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. 2026 Jul 31;46(6):e70276. doi: 10.1002/micr.70276

The Effects of Remote Ischemic Preconditioning on the Inflammatory Cytokine Response to Free Flap Head and Neck Reconstruction: A Randomized Controlled Trial

Kumanan Rune Nanthan 1,2, Julie Brogaard Larsen 3,4, Christine Lodberg Hvas 4,5, Birgitte Jul Kiil 1, Andreas Engel Krag 1,4,✉
PMCID: PMC13426342  PMID: 42536026

ABSTRACT

Introduction

Ischemia–reperfusion injury due to vascular pedicle occlusion is one of the most common causes of free flap failure in head and neck cancer reconstruction. Remote ischemic preconditioning (RIPC) has been proposed as an experimental, non‐invasive intervention to attenuate flap ischemia–reperfusion injury; however, the tissue‐protective mechanisms remain unclear. RIPC may have multiple effects on the inflammatory system, which plays a central part in the pathophysiology of ischemia–reperfusion injury. The aim of the present study was to evaluate the effect of RIPC on inflammatory cytokine plasma levels during free flap reconstruction.

Methods

Head and neck cancer patients (n = 60) undergoing tumor resection and subsequent free flap reconstruction between August 2015 and November 2017 at Aarhus University Hospital, Denmark were randomized 1:1 to RIPC or sham intervention in a single‐center, single‐blinded, randomized controlled trial (RCT) (ClinicalTrials.gov: NCT02548377). The study was approved by The Central Denmark Region Committees on Health Research Ethics (journal no. 1‐10‐72‐140‐15) and reported following the CONSORT guidelines. RIPC was administered intraoperatively as four 5‐min cycles of upper extremity occlusion and reperfusion with an inflatable tourniquet. Blood samples were collected before surgery, 6 h after RIPC/sham intervention, and on the 1st postoperative day. Tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐13, and interferon (IFN)‐gamma were analyzed using a multiplex immunoassay.

Results

The average age of the RIPC group was 67 years (±10) and 64 years (±12) in the sham group. Plasma levels of the pro‐inflammatory cytokines changed significantly between preoperative measures to 1st postoperative day between RIPC and sham; TNF‐α (p < 0.001), IL‐6 (p < 0.001), IL‐8 (p < 0.001), IL‐12p70 (p < 0.001), and IFN‐γ (p < 0.001). No significant differences in cytokine levels were shown between groups at any specific time points.

Conclusion

RIPC does not attenuate pro‐inflammatory cytokine release in head and neck cancer patients undergoing free flap reconstruction.

Trial Registration: ClinicalTrials.gov identifier: NCT02548377

Keywords: cytokines, head and neck cancer, inflammation, microsurgery, remote ischemic preconditioning

1. Introduction

Oncologic and functional outcomes after head and neck cancer surgery have been improved since the introduction of microvascular free flap reconstructions (Hanasono et al. 2009). Several studies have demonstrated that free flap reconstructions in the head and neck are reliable with low morbidity (Wei et al. 2001; Blackwell 1999). However, a meta‐analysis has estimated the free flap failure rate in head and neck reconstructions to be 5% of cases, resulting in increased morbidity, prolonged hospitalization, delay of postoperative radio‐ or chemotherapy, and decreased survival rate (Markiewicz et al. 2015; Chaput et al. 2016; Hyodo et al. 2007; Wang 2009). The most common cause of free flap failure is vascular pedicle occlusion inducing severe flap ischemia–reperfusion injury and subsequently no‐flow phenomenon (Selber et al. 2012).

Remote ischemic preconditioning (RIPC) has been introduced as an experimental intervention targeting flap ischemia–reperfusion injury (Küntscher et al. 2002, 2005; Kolbenschlag et al. 2016). RIPC is administered by inducing brief cycles of extremity ischemia and reperfusion with an inflatable tourniquet prior to expected prolonged target tissue ischemia. Several clinical and experimental studies have demonstrated that RIPC is a promising method to attenuate ischemia–reperfusion injury; however, the exact mechanisms remain unclear (Küntscher et al. 2002; Kolbenschlag et al. 2016; Ballestín et al. 2018; Røpcke et al. 2012; Restifo and Thomson 1998; Krag et al. 2017).

