Purpose: Gemfibrozil (GEM) apart from agonist activity at peroxisome proliferator-activated receptor-alpha (PPAR-α) has antioxidant and anti-inflammatory properties. Accordingly, the present study was designed to investigate the protective effect of GEM on acute liver toxicity induced by acetaminophen (APAP) in mice.Methods: In this study, mice divided in seven groups include, control group, APAP group, GEM group, three APAP groups pretreated with GEM at the doses of 25, 50 and 100 mg/kg respectively and APAP group pretreated with N-Acetyl cysteine. GEM, NAC or vehicle were administered for 10 days. In last day, GEM and NAC were gavaged 1 h before and 1 h after APAP injection. Twenty four hours after APAP, mice were sacrificed. Serum parameters include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver tissue markers including catalase enzyme activity, reactive oxygen species (ROS), malondialdehyde and reduced glutathione (GSH) levels determined and histopathological parameters measured.Results: GEM led to significant decrease in serum ALT and AST activities and increase in catalase activity and hepatic GSH level and reduces malondialdehyde and ROS levels in the liver tissue. In confirmation, histopathological findings revealed that GEM decrease degeneration, vacuolation and necrosis of hepatocytes and infiltration of inflammatory cells.Conclusion: Present data demonstrated that GEM has antioxidant properties and can protect the liver from APAP toxicity, just in the same pathway that toxicity occurs by toxic ROS and that GEM may be an alternative therapeutic agent to NAC in APAP toxicity. This drug has few side effects at therapeutic doses5 but, overdose of APAP can cause liver toxicity.7 The mechanism of toxicity of this drug is because of the production of toxic metabolites, mitochondrial dysfunction and change the innate immune system.11 Liver is a main critical organ that metabolizes APAP in therapeutic doses in the form of glucuronidated and sulfated metabolites and the following metabolites excreted by urine.5 Approximately 2% excreted unchanged in the urine and 5% -10% through cytochrome P450 converted to the N-acetyl-p-benzoquinoneimine (NAPQI) which quickly reduced by GSH and excreted in the urine.12 NAPQI is a strong electrophile oxidizing agent that normally detoxified by GSH in the liver.15 But, in APAP overdose glucuronidation and sulfation pathways become to saturate and APAP metabolism by the cytochrome P450 produces a large amount of NAPQI that leads to rapid depletion of hepatic GSH levels.17 NAPQI causes impairs intracellular calcium homeostasis, increased cell permeability, reduced the integrity of cells membrane.15 It has been reported that released ATP, NAD and damage-associated molecular pattern (DAMP) from damaged hepatocytes can induced liver injury through P2X7 and Toll-like receptors in DAMP sensing cells.18 According the destructive effects of APAP in overdose and poisoning, new potential therapeutics for APAP overdose are routinely investigated in preclinical studies. Many of these studies have shown that pretreatment or simultaneous treatment with diverse agents can provide protection against APAP hepatotoxicity.20 Antioxidant agents have therapeutic potential in drug-induced toxicity.21 NAC is used as present treatment for APAP toxicity, by replacing cellular GSH to prevent of cell damage by NAPQI. Administration of NAC is critical to improving the clinical effect of APAP hepatotoxicity.22 Therefore, investigations on agents that have been therapeutic effect on APAP toxicity are attractive.Liver diseases have become one of the main causes of morbidity and mortality in around the world and hepatotoxicity because of drugs appears to be the most common causal issue.24 The most important mechanism of fibrates is considered as agonists of PPAR-α.25 But, has recently reported that GEM has antioxidant and anti- inflammatory properties in diverse situations.28 In therapy for diabetes has shown that GEM increase the level of paraoxonase activity as an antioxidant enzyme which clean up free radicals and chelate metal ions;29 as well as can inhibit the pathways leading to inflammatory cytokine release,30 and also has shown that decreased atherosclerosis in diabetes via reduction in oxidative stress and inflammation.31 Others fibrates, have shown protective effects against inflammatory colitis,32 inflammatory heart in autoimmune myocarditis33 and neuroprotective effects against stroke.34 In contrast with these studies, there is other reports that GEM weather alone or in combination with statins is associated with the highest risk of myotoxicity as well as GEM causes to increase risk of cholelithiasis and cholestatic jaundice.36 Therefore, considering the importance of APAP toxicity and finding new therapeutic agents against its toxicity, GEM was selected according to its demonstrated properties. With these themes, the present study was designed to investigate the effects of GEM on acute hepatotoxicity induced by APAP in mice, mainly focusing on GEM antioxidant properties.GEM is a fibrate drug that has approved for hyperlipidemia and commonly prescribed for lipid-lowering.Experiments were performed on adult male NMRI mice aged 6-8 weeks old, weighing 23 to 27g. The animals were purchased from the Research Center and Experimental Animal House of Ahvaz Jundishapur University of Medical Sciences (AJUMS). They were retained in standard Plexiglas cages with room temperature (22-25 °C) with a 12 h light/dark cycle. During the study all animal have free access to food and water. The animals were acclimatized to the environment for three days before the initiation of research.DCFDA) were purchased from Sigma-Aldrich . GEM was donated by Dr. Abidi pharmaceutical company , ALT and AST activity assay kits were obtained from . Other chemicals used were of the highest grade available.APAP, thiobarbituric acid (TBA), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and 2’,7’ –dichlorofluorescin diacetate  at a dose of 400 mg/kg/10ml after 16 h overnight fasting to deplete hepatic GSH levels and thereby induce hepatotoxicity better.The mice were divided into seven groups (seven mice in each group).Received vehicle  + a single dose of saline Received vehicle  + a single dose of APAP Treated with GEM  + a single dose of APAP Treated with GEM  + a single dose of APAP Treated with GEM  + a single dose of APAP Treated with NAC  + a single dose of APAP Administered GEM  + a single dose of saline The animals treated with GEM, NAC or vehicle orally once daily for 10 days and then received a single dose APAP. In tenth day, GEM and NAC were gavaged 1 h before and 1 h after APAP. At the end of the experimental period (24 hour after APAP administration), the animals were anaesthetized and blood samples were taken from heart. The serum was separated using centrifuge at 3500 rpm for 20 min for biochemical evaluation. Liver was removed and divided into two portions, one section homogenized for liver tissue biochemical tests and another part fixed in 10% formalin for histopathological assessments.Serum samples were used for measuring AST, ALT activity by using standard assay kits through auto analyzer .g for 30 min at 4°C. The supernatant was used for assessment.Liver tissue was homogenized 10% (w/v) with phosphate buffer , then centrifuged at 12,000×2O2 and 50 µl of tissue extract  were mixed and incubated for 10 min, then 500 µl Ammonium molybdate 4% was added and absorbance was measured at 410 nm. The result was expressed as U/g tissue.37Catalase activity was determined by a slightly modified version. Accordingly, 500 µl of tris-HCl 0.05 mmol was added per one ml H38 For this assessment, 1 ml supernatant was added to 2 ml TBA and placed in 100 °C for 15 min. After cooling, it was centrifuged  and the supernatant separated. Ultimately, the MDA level was reported as nmol/g tissue.39Lipid peroxidation in the liver was measured based on the MDA level. The reaction of MDA with TBA produces a purple color with maximum absorbance at 532 nm.40The GSH level was measured using the Ellman’s reagent. Briefly, trichloroacetic acid 20% along with EDTA 1 mM was added to homogenate tissue. Then, it was centrifuged  and the isolated supernatant (200 μl) was added to 1.8 ml DTNB 0.1 mM. The absorbance was read at 412 nm by spectrophotometer and GSH level was reported as nmol/g tissue.41DCFDA was used for ROS assay in the liver tissue. Cellular peroxides convert DCFDA into highly fluorescent DCF. Briefly, 10% liver homogenate was prepared in ice-cold Tris–HCl buffer 40 mM (pH 7.4). Then homogenate tissue was mixed with 1.25 mM DCFDA in methanol for ROS evaluation. All samples were incubated for 15 min in a water bath at 37 °C. Measurement was determined based on the intensity of fluorescence at 488 nm excitation and 525 nm emission wavelength using a fluorometer  and reported as fluorescence intensity unit (FIU).For evaluation of microscopic changes, the liver was fixed in 10% formalin. Then, it was dehydrated through soaking in alcohol and xylol, respectively. Finally, after preparation of 5μ- tissue sections using rotary microtome, the hematoxylin and eosin (H&E) staining technique was performed. The histopathological changes were examined using light microscope.Statistical analyses were performed with SPSS software, version 16.0 . Continuous variables are expressed as mean ± SEM. Comparison of mean value was performed by one-way analysis of variance followed by with the Tukey’s post hoc test. Graphs were plotted using GraphPad Prism software. Statistical significance was set at p <0.05.The results shown in As shown in  significThe results of lipid peroxidation in  showed tAs shown in  catalaseThe results in  indicateHistopathological findings in control liver tissue indicated that hepatocyte cells, lobules and sinusoid status are normal and do not show any damage. But, in APAP group degeneration of hepatocytes, vacillation of vessels, the necrosis of hepatocytes, infiltration of inflammatory cells, and dilated sinusoids were observed . In mice16 and the protection of liver via antioxidant activity or inhibition of the generation of free radicals are important in the protection against APAP-induced liver injury.42 Furthermore, pretreatment of mice with GEM 25, 50 and 100 mg/kg against APAP–induced acute hepatotoxicity indicates that GEM ameliorates liver damage via augmentation of liver antioxidant function. Suppression of oxidative stress confirmed by catalase activity and ROS level as well as GSH and MDA levels in liver. It has been shown that different doses of GEM have protective effect in brain damage43 as well as indicated that GEM has protective effect against the acute restraint stress-induced disturbances in hippocampus in male rat44 and also demonstrated that GEM causes to decrease atherosclerosis in diabetes via reduction in oxidative stress and inflammation.31In the present study, the liver cell injury by a single dose of APAP was associated with significant increase in serum ALT and AST activities while GEM caused to significant decrease in serum ALT and AST activities. In addition, NAC at the dose 100 mg/kg significant attenuates increasing in activities of these two enzymes due to APAP intoxication. Increasing serum activity levels of transaminases (AST and ALT) have been indicated to liver tissue dysfunctions because