Young Adult Female Mice Research Articles

Comparative Pharmacokinetics of Δ9-Tetrahydrocannabinol in Adolescent and Adult Female Mice.

Introduction: Animal studies suggest that adolescent exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating constituent of cannabis, causes lasting functional alterations in brain and other organs. Those studies often neglect the impact that age- and sex-dependent differences in the distribution and metabolism of the drug might exert on its pharmacological effects. Here, we provide a comparative analysis of Δ9-THC pharmacokinetics in adolescent and adult female mice, which identify significant dissimilarities in distribution and metabolism of Δ9-THC between females of these age groups. Materials and Methods: We administered Δ9-THC (5 mg/kg, intraperitoneal) to adolescent (37-day old) and young adult (70-day old) female mice and quantified Δ9-THC and its first-pass metabolites-11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)-in plasma and brain tissue using liquid chromatography/tandem mass spectrometry. Results: Maximal plasma concentrations of Δ9-THC were 8 times higher in adolescent than adult female mice. Conversely, brain concentrations and brain-to-plasma ratios were 25-50% higher in adults than adolescents. Concentrations of Δ9-THC metabolites were higher in plasma but lower in brain of adolescent compared to adult female mice. Conclusions: The results identify multiple age-dependent differences in the pharmacokinetic properties of Δ9-THC in female mice, which might influence the pharmacological response to the drug.

Translatome analysis in acute ischemic stroke: Astrocytes and microglia exhibit differences in poststroke alternative splicing of expressed transcripts.

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo-Tag-seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT-PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes.

Effects of Repetitive Mild Traumatic Brain Injury on Corticotropin-Releasing Factor Modulation of Lateral Habenula Excitability and Motivated Behavior.

Mild traumatic brain injury (mTBI) is a significant health burden due to mTBI-related chronic debilitating cognitive and psychiatric morbidities. Recent evidence from our laboratory suggests a possible dysregulation within reward/motivational circuit function at the level of a subcortical structure, the lateral habenula (LHb), where we demonstrated a causal role for hyperactive LHb in mTBI-induced motivational deficits in self-care grooming behavior in young adult male mice when exposed to mTBI during late adolescence (at ∼8 weeks old). In this study, we extended this observation by further characterizing neurobehavioral effects of this repetitive closed head injury model of mTBI in both young adult male and female mice on LHb excitability, corticotropin releasing factor (CRF) modulation of LHb activity, and behavioral responses of motivation to self-care behavior and approach versus avoidance behavior in the presence of a social- or threat-related stimulus. We show that mTBI increases LHb spontaneous tonic activity in female mice similar to what we previously observed in male mice, as well as promoting LHb neuronal hyperexcitability and hyperpolarization-induced LHb bursting in both male and female mice. Interestingly, mTBI only increases LHb intrinsic excitability in male mice coincident with higher levels of the hyperpolarization-activated cation currents (HCN/Ih) and reduces levels of the M-type potassium currents while potentiating M-currents without altering intrinsic excitability in LHb neurons of female mice. Because persistent dysregulation of brain CRF systems is suggested to contribute to chronic psychiatric morbidities and that LHb neurons are highly responsive to CRF, we tested whether the LHb CRF subsystem becomes engaged following mTBI. We found that in vitro inhibition of CRF receptor type 1 (CRFR1) within the LHb reverses mTBI-induced enhancement of LHb tonic activity and hyperexcitability in both sexes, suggesting that an augmented intra-LHb CRF-CRFR1-mediated signaling contributes to the overall LHb hyperactivity following mTBI. Behaviorally, mTBI diminishes motivation for self-care grooming in female mice as in male mice. mTBI also alters defensive behaviors in the looming shadow task by shifting the innate defensive behaviors toward more passive action locking rather than escape behaviors in response to an aerial threat in both male and female mice, as well as prolonging the latency to escape responses in female mice. While this model of mTBI reduces social preference in male mice, it induces higher social novelty seeking during the novel social encounters in both male and female mice. Overall, our study provides further translational validity for the use of this pre-clinical model of mTBI for investigation of mTBI-related reward circuit dysfunction and mood/motivation-related behavioral deficits in both sexes while uncovering a few sexually dimorphic neurobehavioral effects of this model that may differentially affect young males and females when exposed to this type of mTBI during late adolescence.

