9–44) ng/mL in the control group and 1.8 (0.67–2.8) ng/mL in the sulodexide group. In the urine, the albuminuria was higher in the control group, although not statistically significant. Glycosaminoglycans were statistically significantly higher in the sulodexide group based on the mixed-effect analysis due to the intervention itself. Histological analysis of the renal biopsies showed necrosis in both groups after reperfusion.

Conclusion

Administering sulodexide significantly reduced the level of endothelial markers of IRI. The study results support further research into using preemptive administration of sulodexide to modulate IRI in clinical medicine.

Keywords

sulodexide ischemia-reperfusion microcirculation syndecan-1 endothelial glycocalyx

Introduction

Ischemia reperfusion injury (IRI) causes twofold damage to the tissues. The first is made by hypoxia and increasing metabolic waste, and the second by oxidative stress.¹ The microcirculation makes an interface where these changes ensue,² resulting in microvascular dysfunction, loss of circulatory coherence,³ and endothelial glycocalyx (EG) shedding.^4,5 EG is particularly sensitive to oxidative stress due to its sugar-based nature. EG has already been shown to play a major role in vascular physiology.⁶ By EG damage, the chain of pathophysiological changes starts. Localized tissue damage might translate into a whole-body reaction with circulatory shock and multi-organ failure. Efforts should be made to prevent or diminish these changes during elective surgeries involving IRI, e.g., vascular surgery where EG shedding has been demonstrated.⁷ Besides EG disruption, endothelial ion channels, purinergic receptors, kinases, and integrin dysfunction mediate the IRI.⁸ Thus, protecting the EG in IRI is of paramount interest. Experimental studies have demonstrated the protection by sevoflurane,⁹ imatinib,¹⁰ acetylsalicylic acid,¹¹ nitric oxide,¹² plasma,¹³ antithrombin III,¹⁴ albumin,¹⁵ hydrogen,¹⁶ hydrocortisone,¹⁷ and the combination of Ruscus extract – ascorbic acid – hesperidin methylchalcone.¹⁸ In the clinical studies, hyaluronic acid¹⁹ and doxycycline²⁰ proved effective in EG protection.

Sulodexide is a routinely prescribed drug in patients with angiopathies (e.g., diabetic vasculopathy, ischemic disease of lower extremities, and venous insufficiency) with clinically proven positive effects. Sulodexide is a compound of heparan sulfate and dermatan sulfate, which are building blocks of the EG. Therefore, one of the possible pathways of sulodexide's positive effect might be EG protection.^21,22 This has been described in experimental research in small animals on models of ischemia-reperfusion^22,23 and in clinical studies.^24,25

We aimed to examine the positive effect of sulodexide in vascular surgery using a porcine model of suprarenal aortic clamping, where IRI occurs inadvertently. We hypothesize that after the application of the sulodexide, the serum concentration of syndecan-1 (a marker of EG damage) will be decreased. Secondly, we hypothesize that the amount of albuminuria (indicative of renal injury) will be reduced, levels of glycosaminoglycans in urine will be increased, and serum concentrations of E-selectin and thrombomodulin, which reflect endothelial cell damage and activation, will increase. Lastly, we hypothesize that the sulodexide will not affect the parameters of sublingual microcirculation.

Materials and method

The experiment was carried out in the animal facility of the Military Faculty of Medicine, University of Defence, Czech Republic. The experiment protocol was approved by the Departmental Animal Protection Committee of the Ministry of Defense of the Czech Republic (protocol number MO 576619/2024-1457). All experimental procedures involving animals adhered to institutional guidelines and followed the Consensus Guidelines for Humane Ethics and Animal Welfare established by the International Association of Veterinary Publishers.