these are normally located in the cytoplasm and leakage occurs into the circulation after cellular damage. Normalization serum activity levels of these two enzymes by GEM indicating protection of liver cells. APAP induced injury by its free radical metabolites27GEM possess profound anti-inflammatory and antioxidant activity which, antioxidant properties dependent or independent to PPAR α/β receptors.45 GSH is the major intracellular defense molecule against ROS and prevents of oxidative stress in cells. In APAP toxicity GSH causes to detoxifying NAPQI and prevents of oxidative damage to hepatocytes.47 Therefore intracellular GSH levels are crucial in protecting from the toxic metabolite of APAP.In APAP toxicity, oxidative stress plays a key role in APAP-induced hepatotoxicity. Increased ROS levels leads to thiol oxidation that as a result decrease cellular GSH level and reduce activity of catalase enzyme function.49 In our study GEM at used doses caused a significant enhance in GSH level compared with APAP group. However, pretreatment with GEM showed an increase in GSH levels that likely inhibits formation of NAPQI or directly reacts with NAPQI and/or prevents of its reaction with proteins  and hydrogen peroxide (H2O2). GEM in all used doses decreased ROS level and this implies antioxidant properties of GEM .47 Catalase enzyme activity significantly decreased in liver 24 hours after APAP administration and GEM in all used doses increased catalase activity in APAP treated mice. This reveals increasing GSH level and alleviation ROS level by GEM  in mice, as shown by a decrease in serum liver enzyme activities AST, ALT levels and also MDA and ROS levels and also increased in GSH level and catalase activity. Moreover, the liver tissue morphology and histopathology findings confirmed the protective activity of this drug against the APAP-induced hepatotoxicity. It is evident by the reversal of degenerative of hepatocytes, vacillation of vessels, the necrosis of hepatocytes, infiltration of inflammatory cells, and dilated sinusoids in hepatic parenchyma by GEM administration. Although this protective effect was dose-dependent, there was no significant difference between doses of 25 and 50. But, according to the results of biochemical and pathological, our study indicates that the highest protective effect is related to GEM at the dose 100 mg/kg. Animal received dose 100 mg/kg GEM and also NAC did not have any differences with control group based on biochemical and histopathological findings and did not cause liver damage. It has been reported that induction of PPAR-α attenuates the extent of oxidative stress in steatohepatitic mice receiving APAP and has hepatoprotective effect due to high repair of liver tissue but not due to suppression of APAP bioactivation.In conclusion, we have shown that GEM has therapeutic effect along with antioxidant activity in APAP-induced hepatotoxicity. Our results propose that GEM with antioxidant properties can protect the liver from the oxidative damage induced by toxic chemicals. GEM may be an alternative therapeutic agent to NAC in APAP toxicity. However, more investigations are needed to realize the mechanism of GEM effects especially modulation and activity of PPAR-α receptors.This paper is issued from a Master of Science thesis in Toxicology of Hojatolla Nikravesh and was financially supported by Toxicology Research Center of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. (Grant No: TRC-9406).The study was performed according to the Animal Ethics Committee guidelines of AJUMS for the use of experimental animals (code of ethics: IR.AJUMS.REC.1394.602).


Intracranial and intraspinal tumours are the most numerous solid tumours in children. Some recently defined subtypes are relatively frequent in childhood. Many cancer registries routinely ascertain CNS tumours of all behaviours, while others only cover malignant neoplasms. Some behaviour codes have changed between revisions of the International Classification of Diseases for Oncology, including pilocytic astrocytoma, downgraded to uncertain behaviour in ICD-O-3.We used data from the population-based National Registry of Childhood Tumours, which routinely included non-malignant CNS tumours, to document the occurrence of CNS tumours among children aged < 15 years in Great Britain during 2001–2010 and to document the descriptive epidemiology of childhood CNS tumours over the 40-year period 1971–2010, during which several new entities were accommodated in successive editions of the WHO Classification and revisions of ICD-O. Eligible cases were all those with a diagnosis included in Groups III (CNS tumours) and Xa (CNS germ-cell tumours) of the International Classification of Childhood Cancer, Third Edition. The population at risk was derived from annual mid-year estimates by sex and single year of age compiled by the Office for National Statistics and its predecessors. Incidence rates were calculated for age groups 0, 1–4, 5–9 and 10–14 years, and age-standardised rates were calculated using the weights of the world standard population.Age-standardised incidence in 2001–10 was 40.1 per million. Astrocytomas accounted for 41%, embryonal tumours for 17%, other gliomas for 10%, ependymomas for 7%, rarer subtypes for 20% and unspecified tumours for 5%. Incidence of tumours classified as malignant and non-malignant by ICD-O-3 increased by 30 and 137% respectively between 1971-75 and 2006–10.Total incidence was similar to that in other large western countries. Deficits of some, predominantly low-grade, tumours or differences in their age distribution compared with the United States and Nordic countries are compatible with delayed diagnosis. Complete registration regardless of tumour behaviour is essential for assessing burden of disease and changes over time. This is particularly important for pilocytic astrocytoma, because of its recent downgrading to non-malignant and time trends in the proportion of astrocytomas with specified subtype. Cancer Incidence in Five Continents have always been restricted to malignant neoplasms [International Incidence of Childhood Cancer [Intracranial and intraspinal tumours are the most frequent solid tumours in children. Age-standardised incidence at age 0–14 years in industrialised countries during the 1980s–90s was typically in the range 30–40 per million . Successeoplasms , 8. By cd Cancer  includedIn the past three decades, new types of intracranial and intraspinal neoplasms have been defined, including several that are relatively frequent in childhood –13 Tabl. Some arThere are few detailed accounts of the occurrence of childhood CNS tumours based on large series from population-based cancer registries using data coded to ICD-O-3 , 18.This study has two principal aims. The first is to give a detailed and up to date account of the occurrence of childhood intracranial and intraspinal tumours based on data from a large, population-based, specialist children’s tumour registry, including information on subtypes that were not assigned specific morphology codes until ICD-O-3. The second is to use data from the same registry to document the descriptive epidemiology of childhood CNS tumours over a longer period during which new entities were accommodated in successive editions of the WHO Classification –12 and rThe population-based National Registry of Childhood Tumours (NRCT) covered the population under 15 years of age throughout Great Britain  during 1962–2010. Sources of notification varied slightly over the years but always included the network of regional and national cancer registries covering the population of all ages in the whole of Great Britain, together with death certificates for children with a neoplasm as underlying cause of death. Cases were also ascertained from specialist children’s tumour registries in certain regions of England. Since 1977 increasing numbers were notified directly from paediatric oncology principal treatment centres –16, withFor this study we included all children who were under the age of 15 years and resident in Great Britain when diagnosed with a tumour included in ICCC-3 Group III ‘Central nervous system and miscellaneous intracranial and intraspinal neoplasms’ or Subgroup Xa ‘Intracranial and intraspinal germ-cell tumours’ during 1971–2010. The main analyses are restricted to cases of a first or only tumour in these ICCC-3 categories, but brief details are also given of the small number of tumours diagnosed in children with a previous cancer diagnosis where it was clear from diagnostic and other reports that these were distinct primary tumours. The detailed description of incidence is based on children diagnosed during 2001–10. Data from the full 40-year study period were used to describe changes in recorded incidence and relative frequency over time and to show the effects of restricting coverage to cases defined as malignant by successive revisions of ICD-O.The population at risk was derived from annual mid-year estimates by sex and single year of age compiled by the Office for National Statistics and its predecessors. The average population at risk was 10.5 million children during 2001–10 and 11.1 million children during 1971–2010.0 + 4r1 + 5r2 + 5r3, where r0, r1, r2 and r3 are the age-specific rates for ages 0, 1–4, 5–9 and 10–14 respectively [Registration rates were calculated per million child-years at risk for age groups 0, 1–4, 5–9 and 10–14 years [Table Ependymomas accounted for 7% of registrations. This subgroup included ependymoblastoma, although it is usually regarded as a type of embryonal tumour, because it shares a morphology code with the more frequently occurring anaplastic ependymoma. Anaplastic ependymomas and ependymoblastoma accounted for 37% of ependymomas, myxopapillary ependymoma for 10% and other specified subtypes for 2%; 51% were of unspecified subtype. Ependymoma was generally most frequent at age 1–4 years, but 68% of myxopapillary ependymomas were diagnosed at age 10–14. Choroid plexus tumours accounted for 3% of registrations. Of these, 56% were benign or unspecified papillomas, 12% were atypical choroid plexus papillomas and 32% were carcinomas. All types of choroid plexus tumour were most often seen in the first year of life.Astrocytomas were the largest diagnostic subgroup, 41% of all registrations. Fifty percent of all astrocytomas were pilocytic. Subependymal giant cell astrocytoma (SEGA), fibrillary astrocytoma and pilomyxoid astrocytoma each accounted for around 2%, pleomorphic xanthoastrocytoma for 1% and other rare low-grade (WHO Grade I and II) subtypes for 0.3%; 5% of astrocytomas were anaplastic, 9% were glioblastoma and 28% were of unspecified subtype. Among the 471 unspecified astrocytomas, 249 (53%) were optic nerve gliomas. If all optic nerve gliomas are assumed to have been low-grade, 73% of all astrocytomas were low-grade, 14% were high-grade (WHO Grade III-IV) and 13% were of unspecified grade. Low-grade astrocytomas predominated in all age groups. The highest incidence of pilocytic astrocytoma and unspecified optic nerve astrocytoma was at age 1–4 years. Pleomorphic xanthoastrocytoma was exceedingly rare before age 10 years, whereas by far the highest incidence of pilomyxoid astrocytoma was in the first year of life. For both subtypes of high-grade astrocytoma, anaplastic astrocytoma and glioblastoma, there was an