Contributed equally
Long-term ketamine abuse has been shown to affect the lower urinary tract and result in interstitial cystitis-like syndrome. However, the causative mechanism of ketamine-induced dysfunction remains unclear. The present study aimed to investigate the physiological, histological and molecular changes on ketamine-associated cystitis (KC) in a mouse model. Both male and female Balb/c mice were separately distributed into the control group (normal saline) and ketamine group, which received ketamine hydrochloride (100 mg/kg/day) daily by intraperitoneal injection for a total period of 20 weeks. In each group, the urine was analyzed by gas chromatography-mass spectrometry to measure the concentration of ketamine and its metabolites. Urinary frequency and urine volume were examined to investigate the urinary voiding functions. Mice bladders were excised for cDNA microarray and hematoxylin and eosin (HE) staining. The ketamine and metabolites were detected only in ketamine-treated mice urine. The voiding interval was reduced in the male mice group after 20 week ketamine administration. Additionally, the result of cDNA array analysis revealed a number of gene expression levels involved in chronic wound healing response and collagen accumulation, which were closely associated with fibrosis progression in the connective tissue. In HE staining of the bladder tissue, the ketamine-injected mice exhibited prominently denser blood vessel distribution in the submucosal layer. Based on the evidence in the present study, a mechanism that delineates fibrosis formation of urinary bladder induced by the pathogenesis of ketamine abuse can be constructed.
Ketamine, a clinical anesthesia, was first synthesized in 1961 (
To date, the underlying mechanisms of ketamine-induced urinary toxicity remain to be completely understood. However, several hypotheses on the association between ketamine and urinary tract damage have been raised based on clinical observations. The most straightforward and important one points out that ketamine and its metabolites in the urine may have a direct toxic effect on the bladder urothelium, leading to a chronic inflammatory response and subsequent interstitial cystitis-like symptoms (
In our previous microarray study, in which male Balb/c mice received 30 mg/kg/day ketamine injection for 2 months, the gene expression of keratin 14, which assembles with keratin 5 to form heterodimers and contribute to the intermediate filament cytoskeleton, was found to markedly decrease in the urothelial tissue (
Male and female (n=20 each) 6-week-old Balb/c mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animals were maintained at the qualified animal care facility of Biotechnology and Health Hall in National Chiayi University (Chiayi, Taiwan) for a 1 week period of acclimation. At 7 weeks of age, 10 male and 10 female mice were administered 100 mg/kg ketamine (Merial Laboratoire de Toulouse, Lyon, France) via intraperitoneal injection daily for 20 weeks. A control group of 10 male and 10 female mice were injected with normal saline. All mice were housed in polycarbonate cages, provided with food and water
Urine was collected three days prior to the final day of the 20th week. Ketamine and its metabolites were excreted primarily by the kidney, with an elimination half-life of around 2 h in rats (
This analysis was made one day prior to the final day of the 20th week. Urinary voiding quantity and interval were determined using the voided stain on paper (VSOP) method (
Following treatment for 20 weeks, the mice were euthanized by carbon dioxide inhalation and the bladders were removed. A total of 24 bladders (n=6/group) were fixed in 10% neutral formalin for 24 h and then embedded in paraffin and cut into 3
The Mouse Whole Genome OneArray® v2 (Phalanx Biotech Group, Hsinchu, Taiwan) contains 27,307 DNA oligonucleotide probes. Each probe is a 60-mer designed in the sense direction. Among the probes, 26,423 probes correspond to the annotated genes in RefSeq v42 (
Fluorescent aRNA targets were prepared from 1
In the present study, two genes [Collagen α-1 (III) chain (Col3a1) and Collagen α-2 (I) chain (Col1a2)] were selectively targeted. Each reaction included 20 ng cDNA, 500 nM forward and reverse primers, and 2X Fast SYBR Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). A total of 10
To interpret the biological functions of the DEGs between the control and ketamine-treated groups, Gene Ontology (GO) enrichment analysis was used to explore the functional distribution. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to analyze the DEGs to identify essential pathways involved in the microarray data.
