Repeated Doses of Ketamine Affect the Infant Rat Urogenital System

Introduction

Ketamine is an N-methyl-D-aspartic acid receptor antagonist and provides loss of consciousness, amnesia, immobility and deep analgesia in anesthesia.¹ Ketamine was first synthesized in 1962 and introduced into clinical medicine for dissociative anesthesia in 1970.² It is used as a short-acting general anesthetic in human and veterinary clinics. As a non-respiratory and non-cardiac depressant, anesthetic doses of ketamine are preferred over other anesthetics.³ Because of this feature, ketamine has been widely used alone in premedication, sedation and postoperative analgesia or combined with a reduced dose of a second analgesic for many years, especially in the pediatric age group.^4–6 Ketamine is even recommended for the treatment of status asthmaticus in children due to its bronchodilator properties.⁷ Ketamine is the most preferred agent, especially in children who receive radiotherapy for a certain period of time due to cancer diagnosis or in children who require intermittent sedation for daily dressing in the burn unit. At subanesthetic doses, ketamine produces a dissociative state, characterized by a sense of detachment from one’s physical body and the external world which is known as depersonalization and derealization.⁸ Due to its hallucinogenic effect, abuse and addiction are also very common in Far Eastern countries such as Hong Kong, Taiwan and in Europe in recent years.^9,10 Long-term ketamine use in adults is known to create a interstitial cystitis-like problem in the bladder. It has been called ketamine cystitis due to its prevalence in ketamine addicts. Characteristic cystoscopic findings and histological changes in bladder biopsies of these patients; a thinner urothelium, mast cell infiltration, tissue inflammation, and fibrosis. Tissue inflammation and fibrosis, an indicator of ketamine-induced cystitis in adults, has been shown in animal studies.^11,12 In recent animal studies, it has been shown that widespread apoptotic neurodegeneration occurs in the developing baby rat brain after exposure to ketamine for a certain period of time.¹³ Various deleterious effects of chronic ketamine intake has been reported, including apoptotic neurodegeneration in the brain, reduction in gray matter, intracranial hypertension, and neuropsychiatric symptoms. Antioxidant systems normally work in integrity, protecting the cell against the toxic effects of free oxygen radicals. They do this by keeping the oxidant and antioxidant systems in the organism in balance. In cases where this balance is disrupted in favor of oxidant capacity, inflammatory mediators and free oxygen radicals are produced.^14,15 Free radicals can contribute to organism disorders by targeting biomolecules of cells such as lipids, carbohydrates, proteins and DNA. Many reasons can lead to the formation of free oxygen radicals in the organism. Animal studies show that long-term administration of ketamine cause lipid peroxidation in the testicles. It has been demonstrated by several mechanisms that increased reactive oxygen species (ROS) negatively affect testicular functions. ROS attacks the sperm plasma membrane and causes lipid peroxidation. Additionally, ROS causes DNA fragmentation and decreases sperm motility.^16,17 In a recent animal study, malondialdehyde MDA level was examined in order to investigate the effect of repeated ketamine treatment on the testis. According to the results, it has been reported that repetitive ketamine treatment may have dangerous effects on reproductive function.¹⁸ MDA has been used for many years as a suitable biomarker for lipid peroxidation of fatty acids in the cell membrane due to its easy reaction with thiobarbituric acid. The thiobarbituric acid test is based on the reactivity of thiobarbituric acid to MDA to obtain an intensely colored chromogen fluorescent color absorbent. CAT enzyme is used as a biomarker to measure antioxidant capacity. CAT is a hemoprotein that contains a single ferriprotoporphyrin in each polypeptide subunit structure. It constantly monitors hydrogen peroxide (H₂O₂) molecules, as a lipid peroxidation product. CAT can break down millions of H₂O₂ molecules in one second. It is important that CAT can effectively limit the H₂O₂ concentration in cells and plays a role as a first-step antioxidant defense enzyme in physiological processes.¹⁹ Studies on testicular tissue has shown that free oxygen radicals cause ischemia reperfusion damage.^20,21 In addition, various studies have been reported on the disruption of the seminiferous epithelium and the loss of germ cells.²²

Long-term ketamine use is known to create an interstitial cystitis-like problem in the bladder. It is known that long-term intermittent ketamine is applied to the children receiving radiotherapy for sedation. This study was planned to investigate whether this effect seen in the bladder causes similar changes in the kidneys, testicles, epididymis and ductus deferens.

