Ischemic Preconditioning on Secondary Arterial and Venous Ischemia in Pedicled Axial Flaps in Wistar Rats

Abstract

Background Microvascular complications, particularly secondary arterial and venous ischemia, pose significant challenges in reconstructive surgery. This study investigates the potential protective effects of ischemic preconditioning on flap survival, anatomopathological alterations, and immunological responses in pedicled axial flaps subjected to secondary ischemia.

Methods Adult male Wistar rats underwent arterial or venous ischemia, with and without ischemic preconditioning. Histological assessments, immunohistochemistry studies, and biochemical analyses were conducted to evaluate the impact of ischemic preconditioning on inflammatory processes and tissue damage.

Results Ischemic preconditioning demonstrated a statistically significant decrease in histological lesions, with reductions of 56% in arterial and 47% in venous ischemia, mainly associated with a reduction of inflammatory changes and necrosis processes. Immunological analyses revealed a significant reduction in IgM levels induced by venous ischemia, and a consistent decrease in inflammatory cytokines (interleukin-1 and tumor necrosis factor alpha) in both arterial and venous ischemia following preconditioning. Furthermore, F2-isoprostane levels indicated a lower production of oxidative stress markers in preconditioned flaps.

Conclusion This study highlights the beneficial impact of ischemic preconditioning on flap viability, providing robust evidence of reduced histological lesions, inflammation, and oxidative stress in both arterial and venous secondary ischemia scenarios. These findings support the potential clinical relevance of incorporating ischemic preconditioning strategies to improve outcomes in microvascular reconstructive surgery.

 

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Keywords

ischemic preconditioning - secondary - ischemia - arterial ischemia - venous ischemia

Ischemia is characterized by insufficient oxygen intake, leading to a shift in cellular metabolism toward anaerobic pathways. Reperfusion is the treatment for ischemic tissues to prevent necrosis.[1] However, when reperfusion occurs after prolonged ischemia, blood entry may be not sufficient for achieving a fully and uniform tissue perfusion. This inadequacy arises due to a significant deterioration of blood flow at the microcirculation level, caused by the accumulation of toxic factors that exacerbate previous ischemic lesions.[1]

Oxidative stress and inflammation are critical elements in the pathogenesis of this microvascular injury.[2] The ischemia–reperfusion (I/R) injury initiates a cascade involving the release of reactive oxygen species (ROS), causing damage to the endothelium and blocking tissue oxygen supply. The formation of ROS during reperfusion triggers lipid peroxidation, protein damage, inflammation, and initiates I/R injury. Inflammation further disrupts the balance between proinflammatory and anti-inflammatory factors and can aggravate ischemic injury.[3] [4]

In clinical practice, large and/or complex defects can be repaired by microsurgical procedures. The free transfer of tissues needs a period of ischemia during surgery, in which arterial and venous anastomosis of the pedicle is performed. Additionally, ischemia is “mandatory” in the treatment of an amputated limb, occurring during the limb's transport to the medical center where it will be reimplanted and the intrasurgical time required until the revascularization process. This is known as termed primary ischemia.[5]

Critical ischemia, defined as the maximum time tissues can tolerate complete ischemia and remain viable after circulation, is restored and is crucial to consider in postoperative period.[6]

During postoperative period, the flaps are exposed to secondary ischemia due to vascular disorders by both external (e.g., torsion of the pedicle, compression) and internal causes (e.g., technical defects, intrasurgical intima lesions, vasospasm). The failure of free flap due to microvascular thrombosis remains a major problem, with recovery rates ranging from 3 to 10%.[6] The consequences of flap failure, including complete flap loss, necessitating reoperation, and increased patient morbidity, they are devastating for both patients and clinicians.[7] [8] [9] [10] Arterial problems manifest as a pale, bloodless flap without bleeding upon puncture, while venous failure presents as a swollen flap with rapid filling and dark bleeding upon puncture.[11] [12] The duration of this secondary ischemia is crucial for the survival of the transplanted or reimplanted area. Although the solution to secondary ischemia is prompt surgical revision, the time until revascularization often proves lengthy, jeopardize tissue survival.[13] [14]

Thus, to mitigate the effects of I/R in various tissues, different concepts have been developed. Among them, studies on ischemic preconditioning (IP) have shown its efficacy in improving survival in skin, muscle, and musculocutaneous flaps subjected to primary ischemia.[15] [16] IP involves cycles of short periods of ischemia followed by reperfusion. The effect of IP develops from an early and a delayed form.[17] The effect of early preconditioning develops within minutes of reperfusion and lasts for 2 to 3 hours, while delayed preconditioning becomes apparent 12 to 24 hours later and persists for 2 to 3 days.[17] However, limited research exists on the action of IP specifically on secondary ischemia. In this pilot study, we aimed to verify the protective effect of IP on secondary ischemia in rat skin flaps, assessing its impact on arterial and venous secondary ischemia separately, through anatomopathological and immunological analyses.

