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ORIGINAL ARTICLE
Year : 2022 | Volume
: 33
| Issue : 4 | Page : 176-181
Using a rat model to translate and explore the pathogenesis of ketamine-induced cystitis
Ying-Che Huang1, Wei-Chia Lee1, Yao-Chi Chuang2, Cheng-Nan Tsai1, Chun-Chieh Yu3, Hung-Jen Wang1, Chia-Hao Su3
1 Division of Urology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
2 Division of Urology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine; Center for Shock Wave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
3 Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Correspondence Address:
Wei-Chia Lee
Division of Urology, Kaohsiung Chang Gung Memorial Hospital, 123 Ta Pei Road, Niao Song Qu, Kaohsiung City
Taiwan
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/UROS.UROS_128_21
Purpose: Ketamine abusers may develop severe ulcerative cystitis along with irritative bladder symptoms. A reliable animal model may benefit the understanding of pathophysiologies and the development of therapeutic strategies for ketamine-induced cystitis (KIC). We used a popular rat model of KIC to validate the micturition behavior, functional brain images, and possible molecular mechanisms of this model. Materials and Methods: Female Sprague–Dawley rats were distributed to control (saline) and ketamine-treated rats (25 mg/kg/day for 28 days). Functional magnetic resonance imaging (fMRI), metabolic cage study, and cystometry were evaluated. Potential bladder transcripts involved in KIC were screened by using next-generation sequencing. Results: In contrast to the control, the ketamine-treated rats developed bladder overactivity accompanied by enhanced fMRI signals in periaqueduct and caudal putamen areas. Alterations in bladder transcripts, including eleven genes involving in regulating NF-κB signaling of bladder inflammation, and Crhr2 gene overexpression associating with vascular endothelial growth factor signaling of bladder ischemia were found in ketamine-treated rats. Both categories could be attributed to neurogenic inflammation induced by the direct toxicity of urinary ketamine and its metabolites. Conclusion: Our study results suggest this animal model could mimic irritative bladder symptoms associated with central sensitization in KIC. Through the bladder transcripts analysis, we highlight the neurogenic inflammation underlying the pathophysiologies of KIC in rats.
Keywords: Central sensitization, ketamine, neurogenic inflammation, rat, ulcerative cystitis
Ketamine, the N-methyl-D-aspartate receptor antagonist, is a general anesthetic agent that has been used in both human and veterinary settings for decades.[1] Ketamine undergoes metabolism through hepatic microsomal enzymes, and its major metabolite is norketamine, which is excreted predominantly in the urine. Street ketamine abusers may administrate ketamine through parenteral, nasal, and oral routes. Younger people abuse ketamine in clubs and parties because of its dissociative effects, low price, and ease of use.[2]
High doses and frequent use of ketamine may develop into ketamine-induced cystitis (KIC).[1],[2] Users with a contracted bladder with ulcerative cystitis could develop severe urgency, frequency, dysuria, and hematuria common in patients with KIC. At present, pathogenesis from KIC is endemic among the United Kingdom, Australia, China, and Southeast Asia.[3],[4] Therefore, establishing experimental animal models mimicking the disease state in humans is crucial to evaluating the pathophysiologies of patients with KIC.
There are current animal models of KIC to explore the pathophysiological mechanisms,[5],[6],[7],[8] novel therapeutic strategies,[9],[10],[11] and translate findings into human clinical trials.[12],[13] A regular daily intraperitoneal injection of a high dose of ketamine in rats can mimic the direct toxicity of ketamine and its metabolites on the bladder.[9],[10],[11]
In this study, the KIC animal model established by Chuang et al.[5] was utilized to reproduce the micturition behavior of KIC rats, which has also been reported in other studies.[9],[10] By using functional magnetic resonance imaging (fMRI) and next-generation sequencing (NGS) technology, the pathophysiologies in central sensitization and neurogenic inflammation of KIC rats were examined. The present study might provide a novel insight into the multidisciplinary collaboration of KIC features between laboratory and clinical researchers.
Materials and MethodsThis study was conducted according to the guidelines of the National Research Council (USA). The Institutional Animal Ethics Committee approved the experimental protocol used in this study (permit number: 2017092604). All surgeries were performed under anesthesia, and every effort was made to minimize the suffering and number of animals used throughout this experiment.
Animal model
In this study, six female Sprague–Dawley rats (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan; weight: 200–240 g) were randomly distributed to two groups (n = 3 in each group) and subjected to an experimental course of 28 days. These rats were maintained under temperature control (24°C ± 0.5°C) and a 12:12 h light-dark cycle. The facility has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The experiments were conducted in two groups: (i) Sham (0.9% saline; control) and (ii) ketamine (25 mg/kg/day ketamine, intraperitoneal injection).