Statistical differences were analyzed using one-way analysis of variance analysis, with the exception of the microarray data. All statistics were calculated using SigmaPlot version 12.5 (Systat Software, Inc., San Jose, CA, USA). Data are presented as the mean ± standard error of the mean with the exception of data in
At the dosage of 100 mg/kg/day, it was found that the mice at first displayed excitation within 1 min following ketamine injection. They gradually turned into anesthetic status in ~2 min and lasted for ~30 min. In awakening from the anesthesia, the mice first entered a semi-conscious state for ~10 min and then became fully awake. The rate of weight gain of the ketamine-treated mice became significantly slower following the second week compared with that of the control mice. This trend continued for the remaining period of the present study (
The concentrations of ketamine and ketamine metabolites (norketamine and dehydronorketamine) in urine were detected by GC-MS. These three types of urinary concentrations were undetectable in the control groups (
The voiding quantity and micturition interval were recorded within 2 h using the VSOP method. At week 20, the voiding interval of ketamine-treated male mice was found to be significantly smaller compared with that of control male mice, while no change was observed in the female group (
To determine the genes and pathways involved in ketamine-induced toxicity on urinary bladder, the total RNA was extracted from the bladders of the 20 week ketamine-treated mice and of the control mice. The bladdes were subsequently analyzed on the Mouse Whole Genome OneArray® v2 microarray chip containing 27,307 probes. The whole microarray data has been deposited in the GEO database (accession number, GSE68539). The standard selection criteria used to identify the DEGs were log2|fold change| ≥0.5 and P<0.05. The male and female mice data were normalized respectively and were included together to do the further calculation. Compared with the control group, 44 upregulated DEGs and 70 downregulated DEGs were identified for the ketamine-treated mice. To interpret the biological functions of the DEGs, GO enrichment analysis, which provides a common descriptive framework and functional annotation of gene sets data, was performed to examine the functional distributions and classification of the gene sets belonging to male and female mice individually. The GO categories were separated into three groups: Biological process, cellular component and molecular function. The top 15 enriched GO terms of the three categories are shown in
After checking the essential DEGs in detail, the present study listed certain critical genes that are all associated with connective tissue fibrogenesis (
Since two angiogenesis-associated genes were found to be upregulated in the result of microarray analysis, the bladder HE stain was used to confirm the vessel distribution. When compared with the control mice, the ketamine-injected mice exhibited prominently denser blood vessel distribution in the submucosal layer (
In the present study, a 20 week ketamine-injection mouse model was used to characterize the effects of long-term ketamine abuse on bladders by examining critical changes of gene expression. The aim was to explore the mechanisms for clinical management or therapeutic development. Through GC-MS quantification, high concentrations of ketamine and its metabolites were present in mice urine following injection (
In general, fibrosis originates from an abnormal wound healing process in which tissue repair is deregulated as a result of excessive ECM accumulation. According to the classical model of wound healing, the cellular course of events can be divided into certain sequential, yet overlapping phases, including inflammatory, proliferative and tissue remodeling stages (
Wound healing is a dynamic and complex process that requires cooperative regulation of angiogenesis and cell migration capacity (
In view of the aforementioned causes coupled to the deregulated wound healing process and excessive mRNA level for collagens, although no obvious occurrence of immune cells infiltration was confirmed, as well as collagen proteins accumulation, it was still concluded that the results of the DEGs clearly demonstrated the existence of fibrogenic actions at an early stage. These findings have important implications of the molecular mechanisms underlying ketamine-induced urinary bladder fibrosis and reveal novel targets for the future development of effective therapies.
differentially expressed genes
gas chromatography-mass spectrometry
gene ontology
hematoxylin and eosin stain
Kyoto encyclopedia of genes and genomes
mouse oligonucleotide DNA microarray
voided stain on paper
The present study was supported by grants from the National Science Council of the Republic of China, Taiwan (no. NSC101-2320-B-415-002-MY3) and from Chiayi Christian Hospital, Taiwan (no. R102-17).
Changes in the body weight of the mice. The weight growth of the ketamine-treated mice was significantly less compared with that of controls after the 2 week treatment. The data are presented by the mean ± standard error of the mean (**P<0.01; ***P<0.001 compared with the control).
A typical total ion current chromatogram derived from the extracted mice urine. The total run time was 14 min. Ketamine and its metabolites appeared around 10–11 min following the order of NK, deNK and K. No detected level was observed in both male and female control groups. NK, norketamine; deNK, dehydronorketamine; K, ketamine.
Voiding quantity and frequency of the mice. The data were recorded following voided stain on paper method at week 20 (n=8 for each group). *P<0.05.
Reverse transcription-quantitative polymerase chain reaction of two upregulated collagen genes. The mRNA samples extracted from four control and three ketamine group mice were assessed. The relative expression value of a gene was normalized against the expression of Actb from mice mRNA at week 20. The data are presented by the mean ± standard error of the mean.
HE staining images of the mice bladders. The ketamine-injected mice exhibited denser blood vessel distribution compared with the controls in (A) male and (B) female mice. The black arrows point the blood vessels. Images of the HE stained bladder tissues were captured by microscopy (magnification, ×400). HE, hematoxylin and eosin.
