Methoxetamine (MXE) is a structural analog of ketamine (KET), which serves as a N
-aspartate receptor antagonist [1
]. MXE binds to serotonin transporter to suppress the re-uptake of 5-hydroxytryptamine and ensure proper levels of 5-hydroxytryptamine in the brain. MXE has also been shown to increase dopamine secretion and suppress dopamine re-uptake [2
]. MXE has only recently been studied as a novel synthetic psychoactive drug, and in 2010, was reported to produce a variety of effects in humans, including feelings of euphoria, calmness, intensive sensations, and physical dissociation, as well as hallucinations and near-death experiences. In 2011, MXE attracted attention for its reported recreational use [3
]. Currently, only a limited amount of MXE-related pharmacologic and toxicology data are available and additional studies of its properties and effects are needed. When considering the similarities between MXE and KET, it may be assumed that current MXE abusers include a large number of former KET abusers. In 2007, ketamine was shown to induce a type of severe cystitis [4
] that showed a poor response to traditional treatments. The lower urinary tract symptoms (LUTS) continued even when a patient had discontinued ketamine. Furthermore, when upper urinary tract involvement occurs, surgical intervention such as augmentation cystoplasty is required if conservative treatment fails [5
]. A series of studies using bladder cell lines, human tissue samples, and animal models have been conducted to examine the biological mechanism of ketamine-induced cystitis. The postulated mechanisms included bladder-epithelial barrier impairment [6
], abnormal neurotransmission [7
], mast cell activation [8
], cell apoptosis [9
], and oxidative stress [11
]. Although results of a survey suggested that MXE use may contribute to LUTS development, all of the MXE abusers in that survey had previously used KET at least once [13
], and thus the impact of MXE use by itself on LUTS occurrence could not be analyzed. Although Dargan et al. reported histology results showing that MXE produced toxic effects in the bladders and kidneys of mice, further research on how MXE affects the urinary system is necessary [14
MXE was originally designed and synthesized to function as an antidepressant [5
], and was advertised as being less toxic than ketamine to the urinary system [15
]. However, additional data are required to verify that MXE is as “bladder friendly” as described. To our knowledge, the effect of MXE on bladder function and inflammation has not been explored in animals. Therefore, we used a rat model of ketamine-induced cystitis to investigate the urodynamic function and histological changes which occur in the bladder after long term MXE and KET treatment.
Our results suggest that MXE and KET cause changes in the urination patterns of rats. Cystometry investigations showed that bladder capacity, and bladder compliance decreased, while the numbers of non-voiding contractions and urination frequency increased in the drug-treated rats. Histologic studies revealed damaged urothelium barriers, reduced amounts of glycosaminoglycans, but increased levels of congestion, inflammatory cell infiltration, and fibrosis in the bladders of drug-treated rats. Moreover, the expression levels of several cytokines in bladder tissue were significantly increased following MXE or KET exposure. In addition to its cytotoxic effects, MXE also stimulated human urothelium cells to generate cytokines. When taken together, these findings explain that MXE induces cystitis and impairs bladder function. In other words, MXE may not be “bladder friendly”.
This study is the first to investigate the impact of MXE on the rat bladder, and is also novel because urodynamic measurements have not been previously employed to measure the effects of MXE in animals. Various researchers have used cystometry to objectively evaluate the status of the lower urinary tract in animal models of ketamine-induced cystitis [11
], and the results found in our KET-treated rats were consistent with the findings in those previous studies. The rats that experienced long term MXE exposure also displayed detrusor over activity and low bladder compliance. Thus, the urodynamic data suggest that MXE induces lower urinary tract symptoms similar to those caused by ketamine exposure.
Ketamine was found to cause damage in the bladder mucosal, submucosal, and muscular layers. Several previous studies have reported that ketamine administration resulted in a thinner or denuded epithelium, inflammatory cell infiltration, and vascular proliferation in the submucosal layer of bladder tissue, as well as collagen deposition in the muscular layer [16
]. We found similar lesions in our MXE-treated rats. Moreover, results obtained in a previous study by Dargan et al. [14
] support our histology findings, and also indicate that MXE is toxic to bladder tissue.
