Reperfusion Interval as a Prevention of Lung Injury Due to Limb Ischemia–Reperfusion After Application of Tourniquet in Murine Experimental Study

Abstract

Background

Tourniquet use is prevalent in the orthopaedic field to achieve a bloodless operating field, but it poses risks of local and systemic complications, including lung injury. This study aims to examine the effect of tourniquet application on the hindlimb of a rat to its lung.

Materials and Methods

This is an experimental study with 48 male Wistar strain rats as samples. The rats were divided into group A (n = 24), killed directly after fracturization and tourniquet application, and group B (n = 24), killed 14 days post-procedure. Each group was divided into four: group A1/B1 (control group, three hours tourniquet application without reperfusion interval), A2/B2 (5-min reperfusion between 2-h and 1-h tourniquet application), A3/B3 (10-min reperfusion), and A4/B4 (15-min reperfusion). The lung tissue was examined histologically within ten high-power fields (400 × magnification). The severity of lung injury was measured using the Lung Injury Score (LIS). The oxidative damage was measured by determining the malondialdehyde (MDA) level, using the TBARS (thiobarbituric acid reactive substance assay) method.

Results

There was a dose-dependent decrease of LIS and MDA in groups A and B with increasing reperfusion interval. Fifteen-minute reperfusion interval caused a 54.55% and 45.33% LIS reduction in groups A and B, respectively. All pair-wise group comparisons (p < 0.05) showed significant differences. Five-minute interval reduced the MDA level by 16.56% and 30.13% in groups A and B, respectively. All possible pair-wise comparisons in both groups A and B also showed a significant difference (p < 0.05).

Conclusions

Reperfusion interval is a possible clinical approach to mitigate the remote organ damage induced by limb ischemia–reperfusion injury.

Introduction

The tourniquet is frequently used in orthopaedic surgical procedures to limit the amount of blood loss. It is especially beneficial in procedures involving a fracture of long bone on the lower extremity [1]. The pressure exerted by this device in the clinical setting is quite high, usually reaching 100 mmHg above systolic blood pressure (SBP), and this pressure is generally sustained for 2 h [2]. This poses some complications in both local and systemic settings. In Norway, the incidence of complications due to tourniquet use in orthopaedic surgery is 0.032%, with significantly higher incidence on the lower extremities compared to upper extremities [3].

The local complications include pain, damage to muscle, nerve, vascular, and skin injuries. Other than that, this device may even cause systemic complications such as hemodynamic changes, lung injury, and coagulopathy [4]. The aforementioned complications are induced by the direct pressure of the tourniquet itself, and the ischemic–reperfusion injury occurring after its deflation. The ischemic–reperfusion injury induces various cellular damages due to the formation of oxygen free radicals and inflammatory cytokines, including high-mobility group box-1 (HMGB1), NOD-like receptor protein 3 (NLRP3), and heat-shock proteins (Hsp). The inflammatory mediators would then cause microvascular dysfunction and induce remote organ injuries such as liver and kidney [5, 6]. Lin et al. demonstrated that tourniquet use in orthopaedic surgery still incurs lipid peroxidation and increases IL-6 and IL-8 levels even in standard allowed duration. Furthermore, it would lead to a disturbance in pulmonary gas exchange [7]. The incidence of post-operative ALI is quite low, only less than 1%, but the mortality rate reaches more than 40% of all cases [8].

Various strategies have been proposed to mitigate the ischemia–reperfusion injury and its sequelae: use of specific anaesthetic agents, antioxidants, and ischemic preconditioning or postconditioning [9]. The experimental murine model showed that ischemic preconditioning might inhibit lipid peroxidation and acute lung injury (ALI).

Another possible method is reperfusion interval which is a brief intraoperative tourniquet deflation period of more than 5 min. Unfortunately, this method does not significantly mitigate nerve damage in the clinical setting. [10] Furthermore, the effect of reperfusion interval on skeletal muscle has been elaborated in previous experimental studies [11, 12]. However, the benefit of this method in preventing distant organ damage due to tourniquet-induced ischemic–reperfusion injury is yet to be elucidated. Thus, this study aims to evaluate the effect of reperfusion interval to tourniquet-induced ischemia–reperfusion lung injury in the murine model.

Methods

This experimental study was conducted during January 2019–March 2019 at the authors’ institution. The acclimatization, maintenance, and intervention of the samples were done in the Parasitology Laboratory of the authors’ institution. The MDA measurement was conducted in the Physiology Laboratory of the authors’ institution. The lung specimen histologic assessment was done in the authors' institution. The Institutional Review Board has reviewed this study with an ethical clearance number of 70A/EC/KEPK—PPDS/02/2019.