Masson's trichrome staining images of the mice bladders. The ketamine-injected mice exhibited no significant difference in the distribution of collagen proteins compared with the controls. Images of the bladder tissue were captured by microscopy (magnification, ×40).
Concentration of ketamine and its metabolites in mice urine.
Gender | K (ppm) | NK (ppm) | deNK (ppm) |
---|---|---|---|
Female | 108±9.9 | 336±17.1 | 1,332±66.0 |
Male | 173±9.1 | 599±8.4 | 1,286±13.7 |
The data are presented as the mean ± standard deviation. K, ketamine; NK, norketamine; deNK, dehydronorketamine.
Top 15 enriched GO terms of DEGs.
Male mice
|
Female mice
| ||||
---|---|---|---|---|---|
Gene set name | No. genes | P-value | Gene set name | No. genes | P-value |
Molecular functions | |||||
Receptor binding | 10 | 6.34E-06 | DNA binding | 15 | 3.29E-06 |
Transmembrane receptor activity | 10 | 1.55E-05 | Enzyme binding | 8 | 1.12E-05 |
Purine ribonucleotide binding | 7 | 3.29E-05 | Identical protein binding | 10 | 1.42E-05 |
Chemokine receptor binding | 4 | 3.62E-05 | Receptor binding | 11 | 1.62E-05 |
Purine nucleotide binding | 7 | 3.95E-05 | Phosphoric ester hydrolase activity | 7 | 3.57E-05 |
Receptor activity | 11 | 5.12E-05 | Ion binding | 9 | 3.75E-05 |
Transcription repressor activity | 6 | 5.45E-05 | Cation binding | 8 | 4.07E-05 |
Identical protein binding | 8 | 5.73E-05 | Transcription factor activity | 10 | 5.15E-05 |
Nucleotide binding | 7 | 5.76E-05 | Protein kinase activity | 9 | 5.22E-05 |
Adenyl ribonucleotide binding | 6 | 8.03E-05 | Protein homodimerization activity | 6 | 8.33E-05 |
Enzyme regulator activity | 8 | 8.74E-05 | Hormone activity | 4 | 1.30E-04 |
G protein coupled receptor binding | 4 | 8.93E-05 | Phosphotransferase activity alcohol group as acceptor | 9 | 1.73E-04 |
Adenyl nucleotide binding | 6 | 9.80E-05 | Receptor activity | 12 | 1.91E-04 |
Carbohydrate binding | 4 | 2.73E-04 | Hydrolase activity acting on ester bonds | 8 | 2.04E-04 |
Glycosaminoglycan binding | 3 | 4.27E-04 | Purine ribonucleotide binding | 7 | 2.22E-04 |
Biological process | |||||
Signal transduction | 39 | 2.71E-18 | Signal transduction | 42 | 8.98E-16 |
Muticellular organismal development | 27 | 1.23E-13 | Multicellular organismal development | 31 | 2.02E-13 |
Response to external stimulus | 14 | 1.10E-10 | Biopolymer metabolic process | 38 | 1.30E-12 |
Negative regulation of biological process | 19 | 1.54E-10 | Positive regulation of cellular process | 24 | 2.19E-12 |
Cell cell signaling | 15 | 3.25E-10 | Positive regulation of biological process | 24 | 7.58E-12 |
Negative regulation of cellular process | 18 | 5.37E-10 | Negative regulation of biological process | 23 | 1.96E-11 |
System development | 20 | 1.31E-09 | Negative regulation of cellular process | 22 | 5.22E-11 |
Anatomical structure development | 21 | 3.69E-09 | Protein metabolic process | 30 | 5.87E-11 |
System process | 16 | 3.85E-09 | System development | 25 | 7.10E-11 |
Intracellular signaling cascade | 17 | 6.20E-09 | Anatomical structure development | 27 | 8.01E-11 |
Immune system process | 12 | 2.70E-08 | Cellular protein metabolic process | 28 | 1.42E-10 |
Regulation of biological quality | 13 | 4.21E-08 | Cellular macromolecular metabolic process | 28 | 1.88E-10 |
Behavior | 8 | 3.74E-07 | Regulation of biological quality | 17 | 6.30E-10 |
Second messenger mediated signaling | 8 | 3.74E-07 | Cell development | 19 | 1.94E-09 |