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of brainstem dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 ( Lrrk2 G2019S ) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in stress-related brain regions.

Modelling lung infection with Klebsiella pneumoniae after murine traumatic brain injury

Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal dose of K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection, in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned to baseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice compared to either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response, primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longer-term consequences of this dual-hit insult.

Effects of age and sex on photoperiod modulation of nucleus accumbens monoamine content and release in adolescence and adulthood

Day length, or photoperiod, is a reliable environmental cue encoded by the brain's circadian clock that indicates changing seasons and induces seasonal biological processes. In humans, photoperiod, age, and sex have been linked to seasonality in neuropsychiatric disorders, as seen in Seasonal Affective Disorder, Major Depressive Disorder, and Bipolar Disorder. The nucleus accumbens is a key locus for the regulation of motivated behaviors and neuropsychiatric disorders. Using periadolescent and young adult male and female mice, here we assessed photoperiod's effect on serotonin and dopamine tissue content in the nucleus accumbens core, as well as on accumbal synaptic dopamine release and uptake. We found greater serotonin and dopamine tissue content in the nucleus accumbens from young adult mice raised in a Short winter-like photoperiod. In addition, dopamine release and clearance were greater in the nucleus accumbens from young adult mice raised in a Long summer-like photoperiod. Importantly, we found that photoperiod's effects on accumbal dopamine tissue content and release were sex-specific to young adult females. These findings support that in mice there are interactions across age, sex, and photoperiod that impact critical monoamine neuromodulators in the nucleus accumbens which may provide mechanistic insight into the age and sex dependencies in seasonality of neuropsychiatric disorders in humans.

Fructooligosaccharides act on the gut-bone axis to improve bone independent of Tregs and alter osteocytes in young adult C57BL/6 female mice.

Targeting the gut-bone axis with probiotics and prebiotics is considered as a promising strategy to reduce the risk of osteoporosis. Gut-derived short chain fatty acids (SCFA) mediate the effects of probiotics on bone via Tregs, but it is not known whether prebiotics act through a similar mechanism. We investigated how 2 different prebiotics, tart cherry (TC) and fructooligosaccharide (FOS), affect bone, and whether Tregs are required for this response. Eight-wk-old C57BL/6 female mice were fed with diets supplemented with 10% w/w TC, FOS, or a control diet (Con; AIN-93M) diet, and they received an isotype control or CD25 Ab to suppress Tregs. The FOS diet increased BMC, density, and trabecular bone volume in the vertebra (~40%) and proximal tibia (~30%) compared to the TC and control diets (Con), irrespective of CD25 treatment. Both prebiotics increased (P <.01) fecal SCFAs, but the response was greater with FOS. To determine how FOS affected bone cells, we examined genes involved in osteoblast and osteoclast differentiation and activity as well as genes expressed by osteocytes. The FOS increased the expression of regulators of osteoblast differentiation (bone morphogenetic protein 2 [Bmp2], Wnt family member 10b [Wnt10b] and Osterix [Osx]) and type 1 collagen). Osteoclasts regulators were unaltered. The FOS also increased the expression of genes associated with osteocytes, including (Phex), matrix extracellular phosphoglycoprotein (Mepe), and dentin matrix acidic phosphoprotein 1 (Dmp-1). However, Sost, the gene that encodes for sclerostin was also increased by FOS as the number and density of osteocytes increased. These findings demonstrate that FOS has a greater effect on the bone mass and structure in young adult female mice than TC and that its influence on osteoblasts and osteocytes is not dependent on Tregs.

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of midbrain dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 (Lrrk2 G2019S) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in some, but not all stress-related brain regions.

A convolutional neural network to characterize mouse hindlimb foot strikes during voluntary wheel running.

