Caffeic acid phenethyl ester (CAPE) protects against acute urogenital injury following pneumoperitoneum in the rat

Abstract

Purpose: Our aim is to determine the biochemical and histologic changes induced in the kidneys, testis and prostate on possible ischemia and reperfusion (I/R) injury caused by pneumoperitoneum (PNP) in a rat model and to evaluate the ability of protective effects of caffeic acid phenethyl ester (CAPE). Methods: Twenty-eight adult male Wistar albino rats were randomly divided to one of three treatment groups, with seven animals in each group. Sham, laparoscopy (L), and laparoscopy plus CAPE (L + C) group were subjected to 60 min of PNP with 15 mmHg one hour before the desufflation period. Total oxidant status (TOS) and total antioxidant status (TAS) levels were determined in kidney, testis, and prostate. Kidney and testis tissues were removed to obtain a histologic score. Also, Johnsen scoring system was used for testicular tissue analysis. Results: L group had significantly higher TOS and lower TAS levels on kidney and testis compared to the other groups. In prostate biochemical analysis, there was not any difference between groups. No difference was found between groups according to kidney and testis tissues’ histologic evaluation. In evaluation of Johnsen scoring, L group showed significant lower score compared to the other two groups. Conclusions: Increased intraabdominal pressure (IAP) had an oxidative effect on kidney and testis but not on prostate in rats. Moreover, it could affect the testicular Johnsen score. All these adverse effects of IAP on both kidney and testis could be prevented by CAPE administration.

• Caffeic acid phenethyl ester
• kidney
• prostate
• pneumoperitoneum
• rat
• testis

Introduction

Laparoscopy has gradually become the gold standard for the diagnosis and effective treatment of various diseases in both adults and children. At the same time, clinical investigation of intraabdominal pressure (IAP) became of interest since it was first described in the late 19th century.1 Several studies have particularly focused on the effects of IAP on intraabdominal blood flow to hollow and solid organs. Most of the studies show that blood flow to intraabdominal organs is reduced by 10% to 80% during laparoscopy but normalizes after desufflation of the abdominal cavity.1,2 In terms of the hypoperfusion and subsequent reperfusion periods, both clinical and experimental studies reveal that the laparoscopic procedure provides a typical model of ischemia-reperfusion (I/R) injury in the organs.1,3

The adverse outcomes of IAP on the kidneys’ wall have been relatively well described.1 Limited studies revealed that IAP could affect germ cell apoptosis and cause testicular hypoperfusion, free radical production, and subsequent testicular damage during laparoscopic procedures in animal models.3,4 In addition, acute I/R injury had an oxidative effect on prostate tissue.5 However, the effect of increased IAP on the prostate has not been studied until now.

The caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antioxidant, immunomodulatory, anti-inflammatory and anticarcinogenic properties, and has been used as a folk medicine for many years. In literature, CAPE is used for protecting kidneys, testis, and prostate against several toxicities.6 The physiological and pharmacological properties of CAPE were investigated both in vitro and in vivo assays. At a concentration of 10 µM, it completely blocks production of free radicals in human neutrophils and in the xanthine/xanthine oxidase system. The structure–activity relationship has been identified by comparing the potency of free radical scavenging.7 The antioxidant activity of CAPE is to limit the anoxia-reoxygenation-induced mitochondria damage in vitro.8 However, the physiological functions and the underlying protective mechanism of CAPE against increased IAP tissue injury remain totally unclear.

In this study, we aimed to determine the histologic and biochemical changes induced in the kidneys, testis, and prostate on possible tissue injury caused by increased IAP followed by abdominal deflation in a rat model and to evaluate the ability of protective effects of CAPE.

Materials and methods

Animals

This experimental study was conducted with 28 adult male Wistar albino rats of the same age, from the same colony weighing between 270 and 320 g, after approval of the Mustafa Kemal University, Faculty of Medicine, Animal Care and Ethics Committee was obtained before the onset of experiments. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. They were housed separately under specific pathogen-free conditions to avoid infections in macrolon cages, with a 12:12-hour light–dark cycle at 22 ± 0.5 °C room temperature and provided standard laboratory rat chow and tap water ad libitum.

The rats were randomly divided to one of three treatment groups, with seven animals in each group. The control group was subjected to a sham operation without pneumoperitoneum (PNP). The laparoscopy (L) group was subjected to 60 min of PNP with 15 mmHg IAP. The laparoscopy plus CAPE (L + C) group was subjected to 60 min of PNP with 15 mmHg and administered a single intraperitoneal injection of CAPE 10 μmol/kg one hour before the desufflation period.