Response to stress | 13 | 3.82E-07 | Biopolymer modification | 20 | 2.29E-09 |
Cellular component | |||||
Extracellular region | 15 | 1.29E-09 | Membrane | 46 | 1.77E-15 |
Membrane | 30 | 2.67E-09 | Cytoplasm | 47 | 4.46E-15 |
Intrinsic to membrane | 23 | 2.39E-08 | Membrane part | 36 | 1.94E-11 |
Membrane part | 25 | 7.07E-08 | Plasma membrane | 32 | 1.04E-10 |
Integral to membrane | 22 | 8.43E-08 | Cytoplasmic part | 30 | 9.19E-10 |
Plasma membrane | 22 | 2.76E-07 | Integral to membrane | 28 | 6.61E-09 |
Extracellular region part | 11 | 2.96E-07 | Intrinsic to membrane | 28 | 8.80E-09 |
Plasma membrane part | 18 | 3.20E-06 | Extracellular region | 15 | 7.94E-08 |
Cytoplasm | 25 | 5.86E-06 | Plasma membrane part | 24 | 1.12E-07 |
Integral to plasma membrane | 15 | 2.51E-05 | Intracellular organelle part | 23 | 7.14E-07 |
Intrinsic to plasma membrane | 15 | 2.95E-05 | Organelle part | 23 | 7.67E-07 |
Extracellular space | 7 | 1.01E-04 | Macromolecular complex | 19 | 3.79E-06 |
Collagen | 3 | 1.31E-04 | Nucleus | 24 | 4.51E-06 |
Cytoplasmic part | 16 | 3.55E-04 | Extracellular region | 11 | 5.85E-06 |
Golgi apparatus | 6 | 4.58E-04 | Nuclear part | 13 | 4.36E-05 |
GO, gene ontology; DEG, differentially expressed genes.
Enriched KEGG pathway of DEGs.
KEGG pathway | No. genes | P-value |
---|---|---|
Male mice | ||
Focal adhesion | 7 | 1.52E-05 |
MAPK signaling | 7 | 9.17E-05 |
Chemokine signaling | 5 | 9.28E-04 |
ECM receptor interaction | 5 | 2.04E-05 |
p53 signaling | 5 | 7.78E-06 |
Vascular smooth muscle contraction | 5 | 9.20E-05 |
Prion disease | 4 | 1.06E-05 |
TOLL-like receptor signaling | 4 | 7.02E-04 |
ABC transporters | 3 | 6.84E-04 |
Sphingolipid metabolism | 3 | 5.16E-04 |
Female mice | ||
Cytokine-cytokine receptor interaction | 10 | 1.27E-06 |
Pathways in cancer | 9 | 5.08E-05 |
Dilated cardiomyopathy | 7 | 4.78E-07 |
MAPK signaling | 7 | 4.58E-04 |
Chemokine signaling | 6 | 4.42E-04 |
Endocytosis | 6 | 3.63E-04 |
Arrhythmogenic right ventricular | ||
Cardiomyopathy | 5 | 4.42E-05 |
Hypertrophic cardiomyopathy | 5 | 7.57E-05 |
Oocyte meiosis | 5 | 3.01E-04 |
p53 signaling | 4 | 4.30E-04 |
KEGG, Kyoto encyclopedia of genes and genomes; DEG, differentially expressed gene; MAPK, mitogen-activated protein kinase; ECM, extracellular matrix.
Potential DEGs involved in fibrogenesis of ketamine-treated mice.
Gene ID | Gene symbol | Official full name | Fold change | P-value |
---|---|---|---|---|
57266 | Cxcl14 | Chemokine (C-X-C motif) ligand 14 | −0.787 | 9.49E-06 |
11687 | Alox15 | Arachidonate 15-lipoxygenase | −0.684 | 2.66E-03 |
59289 | Ccbp2 | Chemokine binding protein 2 | −0.620 | 7.91E-05 |
12772 | Ccr2 | Chemokine (C-C motif) receptor 2 | 0.939 | 1.05E-01 |
20306 | Ccl7 | Chemokine (C-C motif) ligand 7 | 0.820 | 1.04E-01 |
12825 | Col3α1 | Collagen, type III, α1 | 0.676 | 2.35E-03 |
11839 | Areg | Amphiregulin | 0.664 | 1.42E-02 |
12843 | Col1α2 | Collagen, type I, α2 | 0.654 | 4.06E-04 |
68588 | Cthrc1 | Collagen triple helix repeat containing 1 | 0.609 | 2.25E-02 |
13614 | Edn1 | Endothelin 1 | 0.595 | 1.04E-02 |
11504 | Adamts1 | A disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 1 | 0.548 | 5.84E-03 |
12832 | Col5α2 | Collagen, type V, α2 | 0.521 | 2.43E-02 |
18613 | Pecam1 | Platelet/endothelial cell adhesion molecule 1 | 0.517 | 1.10E-02 |
The fold change value is adopted by log2. DEG, differentially expressed gene.