Animal preparation, anesthesia, and monitoring

Twenty animals – piglets (sus scrofa f. domestica, Konarovice, Czech Republic) weighing 33.5 ± 5.4 kg received intramuscular premedication with azaperone (2.0 mg/kg; Stresnil, Sanochemia Pharmazeutika AG, Neufeld/Leitha, Austria), atropine (0.2 mg/kg; Atropin Biotika, HBM Pharma, Martin, Slovak Republic) and ketamine (20 mg/kg; Calypsol, Gedeon Richter Plc., Budapest, Hungary) 30 min before the protocol was started. To prepare for surgery, the animals were shaved and then transported to the operating room. There was no corneal reflex and motoric reaction against nociception to confirm adequate anesthesia. After arrival in the OR, the peripheral venous catheter was introduced through the ear vein in the supine position (Vasofix^® Safety, B. Braun Melsungen AG, Melsungen, Germany, 18 G) and sedation deepened into general anesthesia by propofol (1 mg/kg, Propofol 1%, Fresenius KABI, Bad Homburg, Germany). The trachea was then intubated, and artificial ventilation was initiated by using an anesthesia station machine (Cirrus Trans2/Vent 2, Datex, Helsinki, Finland) with the initial setup: 18 breaths per minute, inspiration fraction of oxygen 0.40 and tidal volume 6 mL per kilogram of the body weight of the piglet. The respiratory rate was changed to maintain the end-tidal carbon dioxide (EtCO₂) within the 4–5.7 kPa range. General anesthesia was maintained by isoflurane 1.5% in the fresh gas (Forane, AbbVie Inc., Chicago, IL, USA).

The intravenous fluid therapy was maintained by infusion of a balanced crystalloid multi-electrolyte solution of Plasmalyte (Baxter SA, Braine l’Alleud, Belgium) at room temperature with an infusion rate of 50 mL per hour. Physiologic functions were monitored, including mean arterial blood pressure (MAP), heart rate, and cardiac rhythm. An arterial catheter was introduced through the common femoral artery (Certofix Duo, B. Braun Melsungen AG, Melsungen, Germany, 7 F, 200 mm) for continuous blood pressure monitoring and blood sampling.

Animal surgery

All surgical procedures were performed by the same team of two surgeons, following an identical protocol. A midline laparotomy was performed. A 14 French urinary catheter with a temperature monitoring probe was inserted via bladder incision and fixed with a circulatory stitch for urine sampling. First, a biopsy of the right kidney was taken after opening the right retroperitoneum. The biopsy site was sutured with a monofilament non-resorbable stitch to ensure complete hemostasis. Afterward, the left retroperitoneum was opened to expose the abdominal aorta at the level of the renal arteries. A 3-centimeter segment of the suprarenal aorta was prepared, and the renal arteries were bilaterally controlled. The aorta was then clamped cranially to the renal arteries, and the clamp was maintained in this position for 30 min. After declamp, the 120 min monitoring interval and sampling followed. At the end of the monitoring time, a final biopsy was taken from the left kidney before euthanizing the animal using intravenous T61 (Intervet International B.V., Boxmeer, The Netherlands).

Study groups and protocol

Ten animals received sulodexide 1200 IU (Vessel Due F, AlfaSigma, Italy) in 100 mL normal saline intravenously over 10 min during the surgery before the aortic clamp was introduced. Ten control animals received normal saline 100 mL intravenously at the same time. The animals were randomized by the envelope method.

Blood and urine samples and sublingual microcirculation recordings were taken at the baseline before the aortic clamp and in the 20^th, 40^th, 60^th, 80^th, 100^th, and 120^th minutes after the declamp.

Biochemical analyses

Activation of the endothelial cells – E-selectin, the shedding portion of the molecule (sE-selectin) (Elisa kit, MyBioSource, San Diego, CA, USA) and damage to the endothelial cells – thrombomodulin (Elisa kit, MyBioSource, San Diego, CA, USA) were assessed. The serum biomarker of EG layer damage – syndecan-1 was measured (Porcine Syndecan SDC1/CD 138 ELISA kit, LS Bio, WA, USA). Albumin in urine was assessed by ELISA (Pig urine albumin Elisa kit, Cusabio, Prague, Czech Republic), and glycosaminoglycans in the urine were quantified (assessment by dimethyl methylene blue,²⁶ spectrophotometer PharmaSpec UV-1700, Shimadzu, Japan) at the same time points as the serum markers.

Histology analysis

One set of samples of the intact kidney was collected before experimentally induced ischemia and one after reperfusion from each pig. In total, 26 samples were histologically examined, from 8 control and five sulodexide-administrated pigs. The samples of the renal cortex were excised and immersed in 4% paraformaldehyde in PBS for 7 days at room temperature, dehydrated, and embedded in paraffin. Five-µm-thick sections were cut, and each tenth slide in the series was stained with hematoxylin-eosin for histological examination of the tissue.