Voluntary wheel running (VWR) is widely used to study how exercise impacts a variety of physiologies and pathologies in rodents. The primary activity readout of VWR is aggregated wheel turns over a given time interval (most often, days). Given the typical running frequency of mice (∼4Hz) and the intermittency of voluntary running, aggregate wheel turn counts, therefore, provide minimal insight into the heterogeneity of voluntary activity. To overcome this limitation, we developed a six-layer convolutional neural network (CNN) to determine the hindlimb foot strike frequency of mice exposed to VWR. Aged female C57BL/6 mice (22months, n = 6) were first exposed to wireless angled running wheels for 2h/d, 5days/wk for 3weeks with all VWR activities recorded at 30 frames/s. To validate the CNN, we manually classified foot strikes within 4800 1-s videos (800 randomly chosen for each mouse) and converted those values to frequency. Upon iterative optimization of model architecture and training on a subset of classified videos (4400), the CNN model achieved an overall training set accuracy of 94%. Once trained, the CNN was validated on the remaining 400 videos (accuracy: 81%). We then applied transfer learning to the CNN to predict the foot strike frequency of young adult female C57BL6 mice (4months, n = 6) whose activity and gait differed from old mice during VWR (accuracy: 68%). In summary, we have developed a novel quantitative tool that non-invasively characterizes VWR activity at a much greater resolution than was previously accessible. This enhanced resolution holds potential to overcome a primary barrier to relating intermittent and heterogeneous VWR activity to induced physiological responses.

The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested.To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr−/−)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment.Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr−/− mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

MP60-20 ROLE OF SHH PATHWAY IN URINARY CONTINENCE AND ITS IMPACT ON CELLULAR FATE IN MOUSE URETHRA

You have accessJournal of UrologyCME1 Apr 2023MP60-20 ROLE OF SHH PATHWAY IN URINARY CONTINENCE AND ITS IMPACT ON CELLULAR FATE IN MOUSE URETHRA Walid Farhat, Priyank Yadav, Marcela Ambrogi, Audrey Spiegelhoff, Chad Vezina, and Kimberly Keil Stietz Walid FarhatWalid Farhat More articles by this author , Priyank YadavPriyank Yadav More articles by this author , Marcela AmbrogiMarcela Ambrogi More articles by this author , Audrey SpiegelhoffAudrey Spiegelhoff More articles by this author , Chad VezinaChad Vezina More articles by this author , and Kimberly Keil StietzKimberly Keil Stietz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sonic hedgehog (Shh) signaling is critical in many developing organs and subject to disruption through gene-environment interactions. Urinary continence is dependent on appropriate development of bladder neck and urethra driven by epithelial-mesenchymal interactions mediated in part by SHH. To further understand the impact of Shh pathway disruption on lower urinary tract and continence, we examined voiding physiology and histology in compound mutant GLI family zinc finger Gli2+/−; Gli3Δ699/+ mice, which have hypomorphic hedgehog signaling. METHODS: Voiding physiology was tested in young adult female mice (n=5-8/genotype) using void spot assay, uroflowmetry and ex vivo bladder bath assays. Urethra and bladder (n=3/genotype) were collected for immunostaining using antibodies against smooth muscle alpha actin to visualize urethral smooth muscle thickness, or uroplakin, transformation related protein 63, and keratin 5 (combinatorial staining resolves basal, intermediate and luminal epithelial cells). RESULTS: Gli2+/−; Gli3Δ699/+ female mice void more urine spots, void more spots of smaller diameter and void a greater percentage of urine in the cage center than wild type mice. Voids from Gli2+/−; Gli3Δ699/+ mice, measured by uroflowmetry, weigh less (indicating smaller volume) and are shorter in duration than wild type mice. Gli2+/−; Gli3Δ699/+ female mouse bladders, isolated in ex vivo bath preparations, are less responsive to electrical field stimulation and potassium chloride than bladders from wild type mice. Our results are consistent with detrusor under activity and potential stress incontinence in Gli2+/−; Gli3Δ699/+ female mice. In search for a physiological mechanism, we conducted histological analysis to reveal a clear anatomic boundary between wild type mouse bladder neck and urethra. Umbrella shaped uroplakin+ cells populate the bladder neck and cuboidal shaped uroplakin- superficial cells populate the urethra. The bladder neck epithelium of Gli2+/−; Gli3Δ699/+ mice does not appreciably differ from wild type, but urethral epithelial cells are uroplakin+, suggesting possible segmental deficits during urethral development that may account in part for incontinence in Gli2+/−; Gli3Δ699/+ mice. CONCLUSIONS: Our data suggest that establishing continent voiding function in mice is dependent on appropriate urethra epithelial cell fate. Future work to understand mechanisms underlying decreased bladder contractile properties, and urethral cell fate will enhance understanding of the continence mechanism. Source of Funding: DK104310 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e851 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Walid Farhat More articles by this author Priyank Yadav More articles by this author Marcela Ambrogi More articles by this author Audrey Spiegelhoff More articles by this author Chad Vezina More articles by this author Kimberly Keil Stietz More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract WP219: Repurposing The K Ca 3.1 Inhibitor Senicapoc For Treatment Of Acute Ischemic Stroke

