Experimental AnimalsPub Date : 2025-01-10Epub Date: 2024-08-22DOI: 10.1538/expanim.24-0017
Yuki Ikai, Goro A Nagura-Kato, Shinsuke H Sakamoto, Akio Shinohara, Chihiro Koshimoto
{"title":"Optimization of inhaled anesthesia for Octodon degus using electroencephalography.","authors":"Yuki Ikai, Goro A Nagura-Kato, Shinsuke H Sakamoto, Akio Shinohara, Chihiro Koshimoto","doi":"10.1538/expanim.24-0017","DOIUrl":"10.1538/expanim.24-0017","url":null,"abstract":"<p><p>Physiological responses to inhaled anesthetics vary among species. Therefore, a precise anesthetic technique is important for each individual species. In this study, we focused on the degu (Octodon degus), a small herbivorous rodent. Degus have recently begun to be used as laboratory models for brain research because of certain human-like characteristics, such as spontaneous development of Alzheimer's disease. In this study, we evaluated appropriate induction and maintenance anesthesia conditions for isoflurane and sevoflurane in degus by a stimulation test, electroencephalography (EEG), minimum alveolar concentration (MAC), and vital signs. During induction, more rapid time to loss of the righting reflex and deeper anesthesia in degus were observed in isoflurane. The MAC value for degus were 1.75 ± 0.0% in isoflurane and 2.25 ± 0.27% in sevoflurane. Whereas some degus were awake during maintenance anesthesia using both anesthetics at concentrations of ≤2%, no rats were awake when using sevoflurane at a concentration of 2%. The duration of the total flat EEG, a measure of the depth of maintenance anesthesia, was longer for isoflurane than for sevoflurane. Furthermore, higher concentrations of both anesthetics suppressed the respiratory rate in degus. These new findings regarding inhalation anesthesia in degus will contribute to future developments in the fields of laboratory animals and veterinary medicine.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"93-103"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742480/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142016861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Experimental AnimalsPub Date : 2025-01-10Epub Date: 2024-08-08DOI: 10.1538/expanim.24-0028
Julio A Almunia, Yoshiko Munesue, Haruka Kawasaki, Kazumichi Takano, Chisato Kayahara, Satoko Noma, Nobuko Morikawa, Shumpei Niida, Noboru Ogiso
{"title":"Hematological and biochemical characterization of aging farm male rat strains in the national center for geriatrics and gerontology.","authors":"Julio A Almunia, Yoshiko Munesue, Haruka Kawasaki, Kazumichi Takano, Chisato Kayahara, Satoko Noma, Nobuko Morikawa, Shumpei Niida, Noboru Ogiso","doi":"10.1538/expanim.24-0028","DOIUrl":"10.1538/expanim.24-0028","url":null,"abstract":"<p><p>Laboratory rats, like mice, are a type of animal commonly used in scientific investigations as well as in basic aging and geriatric research. The selection of a rat strain is an important first step in the planning and design of an experiment due to physiological, anatomical, and ethological variations in each strain, which may significantly modify the expected results. In the present study, we characterized age-related changes, from 3 months old (mo) to 24 mo, in three male rat strains commonly used in medical research: RccHan<sup>®️</sup>:WIST (RccHan:WIST), F344/NSlc (F344), and Slc:SD Rat (SD). The body weight, water/food consumption, and survival rate of each strain were physiologically evaluated. Hematological and biochemical values were analyzed every three months. Hematological results showed a decrease in lymphocytes and increases in other leukocytes from 12 mo in F344 and SD rats. The incidence of hematological disorder was 10-15% in F344 and SD rats from 18 mo. Increases in hepatic biochemical parameters (alanine transaminase (GPT/ALT) and aspartate transaminase (GOT/AST)) and cytopathological parameters (creatine phosphokinase (CPK)) were observed in male F344 rats at 12 mo. Triglycerides (TG) serum levels were significantly elevated in the 12 mo RccHan:WIST rats, while Lipase (LIP) levels were significantly reduced in 24 mo. The present results revealed significant variations in hematological and biochemical values in the different laboratory male rat strains due to genetic and nutritional-metabolic factors specific to each strain.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"66-82"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742475/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Inhibition of tumor necrosis factor receptor-associated factor 6 alleviates secondary brain injury by reducing neuronal pyroptosis after intracerebral hemorrhage.","authors":"Qianxin Hu, Haixin Zeng, Chengao Feng, Wei Tian, Yuxin He, Bing Li","doi":"10.1538/expanim.24-0078","DOIUrl":"10.1538/expanim.24-0078","url":null,"abstract":"<p><p>Secondary brain injury (SBI) is one of the main causes of high mortality and disability rates following intracerebral hemorrhage (ICH). Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a crucial role in the process of pyroptosis, and modulating its expression may present a novel therapeutic strategy for mitigating brain injury. This study aims to explore the mechanisms of TRAF6 in pyroptosis after ICH. C57BL/6J mice were used to establish the ICH model. Brain was collected at different time points for q-PCR and western blot to detect the level of TRAF6. After the C25-140 (the TRAF6 inhibitor) was administrated, the mice were divided into four groups. Then, the neurological deficit, brain water content, and blood-brain barrier (BBB) damage were detected. Immunofluorescence and western blot were used to detect the level of pyroptosis proteins, and ELISA and q-PCR were used to detect the levels of IL-18 and IL-1β. TRAF6 expression was upregulated after ICH and was mainly expressed in neurons. Inhibition of TRAF6 expression with C25-140 alleviated neurological deficits and reduced brain edema after ICH. In addition, inhibition of TRAF6 also reduced the expression of pyroptosis inflammasomes such as GSDMD, NLRP3, and ASC, as well as neurological damage caused by IL-18 and IL-1β after ICH. TRAF6 regulates neuronal pyroptosis in SBI after ICH. Inhibition of TRAF6 may be a potential target for alleviating inflammatory damage after ICH.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"39-48"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742478/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Endothelial GATA3 is involved in coagulofibrinolytic homeostasis during endotoxin sepsis.","authors":"Moyuru Hayashi, Shin'ya Ohmori, Yoshiko Kawai, Takashi Moriguchi","doi":"10.1538/expanim.24-0079","DOIUrl":"10.1538/expanim.24-0079","url":null,"abstract":"<p><p>Sepsis-induced acute lung injury represents a significant threat to human health and is frequently associated with pulmonary thrombosis due to dysregulation of the coagulofibrinolytic system. Plasmin, the major protease that degrades fibrin aggregates, is activated predominantly by tissue-plasminogen activator (tPA), whereas tPA is negatively regulated by plasminogen activator inhibitor (PAI-1). Under septic conditions, the imbalance between coagulation and fibrinolysis results in excessive microthrombosis. Pulmonary capillary endothelial cells serve as a primary source of tPA and PAI-1. The molecular pathways regulating their expression levels depend on the differential activity of transcription factors. In this study, we elucidated the role of the zinc-finger transcription factor GATA3 in response to sepsis-induced pulmonary embolism. Endothelial cell-specific GATA3-deficient mice (G3-ECKO) presented increased susceptibility to bacterial endotoxin-induced pulmonary embolism, which was associated with increased PAI-1 expression levels and decreased tPA expression levels in the lungs. Septic lung extracts from G3-ECKO mice consistently presented decreased plasmin activity, which likely underlies the increased coagulation. These results demonstrate that GATA3 plays a protective role against bacterial endotoxin-induced pulmonary vascular embolism. Our findings will contribute to understanding the molecular mechanisms involving GATA3 in preventing pulmonary embolism.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"104-113"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742476/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142132233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chiaki Sugiura, Mao Sato, Shun Tanaka, Nobuaki Okumura, Akira Terao
{"title":"Royal jelly reduced non-rapid eye movement sleep fragmentation and restored sleep stability in diet-induced obese mice.","authors":"Chiaki Sugiura, Mao Sato, Shun Tanaka, Nobuaki Okumura, Akira Terao","doi":"10.1538/expanim.24-0128","DOIUrl":"https://doi.org/10.1538/expanim.24-0128","url":null,"abstract":"<p><p>Royal jelly (RJ) is recognized due to its high nutritional value and potential health benefits. Previous research showed that RJ supplementation decreased fat accumulation, resulting in weight loss and improvements in hyperglycemia and insulin resistance in high-fat diet (HFD)-induced obese mice. To expand the weight-reducing properties of RJ, this study aimed to investigate the effects of RJ supplementation on HFD-induced obese mice with impaired sleep stabilization. Over a 20-week period, the C57BL/6J mice were divided into the following dietary groups: normal diet (ND), ND supplemented with 5% lyophilized RJ powder (ND + RJ), HFD, and HFD supplemented with 5% lyophilized RJ powder (HFD + RJ) groups. Compared with the HFD group, the HFD + RJ group exhibited a significant reduction in body weight via a decrease in fat mass. Moreover, much like the ND group, the HFD + RJ group demonstrated improvements in the fragmentation of non-rapid eye movement (NREM) sleep and wakefulness. These processes contributed to the reestablishment of sleep/wake continuity and restored the overall stability of sleep. In contrast, the ND + RJ and ND groups exhibited a similar sleep/wake architecture. Thus, RJ supplementation in the ND demonstrated no substantial effect on sleep/wake. According to these findings, dietary RJ improves the sleep/wake architecture and restores sleep stability. Hence, RJ is a promising dietary component for addressing obesity and restoring sleep stability.