Remote ischemic conditioning may have multiple effects on the inflammatory system, including a decrease in the expression of the pro‐inflammatory cytokines tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6, and upregulation of anti‐inflammatory cytokines such as IL‐10 (Pearce et al. 2021). Furthermore, it has been shown that remote ischemic preconditioning reduces neutrophil activation and leukocyte‐endothelial interactions and thereby attenuates the above‐mentioned inflammation‐mediated tissue injury (Pearce et al. 2021). In a porcine model, we have shown that remote ischemic conditioning attenuates secretion of the pro‐inflammatory cytokines IL‐6 and IL‐12p40 from musculocutaneous flaps subjected to ischemia–reperfusion injury, indicating a reduced post‐ischemic inflammatory response (Krag et al. 2017).

The possible attenuation of inflammation by RIPC in head and neck cancer patients should also be explored as emerging studies focus on the role of pro‐inflammatory cytokines in head and neck cancer carcinogenesis and treatment response (Bae et al. 2017; Huang et al. 2017). One study showed that elevated serum levels of IL‐6 before radiotherapy were a strong predictor of the occurrence of a secondary cancer in head and neck cancer patients (Cottin et al. 2018). Another study highlighted the prognostic properties of pro‐inflammatory cytokines in patients receiving treatment for head and neck cancer (Astradsson et al. 2019).

The objective of our study was to investigate the effect of RIPC on inflammatory cytokine release in head and neck cancer patients undergoing tumor resection and free flap reconstruction. We hypothesized that intraoperative RIPC induced an anti‐inflammatory cytokine response.

2. Methods

2.1. Trial Design

This is a sub‐study of a previous single‐center, single‐blinded, randomized controlled trial investigating the effect of RIPC compared with sham intervention on hemostasis and fibrinolysis in head and neck cancer patients undergoing free flap reconstruction (ClinicalTrials.gov: NCT02548377) (Krag et al. 2019). The trial was conducted between August 2015 and November 2017 at the tertiary referral center for microsurgical reconstruction at the Department of Plastic and Breast Surgery, Aarhus University Hospital, Aarhus, Denmark. The study was reported according to the CONSORT 2010 checklist (S1), approved by The Central Denmark Region Committees on Health Research Ethics (journal no. 1‐10‐72‐140‐15) and registered with the Danish Data Protection Agency (journal no. 1‐16‐02‐358‐15). Written informed consent was obtained from all participating patients, and the study complied with all aspects of the Declaration of Helsinki. CONSORT 2010 checklist (S1), Study Protocol (S2) and data set (S3) have been attached as Supporting Information.

Included patients were: (1) patients aged ≥ 18 years, with (2) a histologically verified or clinically suspected malignant tumor in the head and neck region, (3) scheduled for tumor resection and immediate microsurgical reconstruction with a single free flap. Patients were excluded if: (1) diagnosed with arterial and/or venous thromboembolism within the past 3 months and (2) the microsurgical reconstruction was planned with more than one free flap.

2.2. Interventions

Patients were operated under general anesthesia using a 2‐team approach with head and neck surgeons performing the tumor resection and plastic surgeons performing the free flap reconstruction. The microvascular anastomoses were sutured with 9‐0 nylon under the operating microscope. All patients received prophylactic antibiotics on the day of surgery and the following three postoperative days with cefuroxime and metronidazole intravenously. Pharmacologic thromboprophylaxis (dalteparin or tinzaparin) was administered subcutaneously just before surgery, 6 h after surgery, and once daily from the 1st to 28th postoperative day unless contraindicated.

Patients were randomized 1:1 to RIPC or sham intervention in varying block sizes of 2, 4, 6, and 8 performed by a study investigator approximately 35 min before expected free flap ischemia and transfer. The random allocation sequence generation and randomization were carried out as previously described (Krag et al. 2019). RIPC was administered by a study investigator by inducing four 5‐min cycles of upper extremity ischemia and reperfusion with an automated, inflatable tourniquet (autoRIC Device, CellAegis Devices Inc., Toronto, Canada). In sham intervention, the autoRIC Device was attached to the patient but not activated. Hence, patients were blinded to the study intervention, and study investigators were not blinded.