Animal Model

The animal models in this study were 48 male rats (Rattus norvegicus). Male rats were used because the use of male rats is already established in fracture experimental study [13]. Furthermore, the authors used only male rats to reduce variability because the previous study found that female rat fracture model exhibits different mechanical properties [14]. The inclusion criteria were healthy 3-month-old male Rattus norvegicus weighing 180–200 g. The exclusion criteria were an infection, death, and broken/damaged cast. The animals were acclimatised for a week in a controlled condition of 12-h light/dark cycle at the temperature of 23.6 °C; they were fed a standard chow diet with free water intake.

The animals were divided into two large groups: one group (Group A) was killed directly 1 h after the intervention, and the other group was killed after a 14-day period (Group B). The 14 days was chosen because the previous study showed that the peak callus mass was formed after that period [15]. Each group was then further divided to four smaller groups: group A1/B1 was the control group which did not receive any reperfusion period within 3 h of tourniquet inflation, group A2/B2 received 5-min reperfusion interval between 2-h and 1-h tourniquet application period, group A3/B3 received 10-min reperfusion period, and group A4/B4 received 15-min reperfusion period.

Tibia Fracture Model

The right tibia of the rat was fractured using the open bone cutting technique. The open technique was used to minimize surrounding soft tissue damage. It also ensures same fracture configuration across all animal samples [16, 17]. The animals were fasted three hours before the surgical procedure. The animals were anesthetized using intravenous 40 mg/kg ketamine hydrochloride, and the prophylactic antibiotic used was an intramuscular 5 mg/kg cefazoline. Following disinfection and aseptic technique, longitudinal incision on the anterior tibia was made, and both cortices were fractured in the mid-diaphysis using bone cutting forceps after intraperitoneal anaesthesia with ketamine (40 mg/kg BW). The wound was then sutured with a non-absorbable suture. The fracture was immobilized with long leg cast with plaster of paris (POP).

Tourniquet

The tourniquet used was a 4.5 oz orthodontic rubber band with a diameter of 1/8 inch. The orthodontic rubber band was applied to one leg of the rat on the proximal thigh. The rubber was installed for 3 h in the control group (A1 and B1), while the treatment group was given a reperfusion interval of five (A2 and B2), ten (A3 and B3), and 15 min (A4 and B4) after 2 h of tourniquet use. The tourniquet was then reapplied for another hour and only applied on the first day.

Orthodontic rubber band use in the ischemic–reperfusion injury in the murine model has been established in the previous study. Tourniquet is often used in ischemic–reperfusion rats’ model because it provides adequate occlusion to femoral artery and its numerous collateral branches. Orthodontic rubber band is superior because it causes less soft tissue damage [18].

MDA Measurement

MDA represents the end product of lipid peroxidation, one of the markers of oxidative stress (Table 1). The MDA levels in the bone were evaluated using thiobarbituric acid reactive substances (TBARS) method. The right tibia was ground in a porcelain mortar to homogenize the bone sample. An amount of 50 mg bone tissue was weighed and mixed with 1 ml of buffer phosphate, 1 ml of 100% trichloroacetic acid (TCA), 1 ml of 1 N HCl, and 1 ml of 1% Na Thiobarbiturates in a Petri dish. The solution was then heated in the water bath with a temperature of 100 °C for 25 min. It was then centrifuged at 2000–3000 rpm for 15 min. The supernatant was taken and diluted with Aqua Bidest to 3 ml. The absorbance of each sample was measured at 532 nm using spectrophotometry. The results were expressed as nanomolar of MDA per milligram of tissue (nmol/mg).

Table 1.

Lung injury score (LIS) [19]

Indicators	Score/high-power field
	0	1	2
Neutrophil in alveolar cavity	None	1–5	> 5
Neutrophile in interstitial cavity	None	1–5	> 5
Hyaline membrane	None	1	> 1
Protein debris in alveolar cavity	None	1	> 1
Alveolar septal thickening	< 2×	2–4×	> 4×

Histologic Assessment

The lung was taken after killing. The specimen was submerged to 10% formaldehyde then dehydrated with subsequent immersion with 70%, 80%, 90%, 95%, 99%, and 100% alcohol solution, each solution for one hour. The next process was clearing with xylol solution for 20–30 min. The cleared specimen was then embedded in a paraffin block. The embedded specimen was sliced with a microtome, and the resulting slice was dyed with hematoxylin–eosin (HE) stain. The histologic examination was done using the Olympus BX-51 microscope under 400 × magnification in ten high-power-field (HPF). Lung Injury Score (LIS), proposed by the American Thoracic Society (ATS), was used in the lung histologic assessment [19].