Background: Acute ischemic stroke (AIS) is a leading cause of death and long-term disability. Both microglia (MG) and infiltrating macrophages (MP) are critical effector cell types in ischemic brain injury and recovery. K Ca 3.1 is a calcium-activated potassium channel that is upregulated in reactive MG and MP. Studies using either genetic deletion or pharmacological inhibition of K Ca 3.1 demonstrated that this channel is critical for pro-inflammatory activation of MG/MP as well as exacerbation of stroke pathophysiology. Senicapoc (ICA-17043) is a K Ca 3.1-specific inhibitor that has been used in human clinical trials for non-neurological indications (sickle cell anemia, asthma) and was proven safe. Here we evaluate the potential for repurposing senicapoc for AIS. Methods: Young adult male and female mice underwent 60 min middle cerebral artery occlusion (MCAO)/reperfusion. MCAO was monitored by laser doppler. Senicapoc’s pharmacokinetic (PK) profile was determined using high performance liquid chromatography - mass spectroscopy. Drug levels in plasma and brain were quantified at multiple time points post administration. Effects of senicapoc on post-stroke release of cytokines/chemokines was determined by multiplex ELISA. Inflammatory infiltrates were quantified with flow cytometry. Efficacy studies included: (i) infarct volume (MRI T2), white matter integrity (DTI) and longitudinal neurobehavioral outcomes (NBO). In-vitro chromogenic assay was used to assess senicapoc’s effect on proteolytic activity of tissue plasminogen activator (tPA). Results: Administration of senicapoc (40 mg/kg, i.p.) twice daily for seven days starting 12 h after MCAO resulted in ~55% reduction in infarct volume with corresponding improvements in NBO. Free senicapoc levels in brain ranged from 20 - 200 nM in stroked mice at 1, 4 &amp; 12 hours following administration. All values exceeded senicapoc’s IC 50 (11 nM) for K Ca 3.1. Senicapoc, at up to 5 μM, had no effect on tPA’s proteolytic activity. Conclusions: We provide proof-of-concept data that senicapoc, administered in an extended temporal window, can markedly reduce infarct volume and improve NBO in a mouse model of AIS. Senicapoc demonstrated favorable PK and CNS penetration. It did not interfere with tPA.

Neurotensin modulation of lipopolysaccharide induced inflammation of gut-liver axis: Evaluation using neurotensin receptor agonist and antagonist

Lipopolysaccharide (LPS), a toxic component of the cell wall of Gram-negative bacteria, is a potent immune stressor. LPS-induced inflammation of the gut-liver axis is well demonstrated. Neurotensin (NTS), a tri-decapeptide present in the gastrointestinal tract, has anti-inflammatory, anti-oxidative, and growth-promoting properties. This study elucidated the efficacy of PD149163, the type I NTS receptor agonist (NTS1) in the modulation of LPS-induced inflammation of the gut-liver axis of mice. Young-adult female mice (Age: 8 weeks; BW: 25 ± 2.5 g) were maintained in six groups (6/group); Group I as control and Group II, III & IV were exposed to LPS (1 mg/kg BW/Day; i.p.) for five days. LPS pre-exposed Group III and Group IV mice were treated with NTS1 agonist PD149163 (100 μg/kg BW i.p.) and antagonist SR48692 (0.5 mg/kg BW i.p.) respectively for 28 days. Group V and Group VI mice were exposed to only PD149163 and only SR48692 respectively with the doses as mentioned above for 28 days. Group I and LPS-exposed Group II mice were also maintained four weeks without further treatment. Histopathology revealed LPS-induced inflammation of the gut and liver. Significant elevation of plasma TNF-α and IL-6 and serum ALT and AST reflected as biomarkers of inflammation. Oxidative stress on both organs was distinct from decreased glutathione reductase and increased lipid peroxidation. PD149163 but not SR48692 ameliorated LPS-induced inflammation in both gut and liver counteracting inflammatory responses and oxidative stress. The use of NTS agonists including PD149163 could be exploited for therapeutic intervention of inflammatory diseases including that of the gut-liver axis.