Resting-state functional magnetic resonance imaging
As in the previous studies,[9],[10] the control and ketamine-treated rats were anesthetized with zoletil (50 mg/kg) through intramuscular injection at days 0 and 27. fMRI experiments were conducted using the 9.4-T horizontal-bore animal MR scanning system (Biospec 94/20, Bruker, Ettlingen, Germany). The rats were kept in a resting state to capture the images in all sessions. This scanning system was equipped with a self-shielded magnet with a 20 cm clear bore and a BGA-12S gradient insert (inner diameter = 12 cm) that provided a maximal gradient strength of 675 mT/m and a minimum slew rate of 4673 Tm−1 s−1. Resting-state fMRI image data were analyzed using the following procedures: registration to a segmented rat brain atlas and realignment for subtle motion correction with SPM8 software (Wellcome Department of Cognitive Neurology, London, UK), spatial smoothing (FWHM = 1 mm), regressions of motion parameters and white matter/ventricle signals, and band-pass filtering (0.002–0.1 Hz). Variations in the blood oxygen level-dependent signal were calculated using a z-test of different P values (0.2–0.28) with 10 cluster sizes, and at least two contiguous voxels were used as a threshold for the fMRI images and relative to the activation region.[14] The region of interest analysis was based on periaqueductal gray (PAG) detected through REST software (State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, China). PAG's blood oxygen level-dependent images correlated with the caudate-putamen (CPu) of the rats' brains using xjView.
Metabolic cage
On day 23, three rats from each group were placed in individual 3701M081 metabolic cages (Tecniplast, Buguggiate, Italy), as reported previously.[9],[10] After a 24 h familiarization period, the volume of liquid consumed, micturition frequency, and urine output were measured for 3 days. Urine samples were sent for ketamine and norketamine assays using liquid chromatography-mass spectrometry (Super Micro Mass Research and Technology Center, Cheng Shiu University, Taiwan).
Cystometry
As in previous reports,[9],[10] cystometry was performed on day 28. Rats were anesthetized with subcutaneous urethane (1.0 g/kg). A polyethylene-50 catheter was indwelled via the rat's urethra and connected through a T-tube to a pressure transducer and a microinjection pump (infros AG, CH-4130, Bottmingen, Switzerland). Room temperature saline was infused at a rate of 0.08 ml/min, and cystometry was performed using a Gould polygraph (RS3400; Gould, Cleveland, OH). Cystometry was recorded until a stabilized voiding pattern. Thus, reproducible micturition cycles were recorded for a 1 h period. The voiding pressure and intercontractile interval were compared using the Mann–Whitney U test because of the small sample size. An alpha value of 0.05 was considered statistically significant.
Bladder histological study
To characterize histological changes in rat bladders, paraffin sections were deparaffinized and stained with hematoxylin and eosin (H and E) staining and immunohistology. The deparaffinized sections were incubated with 3% H2O2. A primary antibody of uroplakin III (1:500; Abcam) and a secondary antibody conjugated with horseradish peroxidase were utilized. Slides were incubated with 3,3-DAB, counterstained with Mayer's hematoxylin (Novolink; RE7280-K), and mounted with malinol.
Next-generation sequencing and analysis
Urinary bladders were isolated and flash frozen for whole-genome RNA NGS (RNA-Seq) conducted by Welgene Biotech Co., Ltd. (Taipei, Taiwan). All procedures were performed according to the Illumina (San Diego, CA, USA) protocol for generating raw sequences in 30 million reads per sample as previously reported.[15] The open-source Cuffdiff tool from the Cufflinks package was run to calculate expression changes and associated q values (false discovery rate-adjusted P values) for each gene between the control and ketamine-treated rats.
Results[Table 1] and [Figure 1] show the general characteristics of the fMRI images of the brain and micturition behavior of experimental animals. High concentrations of norketamine and ketamine in urine were noted in ketamine-treated rats. The hyperintense signals at the PAG and putamen areas of the brain in fMRI images were observed in the ketamine-treated rats. In addition, in contrast to the controls, the ketamine-treated rats showed a significantly shorter intercontractile interval and an increase in micturition frequency. [Figure 2] shows the histology studies of the ketamine-treated bladder. Compared with the controls, the ketamine-treated rats showed significantly increased suburothelium hemorrhage, mononuclear cells infiltration, and disrupted uroplakin III staining at the apical surface of the urothelium.