early peak of incidence in the first year of life, a nadir at age 1–4 and somewhat higher incidence at age 5 years and over.Other gliomas accounted for 10% of registrations. The great majority, 80%, were of unspecified subtype. Nearly all the remainder were oligodendrogliomas (9%), mixed gliomas (5%) or gliomatosis cerebri (3%). Incidence was highest at age 5–9 years.Embryonal tumours accounted for 17% of registrations. Within this subgroup, 73% were medulloblastoma, 15% were embryonal CNS tumour NOS and 11% were atypical teratoid/rhabdoid tumours (ATRT). Incidence of medulloblastoma was highest at age 1–9 years, whereas both embryonal CNS tumour NOS and ATRT were most frequent in the first year of life. Three quarters of medulloblastoma were of classical or unspecified subtype, 16% were desmoplastic/nodular or with extensive nodularity, and 7% were large-cell/anaplastic. Incidence of the desmoplastic/nodular subtype was highest at 1–4 years of age whereas the age distribution of the large-cell/anaplastic subtypes was similar to that of classical/unspecified medulloblastoma, with highest incidence at age 5–9 years.The subgroup of other specified tumours, excluding germ-cell tumours, accounted for 14% of registrations. Mixed neuronal-glial tumours represented 39% of the subgroup; within this category, 43% were gangliogliomas, 41% were dysembryoplastic neuroepithelial tumours (DNET), and 9% were desmoplastic infantile astrocytoma (DIA). Tumours of the sellar region (craniopharyngiomas) represented 33% of the subgroup, pituitary adenomas 12%, pineal parenchymal tumours 8% and meningiomas 9%. Of the craniopharyngiomas, 41% were adamantinomatous, one (< 1%) was papillary, and 58% were of unspecified subtype. Among pituitary adenomas, 33% were prolactinomas, 9% were basophil adenomas and 58% were unspecified.Germ-cell tumours accounted for 4% of registrations. They were mostly germinomas (70%) or teratomas (25%). Germinomas occurred predominantly in the 10–14 age group whereas teratomas were most frequent in infancy.Finally, 5% of registrations were for tumours of unspecified type, mostly without microscopic verification.The great majority  of tumours in this series were intracranial, but there were also 159 intraspinal tumours, accounting for 3.8% and with an ASR of 1.6 per million. They comprised intramedullary and extramedullary intradural tumours, together with extradural ependymomas since the latter are included in ICCC-3 Subgroup IIIa and occur at midline sites below the head. The most frequent types of intraspinal tumour were astrocytoma (47%) and ependymoma (26%).There were 14,281 registrations during the entire 40-year study period. Table The ASR for total intracranial and intraspinal tumours rose from 25.7 per million in 1971–75 to 41.3 per million in 2006–10, an increase of 60% over 35 years. The corresponding increases in age-specific rates  than for astrocytomas of other or unspecified subtype , and higher for optic nerve astrocytomas  than for other sites . From 1991 onwards, pilocytic astrocytoma was the most frequent single histological type of tumour  of all cases and 6/40 (15%) in children aged under 15 had a final diagnosis of pilomyxoid astrocytoma .DIA, sometimes formerly known as desmoplastic infantile ganglioglioma, is also classified as a glioneuronal tumour. As its name suggests, it is overwhelmingly a tumour of very early life. About 5% of all CNS tumours in infants were DIA in the present series. All other tumour types that were new to ICD-O with the third edition  or its fThe patterns of incidence described here for Britain are broadly similar to those observed in recent national registration data from France , the UniThe excess in the United States was largely attributable to ICCC-3 subgroups IIId (‘Other gliomas’) and IIIe (‘Other specified tumours’) Table . Within In France, incidence of oligodendroglioma was considerably higher than elsewhere and incidence of astrocytoma correspondingly lower, perhaps because French pathologists are more influenced by the St Anne classification in which some diffuse or anaplastic astrocytomas are considered as oligodendrogliomas . Total iIn the four Nordic countries of mainland Europe during 1985–2006, when incidence of childhood CNS tumours was stable over time, the ASR for ICCC-3 group III (i.e. excluding germ-cell tumours) was 42.0 per million , compare. Embryonal tumours accounted for a higher proportion of registrations (21%) than in Britain (17%). Astrocytomas also had a higher relative frequency than in Britain (45% vs 41%), offset by lower relative frequencies of other gliomas (7.5% vs 9.5%) and unspecified tumours (1.4% vs 4.7%). More detailed data on CNS tumours in the GCCR have been published for the periods 1990–9 and 1980–99 [The German Childhood Cancer Registry (GCCR) registered 4270 tumours in ICCC-3 Group III and subgroup Xa during 1998–2007 . Embryon 1980–99 . CompareMany cancer registries, while generally restricting their coverage to malignant neoplasms, also systematically collect data on non-malignant tumours of certain sites because of their pre-malignant status, or because they are potentially lethal and the distinction from malignant tumours can be difficult and opinion as to malignancy has changed over time. The former category is exemplified by tumours of the bladder and cervix. CNS and other intracranial tumours are the most important example of the latter. Some important childhood tumours of these sites, including craniopharyngioma, have always been classed as non-malignant, and will thus have been consistently excluded from presentations of data that are limited to malignant tumours. Many population-based cancer registries in Europe, and all specialist childhood cancer registries, have traditionally registered all CNS tumours regardless of behaviour. By contrast, most state cancer registries in the United States, notably those affiliated to the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute, did not systematically record or publish data on non-malignant CNS tumours . HoweverWhile non-registration of tumours that have always been regarded as non-malignant leads to underestimates of both incidence and survival for total CNS tumours, the effect is at least reasonably constant over time. Comparability and continuity of data over time are disrupted if the definition of malignancy changes. The data presented here illustrate the severity of the problem when pilocytic astrocytoma, the most frequent of all childhood CNS tumours, undergoes a change of behaviour code. It would not be sufficient to report just the tumours that were coded as malignant in ICD-O-3 because unspecified astrocytoma has always been coded as malignant and it is clear that higher proportions of pilocytic astrocytomas were recorded as unspecified astrocytoma in earlier years. The effect of inclusion/exclusion of pilocytic astrocytoma from survival estimates was illustrated in the EUROCARE-4 study, where 5-year survival for children in Europe with astrocytoma diagnosed in 2000–02 was 63% when tumours specified as pilocytic were excluded and 78% when they were included . In EUROThe present study concerns childhood tumours but it is equally important that non-malignant tumours diagnosed at older ages are registered. In England during 1995–2003, when pilocytic astrocytoma was still coded as malignant, 17% of CNS tumours diagnosed at age 0–14 were of uncertain, borderline or benign behaviour, compared with 41% at age 15–24 and 42% at age 25–84 . MeningiIn common with many other countries, the British registration data for childhood CNS tumours showed an increase in recorded incidence over time for most of the study period. While there have been major shifts in assignment of specific tumour types to malignant or non-malignant categories over the years, no entities have switched from neoplastic to non-neoplastic or vice versa in the ICD. Therefore, changes in recorded incidence of CNS tumours overall or of any subtype must be due to changes in underlying risk, changes in the proportion actually diagnosed before the 15th birthday, or changes in completeness of ascertainment of diagnosed tumours by the registration system. A study of time trends in registration rates for all types of childhood cancer in Britain between 1966 and 2005 found that changes in incidence of CNS tumours considered as a single group correlated well with contemporaneous changes in diagnostic and registration practice . It did Within the broad group of astrocytomas, optic nerve tumours increased in incidence more steeply than those of other sites Table . MoreoveThis study has several considerable strengths. The results are derived from a very large population-based data set. Non-malignant tumours were eligible and routinely included throughout the study period. Access to diagnostic reports for the great majority of cases allowed a high degree of specificity in coding, including the possibility of manual recoding of tumours when updating to successive revisions of the coding system. Even so, in some cases it was impossible to obtain evidence regarding whether a tumour was microscopically verified, so the MV percentages in Table The most important limitation is that central diagnostic review is impracticable for such a large national series, hence accuracy of diagnosis is an obvious concern. During 2001–10, childhood brain tumours were diagnosed pathologically in 21 hospitals throughout Britain, and the number in earlier years was much greater (data not shown). The potential effects are illustrated by the results of pathology review in a Children’s Cancer Group study of high-grade gliomas in North America, in which only 69% of tumours with an institutional diagnosis of high-grade glioma were confirmed as high-grade glioma by the review panel, and levels of agreement for specific tumour types such as anaplastic astrocytoma and glioblastoma multiforme were even lower . In a UKWhile this study documents the evolving patterns of recorded incidence of childhood CNS tumours in successive quinquennia over a 40-year period, it is not a formal analysis of time trends and confidence intervals for rates are not provided.In the absence of central review, the data presented here give the most accurate indication of childhood CNS tumour incidence based on histological criteria. Many tumours, however, would likely be reclassified on review using the genotypic criteria of WHO 2016, e.g. some cases of brainstem glioma or diffuse intrinsic pontine glioma could be redefined as diffuse midline glioma H3 K27 M-mutant . UniversTotal incidence of childhood CNS tumours in Britain in the first decade of the new millennium was fairly similar to that in other large western countries. Deficits of some, predominantly low-grade, tumours or differences in their age distribution compared with the United States and the Nordic countries are compatible with delayed diagnosis in Britain. Complete registration of all types of CNS tumour, regardless of behaviour, is essential for assessing burden of disease and changes over time in incidence and survival. This is particularly important for pilocytic astrocytoma, the most common of all childhood CNS tumours, because of the recent change in its behaviour code from malignant to non-malignant and changes over time in the proportion of astrocytomas for which subtype has been specified.