Methods

Animals Study

Male Wistar rats (Rattus norvegicus) originally from SPF animals (SIBA, University of Valladolid, Spain) were bred in the animal facility for the study. Twenty-three healthy animals aged 11 to 13 weeks and with a weight of 382 ± 48 g at the beginning of the study were initially used in experimental procedures. Animals were housed in conventional Eurostandard type III H polycarbonate cages (UNO BV, Zevenaar, The Netherlands), with wood bedding and carton tunnels as cage enrichment. They were fed on a conventional certified diet for rats (A04-Safe, Villemoisson-sur-Orge, France) and tap water ad libitum. Rats were maintained under standard environmental conditions (temperature: 22 ± 2°C; relative humidity: 40–70%; 12:12 hour light:dark cycle).

 

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Welfare Interventions Prior to Procedures

Animals were habituated to experimental conditions by the veterinarian 3 weeks before procedures began. A progressive standard training program was established for all the animals. First, animals were socialized to daily handling: opening the cage, speaking to animals, friendly handling, trickling, weighting, and physical restraining, which simulated the intraperitoneal injection, almost 3 days per week. For animals to become accustomed to new flavors, analgesics were offered in a jelly mixture (own formulation) and drinking water several days prior to surgery. Finally, animals were housed individually 2 days before experiments. Animals that did not follow the growth curve or highly agitated at the end of the training program were excluded from experiments.

Anesthesia and analgesia were standardized using an injectable drug combination ([Table 1]). Selected criteria included agents that minimized interferences with the experiment, ruling out the use of nonsteroidal anti-inflammatory drugs with anti-inflammatory properties. The animals' eyes were protected by application of an ophthalmic ointment (Lipolac 2 mg/g, Angelini Farmaceútica S.A, Barcelona, Spain). Depth of anesthesia was assessed by testing the pedal withdrawal reflex, the pattern and depth of respiration, and the color of mucus membranes. Measures to minimize undesirable side effects during surgery were taken. These included the use of a heating pad to avoid hypothermia, the administration of 100% oxygen through a face mask in the case of respiratory depression and the administration of warmed isotonic fluids to support the circulation. The percentage of surgery complications and mortality within the first 12 hours of procedures was 13% (3/23).

Table 1 Anesthesia and analgesia drug combination

Drug	Dose	Route	Timing	Conditions
Anesthesia combination
Diazepam (Valium  10 mg/2 mL)	10 mg/kg	IP	Once	10 min before ketamine + atropine
Ketamine (Ketolar 50 mg/ml) + atropine (B. Braun 1 mg/mL)	80 mg/kg + 0.05 mg/kg (same syringe)	IP	Once
Redosing	1/3 initial dose of ketamine	IP	Maintenance/if necessary
Preventive analgesia
Buprenorphine (Buprex 0.3 mg/mL)	0.05 mg/kg	SC	Once	15 min before surgery
Fluid therapy
Ringer's lactate (warmed)	5 mL/kg	IP	Twice	15 min before surgery/just after surgery
Postoperative analgesia
Buprenorphine (Buprex 0.3 mg/mL)	0.4 mg/kg	O	12 h; days 1–3 (p.o.)	Voluntary ingestion in 10 mL of jelly (JOF)[a]
Paracetamol (Apiretal 100 mg/mL)	200 mg/kg	O	24 h; days 1–5 (p.o.)	Voluntary ingestion (DW)
Euthanasia
Sodium Pentobarbital (Release 300 mg/mL)	300 mg/kg	IC	Once	Under general anesthesia (the same as for surgery)

Abbreviations: DW, drinking water; IC, intracardiac; IP, intraperitoneal; JOF, Jelly own formulation; O, oral; p.o., postoperative; SC, subcutaneous; SSF, physiological saline solution.