Glycosaminoglycans (GAGs) are distributed on the surface of the bladder mucosal epithelium. Our alcian blue staining studies revealed lower GAG staining intensities in the MXE group and KET group as compared with GAG staining intensity in the control group. As an important component of bladder epithelium barriers, GAGs protect the vesical mucosa from bacteria, molecules, and ions present in the urine. A decreased excretion of GAGs and defects in the epithelial permeability barrier are regarded as mechanisms of bladder pain syndrome/interstitial cystitis (BPS/IC). Moreover, a previous study showed that rats with ketamine-induced cystitis had reduced levels of glycoprotein GP51 and potassium in their urine [19
-cadherin is a glycoprotein found in cell-to-cell adherens junctions, and is crucial for maintaining epithelial morphology and polarity. E
-cadherin expression is often repressed in patients with various bladder disorders associated with LUTS, such as BPS/IC, ketamine-induced cystitis (KC), and recurrent UTIs [20
]. Jiang et al. [21
] proposed that E
-cadherin, involved in bladder dysfunction secondary to bladder outlet obstruction. In the present study, E
-cadherin content in the urothelium became significantly reduced somewhat later after MXE or KET administration. Consistent with KC [12
], the above findings suggest that MXE damages the bladder epithelium barrier by decreasing its GAGs content and thereby affecting cell adhesion.
Another important feature in the examined bladder specimens was evidence of mast cell infiltration. Activated mast cells play a dominant role in the mechanism of BPS/IC [22
]. In previous studies, mast cell numbers were found to be related to bladder capacity in patients with KC, BPS/IC, and especially in patients with KC [9
]. Our results concerning mast cell infiltration in the MXE-treated rats suggest the need for an additional study to investigate the relation between chronic inflammation and clinical symptoms. Strong evidence exists that mast cells undergoing activation and degranulation secrete significant amounts of pro-inflammatory mediators, and especially histamine and various cytokines.
We detected increased mRNA levels of numerous cytokines in bladder tissues obtained from the MXE group. Among those cytokines, IL-1β, IL-6, CCL-2, and NGF are widely regarded as biomarkers for BPS/IC [23
]. Aside from cytokines, the mRNA levels of some representative chemokines such as CXCL-1, CXCL-10, and CCL-2 were also increased. Chemokines are known contributors to symptoms of bladder disorders, and Perters found remarkably reduced levels of chemokines CXCL-1, CXCL-10, and CCL-2 in patients who had received sacral neuromodulation therapy for BPS/IC [24
]. COX-2 is another pro-inflammatory mediator and a key enzyme in the prostaglandin synthesis pathway. Additionally, COX-2 also plays an important role in overactive bladder, bladder inflammation, and BPS/IC. Juan et al. demonstrated that rats with bladder dysfunction had up-regulated COX-2 mRNA levels, and that treatment with a COX-2 inhibitor prevented those changes [11
]. The evidence showing increased levels of pro-inflammatory mediators after long term MXE or KET administration suggests that the type of cystitis induced by MXE or KET is similar to interstitial cystitis.
Numerous animal studies have proven that long term ketamine treatment leads to bladder fibrosis [17
], which is an important factor in bladder abnormalities such as decreased capacity, lower compliance, and impaired detrusor function. This is the first report of mRNA and protein levels being used to verify increased deposition of collagen and fibrotic products in the muscular layer of the MXE-treated rats bladder. Furthermore, the increased extracellular matrix production may be associated with irreversible bladder fibrosis. This change became more severe after a long term exposure to MXE, which could also aggravate LUTS and become a risk factor for upper urinary tract involvement.
No previous study has focused on how MXE affects bladder epithelial cells. In our study, we found that MXE and KET produced toxic effects in SV-HUC-1 cells in a time and dose-related manner. A study by Shen et al. showed that KET was cytotoxic to human urothelial cell lines [10
]. Those findings are consistent with ours, and strengthened our results. We found that treatment with MXE increased the levels of pro-inflammatory mediators in human urothelial cells, and identified a similar trend regarding mediator expression in bladder tissue. These results suggest that the cytokines may not have been released simply as a result of chronic inflammation, but also due to the biological effect of MXE on urothelial cells.