Table 1: General characteristics and cystometry parameter of experimental animals (n=3, in each group)
Figure 1: Resting-state fMRI images and micturition behavior evaluation of rats. (a) T2-weighted image on brain coronal sections of rats with empty bladders in resting-state functional magnetic resonance imaging. In contrast to the control, ketamine-treated rats showed hyperactivated signals of periaqueductal gray and caudate putamen regions on day 27. (The green circle indicates the periaqueductal gray area and the green P indicates the area of caudate putamen). (b) Representative traces of conscious metabolic cage study and anesthetized cystometry. Increased micturition frequency and shorten intercontractile intervals were observed in the ketamine group. (Arrows denote the increased basal tone)
Figure 2: The effect of ketamine on rat's bladder. (a) Hand E: Markedly higher erythrocyte debris (arrowhead) and increased mononuclear cells infiltration (asterisk) under urothelium in ketamine-treated rats. (b) Immunostaining of uroplakin III: Absent staining at the apical surface of the urothelium (arrow) in ketamine-treated rats[Table 2] lists the significant changes of differential expressed genes of rats' bladders between groups in NGS RNA sequencing. These genes can be categorized into two groups through different gatekeeper genes/pathways/mechanisms that affect bladder function. These include (1) 11 genes involved in regulating NF-κB signaling of bladder inflammation and (2) one gene (i.e., Crhr2) relating to vascular endothelial growth factor (VEGF) signaling suggesting bladder ischemia, as illustrated in [Figure 3].
Table 2: Significant gene's expressions (P<0.05) through different gatekeeper genes/pathway of the bladder between the ketamine and control rats (n=3, in each group)
Figure 3: Role of different gatekeeper genes/pathways in neurogenic inflammation. Ketamine may cause the release of prostaglandin D2, nitric oxide and interleukin-6 through NF-kB pathway. Afferent nerves then in turn release pro-inflammation peptide SP and active SP receptors (Tacr3) located on the smooth muscle (vascular and detrusor). On the other hand, under ketamine use, corticotropin-releasing hormone can activate its receptor (Crhr2) on mast cells in lamina propria to release vascular endothelial growth factor, which acts on the remodeling of blood vessels. *AA = Arachidonic acid Discussion The severe bladder pain and storage symptoms of patients with KIC may result from ketamine direct toxicity and bladder ischemia.[1] In the present study, ketamine-treated rats showed an increase in micturition frequency and cystometric bladder overactivity. Meanwhile, there were enhanced signals of fMRI at the PAG and CPu areas of the rat's brain in the ketamine group, which suggests the elicitation of vesical afferent inputs and central sensitization. These findings support the hypothesis that the ketamine-treated rats could mimic the clinical presentations of KIC regarding worse storage symptoms and noticeable central hypersensitivity. By using NGS analysis on rat's bladder, we suggested the NF-κB activation pathway was involved in neurogenic inflammation and the VEGF-mediated ischemia process, which may play important roles in the pathogenesis of KIC.
Symptoms of patients with interstitial cystitis were similar to those of patients with KIC, which may suffer from severe bladder pain and urgency.[1],[2] Chronic bladder pain and sustained urgency of KIC might induce central sensitization through neural plasticity, which could originate from chronic noxious stimuli and repeated neurogenic inflammation from KIC.[16],[17] In this study, we showed hyperintense fMRI signals in the PAG and CPu areas in the brain of ketamine-treated rats, even in the empty bladder status. Healthy bladder function depends on the coordination of afferents and efferents and their integration at the spinal cord and brain.[18] The PAG area is an important passage where bladder afferent signals can emerge. The PAG area signals could help with the understanding of the peripheral sensory inputs (e.g., urgency or transient pain) from the bladder and be a reference for “afferent noise” intensity.[18] Furthermore, the CPu is a crucial site for cortical input into the basal ganglia loops and is frequently activated during pain.[19],[20] The role of the CPu area on the shaping of individual persistent pain by using internal cognition to alter the cerebral cortex activity has been underscored.[20] Measuring CPu signals enables the understanding of sensory aspects of pain in this animal model and can be used as a reference for pain intensity and unpleasantness.
Based on the previous studies,[9],[10],[21] the enhanced NF-κB expression in the bladder of ketamine-treated rats is observed at both urothelium and muscle layers. Furthermore, KIC of rates may elicit monocyte/macrophage infiltration in the suburothelium.[10],[21] The NF-κB overexpression can colabel with ED-1 (macrophage biomarker) in the ketamine-treated rat's bladder.[21] Thus, ketamine and its metabolites may wind up macrophages to express NF-κB and turn on the subsequent inflammatory cascades. Taken together, the overexpression of NF-κB could present at the urothelium, suburothelium, and muscle layer in this model.