Sublingual microcirculation assessment

Video recordings of the sublingual mucosal microcirculation were recorded by a hand-held MicroVision camera (image resolution: 720 × 480 pixels; displayed field: 1044 × 758 μm; software: AVA 4.3C; Microvision Medical, Amsterdam, Netherlands) at baseline and 20 min interval up to 2 h. A detailed description of the Side-stream Dark Field imaging (SDF) technology has already been published elsewhere.²⁷ Video recordings were analyzed according to the second international consensus on the evaluation of the microcirculation²⁸ in the sublingual area. At every time, four recordings were captured – 2 from each side of the pig tongue from different locations in sulcus sublingualis lateralis, laterally from the sublingual bridle where the tip of the MicroVision camera can be easily positioned. The video acquisition process was mediated by a validated automatic algorithm software AVA 4.3C (Microvision, Amsterdam, The Netherlands) to ensure adequate brightness, focus, and stability. The recording was finished after acquiring 60 frames in two seconds of the microcirculatory bed of a diameter of 5–25 μm. Maximum care was carried out to prevent pressure over the mucosa to decrease the capillary perfusion. The quality of the recordings was assessed, and they were analyzed offline by the same automatic software AVA 4.3C, providing parameters according to the consensus:

Number of Crossings – number of vessels crossing three arbitrary horizontal and three vertical equidistant lines,

De Backer score – given in mm-1, calculated as the number of crossings divided by the total length of the arbitrary web lines,

Proportion of Perfused Vessels (PPV) – a percentage of perfused vessels out of all visible vessels.

Statistical analysis

Statistical analysis was done using the software Graph Pad PRISM (version 10.3.1 for macOS, GraphPad Software, San Diego, California, USA). The normality of the data was checked by the Shapiro-Wilk test. Data showing a normal (Gaussian) distribution were expressed as mean with standard deviation (SD). Differences between the groups at baseline and sampling time points were evaluated by the unpaired Student's t-test or Mann-Whitney's U test. Two-way repeated-measures ANOVA was used to separate changes over time from between-group differences or the Mixed-effect analysis based on the normality data testing. The level of significance was set at p < 0.05.

Results

Vital signs

One animal did not complete the protocol, with 10 pigs in the sulodexide group and nine in the control group. Vital signs remained stable in all the animals throughout the experiment. The median MAP was 73 mm Hg in both groups. The lowest values were not below 50 mmHg. None of the animals required vasopressor. The median heart rate was 104 beats per minute in the control group and 101 in the sulodexide. The median EtCO₂ was 36 mmHg in the control group and 37 in the sulodexide group. According to the 2-way ANOVA testing, there were no statistical differences in these vital signs between the animals in the two groups over time.

The biochemistry analysis

The biochemical analysis of the serum concentration of syndecan-1 (Figure 1) showed statistically significantly higher values in the control group based on the 2-way ANOVA testing (p < 0.0001). The highest difference was in the 80^th minute: 7.3 (3.9–44) ng/mL in the control group, 1.8 (0.67–2.8) ng/mL in the sulodexide group, p = 0.0004 (by unpaired t-test).

Figure 1.

Serum concentrations of syndecan-1.

Analysis of E-selectin and thrombomodulin did not show a statistically significant difference based on 2-way ANOVA testing (Figure 2, Figure 3).

Figure 2.

Serum concentrations of E-selectin.

Figure 3.

Serum concentrations of thrombomodulin.

The urine analysis discovered a mild increase in albuminuria in the control group (Figure 4). The 2-way ANOVA was not significant.

Figure 4.

Albuminuria.

In the urine, the glycosaminoglycans were significantly higher in the sulodexide group with mixed effect analysis as the data had a skewed distribution (Figure 5, p = 0.003).

Figure 5.

Urine concentrations of glycosaminoglycans.

The histological evaluation of the renal Cortex

Histological examination of the renal cortex revealed only a small difference between samples of the intact kidneys collected before experimentally induced ischemia (Figure 6(A) and (C)) and samples of the kidneys exposed to ischemia (Figure 6(B) and (D)). The difference was apparent in the proximal tubules, the most sensitive structures to ischemia in the renal cortex, whereas the renal corpuscles, the distal tubules, and the interstitium were not changed.

Figure 6.

Histological analysis of the kidneys.

In the proximal tubules affected by ischemia, the cytoplasm was focally slightly more vacuolized, and some nuclei were disintegrated. Mild degradation of their epithelium was noticed as well (Figure 6(B) and (D)). The structure of the kidneys of ischemia-exposed control (Figure 6(D)) and sulodexide-treated (Figure 6(B)) pigs were also compared, but only a few samples showed a difference. Thus, the degree of such short-term ischemia-induced histopathological changes was independent of the type of infusion solution.