^a Jelly formulation: 4 g of raspberry jelly (Hacendado) + 0.120 g of Agar–Agar powder (Pronagar) + 25 ml hot water. Mix and add 25 mL SSF 0.9% (room temperature). Add 10 mL to each mold + 0.4 mg/kg Buprenorphine. Let cool at 4°C.

 

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Experimental Flap Model

The surgery was performed using a standard aseptic technique and all procedures were conducted by the same surgeon. The abdominal and groin areas were previously shaved and disinfected with a topic solution of 2% alcoholic chlorhexidine gluconate (Despro Sol Color, SAED, Asturias, España, reference: 2028070). The epigastric skin flap, according to Petry and Wortham[18] was the basic procedure in the present study. This flap includes the skin surface, subcutaneous tissue, and panniculus carnosus, leaving the muscle fascia intact. A 3 cm × 4 cm rectangular flap was marked and raised in the left hemiabdomen. Small vessels around the flap were cautiously coagulated with bipolar forceps. The epigastric pedicle was exposed, and vessels were carefully dissected using optical amplification and microsurgery instruments ([Fig. 1]).

Fig. 1 Skin flap photography. This image shows the inferior epigastric flap in the rat with the pedicle with the vessel loop.

I/R injury was induced in the following way: primary ischemia of 1.5 hours by clamping (B-1 microvascular clamp S&T, Neuhausen, Switzerland) the left superficial epigastric artery and vein; reperfusion of 2 hours by removing the microvascular clamp; secondary ischemia of 3 hours by clamping again the epigastric arterial or vein vessels. Preconditioning treatment (P) consisted of three consecutive cycles of 5 minutes of ischemia followed by 5 minutes of reperfusion. A sterilized silicone sheet was placed under the flap for preventing neovascularization from the wound bed. Flap was sutured back into place with 4–0 Ethilon Polyamide 6 interrupted stitches (Ethicon, reference: W1620). Previously, vascular patency was confirmed under the microscope. Finally, flap was protected from self-cannibalism using a “protective shield,” which consisted of a metal mesh adjusted to the size of the flap and attached to the skin with interrupted stitches (3–0, Silkam, reference: 0760412), and two self-adhesive bands (VETER-FLEX) around the shield ([Fig. 2]).

Fig. 2 Skin flap protection. (A) To protect the skin flap from self-cannibalism, a special shield was made with metal mesh adjusted to the size of the flap and (B) fixed to the skin with interrupted stitches (3–0, Silkam, reference: 0760412). In addition, two self-adhesive bandages (VETER-FLEX) were placed around the shield and the abdomen of the animals. This shield, as well as to providing protection, allowed the animals to move freely, as shown in (C) where the animals can be observed ingesting the postoperative analgesia.

 

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Study Design

Animals (n = 20) were randomly allocated into five experimental groups: (1) Sham-operated group (SO) (n = 4): only the epigastric flap was elevated and sutured in situ as previously described; (2) Control arterial group (CA) (n = 4): Secondary ischemia of 3 hours by clamping again the epigastric arterial vessels; (3) Control venous group (CV) (n = 4): Secondary ischemia of 3 hours by clamping again the epigastric venous vessels; (4) Preconditioning arterial group (PA) (n = 4): Before performing 3-hour secondary ischemia by clamping again the epigastric arterial vessels, the preconditioning treatment is performed; (5) Preconditioning venous group (PV) (n = 4): Before performing 3-hour secondary ischemia by clamping again the epigastric venous vessels, the preconditioning treatment is performed ([Fig. 3]).

Fig. 3 Illustration of study design.

 

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Postoperative Care

Animals were monitored until they were awake and ambulatory, at which time they were returned to their cages. Small pellets were deposited on the cage floor in the first 48 hours following the surgery to facilitate food intake. A routine analgesic protocol was established in advance for a minimum of 5 days after procedures to avoid the possibility of animals undergoing unnecessary suffering. Animal welfare was assessed twice a day by the veterinarian using a score sheet with general indicators of pain and distress. If necessary, corrective measures were taken, including the improvement of analgesia, parenteral administration of fluids, and the application of humane endpoints as defined in the monitoring protocol.

 

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Method of Euthanasia

Animals were euthanized at the end of procedures (day 7) by an intracardiac overdose (300 mg/kg) of sodium pentobarbital (euthanasia commercial solution for animals, Release 300 mg/mL, WDT, Garbsen, Germany) under deep anesthesia, following the good veterinary practice. Death was finally ensured by confirmation of permanent cessation of the circulation.