Ageing and obesity independently have been shown to significantly impair isolated muscle contractile properties, though their synergistic effects are poorly understood. We uniquely examined the effects of 9 weeks of a high-fat diet (HFD) on isometric force, work loop power output (PO) across a range of contractile velocities, and fatigability of 79-week-old soleus, extensor digitorum longus (EDL) and diaphragm compared with age-matched lean controls. The dietary intervention resulted in a significant increase in body mass and gonadal fat pad mass compared to the control group. Despite increased muscle mass for HFD soleus and EDL, absolute isometric force, isometric stress (force/CSA), PO normalised to muscle mass and fatigability was unchanged, although absolute PO was significantly greater. Obesity did not cause an alteration in the contractile velocity that elicited maximal PO. In the obese group, normalised diaphragm PO was significantly reduced, with a tendency for reduced isometric stress and fatigability was unchanged. HFD soleus isolated from larger animals produced lower maximal PO which may relate to impaired balance in older, larger adults. The increase in absolute PO is smaller than the magnitude of weight gain, meaning in vivo locomotor function is likely to be impaired in old obese adults, with an association between greater body mass and poorer normalised power output for the soleus. An obesity-induced reduction in diaphragm contractility will likely impair in vivo respiratory function and consequently contribute further to the negative cycle of obesity. Older adults are reported to experience poorer muscular strength and power output (PO) resulting in reduced locomotory function and quality of life . It is sEvidence indicates that obesity and old age independently result in a reduction in muscle quality (force or PO relative to muscle size), which results in the formation of larger muscles of lower quality ,10. For Mechanistically, poorer muscle function in older animals and humans has been attributed to impaired calcium handling, reduced contractile mass, impaired cross-bridge kinetics, a shift in fibre type composition, greater lipid accumulation and chronic inflammation . In obesWe aimed to uniquely determine whether dietary-induced obesity in old age worsens skeletal muscle contractile function at the isolated muscle level via usage of the work loop technique. Previous isolated muscle studies comparing contractile function in old age and in dietary-induced obesity, independently examine isometric force ,21,22,23n = 30) or high-fat diet  group, ensuring each group was matched for body mass .Ethics approval was provided by the Coventry University ethics committee prior to the commencement of this study . 60 female mice  were purchased at 9 weeks old and matured in-house in groups of 8–10 at Coventry University without access to running wheels. Animals were kept in a 12:12-hour light:dark cycle at 50% humidity. Access to standard lab chow (CRM(P); SDS/Dietex International Ltd, Whitham, UK; calories provided by protein 17.49%, fat 7.42%, carbohydrate, 75.09%; gross energy 3.52 kcal/g; metabolizable energy 2.57 kcal/g) and water was provided ad libitum. At 68 weeks of age, mice were divided into cages of 10 and were assigned to either a control (P < 0.05) and work loop PO normalised to muscle mass  have been observed by 78 weeks of age [Animals acclimated to their new groups for 2 weeks, with each dietary protocol commencing at 70 weeks of age for a duration of 9 weeks. This feeding duration was selected as previous work has demonstrated substantial changes in body composition and muscle mechanics following a similar feeding duration of the same dietary sources in younger CD-1 female mice , therefos of age .Animals were sacrificed by cervical dislocation at 79 weeks of age in accordance with British Home Office Animals (Scientific Procedures) Act 1986, Schedule 1. Following sacrifice, animals were weighed to determine body mass, with nasoanal length measured using digital calipers . Body circumference around the abdomen was measured with a textiles tape measure. Body mass and nasoanal length were used to calculate Body Mass Index (Equation (1))  and Lee Equation (1)—The equation used to calculate the Body Mass Index of a rodent . Body maEquation (2)—The equation used to calculate the Lee Index of Obesity of a rodent . Body man = 10 per muscle per group) was dissected from the right hind limb. The proximal tendon was secured via aluminium foil T-clips and a piece of bone left at the distal tendon. A ventral segment of the costal diaphragm (n = 10 per group) was isolated from the right-hand portion of the rib cage, and aluminium foil T-clips wrapped around the central tendon of the diaphragm segment, with the two ribs anchoring the muscle left intact. All dissections were performed in chilled (~5 °C), oxygenated  Krebs-Henseleit solution of composition (mM) NaCl 118; KCl 4.75; MgSO4 1.18; NaHCO3 24.8; KH2PO4 1.18; glucose 10; CaCl2 2.54; pH 7.55 at room temperature before oxygenation.Whole EDL or soleus (L0) was measured using an eyepiece graticule fitted to a microscope, with estimates of mean muscle fibre length measured as 85% of physical soleus length and 75% for EDL [L0 [Each muscle was placed in a flow-through chamber circulated with oxygenated Krebs-Henseleit solution maintained at 37.0 ± 0.2 °C. At one end, the muscle was attached, via crocodile clips, to a force transducer  and a motor arm at the opposing end . The position of the motor arm was detected by a Linear Variable Displacement Transducer . Muscle activation was elicited via parallel platinum electrodes, with electrical currents provided by an external power supply . Stimulation and length change parameters were altered via a custom written programme . Initially, isometric contractions were used to examine the maximal twitch and tetanus kinetics of isolated skeletal muscles as with previous studies of mammalian skeletal muscles ,17,25,30 for EDL . No esti EDL [L0 .L0 which yielded maximal tetanic force, work loop PO was measured by subjecting each muscle to symmetrical sinusoidal length changes around its optimal length whilst being stimulated to produce force during shortening. PO was determined across a range of cycle frequencies (CF), or contractile velocities, in order to produce a power output-cycle frequency curve [Using the stimulation frequency, stimulation amplitude, and cy curve , which aL0, i.e., the muscles lengthened by 5%, shortened by 10%, then re-lengthened by 5% back to L0. At these CF’s, phasic bursts of electrical stimulation were provided for durations of 50 ms, 55 ms and 65 ms to the EDL, diaphragm and soleus respectively. A stimulus phase  of −2 ms, −5 ms and −10 ms were utilised for EDL, diaphragm and soleus respectively. These timings ensured the stimulation was provided just prior to the muscle reaching its maximal length to ensure a rise in force before shortening. The aforementioned values for strain, stimulus phase, and burst duration have been shown to elicit maximal work loop PO at these CF’s [Initially, a CF of 10 Hz for EDL, 7 Hz for diaphragm and 5 Hz for soleus was used as these CF’s typically elicited maximal PO in previous research for 78-week-old locomotor  and younese CF’s ,16,17,25Control sets of work loops were performed using the parameters that elicited maximal net work  every 3 to 4 sets of work loops, and following examination of net work for the final CF of each muscle, to monitor changes in net work over the course of the experiment. Any variation in net work was due to an impairment in force production. Therefore, the PO produced by each muscle at each CF prior to the fatigue run was corrected to the control run that yielded the greatest net work, assuming that alterations in PO were linear over time .Each muscle underwent 10-min of rest prior to the fatigue run. To determine fatigability, each muscle was subjected to fifty consecutive work loops using the optimised stimulation parameters at 5 Hz, 7 Hz and 10 Hz for soleus, EDL and diaphragm respectively. The net work of every second work loop was plotted against time until each muscle produced <50% of the pre-fatigue maximal PO and as such is plotted relative to the pre-fatigue maximal PO. This method has been used previously to examine muscle fatigability ,16,17. TThe experimental protocol for each muscle was ~190 min. Muscle performance prior to the fatigue run declined by 10 ± 2% (S.E.M), indicating that the quality of all muscle preparations was well maintained throughout the experimental protocol as with similar studies utilising this methodological approach .L0 and an assumed muscle density of 1016 kg∙m−3 [2) was calculated as peak tetanic force divided by mean muscle CSA. Absolute PO (Watts) was calculated as the product of net work and CF and was normalised to muscle mass (W∙kg−1 muscle mass) at each CF by dividing absolute PO by muscle mass. Muscle mass, muscle length, muscle CSA and absolute force and power for the diaphragm are not reported due to the slight variations in diaphragm size and mass, despite attempting to remove the same section for each animal. Whilst much of this variation in isolated diaphragm preparation size could be natural, only normalised force and power are presented for the diaphragm as per many previous studies [At the end of the experiment, each muscle was detached from the rig, tendons removed and blotted with tissue paper to remove excess fluid. The muscle was weighed using an electronic balance  in order to determine wet muscle mass. Mean muscle cross-sectional area (CSA) was calculated from muscle mass, 6 kg∙m−3 . Maximal studies ,33,34,35P < 0.05 for all analyses. Initial tests for normality and homogeneity were performed to determine the appropriate