Neonatal antibiotics have long term sex-dependent effects on the enteric nervous system.

Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic-exposed females had significantly longer whole gut transit while antibiotic-treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic-treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5-HT) levels. Antibiotic impacts on microbiota and mucosal 5-HT were not sex-dependent, but we propose that the responses of the host to these changes are sex-specific. This first demonstration of long-term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5-HT has important implications for gut function and other physiological systems of the host. KEY POINTS: Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long-term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex-dependent. Different male and female responses to antibiotic-induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex-specific impacts on gut function.

Angiotensin II differentially affects hippocampal glial inflammatory markers in young adult male and female mice.

Hypertension is a risk factor for neurodegenerative disorders involving inflammation and inflammatory cytokine-producing brain cells (microglia and astrocytes) in the hippocampus and medial prefrontal cortex (mPFC). Here we investigated the effect of slow-pressor angiotensin II (AngII) on gliosis in the hippocampus and mPFC of young adult (2-mo-old) male and female mice. In males, AngII induced hypertension, and this resulted in an increase in the density of the astrocyte marker glial fibrillary acidic protein (GFAP) in the subgranular hilus and a decrease in the density of the microglial marker ionized calcium binding adapter molecule (Iba-1) in the CA1 region. Females infused with AngII did not show hypertension but, significantly, showed alterations in hippocampal glial activation. Compared with vehicle, AngII-infused female mice had an increased density of Iba-1 in the dentate gyrus and CA2/3a region. Like males, females infused with AngII exhibited decreased Iba-1 in the CA1 region. Neither male nor female mice showed differences in GFAP or Iba-1 in the mPFC following AngII infusion. These results demonstrate that the hippocampus is particularly vulnerable to AngII in young adulthood. Differences in gonadal hormones or the sensitivity to AngII hypertension may account for divergences in GFAP and Iba-1 in males and females.

Raloxifene Stimulates Estrogen Signaling to Protect Against Age- and Sex-Related Intervertebral Disc Degeneration in Mice.

Estrogen agonist raloxifene is an FDA-approved treatment of osteoporosis in postmenopausal women, which may also be a promising prophylactic for painful intervertebral disc (IVD) degeneration. Here, we hypothesized that 1) aging and biological sex contribute to IVD degeneration by reducing estrogen signaling and that 2) raloxifene stimulates estrogen signaling to protect against age- and sex-related IVD degeneration in mice. 2.5-month-old (male and female) and 22.5-month-old (female) C57Bl/6J mice were subcutaneously injected with raloxifene hydrochloride 5x/week for 6 weeks (n = 7–9/grp). Next, female mice were ovariectomized (OVX) or sham operated at 4 months of age and tissues harvested at 6 months (n = 5–6/grp). Advanced aging and OVX increased IVD degeneration score, weakened IVD strength, reduced estrogen receptor-α (ER-α) protein expression, and increased neurotransmitter substance P (SP) expression. Similar to aging and compared with male IVDs, female IVDs were more degenerated, mechanically less viscoelastic, and expressed less ER-α protein, but unlike the effect induced by aging or OVX, IVD mechanical force was greater in females than in males. Therapeutically, systemic injection of raloxifene promoted ER-α protein to quell these dysregulations by enlarging IVD height, alleviating IVD degeneration score, increasing the strength and viscoelastic properties of the IVD, and reducing IVD cell expression of SP in young-adult and old female mice. Transcriptionally, injection of raloxifene upregulated the gene expression of ER-α and extracellular matrix-related anabolism in young-adult and old IVD. In vertebra, advanced aging and OVX reduced trabecular BV/TV, whereas injection of raloxifene increased trabecular BV/TV in young-adult and old female mice, but not in young-adult male mice. In vertebra, advanced aging, OVX, and biological sex (females > males) increased the number of SP-expressing osteocytes, whereas injection of raloxifene reduced the number of SP-expressing osteocytes in young-adult female and male mice and old female mice. Overall, injection of estrogen agonist raloxifene in mice normalized dysregulation of IVD structure, IVD mechanics, and pain-related SP expression in IVD cells and osteocytes induced by aging and biological sex. These data suggest that, in addition to bone loss, raloxifene may relieve painful IVD degeneration in postmenopausal women induced by advanced age, biological sex, and estrogen depletion.