2.3. Outcomes

The outcomes of the present sub‐study were the difference in plasma pro‐inflammatory cytokine levels between the RIPC and sham group until the first postoperative day. The following inflammatory cytokines were measured: TNF‐α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐13, and interferon (IFN)‐γ.

The primary endpoint for the main trial was collagen‐induced platelet aggregation of the first postoperative day (Krag et al. 2019).

2.4. Blood Sampling

Blood samples were collected at three time points: (T1) immediately before surgery, (T2) 6 h after RIPC/sham intervention being in the early postoperative phase, and (T3) on the 1st postoperative day at 06:30 AM. Blood was collected in ethylenediamine tetra acetic acid (EDTA) tubes (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The blood samples were centrifuged at 2960 relative centrifugal force for 25 min at room temperature. Subsequently, the platelet‐poor plasma was aliquoted into Sarstedt 2 mL screw cap low protein‐binding microtubes (Sarstedt, Nümbrecht, Germany) and stored at −80°C until batch analysis.

2.5. Laboratory Analyses

The concentration of the 10 inflammatory cytokines was determined in EDTA‐plasma samples using a multiplex immunoassay according to the manufacturer's instructions (V‐PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Diagnostics, Rockville, MD, USA).

The lower limit of detection (LLOD) was a calculated concentration for each cytokine that corresponded to the average signal 2.5 standard deviations above the background (zero) calibrator. Sample concentrations measured below the LLOD were substituted with LLOD/2 in the dataset.

2.6. Statistical Analyses

The sample size was determined by the primary endpoint for the main trial and no sample‐size calculation was performed based on proinflammatory cytokine data.

The analysis was performed as intention‐to‐treat. Data distribution of continuous variables was assessed with quantile‐quantile (QQ) plots grouped after study intervention. Cytokine data underwent logarithmic transformation before analysis. These data are presented as median with interquartile range (IQR). The mixed‐effects linear regression model was used to test whether the interaction between treatment group and time had a significant effect on cytokine levels. The model was validated by assessing normality of residuals with QQ‐plots. Post hoc pairwise comparison of predicted means between treatment groups at each time point was performed.

The statistical analyses were performed in STATA/MP 17.0 (StataCorp LP, College Station, TX, USA). Figures were produced in GraphPad Prism 7.0 (La Jolla, CA, USA). p < 0.05 was considered statistically significant.

3. Results

Sixty patients were included in the study between August 2015 and November 2017 with follow‐up completed in December 2017 (Krag et al. 2019). All planned blood samples were obtained, and no patients were lost to follow‐up. The two groups were similar in preoperative and intraoperative characteristics (Table 1). IL‐2 and IL‐13 were excluded as endpoints since more than 50% of measurements obtained at random time points were below the LLOD.

TABLE 1.

Patient characteristics.