 

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Sampling Collection

Blood samples (5 mL) were collected by cardiac puncture technique under deep terminal anesthesia via the left side of the chest using a 23-G needle. Plasma was obtained by centrifugation at 3000 rpm for 10 minutes and stored at −80°C until analysis. Complete flap was recovered from animals, divided into two longitudinal and equal-size parts, and preserved either at −80°C or in buffered (pH = 7) 4.0% formaldehyde (PanReac AppliChem ITW Reagents, reference: 252931.1315) biopsy container for biochemical and histopathological assays, respectively.

 

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Flap Viability Study

The skin flaps designed in the left hemi-abdomen were photographed before surgery and after 7 days postoperatively in the five groups. In the photographs, the macroscopic viability of the flaps was_studied.

Due to the differences in the establishment of cutaneous necrosis in the case of arterial and venous ischemia, we have divided the groups into the arterial study and the venous study:

• Arterial study: Sham-operated, control arterial group, and group with arterial preconditioning.

• Venous study: Sham-operated, control venous group, and group with venous preconditioning.

In the arterial study, the measurement of necrotic areas was performed using the digital tool Image J, a public domain digital image processing program in Java and developed at the National Institutes of Health.[19] We valued the skin necrosis areas in cm² in each animal of each group, obtaining the percentage of necrotic area by comparison of the areas before and after surgery process and comparing the results between study groups.

In the venous study, the skin changes that occur due to venous congestion are marked by cyanosis of the flap, edema, alteration in hair growth, increased bleeding in the microcirculation, among other symptoms, as well as areas of necrosis. These changes in skin make the flap nonviable. For its evaluation, we apply some items that are presented in [Table 2]. Measurement of various areas is performed using the Image J digital tool as well as the arterial study.

Table 2 Parameters of the viability study of skin flap

Numeric value items	1	2	3	4
Cyanosis of the flap (area, %)	0–25	25–50	50–75	75–100
Presence of hair in the flap (area, %)	100–75	75–50	50–25	25–0
Necrosis of the flap (area, %)	0–25	25–50	50–75	75–100

 

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Histopathological Evaluation

Samples fixed in formalin solution underwent dehydration using an ascending alcohol ladder, followed by rinsing in xylene and were embedded in paraffin for preservation. A Leica TP1020 tissue processor (Wetzlar, Germany) and a Tissue-Tek TEC 5 paraffin dispenser (SakuraSeiki Co., Ltd., Japan) were employed. Histological sections with a thickness of 3–4 μm were prepared from the paraffin-embedded samples using a microtome (Microm model HM325, Thermo Scientific) for the histopathological study through hematoxylin–eosin (H–E) staining. The histological study was performed in another center by a pathologist whose information was limited to achieve greater objectivity in their results ([Table 3]). The pathological findings evaluated were vascular changes like hemorrhages, inflammatory changes as polymorphonuclear and mononuclear infiltrates, as well as tissue damage processes like necrosis. Subsequently, the findings assessed in the flap received an additional numerical score according to the intensity of the lesion found. The presence of each one of these findings was graded as absent or 0% (0), mild or 10 to 30% affected (1), moderate or 30 to 70% affected (2), and severe or 70 to 100% affected (3). All the pathological findings were independently examined by one blinded and experienced observer (M.A.R.).

Table 3 Histological study of the skin flap in the arterial and venous study

Parameter	SO	CA	CV	PA	PV
Hemorrhage	0.25 ± 0.5	7.3 ± 5.4	7.3 ± 0.5	0.25 ± 0.5	1.8 ± 1.7
Polymorphonuclear infiltrate	0.5 ± 1.0	14.5 ± 6.1	6.3 ± 3.9	4.8 ± 6.6	2.8 ± 0.5
Mononuclear infiltrate	2.0 ± 0.8	12 ± 4.2	7 ± 3.5	5.5 ± 1.7	6.3 ± 3.1
Necrosis	0 ± 0	9 ± 3.5	2 ± 2.3	3.5 ± 4.3	1.5 ± 1.0

Abbreviations: CA, control arterial group; CV, control venous group; PA, preconditioning arterial group; PV, preconditioning venous group; SO, sham-operated group;

 

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Analysis of Immunological Mediators of the Inflammation

The expression of plasma interleukin (IL-1) and tumor necrosis factor alpha (TNFα) were measured using enzyme-linked immunosorbent assay (ELISA) kits (RD Systems, Minneapolis, MN). For protein tissue extraction, the tissues were homogenized in an appropriate buffer and centrifuged at 12000 ×g for 20 minutes at 4°C, and the supernatant was assayed according to the manufacturer's instructions.