Our study has several limitations: (1) Clinical investigation and data were not included in present study. The characteristics of MXE abusers awaits further studies; (2) The occurrence and development of bladder inflammation involve various cells. Our study solely estimated the expression of pro-inflammatory mediators in urothelial cells after MXE administration. However, we consider that it is still valuable for our purpose to understand the pro-inflammatory effect of MXE; (3) Only one dose, one preparation and one administration route were tested.
The potential harm resulting from MXE abuse has not been widely recognized. Lawn et al. conducted a survey of 427 MXE abusers who had once used ketamine, and found that 23% of them experienced LUTS [13
]. This was close to the 26.6% incidence of ketamine-associated urinary symptoms found in another study. Furthermore, the frequency of MXE usage, but not ketamine usage, was shown to be associated with LUTS occurrence. Currently, only nine countries (Japan, the United States, and sevencountries in Europe) have unequivocally prohibited the use of MXE [25
]. Purchasing MXE does not require any certificate or authorization, and with the help of the Internet, MXE is even easier to acquire than KET [5
]. Botanas et al. published a report implying that both MXE and KET can be potentially abused by humans [26
]. When considering the extensive popularity of ketamine in Asian and other countries, the potential for MXE or other KET-related substances to be abused as recreational drugs is a matter of serious concern.
4. Materials and Methods
4.1. Animals and Drug Administration
Thirty-six female Sprague-Dawley rats (180–210 g) were purchased from the Laboratory Animal Center of Southern Medical University (SMU) in Guangzhou China. The animals were randomly assigned to three groups which received a single daily intraperitoneal injection of 0.9% saline (control group), 30 mg/kg methoxetamine (MXE group) or 30 mg/kg ketamine (KET group) for a period of either 4 or 12 weeks. Ketamine hydrochloride solution (0.1 g/2 mL; Fujian Gutian Pharmaceutical Co., Ltd., Fujian, China) and methoxetamine solid (Hanxiang Biologicals Inc., Guangzhou, China) were approved for use in this study by the Food and Drug Administration of Guangdong Province, China. The rats were weighed each week to adjust the amount of drug they received. All experimental procedures were performed in accordance with the Guidelines for Laboratory Animal Care, and the study protocol was approved by the Institutional Animal Care and Use Committee of SMU (serial number: SCXK Guangdong 2014-0047, 12 November 2014).
4.2. Micturition Frequency Measurement
Micturition frequency was measured as previously described [20
]. Briefly, each rat was placed alone in a metabolic cage for a period of acclimatization. Next, a piece of modified filter paper which had been immersed in CuSO4
O for >40 min and then dehydrated at 160 °C for 30 min to promote conversion of CuSO4
O to CuSO4
was stretched across the underside of the cage. Direct contact between falling urine and the paper immediately initiated hydration of the CuSO4
, resulting in discoloration of the paper. Micturition frequency was determined by calculating the number of spots on the paper as determined using Photoshop software (version 13.0, Adobe Systems Incorporated, San Jose, CA, USA) in reversed color contrast phase. Overlapped urine spots with legible edges were considered to be a single urination incident. Micturition frequency was measured for a period of 3 h per day, for 3 consecutive days, and the mean value was determined via statistical analysis.
4.3. Urodynamic Investigations
The rats were anesthetized by injection with 20% urethane (1.0 g/kg); after which, they were immobilized in the supine position, and a 1.0 mm inner diameter epidural catheter was transurethrally inserted into the bladder. The other end of the catheter was connected to a pressure transducer and a micro-pump via a T-branch adaptor. After emptying the bladder with a syringe, the bladder was infused with 0.9% sterile saline at a steady rate of 12 mL/h (200 μL/min). When a consistent and reproducible pattern of micturition was established, digital intravesical pressure signals were continuously recorded for 60 min or at least 5 void cycles using a MMS Solar system (Medical Measurement Systems; Enschede, The Netherlands). The measured parameters included VCs, NVCs, bladder capacity, PVR volume (post-void residual volume), ICI, baseline pressure (lowest pressure between adjacent voids), threshold pressure (exact initial pressure that triggered a VC), micturition pressure (peak pressure during urination), and bladder compliance (the dilated bladder volume as determined from baseline/the increase in intravesical pressure).
