Introduction

Lung transplantation has been established as a surgical treatment for a number of terminal lung diseases [1]. Despite significant progress in surgical techniques and immunosuppressive therapies over the last decades, long-term survival of the recipients has not improved similarly, especially when compared to other solid organ transplantations [2]. Lung allograft rejection in humans is still a major issue in transplantation medicine. Although the overall rate of allograft rejection is declining, there is still a high rate of 50% of rejection, which remains clinically undetected but histological evident [3].

The pathogenesis of acute rejection (AR) is a response of the adaptive immune system of the recipient and involves key types of cells such as T-lymphocytes, macrophages and B cells, which infiltrate into the newly transplanted organ and damage it [4]. The endothelial transmigration of these cells is at least in part the result of an increased permeability of the pulmonary endothelium due to ischemia/reperfusion (I/R) injury occurring during transplantation [5].

The amide-linked local anesthetic ropivacaine has been shown to protect and preserve the endothelium in vitro by an attenuation of inflammatory processes, such as the propagation of tumor necrosis factor α (TNF-α)-related signaling events [6]. Subsequently, ropivacaine has also been demonstrated to attenuate experimental acute lung injury as triggered by bacterial lipopolysaccharide with or without high-tidal ventilation in vivo [7, 8].

We therefore hypothesized that ropivacaine might also be able to attenuate AR in a mouse model of allogeneic left lung transplantation due to its ability to preserve endothelial barrier function.

Materials and Methods

Animals

The study was approved by the local animal care committee (License No. ZH103/2016). Specific pathogen-free male in-bred mice C57BL/6 (H2b) and BALB/c (H2d; Charles River Laboratories, Sulzfeld, Germany) received adequate care according to The Principles of Laboratory Animal Care (National Institutes of Health Publication No. 85-23, promulgated in 1985, most recently revised in 1996). Ten- to 14-week-old animals weighing 24–30 g were used. In this allogeneic model of left mouse lung transplantation, BALB/c mice served as organ donors, whereas C57BL/6 mice were used as transplant recipients.

Surgical Technique

Orthotopic, single left lung transplantation between C57BL/6 and BALB/c mice was performed as previously described in detail [9, 10]. Induction of anesthesia was initiated and maintained with isoflurane. In brief, left lungs from BALB/c mice were prepared for transplantation and equipped with tubes for each structure (pulmonary artery 26 gauge, bronchus 20 gauge, pulmonary vein 22 gauge tube). Anesthetized recipients received the donor graft via a left thoracotomy in the 4th intercostal space. The anastomosis was completed by insertion of the preformed tube-equipped donor organ structures into the respective recipient artery, bronchus, and vein. The lung was then re-perfused and re-ventilated.

Intervention

In order to investigate the impact of ropivacaine, donor lungs were flushed prior to surgical removal with 3 ml of cooled (4 °C) normal saline with or without ropivacaine (Ropivacain-HCL Sintetica 2 mg/ml, Sintetica, Mendrisio, Switzerland) at a final concentration of 1 µM via transverse incision at the root of the pulmonary artery trunk with a pressure of 20 cmH2O. Cold ischemia time was 1 h during which the fluid remained in the donor organs.

Functional Analysis of Lung Transplants on Postoperative Day Five

An arterial blood sample was taken from the aorta for blood gas analysis (Epocal Inc, Ottawa, ON, Canada). The oxygenation index was calculated as the ratio of the arterial partial pressure of oxygen over the inspiratory oxygen fraction (PaO2/FiO2). All animals were ventilated with an FiO2 of 1.0. Mice were ventilated at a respiratory rate of 150 per minute, a peak airway pressure of 15 cmH2O and a positive end-expiratory pressure (PEEP) of 2 cmH2O for two minutes (Vent Elite small animal ventilator, Harvard Apparatus, Holliston, MA, USA). The right hilum structure was exposed and ligated. Peak airway pressure in the left lung was increased gradually up to 25 cmH2O for 2 min and lung compliance (ml/cmH2O) was measured.

