The main evidences identified in this study were: (1) Alb and Tf reached similar APR peaks in pneumoperitoneum when the IAP was higher and decreased within 7 and 14 days after the procedure; (2) Apo A1 presented decreasing values to all groups except in G4, which may have occurred due to the metabolic changes inherent to the convalescence period and the postoperative adaptive response; (3) Hp, Cp and α1-AGP levels showed similar patterns, however, Cp had the highest percentage change over time, behaving more characteristically as an APR within 7 and 14 days postprocedure; (4) α1-ATT decreased between days 7 to 14 after the laparoscopy procedure with the highest IAP (G4). These animals had respiratory impairment and the α1-ATT serum decrease possibly reflects its recruitment to lung recovery; (5) Alb, Cp and Hp were the most suitable proteins to characterize APR dynamics in this experimental model.
Negative Acute Phase Proteins
Serum albumin values (Table 1, Fig. 2) showed significant differences only at T0, where G4 was higher than G1 and G2. Yet, it is more important to compare other collection times within each group, since such differences show the behavior of Alb before (T0) and after (T1 to T4) the APR triggering. The same logic can be applied to other APPs, being T0 the control of each group.
In all mammalian species, the APR pattern of serum albumin usually shows a concentration decrease of 10–30% (Mackiewicz 1997; Petersen et al. 2004). In humans, early postoperative albumin drop appeared to reflect the magnitude of surgical trauma and was correlated with adverse clinical outcomes (Hübner et al. 2016).
This decrease is explained by the recruitment of amino acids to be used in the APR synthesis of positive APPs, making it necessary to alter the synthesis process of hepatic proteins (Tothova et al. 2014). Also, the catabolism of albumin occurs in many tissues, especially muscles, liver, and kidneys, where this protein enters cells by pinocytosis to be degraded by proteases. This process differs among species and is also related to the individual’s body size. In ruminants, the clearance half-time of albumin in the serum varies from 14–16 days (Evans 2002; Prinsen and de Sain-van der Velden 2004; Tothova et al. 2016).
Here, the comparison between the Alb serum values within each group over time demonstrated that all groups presented significant lower levels 24 hours after the procedure (Table 1, Fig. 2). This decrease persisted in G3 up to day 7 and in G4 up to day 14. The opposite was observed in G1 and G2, which, at the same periods, had an increase in values that approached their respective initiating values (T0). The percentage change of this biomarker in each group (Table 2, Fig. 3) showed a progressive decrease at T0-T1 – i.e, 17.2%, 14.8%, 20.6%, and 24.0%, respectively – indicating that G4 had the most expressive acute phase response triggering compared to other IAPs. Despite that, it is possible to consider that the APR at T0-T1 was similar in all groups and divergences occurred in the APR persistence. In the following 7 days post-laparoscopy (T0-T3), the percentage change in G3 was 12.7%, while G4 had an even longer APR (12.8%) that lasted up to 14 days (T0-T4).
Sabes et al. (2017), in their study with induced acute ruminal lactic acidosis (ARA) in sheep, had the highest albumin mean peak 20h after oral sucrose administration, coinciding with clinical signs of dehydration. The authors only observed a decrease close to the control rate 6 days after the procedure. Also, Georgieva et al. (2021) recently showed that the serum albumin concentration was lower in sheep with caesarian sections.
Transferrin results showed that G1 presented an increase at all collection times post-laparoscopy when compared to T0. In contrast, G4 had a progressive decrease post-laparoscopy and reached its maximum decrease at T4 (20.4%; Table 2, Fig. 3). Thus, similarly to albumin, Tf also had the longest APR in G4, lasting up to at least 14 days, while in G2 and G3 Tf persisted up to 7 to 14 days.
The antimicrobial activity of Tf might not be limited to simply decreasing free iron levels but is also capable of reducing the adhesion of gram-positive and negative bacteria (Ardehali et al. 2003; Gomme et al. 2005). Free iron can be toxic for promoting free radical formation through lipid peroxidation of low-density lipoproteins (LDL), causing oxidative damage to tissues (Campenhout et al. 2003). When Tf acts minimizing an iron disorder, the progression of a possible cardiovascular disease is also limited (Gomme et al. 2005). Data of a previous study yet to be published (de Gouvêa et al. 2023), demonstrated that the group under IAP of 15 mmHg had fibrinogen and malondialdehyde levels that suggest the persistence of an inflammatory response for 7 and 14 days, respectively. This is compatible with the APR results presented here for the same group (G4).