Levels of 8-iso-PGF2 were quantified by ELISA according to kit instructions (OxiSelect 8-iso-Prostaglandin F2 ELISA kit). To be prepared for the analysis, skin flaps were treated with NaOH at 45°C for 2 hours. In addition, 100 μL of concentrated (10N) HCl per 500 μL of hydrolyzed sample was added. After that, samples were centrifugated 5 minutes at 12000 ×g, and the supernatant was assayed according to the manufacturer's instructions.

 

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Statistical Study

The results were expressed as means ± standard deviations of independent experiments (n = 3). Statistical analysis was performed using Statgraphics Centurion 19, version 19.5.01 (Statgraphics Technologies, Inc., Warrenton, VA). One-way analysis of variance, using Fisher's least significant difference test, was used to determine significant differences (p < 0.05) between different groups.

 

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Results

Preconditioning Improves the Viability of Flaps

In the macroscopic arterial study, the percentage of necrosis in the skin flaps in the CA group was 75 ± 32.03 ([Fig. 4A]), necrosis in PA group was 27.5 ± 21. 73 ([Fig. 4B]), and in the SO group was 25.25 ± 23.31. These results indicated a reduction in necrosis between the CA and PA groups, with a 63% decrease, which was statistically significant ([Fig. 4C]; p < 0.05).

Fig. 4 Skin flap necrosis percentage of arterial study. On postoperative day 7, the macroscopic appearance of skin flap was evaluated for the arterial study. (A) Sham-operated group (SO); (B) Control arterial group (CA); (C) Preconditioning arterial group (PA); (D) percentage of visual necrosis calculated using the program ImageJ. Box plots indicating the 25^th and 75^th percentiles, whiskers specifying maximum and minimum values and medians (horizontal black bar inside the box) are represented. Significant differences (ANOVA, p < 0.05) among SO, CA, and PA are represented with an asterisk (*) and determined from observations of four animals from independent experiments. ANOVA, analysis of variance.

In venous study of necrosis ([Fig. 5]) showed venous congestion, marked by cyanosis of the flap and edema in the CV group ([Fig. 5B]), whereas that in the PV group these findings were light ([Fig. 5C]). The total score in the skin flaps in the CV group showed a of 7.5 ± 1.91. The sum of the items in the PV group, presented a of 4.5 ± 2.4 and the in the SO group was 3.5 ± 0.58. The reduction in sum of the items between the CV and the PV is 40% ([Fig. 5D]).

Fig. 5 Skin flap necrosis from venous study. On postoperative day 7, the macroscopic appearance of skin flap was evaluated. (A) Control venous group (CV); (B) Control venous group (CV) with a hematoma and vascular dilation in the subcutaneous tissue of the flap from; (C) Preconditioning venous group (PV); (D) total score of visual necrosis calculated using the program ImageJ. Box plots indicating the 25^th and 75^th percentiles, whiskers specifying maximum and minimum values and medians (horizontal black bar inside the box) are represented. Significant differences (ANOVA, p < 0.05) among SO, CA, and PA are represented with an asterisk (*) and determined from observations of four animals from independent experiments. ANOVA, analysis of variance.

The Ischemic Preconditioning Reduced the Tissue Lesions on Secondary Ischemia

The histopathological evaluation ([Fig. 6]) evidenced that SO group did not show lesions ([Fig. 6A]). However, CA group showed an intense tissue necrosis, accompanied by a severe mixed infiltrate of mononuclear cells and neutrophils ([Fig. 6B]), which was less intense in the PA group with absence of necrosis ([Fig. 6C]). In the CV group, vascular changes were observed along with the presence of inflammatory cells ([Fig. 6D]). The reduction of hemorrhages in the PA group (0.25 ± 0.5) was 95% compared with the CA group (7.25 ± 5.37) and 76% when we compare the PV group (1.75 ± 1.71) to the CV group (7.25 ± 0.57), both PA (p < 0.01) and PV group (p < 0.001) being at levels close to the SO group and statistically significant. The polymorphonuclear infiltrate in the PA group (4.75 ± 6.6) and PV group (2.75 ± 0.5) showed a decrease of 67.2 and 56%, respectively, compared with the CA (14.5 ± 6.14) and CV group (6.25 ± 3.86). However, the levels of PA and PV are much higher than the SO group. The mononuclear infiltrate in the PA (5.5 ± 1.73) and PV (6.25 ± 3.1) showed a decrease when compared with the CA (12 ± 4.24) and CV (7 ± 3.46), although only the results of PA group was statistically significant (p < 0.030). Necrosis in the PA (3.5 ± 4.3) decreased by 61% compared with the CA (9 ± 3.5), being statistically significant (p < 0.098). Likewise, the score of necrosis in the PV group (1.5 ± 1) decreased by 25% compared with the CV (2 ± 2.3) but was not statistically significant.