Histology and Pathologic Grading

Parts of the transplanted lungs, which had been fixed in formalin, were subsequently embedded in paraffin. Sections of 4 µm thickness were cut and stained with hematoxylin and eosin (H&E). These sections were microscopically graded for rejection pathology (at 100 × magnification, Leica DM6000 B microscope, Leica Microsystems, Heerbrugg, Switzerland) using the standard criteria guidelines proposed by the International Society for Heart and Lung Transplantation (Grades 1–4) [11] by three different investigators blinded for the corresponding treatment group.

Immunohistochemistry (IHC)

IHC staining was performed with BondMax with Refine HRP-Kit DS9800 (Leica Biosystems, Muttenz, Switzerland) in accordance with the manufacturer’s guidelines. Primary antibodies were directed against CD3 (RMAB005; Diagnostic Biosystems, Pleasanton, CA, USA), CD4 (4SM95; eBioscience, San Diego, CA, USA), F4/80 (T-1006; BMA Biomedicals, Augst, Switzerland), and rat CD45 (B220) (RA3-6B2; BD Pharmingen, Allschwil, Switzerland). The number of positive cells in the perivascular and peribronchiolar areas was assessed. Vessels or airways with a diameter of 100 µm or more were selected for counting (five sites on each slide). Immunostained sections were assessed by three different investigators in a blinded fashion, who counted the cells in the corresponding sections manually.

Statistical Analysis

Normal distribution was assessed using a Shapiro–Wilk test. Normally distributed data were analyzed using student’s t test (TNF-α, compliance, oxygenation index) or one-way ANOVA with Bonferroni post hoc testing (Western blot densitometry) and are reported as mean ± standard deviation (SD). Not normally distributed data were compared using a Mann–Whitney U test (interleukin − 6 and − 10, AR score, cell count of CD3 and CD4 positive cells) and are reported as median with corresponding minimum and maximum. Categorical data of B220 and F4/80 positive cells were analyzed with Fisher’s exact test. All analyses were performed using GraphPad Prism for Mac, version 7.0d (GraphPad Software, La Jolla, CA, USA). A p value < 0.05 was considered to be statistically significant.

Results

Ropivacaine Reduces Plasma Levels of TNF-α and Increases Plasma Levels of IL-6 and IL-10 3 h After Transplantation

Three hours after transplantation, mean plasma concentrations of TNF-α were 28% lower in the ropivacaine group than in the control group (84.5 ± 26.9 pg/ml for control vs. 116 ± 18,7 pg/ml for ropivacaine, p = 0.01). Additionally, plasma levels of IL-6 and IL-10 in the ropivacaine group were significantly higher (IL-6: median 46.0 (minimum–maximum 38.1–165.8) pg/ml for control vs. 162.7 (52.6–343) pg/ml for ropivacaine, p = 0.008; IL-10: 2.66 (1.84–3.56) pg/ml for control vs. 6.01 (3.56–15.94) for ropivacaine, p < 0.01, see all Fig. 1).

Fig. 1
figure 1

Plasma concentrations of tumor necrosis factor α, interleukin-6 and interleukin-10 three hours after transplantation. Plasma concentrations of a tumor necrosis factor α (TNF-α), b interleukin-6 and c interleukin-10 in mice 3 h after allogeneic left lung transplantation from BALB/c (donor) to C57BL/6 (recipient) mice after flushing the lungs with either normal saline (control, white bars or boxplots) or ropivacaine (final concentration 1 µM, grey bars or boxplots) prior to transplantation. Data are presented as mean with standard deviation (a) or as median with whiskers indicating minimum and maximum (b, c). N = 9 for both groups. *p < 0.05 compared to control

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Ropivacaine Pre-treatment Attenuates AR Histologically

Allogeneic, orthotopic left-lung transplantation was performed using a major histocompatibility complex class I and II fully mismatch combination strain between BALB/c (donor) and C57BL/6 (recipient). After harvesting the transplanted lungs on postoperative day 5, the macroscopic appearance in the ropivacaine group appeared to show less hemorrhage and improved inflation in the ropivacaine group compared to the control group (representative pictures shown in Fig. 2a). Histology of control lung transplants showed severe AR pathology characterized by dense perivascular and peri-bronchiolar mononuclear infiltration in the interstitium and the ventilated areas (Fig. 2bi). In contrast, the ropivacaine group showed less perivascular mononuclear infiltration into the lung parenchyma. (Fig. 2bii). Quantification of these microscopic findings using the AR score proposed by the International Society for Heart and Lung Transplantation [11], revealed a significantly less severe rejection in the animals treated with ropivacaine (median 3 [minimum–maximum 2–4] for control vs. 2 [1–2] for ropivacaine, p < 0.001, Fig. 2biii).