Statistical Analysis

The data obtained were analyzed using the normality test to check the distribution. One-way ANOVA test was used after the data met the normality test. Further, all pair-wise comparisons were tested using Tukey test. Data analysis was performed by SPSS version 20 software, NY Armonk, USA.

Results

There were 48 Wistar strain rats used in this study. The lung histological examination showed that the reperfusion interval caused a dose-dependent decrease in the LIS. In group A, 15-min reperfusion interval caused a 54.55% LIS reduction. The reduction was less marked in group B (45.33%). The Kolmogorov–Smirnov test showed that the data of all variables were normally distributed (p < 0.05). The difference in group A was statistically significant (p < 0.001), and the post hoc Tukey test showed the difference was significant in all pair-wise group comparisons (p < 0.05). The same significant difference was also observed in group B (p < 0.001) and in all pair-wise group comparisons (p < 0.05). The results of the histologic examination of group A are depicted in Fig. 1, and the histologic result of group B is depicted in Fig. 2.

Fig. 1.

Histopathologic examination of the tibia with haematoxylin–eosin (HE) under ×400 magnification of group A (killed directly 1 h after fracturization and tourniquet application), group a A1 (no reperfusion interval), b A2 (5-min reperfusion), c A3 (10-min reperfusion), d A4 (15-min reperfusion)

Fig. 2.

Histopathologic examination of the tibia with haematoxylin–eosin (HE) under ×400 magnification of group B (killed 14 days after treatment), group a B1 (no reperfusion interval), b B2 (5-min reperfusion), c B3 (10-min reperfusion), d B4 (15-min reperfusion)

The MDA level in group A also showed a dose-dependent decrease with increasing reperfusion interval. Five-minute interval managed to reduce the MDA level by 16.56% and 30.13% in groups A and B, respectively. However, the reductions after this interval point were less marked. The 15-min interval reduced the MDA level by 27.60% in group A and by 37.44% in group B. The difference of the MDA level was significant in both groups A and B (p = 0.003; p < 0.001, respectively). All possible pair-wise comparisons between each group also showed a significant difference (p < 0.05). The details of the LIS of groups A and B are depicted in Table 2, and the details of the MDA of both groups are depicted in Table 3.

Table 2.

The results of lung injury score (LIS) from histopathologic examination

Parameter	Group	N	Mean ± SD	p valueƚ
LIS	A1	6	0.77 ± 0.02aǂ	< 0.001*
	A2	6	0.66 ± 0.03bǂ
	A3	6	0.49 ± 0.03cǂ
	A4	6	0.35 ± 0.02dǂ
LIS-14	B1	6	0.75 ± 0.03aǂ	< 0.001*
	B2	6	0.67 ± 0.02bǂ
	B3	6	0.55 ± 0.02cǂ
	B4	6	0.41 ± 0.03dǂ

Values in a row with different superscript are significantly different

*p < 0.05 was considered statistically significant

^ƚp value from one-way ANOVA test

^ǂPost hoc comparison using Tukey test

Table 3.

The results of MDA level measurement using thiobarbituric acid reactive substrate (TBARS) methods

Parameter	Group	N	Mean ± SD (nmol/mg lung tissue)	p valueƚ
MDA	A1	6	399.30 ± 52.58aǂ	0.003*
	A2	6	333.19 ± 39.09abǂ
	A3	6	305.22 ± 46.43bǂ
	A4	6	289.11 ± 48.00bǂ
MDA-14	B1	6	582.63 ± 36.82aǂ	< 0.001*
	B2	6	407.08 ± 46.15bǂ
	B3	6	401.52 ± 37.75bǂ
	B4	6	364.48 ± 55.52bǂ

Values in a row with different superscripts are significantly different

*p < 0.05 was considered statistically significant

^ƚp value from one-way ANOVA test

^ǂPost hoc comparison using Tukey test

The correlation between the reperfusion interval and LIS showed significant results both in groups A (p < 0.001, r = − 0.98) and B (p < 0.001, r = − 0.98). The correlations between the reperfusion interval and MDA level were also significant in both groups (p < 0.001, r = − 0.67; p < 0.001, r = − 0.79, for groups A and B, respectively).

Discussion

Remote organ injury due to ischemic–reperfusion injury of the limb is already demonstrated in previous studies. Mansour et al. found that the limb ischemia–reperfusion (LIR) injury causes impairment in the oxidative capacity of lung mitochondria in the murine model. On the other hand, the oxidative capacity of the liver is preserved; the renal tissue is even improved [20]. The other mechanism of how LIR induces lung injury is thought to be promoted through endoplasmic reticulum (ER)-mediated apoptosis [21].