Fig. 6 Histopathological study of skin flaps. (A) Sham-operated group (SO) with normal epidermis (arrowhead) and slight mononuclear infiltrate, composed mainly of macrophages and lymphocytes, in the deep dermis (asterisk). (B) Presence of ulcerated epidermis in the skin flap from control arterial group (CA) due to the presence of intense tissue necrosis (arrowhead), accompanied by a severe mixed infiltrate of mononuclear cells and neutrophils of transmural distribution, affecting all layers of the skin (asterisk). (C) Skin flap from preconditioning arterial group (PA) with recovered epidermis (arrowhead), less infiltrate of macrophages and lymphocytes in the superficial dermis, but severe infiltrate of neutrophils in the deep dermis (asterisk). (D) Presence of normal epidermis in the skin flap from venous control group (CV) (arrowhead), accompanied by perifollicular edema and slight mononuclear infiltrate in the superficial dermis, but with a severe infiltrate of neutrophils, macrophages, and lymphocytes in the deep dermis (asterisk). (E) Skin flap from preconditioning venous group (PV) with epidermis and superficial dermis without histopathological lesions, and presence of moderate mononuclear infiltrate, composed mainly of macrophages and lymphocytes, in the deep dermis (asterisk). Staining: hematoxylin–eosin.

In general, the total histopathological score ([Fig. 7]) in the PA (19.25 ± 14.52) and in the PV (27 ± 3.9) was 61 and 30% lower compared with CA (45.5 ± 7.85) and CV (16.25 ± 6.29), respectively, being both reductions statistically significant (p < 0.019; p < 0.027, respectively).

Fig. 7 Total score of histopathological findings in subcutaneous tissue of skin flaps on day 7 of experiment in the sham-operated group (SO), control arterial group (CA), preconditioning arterial group (PA), control venous group (CV), and preconditioning venous group (PV). Box plots indicating the 25^th and 75^th percentiles, whiskers specifying maximum and minimum values and medians (horizontal black bar inside the box) are represented. Significant differences (ANOVA, p < 0.05) among SO, CA, and PA are represented with an asterisk (*) and determined from observations of four animals from independent experiments. ANOVA, analysis of variance.

 

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Analysis of Immunological Mediators of the Inflammation

The inflammatory markers Il-1 and TNF-α were evaluated in plasma and skin flap samples ([Fig. 8]). In plasma, the levels of IL-1 in the CA and CV groups were significantly higher than in the SO group ([Fig. 8A]). IL-1 levels in the PA group were 50.5% lower than in the CA group, and this decrease was statistically significant (p < 0.001). Similarly, a 53.5% decrease was observed in PV compared with the CV (p < 0.05). Plasma IL-1 levels in both PA and PV groups were comparable to the SO group. In the tissue, the levels of IL-1 in the sham, CA and CV groups were similar. However, the PA group had a 40.9% significant lower level (p < 0.05) compared with the CA group. Likewise, tissue IL-1 levels in the PV were 26.7% lower than their CV group (p < 0.05).

Fig. 8 Inflammatory markers in plasma and skin flap rats. (A) Interleukin 1 β (IL-1) (B) tumor necrosis alpha (TNF-α) determined by ELISA assays in plasma and in skin flap sham-operated group (SO), control arterial group (CA), preconditioning arterial group (PA), control venous group (CV), and preconditioning venous group (PV). Box plots indicating the 25^th and 75^th percentiles, whiskers specifying maximum and minimum values and medians (horizontal black bar inside the box) are represented. Significant differences (ANOVA, p < 0.05) among groups are indicated with Latin letters. ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay.