Materials and Methods

This study was conducted in accordance with the guidelines of the National Institutes of Health on animal research and it was approved by the Animal Experiments Local Ethics Committee of Gazi University (G. U. ET-18.092). 3 weeks old 12 Wistar Albino male infant rats were used in the study. All animals were kept in the Animal Care Laboratory of Gazi University. Animals were kept under 21 ± 22 °C at cycles of 12-hour day light and 12-hour darkness and had free access to food and water. A total of 12 rats were equally randomized into 2 groups as; ketamine (K) and saline (S) group. 50 mg/kg of ketamine was administered intraperitoneally (i.p) to the ketamine group and 1 mL/kg (i.p) saline was administered to the saline group during 21 days. For euthanasia, 100 mg/kg of ketamine (i.p) and 10 mg/kg of xylazine (i.p) were administered to all rats and blood was taken from the abdominal aorta. Kidney, epididymis and testicular tissues were removed for and histopathological and biochemical evaluations.

Histopathological Evaluations

Kidney, epididymis and testicular tissue samples were taken in the formalin solution for histopathological analysis. After 12 hours of formol fixation, tissues were embedded in paraffin blocks. Sections obtained from paraffin blocks were stained with Hemotoxylin eosin (H&E). The presence of tubular epithelial degeneration, tubular epithelial edema, vascular congestion, tubule dilation, interstitial edema, interstitial fibrosis, interstitial inflammation, glomerular sclerosis, bowman capsule fibrosis, and tubular atrophy was evaluated in H&E sections. Seminiferous tubule dilatation, stromal fibrosis, epididymal congestion, distortion in the epididymal gland and ductus deferens were evaluated in testis and epididymis tissues. Testicular tissue sections were scanned on the Aperio AT2 device and then seminiferous tubules were marked from each section. Short diameters of the marked tubules were calculated with the metric measurement option after 100 tubule annotations were made on digital slides with Virapath application. Occlusion presence was evaluated at 40 magnification and other parameters at 200 magnification.

Histological Evaluation and Maturation of Seminiferous Tubules

The Johnsen score was used to evaluate spermatogenesis in the seminiferous tubules (Figure 1). 50 seminiferous tubules were examined in cross-section and a score of 1–10 was assigned to each of them according to the following criteria.²³ 10: complete spermatogenesis and perfect tubules, 9: many spermatozoa present but disorganized spermatogenesis,8: only a few spermatozoa present, 7: no spermatozoa but many spermatids present, 6: only a few spermatids present, 5: no spermatozoa or spermatids present but many spermatocytes present, 4: only a few spermatocytes present, 3: only spermatogonia present, 2: no germ cells present, 1: neither germ cells nor sertoli cells present Each score score was multiplied by the number of tubules seen. The results were summed and divided by the number of 50 tubules and the average score was found. In addition, two groups were evaluated according to the presence of spermatozoa. Group 1: scores with spermatozoa between 8–10, Group 2: scores without spermatozoa between 1–7.

Figure 1 The spermatogenesis and spermiogenesis processes are normal and seminiferous tubules with a high proportion of spermatozoa are observed. Spermatogonia-black long arrow, sertoli cell: black short arrow, spermatocyte: yellow long arrow, spermatozoa: yellow short arrows, spermatitis: red arrow (H&E×400).

Biochemical Evaluations

The kidney and testicular tissues were washed with cold NaCl solution (0.154 M) to discard blood contamination and then homogenized in a Diax 900; Heidolph Instruments GmbH&Co KG, Schwabach, Germany at 1000 U for about 3 min. After centrifugation at 10,000 g for about 60 min, the upper clear layer was taken.

MDA levels, thiobarbituric acid reactive substances assay was performed by Van Ye method.⁷ The reaction with thiobarbituric acid at 90–100°C was used to determine the MDA level, as MDA or similar substances react with thiobarbituric acid and produce a pink pigment that has an absorption maximum of 532 nm. To ensure protein precipitation, the sample in room temperature is mixed with cold 20% (w/v) trichloroacetic acid and the precipitate is then centrifuged for 10 min 3000 rpm at room temperature to form a pellet. An aliquot of the supernatant is then placed into an equal volume of 0.6% (wt/vol) thiobarbituric acid in a boiling water bath for 30 min. Following cooling, sample and blank absorbance were read at 532 nm and the results expressed as nmol/mg protein, based on a graph where 1,1,3,3-tetramethoxypropane has been used as the our MDA standard.