statistical analyses. Differences in animal anthropometrics and isometric properties between the control and HFD groups were measured using an independent t-test . Comparisons of the absolute and normalised power output-cycle frequency data were assessed using two-way analysis of variance (ANOVA) using SPSS v.23 , with diet and cycle frequency as factors. Correlations between animal body mass and maximal normalised work loop power output were performed to determine whether larger animals generated lower power relative to muscle size. Correlations were performed in Excel.All data are presented as mean ± S.E.M. The level of significance was set at t-test were used to determine significant differences in time taken to reach <50% of the pre-fatigue maximal PO for all muscles of each dietary group. Recovery was assessed by a two-factor ANOVA with time and diet as the factors. An independent samples t-test was used to determine whether there was a significant difference between diet groups in PO normalised to muscle mass after 30-min of recovery.Independent samples d was calculated and corrected for bias using Hedge’s g due to the small sample sizes for each experimental group [Effect size (ES) was calculated to determine the magnitude of the effect of the HFD on animal morphology and contractile performance in comparison to the control animals. Cohen’s al group . Threshoal group .p-values to provide a p-value for each group of multiple hypothesis tests to assess whether these values were biased via multiple hypothesis testing. The truncated product method p-value was <0.001, demonstrating that the results were not biased based on multiple hypothesis testing.The truncated product method  was usedP < 0.001). Furthermore, the HFD group nasoanal length, Lee Index of Obesity and Body Mass Index was 5%, 2% and 13% greater respectively (P ≤ 0.04). The HFD group had 16% greater soleus muscle mass and 18% greater EDL muscle mass (P < 0.04), and soleus and EDL muscle CSA was 13% and 17% greater in the HFD group than the control group (P ≤ 0.05). However, soleus and EDL muscle length were not significantly different between each group (P > 0.30). The FPM of the HFD group accounted for a greater percentage of the total BM than the control group (P < 0.001). However, when soleus and EDL muscle mass was expressed as a ratio to animal body mass, there were no significant differences between the control and HFD groups for either muscle (P > 0.66).The HFD diet group had 24% greater body mass, 21% greater body circumference and 119% greater gonadal fat pad mass than the control group . However, there was a tendency for isometric stress to be lower for the HFD group than the control group . There were no significant differences between the controls and HFD groups in tetanus activation and relaxation times (P > 0.12).Maximal absolute tetanus force and maximal tetanus stress were unaffected by a HFD for soleus and EDL  and 15%  respectively. Whilst there was a significant change in optimal CF for PO in HFD soleus (P < 0.001), this was not the case for EDL (P > 0.16). There was no diet*CF interaction for either muscle (P = 1.00). When PO was normalised to muscle mass, differences were not apparent between control and HFD in soleus or EDL . In contrast to the locomotor muscles, PO normalised to muscle mass for the diaphragm in the HFD group was significantly lower by an average of 27% across all CF’s in comparison to the control group . CF had a significant effect on normalised and PO for all groups (P < 0.05). There was no interaction between diet & CF for all muscles indicating no alteration in the shape of the normalised power output-cycle frequency curves between each group (P = 1.00).Absolute PO of the soleus and EDL was significantly higher in the HFD group than the control group, increasing on average by 13%  however, there was no significant relationship between maximal normalised PO and body mass for the EDL . There was a tendency for obese diaphragm isolated from heavier animals to produce lower normalised PO .For the obese group, soleus isolated from obese animals that were heavier produced significantly lower maximal normalised work loop PO , however, force during muscle shortening was typically lower for the HFD diaphragm. Additionally, the passive work during lengthening and re-lengthening appears to be greater for the HFD diaphragm than the control, increasing the work done on the muscle to lengthen it, consequently decreasing the net work . However, diet did not significantly affect the time-course of fatigue for each muscle (P > 0.29), nor time to reach 50% of the pre-fatigue maximum for all muscles .Fifty consecutive work loop cycles resulted in a significant reduction in PO, over time for all muscles , diet significantly reduced the recovery of PO for the diaphragm from HFD individuals when compared to controls (P = 0.01) where PO was significantly different after 30 min of recovery . There was no time effect on the recovery of PO for all three muscles (P > 0.17), nor was a diet*time interaction observed (P > 0.37).Whilst diet did not significantly impair the ability of soleus and EDL to recover from the fatigue protocol . These results differ to findings from younger models of obesity induced via diet which have investigated isolated muscle performance where, locomotory muscle contractile performance is generally poorer ,21,22, a2–280 kN∙m2 for soleus [2–332 kN∙m2 for EDL [2–169 kN∙m2 for diaphragm [−1–34 W∙kg−1 at 5 Hz for soleus [−1–107 W∙kg−1 at 10 Hz for EDL [−1–60 W∙kg−1 at 7 Hz for diaphragm [When comparing the differences in contractile performance between young CD-1 mice in previous studies and 79-week-old CD-1 mice in the present study, a substantial age-related reduction in contractile performance can be observed for all skeletal muscles examined. Isometric stress for 10-week-old CD-1 females ranges from 199 kN∙mr soleus ,40,41,42 for EDL ,31,39,42 for EDL ,31,39, aProvision of a calorie-rich diet resulted in the excessive accumulation of gonadal fat and elevated skeletal muscle mass, which ultimately contributed to an increase in animal morphology and body mass . The incThe increased absolute PO of the soleus A muscle By contrast, a HFD in older animals caused a significant reduction in diaphragm PO relative to muscle mass E. As witDietary-induced obesity did not cause a significant reduction in the ability to sustain PO over repeated work loops for old obese soleus, EDL or diaphragm A,C,E, noThe added load of an increased bodily inertia in older adults could be a significant contributor towards reduced muscular endurance in older adults . It is eWhilst obesity studies in young rodents share similar characteristics with the present study, such as inducing obesity via diet, and comparing the soleus and EDL to represent phenotypic differences, comparisons are difficult due to the different methodological approaches including feeding duration and diet composition, a lack of classification of what is considered obese for rodent models, and different test temperature for isolated skeletal muscles .For soleus, absolute force, isometric stress, and absolute and normalised PO remain unchanged or even improve following an obesogenic diet in young female CD-1 mice fed the same HFD  which doWe have outlined the limitations of normalising contractile performance to muscle mass in detail in our previous work ,16,17. WThe onset of obesity does not suddenly occur in old age, rather an old obese adults’ high body fat content is more likely due to a prolonged duration of poor dietary choices, which may alter body composition and muscle contractile function in a muscle-specific and sex-specific manner. However, the present study has given further insight into the relative effects of aging and obesity in old mice. Other studies have indicated that there is further complexity in the interaction between obesity and aging. For example, DeNies et al.  reportedThe current study utilises an isolated muscle approach to examine the impact of inducing obesity in old age on contractile function of skeletal muscle independent of any potential effects on the central nervous system. It is well documented that denervation of skeletal muscles is an inescapable consequence of the ageing process . The impThe reversibility of obesity in older adults is of importance due to the numerous benefits of physical activity on body composition and quality of life. Rodent models have shown that 8 weeks of voluntary exercise in 38-week-old CD-1 mice can evoke an improvement in soleus and diaphragm stress, normalised PO and body mass compared to untrained age-matched controls . As animAbsolute force and PO, muscle quality, and fatigability of locomotor muscles is well maintained following a HFD in old animals. By contrast, muscle quality of old obese diaphragm is significantly lower compared to lean, age-matched counterparts. These findings differ to that of young obese skeletal muscles, where both force and PO normalised to muscle mass generally declines for EDL and diaphragm, though soleus muscle quality is well maintained irrespective of age. An elevated body mass in old obese adults is likely to act as a training stimulus on the soleus and EDL, as demonstrated by an increase in the absolute PO for the locomotor muscles. However, the increased bodily inertia will mean acute and sustained in vivo locomotor performance is likely to be substantially affected due to a larger limb mass and body mass creating a greater work demand on the skeletal muscle. The elevated fat mass loaded on the diaphragm in vivo could be a plausible contributor explaining the reduction in muscle quality as found in vitro. This may potentially elevate respiratory disease risk and further contribute to the negative cycle of obesity.