Surgical procedure

The rats were anesthetized by intraperitoneal administration of ketamine hydrochloride (Ketalar, Pfizer, Turkey) 50 mg/kg and xylazine hydrochloride (Rompun, Bayer, Turkey) 3 mg/kg and then placed at a supine position on the operating table. During the procedure, additional doses were administered if necessary. The experiments were performed in a position allowing spontaneous breathing under sterile conditions. The body temperature was maintained at 37.0 °C with a heat pad to prevent the effects of hypothermia and to maintain the stability of hemodynamic parameters. After proper preparation of the surgical field, an 18 G Abbocath catheter was placed supraumbilically into the abdominal cavity and connected to an insufflator tube.9 PNP was automatically established up to a 15 mmHg pressure for 60 min via the insufflation of CO₂ by an abdominal CO₂ insufflator (Karl Storz GmbH, Tutlingen, Germany), and followed by immediate desufflation.

Tested drugs

CAPE (Sigma, St. Louis, MO) was administered intraperitoneally to rats in doses of 10 μmol/kg, prior to use, CAPE was dissolves with 10 mL of 70% ethanol.7

Biochemical analysis

Immediately following desufflation, first kidneys, testicles, and prostate were immediately removed, respectively. The excised left kidney, left testis, and prostate tissue were weighed, immediately stored at −80 °C. For biochemical analysis, tissues perfused with 1.15% ice-cold KCl, minced, then homogenized in five volumes (w/v) of the same solution. Assays were performed on the supernatant of the homogenate that was prepared at 14,000 rpm for 30 min at +4 °C. The protein concentration of the tissue was measured by the method of Lowry.10

The levels of the total oxidant status (TOS) and total antioxidant status (TAS) were determined in the supernatants. TAS was determined using a novel automated colorimetric measurement method developed by Erel.11 The TAS results were expressed as mmol Trolox equivalent/g protein for kidney, prostate, and testis tissue.

TOS were measured by Erel’s methods.12 The TOS results were expressed as nmol H₂O₂ equivalent/mg protein for all tissues.

Histological evaluation

For light microscopic examination, right kidney and right testis tissues of each rat were removed and tissue pieces were fixed in 10% neutral buffered formalin solution, embedded in paraffin, sectioned at 5 µm, and then stained with hematoxylin and eosin (H&E). For statistical analysis, seven sections of kidney tissue and seven sections of testis tissue from each group were prepared. Totally, 48 sections of kidney and testis tissues were examined and photographed with DP 20 camera attached Olympus CX-41 photomicroscope (Olympus Corporation, Tokyo, Japan) for histopathological changes. The prostate tissues were not adequate for histological examination; therefore, only biochemical assessment was done.

To obtain a histologic score of kidney tissues, histopathological changes were graded according to cortical involvement and the severity of the lesions as shown in Table 1.13

Table 1. Histologic kidney grading scale.

0 point	No pathological changes
1 point	Slight degenerative changes in tubuli and glomeruli with cortical involvement less than 25%.
2 point	Mild degenerative changes with cortical involvement of 25–50%.
3 point	Tubular and glomerular necrosis at different foci throughout the cortex with cortical involvement of 50–75%.
4 point	Extensive and marked necrosis throughout the cortex with cortical involvement more than 75%.

For testicular tissue analysis, Johnsen scoring system14 was used. Using 10X objective lens, 10 tubules were scored for each rat. Totally, 50 tubules were evaluated for each group at least. Each seminiferous tubule profile was given a score from 1 to 10 on the basis of the criterion according to Johnsen.

The testis was evaluated histologically with a 4-level grading scale, similar to that described by Erpek et al.15 for the presence of hemorrhage, polymorphonuclear leukocyte infiltration, degeneration of seminiferous epithelium, tubular atrophy, and necrosis in 50 sections per group as shown in Table 2.

Table 2. Histologic testes grading scale.

Grade 0	Absent (no finding)
Grade 1	Slight (<25% of the fields contained evidence of any finding)
Grade 2	Moderate (from 25–50% of the fields contained evidence of any finding)
Grade 3	Severe (>50% of the fields contained with abovementioned histological parameters).

Statistical analysis

The statistical analyses of results were performed using the SPSS (Statistical Package for Social Sciences, Chicago, IL) software version 16. One-way ANOVA was used for the statistical evaluation of TAS and TOS results. When an overall significance was observed, pairwise post-hoc tests were performed using Turkey’s test. Chi-square test was conducted to compare among groups for renal histologic score and testis histopathological findings. A p value less than 0.05 was accepted as statistically significant result.