The activation of the NF-κB pathway in the ketamine-treated rat's bladder disclosed the entity of severe inflammation, especially for neurogenic inflammation. Neurogenic inflammation of the bladder mucosa is a complex process triggered by the release of inflammatory peptides, such as substance P, calcitonin gene-related peptide, and neurokinin from the afferents in response to the noxious stimuli.[22] In our previous study, we reported that the ketamine and its metabolites could induce substance P and TRPV1 receptor overexpression in the bladder mucosa layer of this rat model.[9],[10] By observing patients with ketamine cystitis, Yang et al. suggested that the elevated TRPV1 and TRPV4 receptors could be associated with the severity of bladder dysfunction in KIC.[12] Transient receptor potential channels in the bladder involved in nociception and mechanosensory transduction, which can interact with bradykinin, neurokinin, and α-and-β adrenergic receptors to enhance neurogenic inflammation.[22] This study demonstrated that the increase of gene expression in Trpv3, Trpm1, Tacr3, Bdkrb1, Adra2b, and Adra2c of the bladder might be involved in the neurogenic inflammation of this rat model. After the activation of NF-κB, the downstream inflammatory signaling could enhance the gene expression on Il6, Ptgs2 (encoding cyclooxygenase [COX]-2), Ptgds (encoding prostaglandin D2 synthase), and Nos2 (encoding inducible nitric oxide synthase [iNOS]). Researchers suggested that the translocation of NF-κB and upregulation of COX-2 and iNOS play a crucial role in developing the ulcerative cystitis of KIC in rats.[21] Fan et al. also reported that serum interleukin-6 level is an activator of immune response in patients with KIC.[23] Our findings are consistent with the previous reports. In contrast to the early presentation of KIC in neurogenic inflammation and submucosa hemorrhage in the present study, Shen et al. reported that the fibrogenesis mechanism is prominent in the long-term KIC mouse model.[24]
The results of this study support the hypothesis that microvascular injury of the bladder is another pathogenesis of KIC.[1] Lin et al. suggested that ketamine could directly act on the N-methyl-D-aspartate receptor of bladder vessels to impair the microcirculation of the bladder.[25] The vesical vascular damages (i.e., tortuous in shape and thickness of basal membrane) of patients with KIC can be observed through electron microscopy. In the present study, an increase in Crhr2 gene expression in the bladder of ketamine-treated rats was shown. Acute bladder stress may stimulate hypothalamic–pituitary–adrenal axis through the release of corticotropin-releasing hormone, which may increase bladder vascular permeability and induce VEGF release.[26] In addition, Lu et al. suggested that sufficient VEGF might help the angiogenetic remodeling and bladder repair in ulcerative cystitis of ketamine-treated rats.[27]
This study has some limitations. First, this study has a small sample size (n = 3). The purpose of this study is to reproduce and validate the observations of previous studies. Thus, we demonstrated the traits of KIC in rats in a small number. Second, we did not validate the statistical results of NGS using quantitative real-time polymerase chain reaction. To keep the homogeneity and consistency of NGS results, the bladders were combined in each group for analysis, and a cut-off value of twofold was set. Third, someone may question the reliability of a preclinical study to represent the clinical features of KIC. Indeed, an animal model cannot completely represent the same features of human disease, especially for an uncertain dosage and period of ketamine abuse in patients with KIC.
ConclusionUsing daily intraperitoneal injection of ketamine for 28 days, this study reproduced ulcerative cystitis in rats. It is a reliable KIC model that can induce bladder overactivity and central sensitization, which mimic the clinical presentation of patients with KIC. This study further highlights the role of neurogenic inflammation and microvascular injuries in the pathogenesis of KIC.
Acknowledgment
We would like to thank the Department of Urology, Kaohsiung Chang Gung Memorial Hospital, for data collection.
Financial support and sponsorship
This work is supported by Grants MOST 104-2314-B-182A-081 and MOST 108-2314-B-182A-033-MY3 from the Ministry of Science and Technology of the Republic of China, and CMRPG8F1211, CMRPG8G1461, and CMRPG8J0271-73 from Chang Gung Medical Foundation and Chang Gung Memorial Hospital.
Conflicts of interest
Prof. Yao-Chi Chuang, an editorial board member at Urological Science, had no role in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.
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