Thermoregulation was also more efficient in males. In females, proestrus increased exercise 2 and 2 at low running speeds  and estrus worsened thermoregulation. These differences translated into different absolute and relative workloads on the treadmill, even at equal submaximal 2 and belt speeds. In summary, our results demonstrate the better muscle strength, running power and economy, and exercise-induced thermoregulation of males compared to females. Proestrus and estrus still undermined the running economy and exercise-induced thermoregulation of females, respectively. These results demonstrate an important exercise sex gap in mice.Exercise physiology is different in males and females. Females are poorly studied due to the complexity of the estrous cycle and this bias has created an exercise sex gap. Here, we evaluated the impact of sexual dimorphism and of the estrous cycle on muscle strength and running power of C57BL/6 mice. Like men, male mice were stronger and more powerful than females. Exercise-induced increase of O The historical gender differences in motivation/opportunity to practice physical activity  limited the best women exercise/sport performance, a phenomenon known as exercise gender gap in humans2. For instance, women are more prone to physical inactivity3, a risk factor for many diseases5. The historical evolution of exercise gender gap in modern Olympic Games (World Record and 10 best performances) also reveals a systematically lower sport performance of females compared to males; nowadays, the differences varies between 10.7% for running and 36.8% for weightlifting2. The exercise gender gap is greatest in sports that require running economy, muscle strength, and exercise power2. Running economy is the energy demand for a submaximal running speed6, higher in men8 but it is unknown if this sex difference is also present in laboratory animals.The importance of differences between sexes/genders is recognized in biology and medicine. Sex describes biological differences, while gender includes social, cultural and economic aspects9. However, sex is a major determinant of exercise performance through the impact of anthropometry , aerobic power and anaerobic threshold, besides genetic and hormonal factors10. The minor representation of females also translates into less knowledge about the biology of exercise in this sex. So far, the main features of sexual dimorphism important for exercise described in rodents are differences in skeletal muscle kinetics and fiber-type composition10 and energy metabolism12. In fact, the biological mechanisms underlying the benefits of exercise were investigated in numerous animal studies in a laboratory setting, with a strong tendency to only use males probably to avoid dealing with the possible influence of the menstrual/estrous cycle13. Exercise-induced thermoregulation, submaximal and maximal 2 and 2, and running economy are gold physiological indexes for exercise, but have never been studied in females at different phases of the estrous cycle.A review of ≈1400 manuscripts involving more than 6 million people revealed an under-representation of women in studies of exercise and sports (35–37%)In humans, the exercise sex gap is greatest in sports that require running economy, strength, and power. Similarly, we investigated the role of sex and estrous cycle in maximum  muscle strength and running power/economy of mice. We also evaluated exercise-induced thermoregulation. This knowledge is essential to advance the knowledge of exercise physiology in female sex. We will demonstrate that the simple extrapolation of male knowledge is not correct.4,51 = 12.6, P < 0.05, Fig. 16. The length of the tail of the males (6.5 ± 0.2 cm) was shorter than females . These parameters were independent of the estrous cycle, which was devoid of effects on body weight . The prominent estrous cycle was estrus . The higher intensities  presented similar kinetics for 2 and 2 in the different phases of the estrous cycle.The submaximal 7,126 = 264, P < 0.05; Fig. 7,94 = 0.32, P > 0.05; Fig. Thermoregulation requires the dissipation of heat produced during exercise. Exercise increased the heat production of males and females .Resting body and tail infrared temperatures did not differ between sexes or in females at different phases of the estrous cycle . This had already been described for muscle strength21. Likewise, normalization of exercise performance by specific muscle mass (rather than body mass) makes sexual dimorphism disappear21. Male mammals are larger, with larger cross-sectional muscle area10. Several studies also showed that muscle length (and the length of the long bones) is also higher in male mammals, important for greater tetanic strength of the anterior masseter muscle8. Larger levers determine higher torques and muscle strength. Sex is also important for muscle fiber-type composition, especially the myosin IIB gene (fast muscle fiber)10. Evidence shows threefold more IIB muscle fibers in the masseter of male mice22. In addition, testosterone signals hypertrophy in this musculature20. Conversely, estrogen decreases muscle contractile force in female mice24. Thus, muscle strength and running power depends on size and sex: males have large muscles and bones, responsible for great muscle strength; this difference is further amplified by the anabolic effects of testosterone, resulting in larger muscle strength, speed and power.Skeletal muscle mass and strength are lower in females25, a model of submaximal physical activity. Testosterone replacement completely reversed this impairment25. The antiandrogen Flutamide decreases the treadmill endurance of rats, but does not change 2max and running economy26. Here, the exercise-induced submaximal 2 and 2 up to 2max were similar between sexes, as previously described27. Only one study demonstrated increased female submaximal 2 during treadmill test, which further reinforces the hypothesis of females’ worst running economy27. These testosterone evidences support the best physical performance (power and endurance) of running male mice, but not the best running economy.The testosterone also seems to influence running endurance, but not the running economy. Castration of mouse testicles deplete blood testosterone and impair running wheel endurance 2 and possibly the running economy of mouse. Similar to our results, submaximal 2 was higher in female rats during the estrogen-dominant proestrus at low treadmill speeds 5–12 m/min 17, which may be considered as a low intensity exercise. We also found these differences at near speeds 9–12 m/min. A possibility is the effect of estradiol in the lung gas-exchange surface area (GSA). 2 is directly proportional to GSA29; which increases during proestrus with high estradiol levels29. Estradiol also increases lung’s GSA and 2 in ovariectomized rats29. Our results suggest that estrogen can increase 2 during exercise, and worsen the running economy, especially at proestrus.On the other hand, estrogen seems to influence 30. We now report that sex and the estrous cycle do not modify the calories consumed by exercise, another important variable for running economy; however, our results showed that male thermoregulation was more efficient, since the infrared dissipation of males was more effective. Literature suggests two important points for mouse thermoregulation: body surface area (BSA) and tail dry heat loss. BSA is estimated by the Meeh’s formula (BSA = body weight0.667)25. The greater body mass of males assists in better heat dissipation during/after exercise. Moreover, tail size seems to be related to thermal stress31, with animals that live in warm environments having longer tails32. Female tails, even longer, warmed up more during exercise than that of males. The tail length of C57BL/6 female mice was similar to that described in female BALB/c mice15. Thus, a longer tail length in female mice is suggestive of a required adaptation to compensate for their lower body mass (and area).Exercise-induced hyperthermia is a biological response due to greater muscle activation, mitochondrial uncoupling and proton leak33 demonstrated that body warming during exercise was higher in female mice at estrous. We saw it in the tails. Progesterone promotes heat conservation and higher body temperatures at rest35. Bilateral ovariectomy eliminated this estrous-associated change33. We suggest that this may apply to body temperature of running female mice during estrus, characterized by high progesterone levels. Thus, sex seems to be a crucial factor also for the exercise-induced thermoregulation of mice.Sanchez-Alavez37. The woman’s menstrual cycle is divided into three phases: follicular, ovulation and luteal. The follicular phase can be divided into initial and late, corresponding to metestrus and diestrus, respectively. Ovulation corresponds to proestrus, and the luteal phase to estrus. The woman’s follicular and luteal menstrual cycle does not seem to influence muscle strength, power, and 240. Human studies still allow evaluating rate of perceived effort (RPE), which also does not differ in the different menstrual phases41. However, the differences we found are close to ovulation, virtually impossible to evaluate in women. We demonstrated that the mouse proestrus (or human ovulation) increased 2 and heat production at light exercise.Some of our results are similar to those reported in humans, since the physical performance of women is generally lower than in men, in accordance with the exercise gender gap2 and 2, implying a finest running economy in males. Males also presented better thermoregulation after exercise. The estrous cycle played a subtle role in mouse physical performance: proestrus impaired running economy and estrus impaired exercise heat loss. This implies that the impact of the estrous cycle on the performance of females should not be considered a limiting factor for their use in experimental designs. In fact, size is the main factor that should be considered in the construction of experimental designs involving exercising male and female mice. For running, a light-intensity exercise seems similar between the sexes (except proestrus), but the performance of females at moderate-intensity running corresponded to the performance of males at low-moderate intensity; the performance of females at high-intensity running corresponded to the performance of males at moderate-high for males, and male high-intensity running was supra-maximal for females. Failure to consider these differences by measuring only the running speed, as done in most studies, introduces an error to compare performance between sexes. These results are of particular interest to counteract the underrepresentation of females in exercise experimental designs.In summary, our results highlight differences