Several pharmacologic agents have been proposed as a possible measure to reduce the severity of lung injury induced by LIR. These include N-acetylcysteine, methylene blue, tramadol, melatonin, curcumin, and others [22–26]. Another method of diminishing lung injury is mechanical: ischemic preconditioning and postconditioning. These terms refer to application to a brief application of an acceptable ischemic period to the organ before and after the definite ischemic period, respectively [27]. Ischemic preconditioning of the limb is proven to mitigate lung injury in the murine model by decreasing plasma IL-6 and improving oxygenation [28, 29]. On the other hand, ischemic postconditioning has also been proven to reduce the negative effects of LIR [30].

The reperfusion interval applied in this study is different from ischemic preconditioning in previous studies. Ischemic conditioning refers to a bout of intermittent ischemia and reperfusion periods before or after the tourniquet-induced ischemia and the following reperfusion [9, 27]. In this study, the authors used only a brief reperfusion period in the middle of two tourniquet-induced ischemic periods; this method aimed to reflect a possible clinical implementation intraoperatively.

Ischemic–reperfusion injury is caused by the formation of reactive oxygen species (ROS) and the systemic inflammatory response [31]. LIR increases the expression of inflammatory cytokines, including IL-1, IL-6, and TNF-α [32, 33]. The lung injury due to LIR is characterized by the neutrophil infiltrations and damage by ROS, and neutrophil is known as one of the leading players in ischemia–reperfusion injury [34–37]. Said inflammatory cytokines are also known to promote leukocyte infiltration into the lung [38]. The Lung Injury Score (LIS) used in this study evaluated the presence of neutrophils, hyaline membranes, proteinaceous debris, and alveolar septal thickening [19] The present study showed a dose-dependent decrease of LIS with a longer duration of reperfusion interval. Moreover, there was a significant inverse correlation between the length of the reperfusion interval and the LIS. This suggests reperfusion interval may attenuate the ischemic–reperfusion injury of the remote organ due to tourniquet use.

This effect was observed both in the direct measurement groups and in the post-14-day groups. The increase of the post-14-day LIS signified that the lung injury induced by LIR is an ongoing process and persists after that period. The interval reperfusion benefit on mitigating the lung injury also persisted until after 14 days. Another method, postconditioning, also reduces lung injury severity in the long term. However, Song et al. only observed the effect until 72 h after their intervention [30].

Malondialdehyde (MDA) is a product of lipid peroxidation which can be used as a mean to measure oxidative stress [39]. The previous study in the murine model showed that LIR would increase the MDA in lung tissue [40]. Higher MDA level is correlated with more severe lung injury and worse pulmonary function [41, 42]. The MDA level measured after 14 days in the control group also showed a higher value compared to the value in the direct measurement (582.63 nmol/mg lung tissue and 399.30 nmol/mg lung tissue, respectively). This result suggested that the lung injury process induced by LIR persisted in 14 days. There was also a reduction of the MDA level both in the direct measurement group and in the post-14-day-group. This may indicate that the reperfusion interval also decreased the oxidative stress occurring in the lung due to LIR. This result was in line with the LIS observed in both groups. The oxidative stress is proportionate to the ischemic duration, and this also applies to orthopaedic surgery [33, 43]. The positive effect of reperfusion interval in this study might be related to the interruption of ischemic duration.

Reperfusion interval by 5 or 15 min may increase surgery duration. However, the results of our study showed this brief duration may result in positive impact. Future study is needed to achieve a more detailed explanation regarding the impact of reperfusion interval on postoperative outcome.

In conclusion, reperfusion interval is a possible clinical approach to mitigate the remote organ damage induced by LIR. This is reflected by the dose-dependent decrease of the LIS and the MDA level of both intervention groups (direct and post-14-day measurement). However, this study still had some limitations. Future studies should include more variables regarding LIR-induced lung injury, such as the measurements of the inflammatory cytokines level and other parameters to measure oxidative stress such as superoxide dismutase (SOD). Another limitation is that there was a lack of surgical insult in the animal models in this study, unlike the ones observed in clinical trauma setting. Furthermore, ensuing studies should try to elaborate on the molecular mechanism of the protective effect of reperfusion interval. On the other hand, to the authors' knowledge, this was the first study to examine the effect of reperfusion interval to lung injury due to tourniquet-induced ischemic–reperfusion injury of the limb.

Funding

This study was solely funded by the authors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard statement

All institutional and national guidelines for the care and use of laboratory animals were followed. This study has been reviewed and approved by the authors' Institutional Review Board.

Informed consent

For this type of study informed consent is not required.