CAT activity is based on the measurement of absorbance decrease due to H₂O₂ consumption at 240 nm by Aebi H method.⁸ PON-1 activity was measured the rate of hydrolysis of paraoxon by monitoring the increase of absorbance at 405 nm and at 25 °C. The basal assay mixture included 1.0 mM paraoxon and 1.0 mM CaCl2 in Tris/HCl buffer (pH: 8.0, 100 mM). The definition of 1 unit of paraoxonase activity was taken as 1 millimole of p-nitrophenol formed per minute.⁹

CAT and PON-1 activities were given in IU/mg protein. Samples protein amount was determined by Lowry O method, BSA was used standard protein.¹⁰

Statistical Analysis

All the data analyses were performed using SPSS 20.0 software (SPSS Inc., Chicago, IL). Data are presented as means ± SEM. Histopathological data of rat kidney tissue, testicular tubule and CAT, PON-1 enzyme activities and TBARS level of rat kidney and testicular tissues the groups were analyzed using students t test. Histopathological data of rat testicular tissue between the groups were analyzed with Fisher’s test and χ2 test. P<0.05 was considered significant.

Results

Histopathological Results

In testicular tissue, congestion and distortion in the epididymal gland were found to be different in the ketamine group compared to the saline group (p=0.015, p=0.015, respectively) (Table 1). Testicular tubule diameter increased significantly in the ketamine group compared with the saline group (p=0.026) (Table 2) (Figure 2). In staining with H&E×100, congested vascular structures were observed in the testicular tissue in the ketamine group (Figure 3). Johnsen scores of the saline and ketamine groups were given in Table 4. Johnsen scores were found to be significantly lower in the ketamine group compared to the saline group (p = 0.013). The mean seminiferous tubule containing spermatozoa was found to be significantly lower in the ketamine group compared to the saline group (p = 0.001). However, the mean of seminiferous tubules without spermatozoa was found to be significantly higher in the ketamine group compared to the saline group (p <0.0001). While maturation was observed until the stage of spermatitis, a loss was observed in the formation of spermatozoa in the subsequent spermiogenesis. Loss of maturation was detected in the ketamine group (Table 3).

Table 1 Histopathological Data of Rat Testicular Tissue [N%]

Table 2 The Diameter of Testicular Seminiferous Tubules [Mean ± SEM]

Table 3 Johnsen Score Spermatogenesis in the Seminiferous Tubules [Mean ± SEM]

Table 4 Histopathological Data of Rat Kidney Tissue [Mean ± SEM]

Figure 2 In ketamine group spermatozoa were not observed, only spermatids were seen in the seminiferous tubules. Seminiferous tubule diameters were measured in transverse tubules (H&E×400).

Figure 3 Congestion was observed in the vessels between the seminiferous tubules in the ketamine group (H&E×100).

Vascular congestion were significantly increased in the ketamine group in kidney tissue (p=0.049) (Table 4), (Figures 4 and 5).

Figure 4 Congested vascular structures were observed focal in the saline group (H&E×100).

Figure 5 Congested vascular structures were more common in the ketamine group (H&E×100).

Biochemical Results

TBARS level was found to be significantly higher in the ketamine group compared to the saline group in the testicular and kidney tissue (p=0.027, p=0.025, respectively) (Tables 5 and 6). CAT enzyme activity was found to be significantly lower in the ketamine group in both tissues (p=0.017, p=0.025, respectively) (Tables 5 and 6). PON-1 enzyme activity was found to be significantly higher in the ketamine group compared to the saline group (p=0.049, p=0.012, respectively) (Tables 5 and 6).

Table 5 TBARS Level and CAT, PON-1 Enzyme Activities of Rat Kidney Tissues [Mean ± SEM]

Table 6 TBARS Level and CAT, PON-1 Enzyme Activities of Rat Testicular Tissues [Mean ± SEM]

Discussion

Because of the low incidence of cardiovascular depression, ketamine is considered a safe drug, especially for children. Ketamine is the most preferred agent by anesthesiologists, especially in procedures such as angiography, endoscopy, bronchoscopy, and radiotherapy.^24–27