Results regarding our Apolipoprotein A1 values showed variation between groups, but G1, G2, and G3 behaved similarly. In these latter groups, the Apo A1 serum levels at T4 varied less than 10% in respect to T0, while G4 persisted with a 56.0% and 94.3% increase at T3 and T4, respectively, when compared to T0 (Table 2). In sheep, periods that cause a metabolic shift may be identified by the increase in Apo A-1 serum concentrations, suggesting that this biomarker could be used to monitor adaptive responses and metabolic changes in this species (Chiaradia et al. 2012). Therefore, the convalescence period and the postoperative adaptive response were likely longer in the group with the highest IAP (G4), persisting for 7 and 14 days after surgery.
Positive Acute Phase Proteins
In G1 and G4, haptoglobin serum concentration (Table 1, Fig. 2) was higher at T1 when compared to T0. Yet, the between-groups comparison at T1 shows that G1 had the highest value despite its non-significant percentage change (Table 2, Fig. 3). In sheep, Hp is considered as an outstanding APP, with a value almost negligible in healthy individuals but increasing in about 80% during an inflammatory process (Pfeffer and Rogers 1989; Ceciliani et al. 2012). Our values were higher than the baseline value at all collection times (Table 1) and surpassed 80% at T0-T1 in G1, and at T0-T3 and T0-T4 in G4. Also, considering that an increase beyond 25% is considered an APR, at T3, G3 and G4, presented this increase. Even though, Hp serum concentration can be influenced by other factors other than the acute phase response, such as free hemoglobin (Petersen et al. 2004). Hematological parameters were also evaluated and showed no alterations in all groups at all collection times (data to be published) (de Gouvêa et al. 2023).
As Murata et al. (2004) compiled in their research on APPs as diagnostic tools in veterinary medicine, Hp has a variety of immunomodulatory effects. Immunomodulation is partially mediated by Hp binding to the CD11/CD18 receptor of effector cells, having an inhibitory effect on granulocyte chemotaxis, phagocytosis, and bactericidal activity. This protein may also inhibit mast cell proliferation, prevent spontaneous maturation of epidermal Langerhans cells and suppress T cell proliferation (Murata et al. 2004). Such effects can be influenced by several physiological and pathological alterations, which can explain how Hp serum levels vary according to the type of injury – here represented by different degrees of peritoneal distension.
Miglio et al. (2018) showed that, clinically healthy Lacaune sheep aged from 2 to 6 years had mean Hp levels of 0.29 mg/mL. Concentrations above 1 mg/mL are considered the approximate cut-off value for severe inflammation (Wells et al. 2013; Miglio et al. 2018). The G4 Hp value at T3 (1.04 mg/mL) was sustained at T4 (0.97 mg/mL), showing a percentage change increase of 118% and 56.7%, respectively, when compared to T0 (Table 2, Fig. 3). Yet, G1 reached 1.3 mg/mL at T3, but increased to its highest value at T4 (2.3 mg/mL). This last increase in G1 may be due to individual variation within the group or other triggering effects. Also, the fact that G4 initiated with 0.56 mg/mL, – the lowest value among groups at T0 – that consistently increased at T3 and T4, shows a progressive APR with its peak at 7 days post-surgery.
Additionally, in a study on pulmonary damage in sheep (Pfeffer and Rogers 1989), peak serum haptoglobin concentrations were significantly higher in animals with bronchial obstruction (2.4mg/mL on day 5) than in those with sham bronchial obstruction (0.53 mg/mL on day 3). However, in sheep with experimental-induced Caseous Lymphadenitis, Hp levels raised significantly seven days post-infection (1.65 ± 0.21 g/L) and remained significantly high 15 days later when compared to the serum levels from the unchallenged sheep (Eckersall et al. 2007). Sabes et al. (2017) also demonstrated increased Hp serum levels 8h after ARA induction, which remained altered until the second week, when values began to return to those of the control moment. These latter data are compatible with the Hp percentage change (Table 2, Fig. 3) observed in G4 at 7 and 14 days postprocedure.
Furthermore, despite the group or collection time, our Hp serum concentrations were higher than most values reported by Iliev and Georgieva (2018). This effect may be explained by a possible overlapping inflammatory response due to the short period between trials (15 days). However, it was possible to assess Hp alterations and this protein showed to be useful to compare the APR between different IAP.