Variable RIPC (n = 30) Sham (n = 30)
Sex (male/female) 18/12 19/11
Age (years) 67 ± 10 64 ± 12
Body mass index (kg/m2) 25 ± 4 23 ± 4
Smoking status
Active 12 (40%) 16 (53%)
Former 15 (50%) 10 (33%)
Never 3 (10%) 4 (13%)
Alcohol consumption (1 unit = 12 g)
< 21 units per week 21 (70%) 23 (77%)
≥ 21 units per week 9 (30%) 7 (23%)
American society of anesthesiologist classification
I 1 (3%) 0
II 13 (43%) 19 (63%)
III 16 (53%) 11 (37%)
Surgery time (min), mean ± SD 398 ± 78 417 ± 95
Tumor resection
Composite resection 14 (47%) 12 (40%)
Mandibulectomy 7 (23%) 11 (37%)
Maxillectomy 3 (10%) 3 (10%)
Oral cavity 4 (13%) 0
Laryngopharyngectomy 2 (7%) 3 (10%)
Partial glossectomy 0 1 (3%)
Neck dissection
Not performed 9 (30%) 8 (27%)
Ipsilateral 15 (50%) 16 (53%)
Bilateral 6 (20%) 6 (20%)
Free flap type
Fibula 10 (33%) 10 (33%)
Radial forearm 10 (33%) 5 (17%)
Anterolateral thigh 6 (20%) 5 (17%)
Rectus abdominis 2 (7%) 6 (20%)
Jejunum 2 (7%) 3 (10%)
Latissimus dorsi 0 1 (3%)
Free flap composition
Fasciocutaneous 16 (53%) 10 (33%)
Osteocutaneous 9 (30%) 8 (27%)
Bone 1 (3%) 2 (7%)
Musculocutaneous 0 3 (10%)
Muscle 2 (7%) 4 (13%)
Bowel 2 (7%) 3 (10%)
Free flap ischemia time (min), median (IQR) 58 (49–94) 58 (41–70)
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Note: Preoperative and intraoperative characteristics of study groups presented as number of patients with frequencies, mean and standard deviation, or median with interquartile range.

Abbreviations: IQR; interquartile range, SD; standard deviation.

The following pro‐inflammatory cytokines had a different development in plasma levels over time between the RIPC and sham group: TNF‐α, IL‐6, IL‐8, IL‐12p70 and IFN‐γ (all, p < 0.001) (Figure 1, Table 2). IL‐1β plasma levels did not develop differently over time between the two groups (p = 0.18). When visually assessing median cytokine levels over time, the notable patterns were: (1) TNF‐α levels were decreasing during and after surgery (T1 to T2 and T2 to T3) in the RIPC group versus a small increase in the postoperative period (T2 to T3) in the control group; 1.20 (1.02–1.49) to 0.87 (0.72–1.45) and 1.22 (1.01–1.49) to 0.98 (0.76–1.17) (p < 0.001), (2) IL‐6 levels showed a profound increase during surgery (T1 to T2) in both groups, with a much wider IQR for the RIPC group at T2, but subsequently decreased to similar levels in the two groups on the first postoperative day (T3); 2.23 (1.32–4.04) and 1.26 (0.94–2.39) to 29.64 (18.89–58.10) and 30.54 (19.67–47.05) (p < 0.001), (3) IL‐12p70 remained at the same level during surgery (T1 to T2) in the RIPC group but increased twofold in the control group during surgery, followed by an increase in the RIPC group and decrease in the control group during the postoperative period (T2 to T3); 0.045 (0.017–0.077) to 0.073 (0.017–0.112) and 0.060 (0.017–0.13) to 0.083 (0.022–0.130) (p = 0.04) (Figure 1).

FIGURE 1.

FIGURE 1

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Cytokine plasma levels presented as medians with interquartile range. T1: Before surgery. T2: 6 h post RIPC/Sham. T3: 1st postoperative day. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

TABLE 2.

Inflammatory cytokines during and after surgery in the RIPC and sham group.