The values of TNF-α in plasma and tissue are shown in [Fig. 8B]. In plasma, TNF-α levels decreased by 29.9% in the PA group compared with the CA group (p < 0.05). In the PV group, there was a 28.6% decrease compared with CV (p < 0.05). The data obtained in the sham group were very similar to those obtained in the PV. In tissue, the reduction of TNF-α levels in PA group compared with CA was 24. 8%, and the reduction in PV group compared with the CV group was 20.14%, but this reduction was not statistically significant.

As a measurement of lipid peroxidation, isoprostanes were evaluated in the skin flap. Significant increase was observed in the CA and CV groups compared with SO group reflecting the extent of tissue damage. The levels of isoprostanes decrease by 60.23% in PA versus CA (p < 0.001). In the PV, there was a 57.40% decrease compared with CV (p < 0.001; [Fig. 9]).

Fig. 9 Isoprostanes levels in rat skin flaps. Box plots indicating the 25^th and 75^th percentiles, whiskers specifying maximum and minimum values and medians (horizontal black bar inside the box) are represented. Significant differences between the sham operate group (SO) and the control arterial group (CA), preconditioning arterial group (PA), control venous group (CV), and preconditioning venous group (PV) are indicated with asterisk (*p < 0.05; **p < 0.05) and determined from observations of four animals from independent experiments.

 

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Discussion

This study aimed to evaluate the impact of IP on arterial and venous secondary ischemia separately in pediculate axial flaps of the lower epigastric in Wistar rats, mimicking scenarios encountered in clinical free flap procedures where secondary arterial or venous failure may occur.

Arterial ischemia has been extensively studied, and its pathophysiology involves an inadequate oxygen supply and a deficit in clearing toxic metabolites. In contrast, venous congestion has received less attention in the literature. Persistent, arterial flow in venous congestion leads to increased intravascular pressure and subsequent hemorrhage of the microvasculature into the extravascular space.[20] [21] [22] The elevated extravascular pressure results in external compression and collapse of vessels, whereas edema in the interstitial tissue acts as a barrier to the diffusion of oxygen, further contributing to tissue damage.[23] [24] When veins are completely thrombosed, the flaps exhibit gross edema, cyanosis, and warmth compared with surrounding nonischemic tissue.[25] Flaps with arterial failure, however, appear to maintain a constant size, pale color, and cool temperature. These observations underscore the profound differences in the gross phenotype, histology, and flap survival outcomes between arterial ischemia and venous congestion,[26] [27] [28] [29] justifying the different way of measuring the results in the arterial and venous groups in our study. Here, we observed a very low presence of necrosis in the case of venous ischemia considering our ischemia times, as well as higher inflammatory findings in arterial ischemia results that in venous ischemia when these groups are compared with the sham groups, coinciding with the results presented by Litrico et al.[25]

Different study groups have focused on the implementation of strategies against tissues damaged by prolonged ischemia and I/R, as the concept of retardation has been widely used to improve circulation, there are several ways to retard flaps such as parallel incisions, intermittent sectioning or clamping of the pedicles.[30] [31] [32] [33] IP is defined as repetitive, short periods of ischemia, separated by intermittent reperfusion periods. Murry et al[34] demonstrated that preconditioning of the dog's myocardium significantly reduced the size of the infarction. Subsequent studies have extensively explored the impact of IP on various tissues, including gastric mucosa,[35] small intestine,[36] myocardium,[37] [38] [39] strokes[40] [41] [42] kidney[43] [44] and liver transplants.[45] Studies of IP in cutaneous, muscular, or musculocutaneous flap have predominantly focused on evaluating effects on primary ischemia demonstrating an improvement in flap survival.[46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] The preconditioning can also be pharmacological, using various substances with different points of action. Piracetam[65] is a nootropic agent used mainly in neurological diseases because of its rheological, antithrombotic, neuroprotective activity, and its effects on microcirculation, whereas C1 esterase inhibitor (C1-Inh),[66] appears to be effective in preserving the ischemic myocardium from I/R injury. C1-Inh has an important role inhibiting several of the major cascade systems, including the complement (C1r, C1s, and MASP2), the intrinsic coagulation (factor XII and plasma kallikrein), and the fibrinolytic systems (plasmin and tissue plasminogen activator) with different results among them when compared with IP.