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142978095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Experimental AnimalsPub Date : 2025-01-10Epub Date: 2024-09-07DOI: 10.1538/expanim.24-0050
Mao Sato, Chiaki Sugiura, Nobuaki Okumura, Akira Terao
{"title":"Melinjo (Gnetum gnemon L.) seed extract for treatment of sleep/wake fragmentation in diet-induced obese mice.","authors":"Mao Sato, Chiaki Sugiura, Nobuaki Okumura, Akira Terao","doi":"10.1538/expanim.24-0050","DOIUrl":"10.1538/expanim.24-0050","url":null,"abstract":"<p><p>Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been an integral part of an anti-obesity therapeutic regimen. To examine the relationship between anti-obesity and sleep, we explored the effect of MSE on sleep structure in high-fat diet (HFD)-induced obese mice. Although HFD did not alter the total amount of daily sleep, it significantly reduced the average duration of non-rapid eye movement (NREM) sleep and wakefulness episodes and significantly increased the number of these episodes. These findings indicate fragmented NREM sleep due to repeated brief awakenings in the HFD-fed mice. When 1% (w/v) MSE was given to HFD-fed mice, their weight or sleep structure were comparable to those of ND-fed mice, proving that dietary MSE completely hindered HFD-induced weight gain and sleep/wake fragmentation. Our data provide compelling evidence that MSE is a novel and promising dietary supplement that restores obesity-induced sleep architecture changes in mice.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"114-121"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742479/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142153497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Identification of gene mutations associated with the phenotype of short-limb mice emerging from a foundation colony of severely immunodeficient mice.","authors":"Yui Kaneko, Kayo Tomiyama, Masahiko Yasuda, Yuji Komaki, Tomoyuki Ogura, Riichi Takahashi, Masafumi Yamamoto","doi":"10.1538/expanim.24-0045","DOIUrl":"10.1538/expanim.24-0045","url":null,"abstract":"<p><p>Here, we report the identification of causative genes for limb-shortening in individuals repeatedly found in a population of severely immunodeficient NOG mice maintained via sibling mating. First, we conducted a pedigree survey to determine whether limb-shortening was a recessive genetic trait and then identified it using a crossing test. Simultaneously, the symptoms were identified in detail using pathological analysis. Accordingly, a mouse strain exhibiting a recessive trait caused by a single gene trait and similar symptoms was identified, suggesting growth differentiation factor 5 (Gdf5) as a causative gene. Genome walking via PCR and sequence analysis of Gdf5 revealed a deletion of approximately 1.1 kb from the latter half of exon 2 of Gdf5. Furthermore, we established NOG-Gdf5<sup>bpJic</sup> by removing other modified genes and confirmed that the inheritance pattern was reconfirmed semi-dominant. In recent years, regenerative medicine research using immunodeficient mice has been actively conducted, and this murine strain is expected to contribute to niche stem cell analysis and transplantation research.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"122-131"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742472/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142282552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Experimental AnimalsPub Date : 2025-01-10Epub Date: 2024-08-28DOI: 10.1538/expanim.24-0029
Xiaoqi Chang, Jiping Gao, Junting Yang, Yunhui Ma, Guohua Song