In recent years, the widespread use of ketamine in pediatric anesthesia, especially in repeated applications, has brought many negative effects on metabolism. Ketamine-induced neuroapoptosis has been extensively studied in mice, rats, and rhesus monkeys. Ikonomidou et al administered ketamine to infant rats at equal intervals for 9 hours and stated that this caused a strong apoptotic neurodegeneration pattern in the brain. Neurotoxicity was observed when ketamine was administered to 7-day-old rats at a dose of 20–25 mg/kg 7 times every 90 minutes.^28,29 Signs of neurotoxicity were seen only in the long-term exposed group. Decreased learning, motivation, color discrimination and short-term memory were observed in subjects taken ketamine for 24 hours.³⁰ These findings confirmed that ketamine-induced neurotoxicity was both dose and time dependent. The dose of ketamine used in these studies is about 10 times the amount required to anesthetize humans, and also almost 10 times the therapeutic levels of ketamine.³¹ It is thought that sensitivity to anesthesia-induced neurotoxicity in children covers the period between the last three months of pregnancy and the age of 3 years.³² Studies have shown that ketamine and its metabolites in urine lead to a chronic inflammatory response with a direct toxic effect on the bladder urothelium and subsequently to symptoms similar to interstitial cystitis.³³ In an in vitro study in animal bladder, ketamine has been shown to induce cytotoxicity and increase barrier permeability in these cells in a dose and time dependent manner. Long-term abuse of ketamine has been characterized in all studies that cause ketamine-associated cystitis, and based on this, experimental animal studies have been conducted. Shen et al administered high-dose daily ketamine injections to mice for 20 weeks to demonstrate the effects of long-term ketamine abuse on the bladder. In particular, it has been shown that male mice injected with ketamine were adversely affected.³⁴ Di Gu et al showed that ketamine disrupts the proliferation of bladder epithelial cells and causes cystitis by causing defects in the bladder epithelial barrier.³⁵ It has been stated that long-term ketamine treatment may affect the sperm environment, causing a decrease in sperm motility that may affect fertility, and this may impair male reproductive function.⁸ In a similar study, Paolıs et al administered 50 mg/kg ketamine to adult rats for 6 weeks. In conclusion, it was shown that ketamine administration caused a significant decrease in seminiferous tubule diameter, lumen diameter and epithelial height. Significant deterioration was observed in sperm parameters (number, motility, etc.) of rats receiving ketamine. It has also been shown that chronic ketamine administration causes a significant increase in MDA levels.¹⁸ Soltani et al investigated the effect of hydroalcoholic extract of Matricaria chamomile (MC) on torsion/detorsion (T/D) injury.²⁰ In this study, they applied only T/D to the control group and applied MC treatment before T/D to the study group. They showed that the levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase and testosterone decreased in the control group but increased in the MC group. Increased MDA levels during ischemia have also been shown to decrease with MC treatment. In the same study, the average Johnson score was found to be lower in the T/D and T/D + MC groups compared to the control group. It was observed that the MC and control groups did not differ significantly in terms of the mean Johnson score, and the seminiferous tubule diameter was significantly reduced in the T/D group compared to the control group. It was observed that the diameter of the seminiferous tubule increased significantly in the group receiving MC extract compared to the T/D group.²⁰ Similar effects were seen in verapamil/tadalafil combination therapy.²¹ Shoorei et al demonstrated that oral administration of Fumaria parviflora (FP) 14 days before detorsion in adult male rats significantly decreased the apoptosis index, bax gene expression and MDA levels.³⁶ In another study with onion juice, it was shown that the use of onion juice after testicular T/D can increase sperm quality and fertility.³⁷ Our study was conducted on infants who took ketamine for a shorter period of 3 weeks and oxidative stress parameters in metabolism were measured. It was determined that TBARS level, which is an indicator of oxidative stress significantly increased, and antioxidant CAT enzyme activity significantly decreased in the ketamine group in testicular and kidney tissues. In rat testicular tissue treated with Syzygium aromaticum extract, Johnson’s score, seminiferous tubule diameter, and seminiferous tubule epithelial thickness were shown to increase significantly compared to the untreated group.²¹

In this study we observed that vascular congestion in the kidney significantly increased in the ketamine group compared to the saline group. We also found vascular congestion in the testicle and distortion in the epididymal glands. Johnsen scores were found to be significantly lower in the ketamine group compared to the saline group. The mean of seminiferous tubules containing spermatozoa was significantly lower in the ketamine group than in the control group. Maturation was observed in the ketamine group until the stage of spermatitis, while loss in spermiogenesis was observed in the formation of spermatozoa. Loss of maturation was detected in the ketamine group. In the ketamine group, vascular congestion in the testicle and distortion in the glands may have led to nutritional disorders and loss of maturation in spermatogenesis. The time to onset of clinical symptoms after ketamine abuse can range from a few days to a few years. In our study, ketamine was administered to infant rats during 21 days to characterize the effects on the urogenital system and to examine the critical changes in infants who were sedated with ketamine for an average of 21 days for radiotherapy. In the literature, we did not find any other study about ketamine based on the pediatric age group for long-term sedation. Compared to other studies, ketamine administration was applied for a short time. Nevertheless, we observed that repeated ketamine administration caused significant histological changes and oxidative damage in epididymis, testicular and kidney tissues.

There are 2 limitations in our study. First one is low number of animals used in groups, second is power analysis was not performed in the study.

We concluded that the results we obtained will be a guide for repeatedly administered ketamine in daily anesthesia practice, especially in the pediatric age group.