in exercise performance and metabolism between male and female mice. Sex influences size, which appear to be the main factor for mice exercise sex gap. Mouse sexual dimorphism also influenced exercise workload, but not ad libitum access to food and water. Animals were housed and handled according to European Union guidelines and the study was approved by the Ethical Committee of the Center for Neuroscience and Cell Biology (University of Coimbra).Male and female C57BL/6 mice (10–12 weeks old) were obtained from Charles River . Mice were housed under controlled environment  with th day in independent groups of animals. Ergospirometry was performed on the 5th day. All tests were carried out between 9:00 and 17:00 hours in a sound-attenuated and temperature controlled observation room under low-intensity light (≈10 lux), where mice had been habituated for at least 1 hour. The apparatuses were cleaned with 10% ethanol between animals. Within the time window of the tests, we did not record any significant impact of the time of day (morning vs. afternoon) on the treadmill vertical power, 2max and temperature of the tail at rest in either males or females (data not showed).The animals were accustomed to the treadmill for 3 days. The open field or grip strength test was performed on the 42O) then mounted on gelatinized slides (76 × 26 mm). These procedures lasted no more than 3–5 minutes, and there were no major temporal delays between behavioral experiments and fluid collection for vaginal cytology.We evaluated the estrous cycle immediately after the behavioral and exercise experiments, through 4–5 consecutive vaginal lavages (with 40–50 μL of distillated H2O and desiccated at room temperature. The slides were mounted with Eukitt medium (Sigma-Aldrich) and evaluated under an optical microscope at 1x, 5x and 20x (Zeiss Axio Imager 2). The characterization of the estrous cycle was performed according to literature42. Females were categorized for initial (metestrus) or late (diestrus) follicular phase, ovulation (proestrus), or luteal phase (estrus)42.The vaginal smear were desiccated at room temperature and covered with 0.1% crystal violet for 1 min, then twice washed with 1 mL H41.The exploration of an open field (38 × 38 cm) was analyzed for 15 min using the ANY-maze™ video tracking system (Stoelting Co.)41. The computed result was the average of 3 trials, expressed in kgf.The animal was hung with its forepaws to the central position of a 300 g metal grid and the grip strength was determined as the weight pushed (in grams)Mice were accustomed with a single-lane treadmill  for 3 consecutive days , with 24 h interval between each habituation session.th day, 48 hours after the last habituation session. The incremental protocol started at 15 cm/s with an increment of 5 cm/s every 2 min, with a constant inclination of 8.7% (5° for the LE8710 model). The exercise test lasted until running exhaustion, defined by the inability of the animal to leave the electrical grid for 5 seconds44. We estimated the power output for treadmill running based on a standard conversion of the vertical work, body weight and running speed46. Power is the 1st derivative of work relative to time (run time at each stage).The ergospirometry test was carried out on 52) and carbon dioxide production (2) were estimated during treadmill running in a metabolic chamber47  coupled to treadmill. The animals remained in the chamber for 15 min prior to exercise testing. Atmospheric air  was renewed at a rate 120 mL/min, using the same sampling rate for the LASER oxygen sensor  and infrared carbon dioxide sensor . Heat  was estimated according to the equations of Lusk48.Oxygen uptake  camera  placed overtop (25 cm height) of a plastic tube (25 cm diameter) was used to acquire a static dorsal thermal imageest Fig.  and recoest Fig.  periods,The FLiR C2 camera also captures digital pictures (640 × 480 pixels) that were loaded and calibrated  in the ImageJ software  for tail length measurement of live animals (ImageJ software).2 and 2, and body and tail temperature was evaluated using on-way ANOVA. The repeated measures of ANOVA were performed to evaluate the effect of different treadmill speeds, sex and estrous cycle on the vertical power, 2 and 2, and heat. The Bonferroni post hoc test was applied for significant F values. The accepted level of significance was p < 0.05. Statistics were performed using Dell Statistica , version 13.Data are presented as mean ± Standard Error of the Mean (SEM). A test for normality was performed by Kolmogorov–Smirnov test. For each test, the experimental unit was an individual animal. The frequency of the estrous cycle was assessed using the Kruskal-Wallis test. The role of sex and estrous cycle in the dependent variables body mass, open field, grip strength and vertical power, The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.


Abdominal aorta calcification (AAC) has been associated with clinical outcomes in peritoneal dialysis (PD) patients. Heart rhythm complexity analysis has been shown to be a promising tool to predict outcomes in patients with cardiovascular disease. In this study, we aimed to analyze the association between heart rhythm complexity and AAC in PD patients. We prospectively analyzed 133 PD patients. Heart rhythm complexity including detrended fluctuation analysis and multiscale entropy was performed. In linear analysis, the patients in the higher AAC group (AAC ≥15%) had a significantly lower standard deviation of normal RR intervals, very low frequency, low frequency, high frequency and low/high frequency ratio. In non-linear analysis, DFAα1, slope 1–5, scale 5 and area 6–20 were significantly lower in the patients with higher AAC. Receiver operating characteristic curve analysis showed that DFAα1 had the greatest discriminatory power to differentiate these two groups. Multivariate logistic regression analysis showed that DFAα1 and HbA1c were significantly associated with higher AAC ratio. Adding DFAα1 significantly improved the discriminatory power of the linear parameters in both net reclassification improvement and integrated discrimination improvement models. In conclusion, DFAα1 is highly associated with AAC and a potential cardiovascular marker in PD patients. Because of the high cardiovascular mortality rate in dialysis patients 3, the burden of CVD on patients starting dialysis and its impact on the survival of dialysis patients has recently received increasing attention3.Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in end-stage renal disease (ESRD) patients4. Abdominal aorta calcification (AAC) has been reported to predict CVD events and mortality in ESRD patients, including those undergoing peritoneal dialysis (PD)7. The abdominal aorta calcification can be measured via X ray with grading systems8 or computed tomography (CT) with direct measurement of the percentage of AAC (%AAC)9. The %AAC has been shown to be independently associated with mortality and hospitalization in PD patients7. The cutoff value of 15% AAC in CT has been reported that predicting clinical outcomes in PD patients7. However, the clinical use of %AAC measurement is limited by radiation exposure and medical cost.Possible explanations for the high rates of CVD mortality and mobility in ESRD patients include atherosclerosis-related vascular complications and autonomic nervous system dysfunction10. HRV has also been commonly used to predict CVD outcomes11. Newer biological signal analysis methods based on nonlinear signal modeling and complexity evaluation including detrended fractal analysis (DFA) and multiscale entropy (MSE) have been developed in recent years12. Compared to traditional HRV parameters, nonlinear heart rhythm complexity analysis has a better prognostic power in patients with CVD. In addition, both DFA and MSE have been shown to be useful in predicting survival of heart failure patients14.Analysis of beat-to beat variation of heart rate, also known as heart rate variability (HRV), is commonly used in cardiovascular researches as a simple and noninvasive approachTo the best of our knowledge, no previous study has investigated the association between heart rhythm complexity and AAC. Therefore, the aim of this study was to analyze the association between heart rhythm complexity and the severity of AAC in PD patients.A total of 133 PD patients (61 men) were enrolled in this study, including 59 (26 men) with AAC ≥15% (higher AAC group) and 74 (35 men) with AAC <15% (lower AAC group). The clinical data are shown in Table In linear analysis, the patients in the higher AAC group had a significantly lower standard deviation of normal RR intervals (SDRR), very low frequency (VLF), low frequency (LF), high frequency (HF) and low frequency to high frequency ratio (LH/HF ratio) than those in the lower AAC group. In non-linear analysis, DFAα1 was significantly lower in the higher AAC group. The value of DFAα2 was comparable between the two groups. In MSE analysis, the patients in the higher AAC group had significantly lower slope 1–5, scale 5, and area 6–20 than the patients in the lower AAC group (Table Receiver operating characteristic (ROC) curve analysis showed that DFAα1 had the greatest discriminatory power to differentiate the two groups compared to other linear, non-linear and clinical parameters  of HRV parameters including SDRR, the percentage of absolute differences in normal RR intervals greater than 50 ms (pNN50), pNN20, VLF, LF, HF, LF/HF ratio DFAα1, DFAα2, slope 1–5, scale 5, area 1–5, and area 6–20 were 0.663, 0.457, 0.548, 0.752, 0.721, 0.605, 0.723, 0.781, 0.509, 0.707, 0.657, 0.578 and 0.667, respectively.The AUC of clinical parameters including age, DM, fasting serum glucose, HbA1c, creatinine, CRP and LVEF were 0.641, 0.651, 0.683, 0.729, 0.580, 0.622 and 0.619, respectively.p < 0.001) and HbA1c  remained in the model, and both were associated with higher AAC , LF/HF ratio , age  and HbA1c  were significantly associated with the percentage of AAC  and integrated discrimination improvement (IDI) models. In addition, area 6–20 significantly improved the discriminatory power of SDRR, LF, HF and LF/HF ratio in the IDI model, and SDRR and HF in the NRI model. Scale 5 significantly improved the discriminatory power of SDRR, HF and LF/HF ratio in the IDI model and HF in NRI model  have also been reported to contribute to vascular calcification22. Several studies have reported significant associations between qualitative or semiquantitative evaluations of arterial calcification and all-cause and cardiovascular mortality in hemodialysis patients25. In our study, HbA1c and age were significantly associated with %AAC in multivariate linear regression model. Age and HbA1c are known risk factors of vascular calcification27. In addition, HbA1c levels are associated with