Results

Biochemical results

In kidney biochemical analysis, sham group had significantly higher TAS value than L group (p = 0.003, Turkey’s test). When L + C group was compared with L group, TAS value was higher (p = 0.048, Turkey’s test). Of TOS analysis, L group had higher oxidative value on kidney when compared with the other groups (p < 0.0001, Turkey’s test).

In testis, TAS level was significantly higher in sham group than the other groups (p < 0.0001, Turkey’s test), and L group had significantly diminished values than the other groups (p < 0.0001, Turkey’s test). Of TOS levels, sham group had significant lower values when compared with L group (p = 0.0001, Turkey’s test), and L group had significantly higher value when compared with L + C group (p = 0.004, Turkey’s test).

In prostate analysis, there was not any difference between groups according to biochemical TAS and TOS results. TAS and TOS findings were summarized in Figure 1.

Figure 1. Tissues’ biochemical total antioxidant status (TAS) and total oxidant status (TOS) (B) results. S: Sham, L: Laparoscopy, L + C: Laparoscopy and CAPE. †: p < 0.05 compared to S and L + C groups. ∫: p < 0.001 compared to S and L + C groups. Δ: p < 0.01 compared to S and L + C groups.

Histologic results

In histologic evaluation of kidney tissues, normal kidney tissue was observed in sham group (Figure 2A). Tubular degeneration and glomerular necrosis were observed in L group (Figure 2B). But these findings were not statistically significant compared to sham. CAPE administration reversed the unsignificant findings seen in L group (Figure 2C). According to kidney histologic score, no difference was found between three groups (p = 0.730).

Figure 2. Normal kidney histology in sham group (A). Glomerular necrosis (arrow) in laparoscopy group (B). Improved morphology in CAPE group (C). Normal germinal epithelium in sham group (D). Minimal disorganization and blood (*) in interstitial area in lap group (E). Improved epithelial structure in cape group (F).

Based on Johnsen criteria, testicular tissue evaluation revealed normal spermatogenesis in S group (Figure 2D). According to statistical analysis of Johnsen scoring, S group showed significant difference compared to L group (p < 0.0001). There was a significant difference between L and L + C group (p = 0.046, Figure 3). There was not any difference between L + C and sham groups.

Figure 3. Testis Johnsen score results. †: p < 0.05 compared to S and L + C groups. S: Sham, L: Laparoscopy, L + C: Laparoscopy and CAPE.

In histologic evolution of testis tissue, minimal disorganization and blood in interstitial area were observed in L group (Figure 2E). CAPE administration improved tubular histology (Figure 2F). When a scoring system was used, no significant difference was recorded between groups.

Discussion

Laparoscopy is a recognized surgical approach that has great benefits for patients, significantly reduces operative time and morbidity, and results in a better postoperative recovery. However, a few physiologic complications are closely associated with gas insufflation under pressure in the abdominal cavity of mammals.16 Sudden and erratic elevations of IAP can lead to various adverse events during laparoscopic abdominal surgical procedures such as subcutaneous emphysema, decreased venous return, impaired ventilation, risk of gas embolism, and even abdominal compartment syndrome,17 especially in small mammals.18,19

Chiu et al.19 performed a prospective controlled animal study on a pig model of PNP with 15 mmHg pressure. They found that superficial renal cortical blood flow reduction decreased by an average of 60% in the compressed kidney, and blood flow returned to the pre-insufflation level after the pressure was released. In animal studies, comparing CO₂ to argon gas for PNP, no significant change in urine output was noted for IAPs less than 15 mmHg. For IAPs greater than or equal to 15 mmHg, a similar impairment in urine output and glomerular filtration rate was seen for both types of gas.18 As a result, the major effect of IAP is I/R injury for kidneys.1,16,18,19 Our findings showed that 15 mmHg IAP generated an oxidative stress on kidney in terms of possible ischemia injury in a rat model as supported by the literature (Figure 1).

Many studies have demonstrated that the major component of the adverse effects in I/R injury is not initiated by hypoxia, but rather by the return of the oxygenated blood to ischemic tissue.20 Several endogenous substances, including free oxygen radicals, platelet activating factor, arachidonic acid metabolites, and bacterial endotoxins have been implicated in the pathogenesis of renal reperfusion injuries.21 The amount of reactive oxygen species is under the control of antioxidant defense mechanisms.22 In our study, similar to previous literature, we demonstrated that increased IAP caused oxidative stress to the kidney tissue in terms of TOS levels in L group compared to sham group. Also, sham group had higher TAS when compared with L group (Figure 1). In addition, CAPE administration had protective effect for renal tissue against I/R injury induced by increased IAP.