The ceruloplasmin serum concentrations in our study (Table 1, Fig. 2) had at T0, the highest values in G1 and G2, which were also the only groups with a decrease at T1. Additionally, G1 also presented a significant decrease at T4, while G2 increased progressively until T4. The G3 values were sustained over time, while G4 had a significant progressive increase from T3 onward. In a study with induced pulmonary damage, Cp increased up to 140% between 3 and 5 days after the procedure (Pfeffer and Rogers 1989). More recently, Georgieva et al. (2021) showed that the Cp plasma concentrations in sheep submitted to cesarean section, levels markedly increased 48 hours after the surgery, reaching its peak at day 10. These findings are compatible with the ones presented here, where the Cp levels of all groups increased significantly within seven days. On day 14, G4 had the highest value, while G1 (0 mmHg of IAP) presented the lowest value. The percentage change among these groups also shows that only G1 had a pronounced decrease (74.7% at T4). The remaining groups showed an increase, with G4 having the highest values (344% at T3, and 282% at T4).
Although our Cp levels were above normal range in all collection times, also reflecting overlapping APRs, these findings show the importance of this protein as a biomarker that had a meaningful increase in the group that received the highest IAP. This result is in accordance with the outcomes of the increased pulmonary dead space effect and circulatory impairment caused by pneumoperitoneum and mechanical ventilation (Perrin and Fletcher 2004; Atkinson et al. 2017; do Nascimento et al. 2022).
Regarding the α1 acid glycoprotein, in a study involving acute and chronic inflammatory reactions in cattle, Alsemgeest et al. (1995) showed that increased levels of this APP occurred more frequently during chronic inflammatory states. The α1-AGP is considered as a protective biomarker against ischemia-reperfusion in kidneys (Daemen et al. 2000; Janciauskiene et al. 2011). In an experimental model of ovine caseous lymphadenitis, Eckersall et al. (2007) showed that the mean serum concentration of α1-AGP had a significant increase by day seven post-infection, and continued to gradually increase until day 13, when it reached a mean concentration of 0.38 ± 0.05 g/L (3800 ± 5 mg/dL).
Both our Hp and α1-AGP values in G4 showed the higher percentage change at T3 and T4, but only α1-AGP presented a significant concentration value and percentage change at T3. These two APPs had high percentage changes, greater than 30% for all groups, but were only sustained for 7 and 14 days postprocedure in G4. Although the group that received the lowest IAP (G1) had the highest α1-AGP values, this response was likely due to individual variation and the many functions of α1-AGP when binding to drugs, inflammatory mediators, and bacterial derived molecules (Iliev and Georgieva 2018).
The serine protease inhibitor α1 antitrypsin protects tissues from neutrophil proteolytic enzymes (mainly elastase and proteinase) at the site of inflammation (Murata et al. 2004; Tothova et al. 2014; Iliev and Georgieva 2018). This protease is well known to play an important role in lung homeostasis (Chakraborti et al. 2017) and had been used as a pretreatment (120 mg/kg) to attenuate acute lung injury induced with endotoxin in rabbits (Jie et al. 2003). In mice with LPS-induced lung injury, α1-ATT was also shown to attenuate the deterioration of oxygenation and other parameters, to reduce compliance, to inhibit LPS-induced pro-inflammatory genes and enhanced the expression of genes associated with tissue repair and regeneration (Janciauskiene et al. 2011). The decrease of this protein in G4 only occurred from 7 to 14 days after the procedure, when respiratory impairment was observed (do Nascimento et al. 2022), could be explained by α1-ATT consumption aimed at lung recovery.
The 15-day period between procedures showed an overlapping inflammatory effect, detected in the values of all biomarkers at T0. However, despite this study limitation, our aim was not to investigate whether measurements would return to baseline levels, but rather to understand the behavior of the chosen APPs in a normal postoperative period for clinical discharge. Therefore, we were able to measure the post-laparoscopy alterations in all groups.
The post-laparoscopy acute phase response was observed from 7- to 14-days after the procedure especially in the group with the highest IAP (15 mmHg; G4). This effect was interpreted as a consequence to pneumoperitoneum and mechanical ventilation, since no other surgical procedure was performed, and all individuals were healthy. Therefore, postoperative care longer than this period is recommended, especially when other invasive procedures are performed concomitantly. Also, albumin, ceruloplasmin and haptoglobin were the most useful APPs to characterize the dynamics of the APR in this experimental model.
Hence, the results described here are unprecedented to the authors' knowledge, may be used in other translational studies, and will help improve post-laparoscopic techniques and care in sheep.