mortality in ESRD patients29. Even in PD patients without diabetes, higher HbA1c is still associated with higher cardiovascular events30.In daily practice, predicting the clinical outcomes of PD patients is a challenge. Atherosclerosis-related vascular calcification has been highly associated with morbidity and mortality in ESRD patients8 and CT with direct measurement of %AAC9. Previous study supported that CT appeared to be more sensitive than plain X-rays at detecting peripheral and aortic vascular calcifications in hemodialysis patients31. Tsushima et al. developed a method to measure the percentage of calcified volume against whole vascular volume using CT32 and CT remains the reference standard in AAC evaluation33. AAC was reported to be an important predictor of vascular morbidity and mortality in the Framingham Heart Study34, and it has also been reported to be associated with clinical outcomes in ESRD patients7. The percentage of AAC has been shown to be independently associated with mortality and hospitalization in PD patients7. In addition, patients with AAC ≥15% had more cardiovascular events than those with AAC <15%7. However, despite the usefulness of %AAC by CT, the radiation exposure and cost were limited the use of this tool.The AAC severity measurements include X ray with Kauppila scoreIn contrast, electrocardiography (ECG) is an easy, low cost and radiation-free examination. In the present study, we found high correlations among the HRV parameters  and AAC. This indicates that ECG recording followed by HRV analysis using linear and non-linear parameters has the potential to be an alternative to AAC in clinical practice.36. Despite the reported association between autonomic dysfunction and atherosclerosis37, the mechanisms linking autonomic imbalance to atherosclerosis are still unclear. In addition to atherosclerosis-related autonomic dysfunction, uremic autonomic neuropathy in ESRD patients has frequently been associated with parasympathetic damage and sympathetic nerve overactivity38, both of which have been associated with worse clinical outcomes in ESRD patients39. In our previous study, PD patients had significantly lower values of several linear and nonlinear parameters than those with normal renal function, and this also supports the hypothesis of prominent autonomic dysfunction in ESRD patients40.Previous studies have reported an association between the progression of coronary and carotid artery atherosclerosis and autonomic dysfunction13, outcome of acute stroke41, primary aldosteronism42, critical illnesses requiring extracorporeal life support43, and post-myocardial infarction heart function44. Furthermore, long-time scale parameters (area 6–20) in heart failure patients have been shown to have the best prognostic predictive power13, which is similar to our MSE results. DFA as a scaling analysis method to determine the statistical self-affinity of a signal can be used for the evaluation of the fractal behavior in the heart beat dynamics. The short-term  and long-term  fractal correlation exponents have been shown to provide a clearer understanding of the fractal correlation property in a physiological system45. DFA has also been associated with the interaction between sympathetic and vagal systems46. In the DIAMOND-CHF trial, after adjusting for clinical parameters, DFAα1 but not linear parameters remained to be an independent predictor of mortality14. Taken together with our findings, non-linear HRV analysis may be a useful tool to evaluate the risk of cardiovascular events.In the present study, DFAα1 had a better correlation with AAC than linear parameters, which implies that non-linear parameters provide more useful information. The non-linear analysis of HRV including MSE and DFA has been reported to be a better predictor of clinical outcomes than traditional linear analysis. MSE has been associated with the prognosis of heart failure48. We also found similar results in that linear HRV parameters including SDRR, VLF, LF, HF and LF/HF ratio were also significantly associated with AAC. In addition, combining linear and non-linear analysis further significantly improved the discriminatory power of the severity of AAC. Combining these linear and non-linear HRV parameters can provide more accurate information to build a ROC curve model to predict the severity of AAC.The traditional linear HRV parameters have also been positively associated with CVD risk factors and multiple cardiovascular outcomes including coronary artery disease and cardiovascular mortalityThere are several limitations to this study. First, this is a small pilot study and the findings should be confirmed by a larger clinical study with long-term follow-up data. Second, our study group is limited to PD patients, and further studies are needed to elucidate whether the same association between AAC and heart rhythm complexity exists in hemodialysis patients.In conclusion, heart rhythm complexity analysis can predict the severity of AAC in PD patients. DFAα1 had the greatest discriminatory power to differentiate PD patients with higher or lower AAC. In addition, DFAα1 and MSE slope 1–5 significantly improved the discriminatory power of the linear parameters, which suggests the advantage of combining linear and non-linear parameters.We prospectively enrolled 133 patients who received PD with conventional glucose-based lactate-buffered solution  for more than 6 months. Patients with chronic atrial fibrillation, clinical signs of acute infection, and those with a prior renal transplant were excluded. The baseline characteristics, medical history and medication usage were carefully recorded, and biochemical parameters were measured during initial evaluation. All patients received 24-h ambulatory ECG Holter recording . This study was approved by the Institutional Review Board of National Taiwan University Hospital, and all research was performed in accordance with relevant guidelines and regulations. All subjects provided written informed consent including for storage of their information in the hospital database and usage for research.A stable 4 hours segment of daytime RR intervals (between 9AM and 5PM) was selected for analysis. The selected electrocardiograms were automatically annotated via an algorithm and carefully examined by two experienced technicians.10. SDRR and the percentage of absolute differences in normal RR intervals greater than 50 ms (pNN50) were calculated to represent the total variance and vagal modulation of heart rate. The frequency domain parameters including high frequency , low frequency , and very low frequency  power, were computed by averaging the absolute powers (ms2) after Fourier transformation.All parameters were calculated according to the recommendations of the North American Society of Pacing Electrophysiology and the European Society of CardiologyDFA is used to evaluate the fractal behavior beneath seemingly nonstationary RR dynamics by eliminating extrinsic trends to remove spurious long-term correlations. The external trends were assumed to be the linear or polynomial fitted trends over different scales, and by removing these trends from the integrated time series, the intrinsic fractal behavior could be better quantified. Detrended fluctuations were calculated by adding up the detrended integrated time series in individual scales. Then, the logarithmic plot of fluctuations against time scales were further constructed. The slope (α exponent) of the log-log plot was used to indicate the fractal correlation characters of time series.While the respiratory sinus arrhythmia is responsible for most of the short-term RR dynamics in normal subjects, the crossover phenomenon of α exponents of RR dynamics over short  and long  time scales are of importance. We calculated both short- and long-term α exponents for better probing the fractal characters of the biological system.49. The estimated entropy over different time scales can then be used to represent the complexity (meaningful information richness) of the physiological signals. The linear-fitted slope of scale 1 to scale 5 (slope 1–5), the sum of entropy values of scales 1 to scale 5 (area 1–5) or scale 6 to scale 20 (area 6–20) were calculated to quantify the complexity of the beat-to-beat dynamics exhibited in short and long time scales.MSE takes the predictability of multiple time scales into account and extends the entropy of a single timescale to the information richness structure embedded over different time scales. The profile of the sequential changes of the entropies over different time scales can be further quantifies. In brief, the time series of different time scales were derived by using a coarse-graining process (i.e. averaging consecutive beats to form a new time series), and the sample entropy was adopted to estimate the predictability over different time scales9.Transthoracic echocardiography  was performed in all patients. The LVEF was quantitated by M-mode measurements or area-length methods32.A standard 64-MDCT scan  was performed in all patients. The calcified area was quantified based on an attenuation range of >150 Hounsfield units using image analysis software . The percentages of the area of the whole aorta affected by aortic calcification were calculated from the images of four consecutive slices just above the iliac bifurcation levelth and 75th percentiles). Comparisons of data between the higher and lower AAC groups were performed by the Mann-Whitney U test. Differences between proportions were calculated by the chi-square test or Fisher’s exact test. Logistic regression analysis was used to validate associations between parameters and the presence of high AAC. Significant determinants in univariate logistic regression analysis (P < 0.05) were then tested in multivariate logistic regression analysis with stepwise subset selection to identify independent factors to predict the presence of high AAC. Linear regression analysis was used to validate associations between parameters and percentage of AAC. Significant determinants in univariate linear regression analysis (P < 0.05) were then tested in multivariate linear regression analysis with stepwise subset selection to identify independent factors to predict the percentage of AAC. The goodness-of-fit of a logistic model was indicated by R2, while the discriminatory power of the model was assessed by the area under the ROC curve (AUC).Data were expressed as median  and SPSS version 25 for Windows . The significance level of the statistical analysis was set at 0.05.Two statistics, net reclassification improvement (NRI) and integrated discrimination improvement (IDI), were used to evaluate improvements in the accuracy of the prediction after adding a single nonlinear parameter into a logistic regression model using only linear parameters