In histological evaluation of kidney tissues, laparoscopy did not cause significant degeneration. Our histologic results were supported by Farias et al.,23 Hazebroek et al.,24,25 and Lee et al.26 who reported no significant histological changes in kidney tissue. In L group, we observed insignificant findings such as focal glomerular necrosis and signs of tubular degeneration like vacuolization, bleeding, and vascular congestion. Ozmen et al.27 found similar findings of reversible hypoxic cell injury including cellular swelling, vascular congestion, and focal parenchymal bleeding. Those findings were reported to increase in 20 mmHg pressure. The significant degeneration in Ozmen’s study could be due to increased pressure.27

Imamoglu et al.3 initially investigated the effects of IAP on testicular blood flow (TBF) in a rat model. In their study, PNP caused a significant decrease in TBF at the 10th and 50th minutes of PNP in both 10 and 20 mmHg IAP groups. They also demonstrated that abdominal deflation after IAP of 10 and 20 mmHg for 60 minutes caused testicular free radical production. They concluded that the direct pressure effect of critically increased IAP on the testicular vessels reduced TBF, which might cause testicular hypoperfusion. IAP changes affecting the abdomen might also affect the testis due to I/R injury. Istanbulluoglu et al.4 showed that IAP resulted in marked increases in inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in the testis in a pig model. However, there are limited studies which demonstrated the oxidative stress caused by IAP in testis. In our study, L group was found to have higher TOS and lower TAS levels than S group in testis tissue similar to literature (Figure 1). Moreover, CAPE administration significantly inhibited oxidative stress due to increased IAP.

The effects of PNP on testicular histology were initially evaluated by Imamoglu et al.3 and significant histologic degeneration was reported in 10 and 20 mmHg pressure groups for 1-hour insufflation period. Our results were partially supported by Istanbulluoglu et al.4 who evaluated the effect of 20 mmHg PNP on testicular tissue in terms of apoptosis and spermatogenesis in 4 hours CO₂ insufflation period. They reported marked germ cell apoptosis in study group and no significant difference in Johnsen’s score between groups. In addition, they found histologic evidences of testicular degeneration. However, they performed no quantification method for histologic degeneration. We evaluated testicular tissue according to both Johnsen criteria and a histologic quantification method. We found that laparoscopy did not make any significant changes in 1 hour with 15 mmHg CO₂ insufflation with regard to histologic scoring system in contrast to Johnsen scoring system. The reason why Istanbulluoglu et al. reported marked apoptosis could be higher pressures for longer periods. In addition, S group had no significant difference compare with L + C group. It revealed that CAPE had a protective effect against toxic effect related to increased IAP on testis (Figure 3).

Prostatic infarction has been reported to be significantly increased in patients with acute urinary retention.28 Mentler et al.29 retrospectively reviewed 111 inpatient records and found a 21.6% (n = 24) incidence of acute urinary retention after laparoscopic Nissen fundoplication. These reports suggested that blood flow and I/R played an important role in the normal functioning prostate. Saito et al.5 investigated the expression of heat shock protein (HSP) 70 and its mRNAs during I/R in the rat prostate. In their study, the abdominal aorta was clamped just above its bifurcation using a small clip for 30 minute and reperfusion of the prostate was accomplished by removing the clip. They found that the expressions of HSP 70 proteins and their mRNAs were dramatically changed during I/R in the rat prostate. In our animal model, we suggested that IAP might affect the prostate vascular supply via compressing the large vessels such as abdominal aorta. Based on our findings, there was not any significant oxidative stress on prostate tissue in one hour with 15 mmHg IAP in rat model. In our opinion, higher pressures and longer periods are needed to figure out the effect of the PNP on prostate tissue. To our knowledge, this is the first study, which investigates the effect of the PNP on prostate tissue.

The limitation of our study was the lack of significant evidences in humans. There is significant difference in perfusion pressure of rat kidneys and other intraabdominal organs compared to large animals and humans. However, in patients, treated with laparoscopic partial nephrectomy, especially in an off-clamp setting, PNP was raised to 20 mmHg during excision time (usually 30–45 minutes or longer), the effect of increased IAP on kidney, testis, and the other organs remain unclear.30

Conclusion

In rat model, increased IAP had an oxidative effect on kidney and testis but not on prostate. Moreover, it could affect the testicular Johnsen score. According to kidney and testis tissues’ histologic evaluation, no significant alteration was obtained in 15 mmHg pressure groups for 1-hour insufflation period. All these adverse effects of IAP on both kidney and testis could be prevented by CAPE administration. Further studies are needed to show oxidative effect of IAP against the tissues with more detailed morphological and biochemical analysis.

Declaration of interest

The authors report no conflicts of interest.