Fig. 2
figure 2

Transplanted mouse lung allografts and microscopic AR on postoperative day five. a Representative macroscopic posterior views of lungs harvested on postoperative day five after allogeneic left lung transplantation from BALB/c (donor) to C57BL/6 (recipient) mice after flushing the lungs with either normal saline (i control) or ropivacaine (ii final concentration 1 µM) prior to transplantation. The transplanted lung is marked with an asterisk (*). b Representative microscopic image (magnification × 100) of left mouse lung slides stained with hematoxylin/eosin five days after allogeneic left lung transplantation from BALB/c (donor) to C57BL/6 (recipient) mice after flushing the lungs with either normal saline (i control) or ropivacaine (ii final concentration 1 µM) prior to transplantation. iii Quantification of AR using the AR score proposed by the International Society for Heart and Lung Transplantation (Grades 1–4, N = 9 for both groups) [11]. Data are presented as median with whiskers indicating minimum and maximum. *p < 0.05 compared to control

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Ropivacaine Reduces Graft Infiltration of T-Lymphocytes

Immunohistochemical staining revealed a significant decrease in the number of CD3+ (Fig. 3a) and CD4 + positive (Fig. 3b) T-lymphocytes after treating the donor lungs with ropivacaine (p = 0.02 for CD3+, p = 0.02 for CD4+). No significant differences were detected regarding B220 + positive cells (CD45, B cell marker, p = 0.56, Fig. 3c) or F4/80 + positive cells (murine macrophage marker, p = 1, Fig. 3d).

Fig. 3
figure 3

Immunohistochemical analysis of different subtypes of leukocytes in lung allografts on postoperative day five. a Representative immunohistochemistry sections of CD3-positive cells (brown, arrows indicating target cells) in lungs from control (i) or ropivacaine (ii) animals. (iii) Quantification of CD3-positive cells in sections from the control (white squares) or ropivacaine group (grey squares). Cell count was binned into categories as follows: 1 = 0–49 cells, 2 = 50–99 cells, 3 = 100–149, 4 = 150–199, 5 = 200–249 cells, 6 = 250–299, 7 = 300–349 cells. N = 7 for both groups. *p < 0.05 compared to control. b Representative immunohistochemistry sections of CD4-positive cells (brown, arrows indicating target cells) in lungs from control (i) or ropivacaine (ii) animals. (iii) Quantification of CD3-positive cells in sections from the control (white squares) or ropivacaine group (grey squares). Cell count was binned into categories as follows: 1 = 0–49 cells, 2 = 50–99 cells, 3 = 100–149, N = 7 for both groups. *p < 0.05 compared to control. c Representative immunohistochemistry sections of B220 (CD45, B-lymphocyte marker) positive cells (brown, arrows indicating target cells) in lungs from control (i) or ropivacaine (ii) animals. (iii) Quantification of B220-positive cells in sections from the control (white squares) or ropivacaine group (grey squares). Cell count was classified as category 1 with 5–9 cells present and as category 2 when 10–19 cells were present. N = 7 for both groups. d Representative immunohistochemistry sections of F4/80 (murine macrophage marker) positive cells (brown, arrows indicating target cells) in lungs from control (i) or ropivacaine (ii) animals. (iii) Quantification of F4/80-positive cells in sections from the control (white squares) or ropivacaine group (grey squares). Cell count was classified as category 1 with 5–9 cells present and as category 2 when 10–19 cells were present. N = 7 for both groups

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Ropivacaine Does Not Significantly Improve Lung Function

There was a tendency toward a better oxygenation index in the ropivacaine group (246 ± 117 mmHg for control vs. 303 ± 164 mmHg for ropivacaine, Fig. 4a). However, this difference did not reach statistical significance (p = 0.43). Similar results could be obtained for the compliance of the left lung, again with no significant differences (0.012 ± 0.001 ml/cmH2O for control vs. 0.013 ± 0.003 ml/cmH2O for ropivacaine, p = 0.2, Fig. 4b).