{"title":"The Chinese hamster as an excellent experimental animal model.","authors":"Xiaoqi Chang, Jiping Gao, Junting Yang, Yunhui Ma, Guohua Song","doi":"10.1538/expanim.24-0029","DOIUrl":"10.1538/expanim.24-0029","url":null,"abstract":"<p><p>Hamsters are valuable rodent models that are distinct from mice and rats. Currently, the main hamster species used for experimental research are the Syrian golden hamster and Chinese hamster, in addition to hamster species from other countries. Chinese hamsters are small, easy to run and feed, and inexpensive. They are prominent species found only in China and are part of the experimental animal resources of Chinese specialty. Chinese hamsters are distinguished by a black stripe on their back, short tail, pair of easily retractable cheek pouches, and pair of large drooping testes in males with 22 chromosomes. Due to their unique anatomical structure and biological features, Chinese hamsters have been used as a model in biomedical research. Moreover, the breeding and use of Chinese hamsters was comprehensively studied in 1958, with significant breakthroughs. We present a thorough review of the current developments and applications of Chinese hamsters and support the use of this species as a suitable and innovative experimental research model. With the success of Chinese hamster transgenic technology, this species will become more commonly employed in biological and medical research in the future.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"1-15"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742471/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142092562","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Clodronate liposome treatment contributes to the nerve regeneration in corneal nerve involvement of diabetic mice.","authors":"Hiroki Ueno, Takaaki Hattori, Hsi-Hua Chi, Yoshishige Miyabe, Masanori A Murayama","doi":"10.1538/expanim.24-0063","DOIUrl":"10.1538/expanim.24-0063","url":null,"abstract":"<p><p>The dense nerve and thin vascular structure of the corneal tissue provide the refractive function in healthy eyes. Diabetes mellitus causes ocular complications including corneal opacification because of corneal nerve degeneration. Diabetic neurotrophic keratopathy is characterized by reduced corneal sensitivity, delayed corneal wound healing, and nerve degeneration. Neurotization and vascularization inhibit each other in the cornea. Macrophages contribute to the corneal neovascularization. To investigate the role of macrophage in neurotrophic keratopathy, clodronate liposome was subconjunctivally injected into diabetic db/db mice with neurotrophic keratopathy. The clodronate liposome treatment decreased F4/80<sup>+</sup> macrophage infiltration into the corneal epithelium, and improved corneal nerve involvement in diabetic db/db mice. Furthermore, we found that Il1b and Il34 mRNA expression was increased in the corneal epithelium of clodronate-treated diabetic db/db mice. These cytokines contribute to the maintenance of nerve tissues via microglia and nerve regeneration; however, their role in corneal nerve involvement remains unknown. Notably, the intraocular injection of recombinant IL-1β and IL-34 promoted nerve regeneration in the cornea of diabetic db/db mice. These results suggest that clodronate liposome treatment contributes to nerve regeneration during corneal involvement via IL-1β and IL-34 signaling.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"58-65"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742477/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Experimental AnimalsPub Date : 2025-01-10Epub Date: 2024-08-08DOI: 10.1538/expanim.24-0027
Xiaolong Zhao, Longqi Shang, Chunjian Shen
{"title":"Daphnetin ameliorates diabetic cardiomyopathy by regulating inflammation and endoplasmic reticulum stress-induced apoptosis.","authors":"Xiaolong Zhao, Longqi Shang, Chunjian Shen","doi":"10.1538/expanim.24-0027","DOIUrl":"10.1538/expanim.24-0027","url":null,"abstract":"<p><p>Daphnetin has been demonstrated to exert beneficial effects on diabetes mellitus and renal complications. However, the role and molecular mechanism of daphnetin in diabetic cardiomyopathy (DCM) remain unclear. In this study, rats were injected with streptozotocin (STZ) to induce diabetes. The diabetic rats were then administered daphnetin (1 and 4 mg/kg) or dimethyl sulfoxide (DMSO) daily for 12 weeks. The results demonstrated that the diabetic rats exhibited elevated blood glucose levels, which were dose-dependently ameliorated by daphnetin. At 13 weeks following STZ injection, the rats exhibited typical diabetic signs, cardiac dysfunction, and evident pathological alterations in myocardial tissues. The administration of daphnetin to diabetic rats resulted in improvement in cardiac function, reductions in myocardial injury biomarkers, and the inhibition of myocardial fibrosis. Furthermore, daphnetin treatment suppressed inflammation and endoplasmic reticulum stress-induced apoptosis in a dose-dependent manner. Additionally, daphnetin exhibited partial blockade of the activation of mitogen-activated protein kinase pathways induced by diabetes. These findings indicate that daphnetin may be a promising therapeutic agent for the treatment of DCM.</p>","PeriodicalId":12102,"journal":{"name":"Experimental Animals","volume":" ","pages":"49-57"},"PeriodicalIF":2.2,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742473/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}