Microscopic analysis of the sublingual microcirculation

The analysis of the capillary density (Number of Crossings and DeBacker score, Figure 7) and capillary perfusion (PPV, Figure 8) did not show any significant differences between the groups based on the 2-way ANOVA testing.

Figure 7.

Number of crossings.

Figure 8.

Proportion of perfused vessels.

Discussion

In our experimental study, we demonstrated that sulodexide significantly decreased the concentration of syndecan-1 in the serum, a marker of EG damage, after 30 min of ischemia followed by 2 h of reperfusion on a porcine model of supra-renal aortic clamp. This may signify protection to the EG and thus to the microcirculation from the IRI. Moreover, the urinalysis showed decreased albuminuria in the sulodexide group of animals, although not statistically significant. Sulodexide is composed of two sulfated glycosaminoglycans (heparan sulfate and dermatan sulfate), so after their administration to the animals, the concentration in urine statistically significantly increased. This is to confirm that our intervention was effective in a pharmacokinetic way. Finally, the histological analysis described slight changes in proximal tubules in the kidneys caused by IRI, so our model was biologically plausible.

There is more than a pharmacological way of protecting against IRI. A physical method, remote ischemic preconditioning, which involves intermittent arm compression with a blood pressure cuff, proved effective.²⁹ However, the mortality in clinical studies remained unchanged³⁰ even in vascular surgery.³¹

Among the drugs used for IRI prevention, sulodexide is promising by its nature as the building block for EG restoration.²² It has been successfully used in a carotid balloon occlusion in rats²² and in a 45-minute bilateral kidney ischemia model in rats.²³ Sulodexide also prevented renal failure in limb ischemia in rats.³² In rabbits, Lauver et al. had positive results with sulodexide on a model of myocardial infarction made by 30 min of coronary ischemia.³³ However, to our knowledge, no study on the porcine model of IRI has been published. Therefore, sulodexide might be used in human medicine for IRI prevention in elective cardiovascular surgery³⁴ transplantation³⁵ and even in emergency medicine during resuscitation procedures such as an emergency department thoracotomy with aortic cross-clamping or application of so-called REBOA catheter to the thoracic aorta which is performed during traumatic cardiac arrest. The main limitation of this bridging therapy is a time of about 25 min due to IRI. Applying sulodexide might prolong this period and increase the time for surgical hemostasis.³⁶

Our study has some limitations. First, we used 30 min of ischemia and 2 h of reperfusion. These times might have been longer, but we intended to be close to the human clinical practice, where the aortic clamp should be no longer than 30 min. Second, we did not investigate the acid-base status with lactate. These would have brought up valuable information about the dynamics of the internal environment disequilibria and restoration.

Third, other markers of EG damage (like hyaluronan, not heparan sulfate because the sulodexide would interfere with this analysis) and endothelial cell activation and damage (like angiopoietin-2) could have been used. Fourth, the total amount of excreted albumin might give a better view of the renal injury than a concentration from a series of urine samples.

IRI represents an important pathogenetic mechanism during various procedures in clinical and perioperative medicine⁷ and contributes to the morbidity and/or mortality of many patients with IRI. In this context, searching for procedures or pharmaceuticals is an important research topic, and several pharmaceuticals or procedures have been tested to date.³⁷ Our work is original in that it tests for the first time the possible protective effect of sulodexide, and the obtained results support its further research in the field of pretreatment in elective procedures that are associated with IRI and where the modulation of IRI could be associated with influencing the manifestations of IRI and as a tool of IRI modulation.

Conclusion

Footnotes

List of abbreviations

Acknowledgments

Robert Eisenbruk did a language check as a native speaker.

Institutional review board statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Departmental Commission for Animal Protection of the Ministry of Defence of the Czech Republic (protocol code MO 576619/2024-1457).

Author contributions

LH – experimental protocol preparation, vascular surgery, manuscript writing, KT – part of the team during the experiment, DC – histological analysis, AT – biochemical analysis, CHL – manuscript writing, editing, language check, VC – formal analysis, writing – review and editing, RGH – formal analysis, writing – review and editing, JK – formal analysis, writing – review and editing, DA – conceptualization, methodology, project administration, conducting and leading the experiment, investigation, data curation and analysis, statistics, writing original draft, review, and editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research was supported by Cooperatio UK, section Intensive Care.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

Data are available at the corresponding author on a reasonable request.

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