Fig. 4
figure 4

Functional data of mouse lung allografts on postoperative day five. Oxygenation index (a) and compliance (b) of left lungs on day five after allogeneic left lung transplantation from BALB/c (donor) to C57BL/6 (recipient) mice after flushing the lungs with either normal saline (control, white bars) or ropivacaine (final concentration 1 µM, grey bars) prior to transplantation. Data are presented as mean with standard deviation. N = 9 for both groups

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Ropivacaine Decreases the Phosphorylation of Caveolin-1 in Transplanted Lungs

Representative Western blots of lung homogenates probing for phosphorylation and expression of Src and caveolin-1 are shown in Fig. 5. Densitometry analysis revealed no significant impact on Src phosphorylation in both treatment groups (p = 1, Fig. 5a). However, a significant reduction in caveolin-1 phosphorylation by ropivacaine compared to control was observed (p = 0.004, Fig. 5b).

Fig. 5
figure 5

Src tyrosine protein kinase and caveolin-1 in mouse lung allografts on postoperative day five. a (i) Representative Western blots of Src tyrosine kinase, phosphorylated at tyrosine 419 (pY419 Src) or the total amount of the enzyme (total Src) in lungs 5 days after allogeneic left lung transplantation in mice after flushing the lungs with either normal saline (control) or ropivacaine prior to transplantation. Baseline values were obtained from animals without any treatment. (ii) Quantitative analysis of densitometry of Western blots showing the ratio of analysis pY419 Src over total Src in lungs treated as explained above (baseline: striped bars, control: white bars, ropivacaine: grey bars). Data are presented as mean with standard deviation. N = 4 for all groups. b (i) Representative Western blots of caveolin-1, phosphorylated at tyrosine 14 (pY14 Caveolin-1) or the total amount of the protein (total Caveolin-1) in lungs 5 days after allogeneic left lung transplantation in mice after flushing the lungs with either normal saline (control) or ropivacaine prior to transplantation. Baseline values were obtained from animals without any treatment. (ii) Quantitative analysis of densitometry of Western blots showing the ratio of analysis pY14 Caveolin-1 over total Caveolin-1 in lungs treated as explained above (baseline: striped bars, control: white bars, ropivacaine: grey bars). Data are presented as mean with standard deviation. N = 9 for control and ropivacaine, N = 4 for baseline. #p < 0.05 vs. baseline, *p < 0.05 versus control. c (i) Representative Western blots of Src tyrosine kinase (Src), caveolin-1 and GAPDH in BALB/c lungs 5 days after allogeneic left lung transplantation from BALB/c (donor) to C57BL/6 (recipient) after flushing the lungs with either normal saline (control) or ropivacaine prior to transplantation. Baseline values were obtained from animals without any treatment. Quantitative analysis of densitometry of Western blots showing the ratio of analysis Src over GAPDH (ii) or caveolin-1 over GAPDH (iii) in BALB/c lungs treated as explained above (baseline: striped bars, control: white bars, ropivacaine: grey bars). N = 9 for control and ropivacaine, N = 4 for baseline. Data are presented as mean with standard deviation. #p < 0.05 versus baseline

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Additionally, expression of both proteins compared to baseline values obtained from untreated (non-transplanted) lungs were reduced as well (Fig. 5c), but the comparison of both treatment groups (control vs. ropivacaine) with each other did not show any statistically significant differences (p = 1 for both comparisons).

Discussion

The results of this study demonstrate beneficial effects of the amide-linked local anesthetic ropivacaine on AR 5 days after allogeneic lung transplantation in mice. Amide-linked local anesthetics such as ropivacaine and lidocaine have been demonstrated to possess profound anti-inflammatory properties [12,13,14]. As inflammatory signaling processes, leukocyte infiltration and subsequent damage to the transplanted organ are hallmarks of AR, we hypothesized that the drug would also be able to attenuate AR due to its anti-inflammatory effects, e.g., by preserving endothelial barrier function [6].

Naidu and colleagues previously demonstrated that TNF-α release from pulmonary macrophages 4 h after reperfusion might be critical for the development of lung I/R injury [15]. Therefore, the reduced plasma levels of TNF-α found in the ropivacaine animals 3 h after transplantation in the current study support the hypothesis that ropivacaine might be able to blunt I/R injury occurring during the process of transplantation and subsequently preserve endothelial barrier function [4, 16, 17].