Cytokine Group Before surgery 6 h post‐intervention 1st postop. day p
TNF‐α RIPC 1.20 (1.02–1.49) 1.10 (0.71–1.37) 0.87 (0.72–1.45) < 0.001
Sham 1.22 (1.01–1.49) 0.93 (0.75–1.16) 0.98 (0.76–1.17)
IL‐1β RIPC 0.054 (0.009–0.130) 0.043 (0.009–0.110) 0.063 (0.0088–0.10) 0.18
Sham 0.017 (0.088–0.050) 0.05 (0.02–0.12) 0.056 (0.016–0.110)
IL‐6 RIPC 2.23 (1.32–4.04) 48.70 (20.02–110.48) 29.64 (18.89–58.10) < 0.001
Sham 1.26 (0.94–2.39) 44.73 (31.26–76.11) 30.54 (19.67–47.05)
IL‐8 RIPC 10.98 (8.42–13.28) 20.37 (9.25–34.86) 10.58 (7.85–19.58) < 0.001
Sham 10.66 (9.55–13.14) 18.45 (12.45–23.93) 10.81 (8.75–14.24)
IL‐12p70 RIPC 0.045 (0.017–0.077) 0.044 (0.017–0.230) 0.073 (0.017–0.112) 0.04
Sham 0.060 (0.017–0.13) 0.12 (0.040–0.190) 0.083 (0.022–0.130)
IFN‐γ RIPC 7.34 (4.07–13.81) 1.73 (1.00–5.47) 1.54 (0.91–5.32) < 0.001
Sham 6.81 (4.68–9.65) 1.43 (0.93–4.06) 2.33 (0.77–4.54)
IL‐4 RIPC 0.0050 (0.0029–0.0068) 0.020 (0.006–0.074) 0.015 (0.007–0.032) < 0.001
Sham 0.0045 (0.0029–0.0068) 0.024 (0.009–0.045) 0.017 (0.010–0.030)
IL‐10 RIPC 0.21 (0.15–0.37) 2.29 (1.27–5.21) 0.58 (0.40–1.50) < 0.001
Sham 0.23 (0.17–0.34) 2.10 (1.17–4.90) 0.90 (0.55–1.42)
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Note: Cytokine plasma levels presented as medians with interquartile range. p‐value from mixed‐effects linear regression model.

Abbreviations: IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

The anti‐inflammatory cytokines IL‐4 and IL‐10 showed a different development in plasma levels over time between the RIPC and sham group (both p < 0.001). There were no notable differences between groups when visually assessing these anti‐inflammatory median cytokine levels over time.

Post hoc comparison of predicted means between the RIPC and sham group did not show any significant differences in cytokine levels at specific time points.

No patients were lost to follow‐up. No adverse events were observed related to the study intervention.

4. Discussion

Our study demonstrates that RIPC induced an altered inflammatory cytokine response during and after surgery in head and neck cancer patients undergoing free flap reconstruction. The development in plasma inflammatory cytokine levels over time differed between the RIPC and sham groups, but not in a consistent anti‐ or pro‐inflammatory direction.

IL‐12p70 median plasma levels remained low in the early postoperative period in RIPC patients compared with sham patients, which on the contrary demonstrated increased median levels during this period. This attenuation of IL‐12 expression 6 h after RIPC administration corresponds well with our porcine model, where remote ischemic conditioning reduced secretion of IL‐12 from musculocutaneous flaps 5 h after ischemia–reperfusion injury measured by the IL‐12p40 subunit of the heterodimeric IL‐12 (Krag et al. 2017). Beyond the translation from a porcine model to the present human study, our porcine model also differed by measuring the net secretion of IL‐12 directly from the flap by calculating a concentration gradient between systemic venous blood and flap venous blood samples. Orlandi et al. conducted a randomized controlled trial on the effect of RIPC on inflammatory cytokines in patients with periodontitis and found an increase in IL‐12 levels in the sham group compared to the RIPC group at day 1 after the intervention. However, IL‐12 levels were conversely higher in the RIPC group compared with a decrease in the sham group at 7 days after the intervention (Orlandi et al. 2021).

IL‐12p70 is the active heterodimeric form of IL‐12 (Zhang and Wang 2008). IL‐12 is a key pro‐inflammatory cytokine involved as a potent T‐helper type 1 cell regulator, inducer of IFN‐γ by stimulating T‐cells and NK cells, and it enhances cytolytic activity of macrophages, T cells, and NK cells (Waller 2012). Studies on periimplantitis have shown a correlation between elevated IL‐12 levels and plaque accumulation, pocket depth, bleeding, and suppuration (Duarte et al. 2009; Bordin et al. 2009). Further, a study on atherosclerotic plaques in patients with acute coronary syndrome found that IL‐12, through its effects on T cells, induced atherosclerotic plaque inflammation which is associated with plaque vulnerability and plaque rupture (Zhang et al. 2006). Hence, these properties of IL‐12 may play an important role in driving the post‐ischemic inflammatory injury of free flaps after transfer, which ultimately can lead to the no‐reflow phenomenon (May et al. 1978).