There are limited studies assessing the outcomes of ischemic and pharmacological preconditioning in secondary ischemia.[67] [68] [69] With respect to the technique, although randomized models are widely employed due to their technically simplicity, the unpredictable vascularization of the distal portion poses a limitation. Therefore, we opted for an axial vascularization model, specifically the epigastric flap, which offers greater predictability in vascularization.[70] In determining the I/R protocol, some authors have considered varying numbers of cycles (one, two, or three) and different durations for both ischemia and reperfusion processes.[71] The specific ischemia times required for protection vary among species and organs. Given the lack of a standardized protocol regarding the number of cycles, as well as the minutes of ischemia and reperfusion, we based our approach on the reviewed literature, choosing to conduct three cycles of 5 minutes each for ischemia and reperfusion.[56]

In the present study, a statistically significant increase in the survival of treated flaps IP in secondary arterial and venous ischemia separately, concurring with other studies[66] [67] and with the action of pharmacological preconditioning with lidocaine[64] and botulinum toxin[65] in secondary ischemia with arterial and venous ischemia. Moreover, in our study, we found a more effective action of IP in the arterial group than in the venous group.

Our assessment included an examination on histological and immunological changes in both tissue and serum to comprehensively evaluate the outcomes. In the preconditioning groups, both the arterial and venous groups showed a significant decrease in histological lesions by 56 and 47%, respectively. Among the histological effects observed in various tissues subjected to ischemia and reperfusion processes, the infiltration by neutrophils is commonly studied, being considered key in ischemic–reperfusion lesions.[71] In our study, we observed a more intense leukocyte infiltration in the arterial control group compared with the venous group, as well as the action of IP appears to be more crucial in arterial than in venous ischemia in the reduction of infiltration of neutrophils. These results are supported by many studies that associate a reduced infiltration of polymorphonuclear cells with enhanced flap survival.[56] [72]

Furthermore, we evaluated whether IP is accompanied by changes in the inflammatory process. The preconditioning of both arterial and venous ischemia effectively reduced the levels of inflammatory cytokines like IL-1 and TNFα involved in the leukocyte adhesion to endothelial cells.[73] This activation could decrease due to the accumulation of leukocytes in the ischemic area, reducing flap ischemic damage,[74] [75] consistent with the observed reduction in leukocyte infiltration and the preservation of viability in flap tissues observed in the present study.[76] [77] However, no significant differences were observed between arterial and venous ischemia.

The preconditioning study also revealed a lower production of F2-isoprostanes, an important biomarker of oxidative stress,[78] indicating the protective effect of this treatment in flap tissues. These results can reflect a low oxidative status of tissues[79] [80] and are correlated with the observed reduction in inflammation and necrosis in flap tissue after preconditioning.[81] [82] [83]

Finally, we have taken meticulous care in addressing variables that could potentially impact our results in this study. Specifically, we used only adult male animals to avoid possible estrogen interaction in flap survival. Additionally, we have given considerable attention to the comprehensive management of animals from birth until the initiation of surgery. Our training approach aimed to minimize stress in animals, recognizing the potential influence on study outcomes. This adaption period is of great importance for obtaining reliable results that are not compromised by the presence of systemic substances secondary to stress.

 

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Conclusion

In conclusion, our findings demonstrate a significant positive impact of preconditioning on both arterial and venous ischemia when studied independently in pedicled axial flaps of the lower epigastric region in Wistar rats. Arterial ischemia exhibited a higher histological burden compared with venous ischemia, with reductions of 56 and 47%, respectively, in the preconditioning groups. Furthermore, our investigation revealed alterations in the inflammatory processes associated with I/R injury. The significant reduction in inflammatory cytokines, IL-1, TNF-α, and F2-isoprostanes, in both arterial and venous ischemia following preconditioning further supports its protective role. These results collectively underscore the potential clinical relevance of IP in enhancing the viability of flaps subjected to secondary arterial and venous ischemia. Despite the limitation of a relatively small sample size in each group, our study contributes valuable anatomopathological and immunological insights, laying the groundwork for future research in this critical area of microvascular surgery.

 

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Conflict of Interest

None declared.

Note

Animal use described here complied with EU Directive 2010/63 and Spanish Government RD 53/2013 on the protection of animals used in experimentation with the permission from the Animal Welfare Committee (University Hospital of Burgos, reference: CEBA 22).

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Publication History

Received: 13 October 2024

Accepted: 20 March 2025

Article published online:
16 June 2025