4.4. Histological and Immunohistochemical Staining
After completing the urodynamic investigations, the rats were sacrificed and their bladder tissues were removed. Specimens of bladder tissue for histologic staining were fixed in 4% paraformaldehyde; after which, they were embedded in paraffin and cut into 4 μm sections for analysis. Sections for histopathologic evaluation were stained with hematoxylin and eosin (H&E, Servicebio, Inc., Wuhan, China), alcian blue, masson’s trichrome and toluidine blue. Epithelial integrity was determined by E-cadherin expression. Staining for CD68 was performed to analyze macrophages aggregates present in the urinary bladders. Briefly, the tissue sections were deparaffinized and immersed in 3% H2O2 for 30 min to quench endogenous peroxidase activity. After being blocked with 2% BSA, the sections were incubated with E-cadherin (1:100, Santa Cruz Biotechnology; Santa Cruz, CA, USA), CD68 antibody (1:200, Santa Cruz Biotechnology), collagen I antibody (1:100, Boster; Pleasanton, CA, USA ), fibronectin antibody (1:500, Abcam, Cambridge, UK) or α-SMA antibody (1:200, Abcam) overnight at 4 °C. Following incubation with the primary antibodies, the tissue sections were incubated with the appropriate biotinylated secondary antibody (1:5000, Cwbio, Beijing, China) for 30 min; after which, they were incubated with 3,3′-diaminobenzidine and lightly counterstained with hematoxylin. Quantitative digital image analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and representative images were selected for presentation in this report.
4.5. Cell Culture and Treatment
SV-HUC-1 cells (an immortalized human uroepithelial cell line infected with the SV40 virus) were cultured in DMEM/F12 medium containing 10% at 37 °C in a 5% CO2 atmosphere. Prior to treatment, the cells were transferred to serum-free medium and incubated overnight to synchronize their growth; after which, they were treated with ketamine or methoxetamine solution at the indicated concentration for the designated time period.
4.6. CCK-8 Assay
SV-HUC-1 cells (100 µL containing 5000 cells) were seeded into individual wells of a 96-well plate, and then pre-incubated for 24 h to allow their adherence. The cells were then incubated with 0.25 mM ketamine or methoxetamine for 24 or 48 h, respectively. Following incubation, 10 μL of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan), which serves as a tetrazolium salt colorimetric assay, was added to each well of the plate and incubated with the cells for 2 h. Next, a microplate reader (Tiangen Biotech Co., Ltd., Beijing, China) was used to measure the absorbance of each well at 450 nm.
4.7. Real Time-PCR
Total RNA was extracted from rat bladder tissue and SV-HUC-1 cells using RNAiso Plus reagent (Takara Bio Inc., Otsu, Shiga, Japan). Complementary DNA (cDNA) was synthesized using the PrimeScript™ RT reagent Kit (Takara Bio Inc.), and genomic DNA was eliminated according to the manufacturer’s instructions. Next, qRT-PCR was performed using an Applied Biosystems 7500 Real Time PCR System and SYBR®
Premix Ex TaqTM II (Takara Bio Inc.). The sequences of the primer pairs were designed by Invitrogen (Paisley, UK) (Table 1
), and primer-blast programs were used to ensure the specificity of the amplified fragments. The cycle threshold (Ct
) of various genes was determined, and changes in gene expression were calculated using the delta-delta Ct
method. The results are expressed as a ratio relative to GAPDH. Allof the PCR experiments were repeated individually at least 3 times.
4.8. Statistical Analysis
All data were analyzed using SPSS for Windows, Version 13.0. Chicago, IL, USA: SPSS Inc., and results are expressed as the mean ± SD. Multiple group comparisons were performed using analysis of variance (ANOVA), and the LSD test was used for pair-wise comparisons (equal variances assumed). Otherwise, the Dunnett’s t3 test was preferred. For all analyses, two-sided p-values < 0.05 were considered statistically significant.