On the other hand, the pro‐inflammatory properties of IL‐12 have also been studied as a potential immunotherapy against cancer (Nguyen et al. 2020). Animal studies have shown that IL‐12 combined with chemotherapy inhibited tumor growth in head and neck squamous cell cancer (Fabian et al. 2024). These potential anti‐cancer properties of pro‐inflammatory cytokines should also be considered, especially in head and neck cancer patients.

IL‐6 levels showed a similar increase in median plasma levels in both groups in the early postoperative period whilst decreasing to similar levels at the 1st postoperative day. This conflicts with our porcine model, where remote ischemic conditioning reduced the secretion of IL‐6 from our musculocutaneous flap 5 h after ischemia–reperfusion injury (Krag et al. 2017). Orlandi et al. reported a significant attenuation of IL‐6 in the RIPC group on day 1, but no difference on day 7 in periodontitis patients (Orlandi et al. 2021). Several studies have shown an association between elevated plasma levels of IL‐6 and seroma and other post‐surgical complications (Oka et al. 1992; Miyaoka et al. 2005; Roch et al. 2024; Van Bastelaar et al. 2019). A review from 2020 concluded that remote ischemic conditioning attenuates pro‐inflammatory cytokine release, including IL‐6, in a wide range of pathologies in animal studies. However, clinical translation of this matter is still lacking (Pearce et al. 2021).

This randomized controlled trial adds to the limited evidence regarding the effect of RIPC on inflammatory cytokines in patients undergoing free flap reconstruction. Data was collected prospectively, and all patients completed the follow‐up. The study provides novel data on the inflammatory cytokine response to free flap reconstruction contributing to the understanding of the perioperative physiologic response to these procedures.

The study is limited by only investigating the early postoperative period until the morning of postoperative day 1. Flap failure might occur up to 48 h and beyond (Wang et al. 2020). The RIPC protocol used in the present study with four 5‐min periods of upper extremity ischemia and 5‐min reperfusion was based on previous protocols that demonstrated improved clinical and biochemical outcomes in elective surgery (Kolbenschlag et al. 2016; Corcoran et al. 2018; Hou et al. 2017; Sales et al. 2017; Thielmann et al. 2013; Hausenloy et al. 2007; Hoole et al. 2009). However, 10 min extremity ischemia–reperfusion cycles have also been used in experimental models to attenuate flap ischemia–reperfusion injury (Krag et al. 2017; Addison et al. 2003). This study did not include direct measurements of inflammatory cytokine levels in the flap with e.g., microdialysis or histologic examination, which could have shown a protective effect of the RIPC in the flap tissue, which could not be measured systemically. Another limitation was that only patients were blinded to the study intervention.

5. Conclusion

RIPC does not affect inflammatory cytokine release in head and neck cancer patients undergoing free flap reconstruction in a clear pro‐inflammatory or anti‐inflammatory direction. Further clinical research is warranted to assess the protective and beneficial role of RIPC in free flap reconstruction.

Funding

The authors have nothing to report.

Ethics Statement

The study was approved by The Central Denmark Region Committees on Health Research Ethics (journal no. 1‐10‐72‐140‐15) and registered with the Danish Data Protection Agency (journal no. 1‐16‐02‐358‐15).

Consent

Written informed consent was obtained from all participating patients, and the study complied with all aspects of the Declaration of Helsinki.

Supporting information

Supporting Information S1: CONSORT 2010 checklist (S1).

MICR-46-e70276-s002.doc (217KB, doc)

Supporting Information S2: Study Protocol (S2).

MICR-46-e70276-s003.pdf (108.2KB, pdf)

Supporting Information S3: Data set (S3).

MICR-46-e70276-s001.xlsx (73.4KB, xlsx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information S1: CONSORT 2010 checklist (S1).

MICR-46-e70276-s002.doc (217KB, doc)

Supporting Information S2: Study Protocol (S2).

MICR-46-e70276-s003.pdf (108.2KB, pdf)

Supporting Information S3: Data set (S3).

MICR-46-e70276-s001.xlsx (73.4KB, xlsx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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