IL-6 is considered to be a primarily pro-inflammatory and IL-10 an anti-inflammatory cytokine [18]. However, IL-6 is also known to be able to promote pro- and anti-inflammatory responses depending on the type of injury and the organ in which it is examined, e.g., by directly increasing IL-10 levels [19]. It is also known that lidocaine, another common amide-linked local anesthetic, might be able to decrease the release of pulmonary TNF-α and IL-1β after an inflammatory stimulus, while at the same time leaving the secretion of IL-6 unaffected [20]. The increase in IL-10 after treatment with ropivacaine might therefore again serve as a surrogate marker for the anti-inflammatory properties of the drug. Additionally, the results of the current study are at least able to show that the raised IL-6 levels did not seem to have a negative impact on the outcome regarding AR of the ropivacaine-treated transplants.

The number of CD3 + and CD4 + positive T-lymphocytes was significantly reduced in the lung allografts pre-treated with ropivacaine. It is well known that the immune response during AR and graft failure is at least in part due to T-lymphocyte activation [4, 21, 22]. Therefore, the reduced number of CD3 + and CD4 + positive cells might not only be able to serve as a surrogate measure for the attenuated AR in the ropivacaine group but might also be part of the explanation of this observed effect.

There was a trend toward a better functionality of the transplanted organ after treating the graft with ropivacaine as assessed by the oxygenation index and the compliance of the transplanted lung. Unfortunately, these results did not reach statistical significance. This is in accordance with previously reported findings that an attenuation of AR does not necessarily have an impact on the functionality of the transplanted organ on postoperative day 5 in this particular model [16]. However, the occurrence of AR has been linked to the development of chronic rejection, which is known to be one of the most important life-limiting factors after lung transplantation [3, 23, 24]. Thus, a significant attenuation of AR as shown in the current study might have a strong impact on the patients’ overall outcome and survival.

Previous data suggested a potential mechanism involving Src tyrosine protein kinase and its primary target caveolin-1, by which ropivacaine might exert its anti-inflammatory properties in lung endothelial cells, thus preserving endothelial barrier function [6, 7, 25]. These signaling pathways might also be crucial during the pathogenesis of AR and primary graft dysfunction after lung transplantation due to their participation in I/R injury [26]. Caveolin-1 is also known to be involved in several inflammatory processes in the lung [27,28,29,30] and serves as a binding protein for CD26, a glycoprotein expressed on the surface of T-lymphocytes [31]. In case CD26 is blocked or absent, allograft rejection might be attenuated [32]. In the current study, we observed a significant reduction in caveolin-1 phosphorylation in the ropivacaine group, which might be a first hint for a potential mechanism by which the local anesthetic exert its anti-inflammatory effects in the current experimental setting, being in accordance with previous findings [6, 7]. The fact that there was no difference regarding Src phosphorylation and expression patterns might at least in part be explainable by the late time point for the evaluation on postoperative day five. However, we cannot exclude the possibility that the observed beneficial effects of the drug might not only be related to its impact on inflammatory signaling events and that additional pathways, e.g., the well-known anti-apoptotic properties of local anesthetics [33], might be involved as well.

The mouse model of lung transplantation provides the best and the only method to study AR in a physiological condition, namely a perfused and ventilated graft, that is superior to all other previous models proposed in research. To our knowledge, this is the first study evaluating an effect of preconditioning of donor organs with local anesthetics. In transplant medicine, little is known about the impact of the type of anesthetic applied to the donor on outcome after organ transplantation [34]. The encouraging results of the current study might therefore have a significant impact on the human transplant situation, which might include an improved transplant acceptance.

In conclusion, the current study shows that pre-treatment of donor lungs with the local anesthetic ropivacaine bears the potential to diminish AR after lung transplantation in mice by attenuating influx and damage by infiltrating leukocytes. A potential mechanism could be the reduction in caveolin-1 phosphorylation. The addition of local anesthetics to the organ preservation solution might therefore be a simple intervention with a potentially large impact on organ acceptance and therefore also on survival after lung transplantation.