Age-dependent aneuploidy in mammalian oocytes instigated at the second meiotic division.

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.

The role of the hot foot gene in the fertility of The in vitro fertilization and embryonic development of young adult and old mice as a model for assisted reproductive technology

The hotfoot mutation first occurred in strain of mice C57BL/Ks in 1964. The homozygous (ho/ho) hotfoot mutation show a quick pattern motion that produces a progressive neuromuscular disability of the hind legs. The (ho/ho) is an autosomal recessive mutation affects fertility and neuromuscular system in mice. ObjectiveThis study was implemented to determine the nature and causes of infertility, to ascertain potential value as an animal model due to the presence of similar mutation in human and other mammals. MethodsThe experimental design to evaluate the fertility and sterility of young and old adult female and male hotfoot mice by utilizing the assisted reproduction technology (ART) via the in vitro fertilization, the in vitro embryo development, and the normal matting, the growth of the new born hotfoot mice. ResultsDemonstrated that the young adult and the old hotfoot females produce about similar rate of ova number following superovulation. In addition, they yielded similar rate of embryo development in vitro form 1 cell to the 16 cell stage, morula and blastula stage compared to normal females. The old hotfoot females show better rate in the in vitro fertilization IVF and embryo development 53.6%, and the rate of the degenerating ova (46.4%) compared to the young adult females 38.5%, 61.35, respectively. The hotfoot males show sterility (22%) of the homozygous mutant males compared to phenotypically normal males. Regarding the normal matting, the hotfoot female mating with males had less litter size compared to normal mice mating. ConclusionThe hotfoot gene had a noticeable effect on the in vitro fertilization IVF of young adult compared to old adult hotfoot female, also the body weight growth and litter size of hotfoot less than normal mice.

Haploinsufficiency of Col5a1 causes intrinsic lung and respiratory changes in a mouse model of classical Ehlers-Danlos syndrome.

The Ehlers‐Danlos syndromes (EDS) are inherited connective tissue diseases with primary manifestations that affect the skin and the musculoskeletal system. However, the effects of EDS on the respiratory system are not well understood and are described in the literature as sporadic case reports. We performed histological, histomorphometric, and the first in‐depth characterization of respiratory system function in a mouse model of classical EDS (cEDS) with haploinsufficiency of type V collagen (Col5a1+/−). In young adult male and female mice, lung histology showed reduced alveolar density, reminiscent of emphysematous‐like changes. Respiratory mechanics showed a consistent increase in respiratory system compliance accompanied by increased lung volumes in Col5a1+/− compared to control mice. Flow–volume curves, generated to mimic human spirometry measurements, demonstrated larger volumes throughout the expiratory limb of the flow volume curves in Col5a1+/− compared to controls. Some parameters showed a sexual dimorphism with significant changes in male but not female mice. Our study identified a clear respiratory phenotype in the Col5a1+/− mouse model of EDS and indicated that intrinsic respiratory and lung changes may exist in cEDS patients. Their potential impact on the respiratory function during lung infections, other respiratory disease processes, or insults may be significant and justify further clinical evaluation.

CHL1-Deficient and Wild-Type Male Mice do Not Differ in Locomotor Recovery from Spinal Cord Injury.

CHL1 is a close homolog of L1, a cell adhesion molecule that plays major roles in neural and tumor cell functions. We had found that young adult female mice deficient in CHL1 recovered better than their wild-type female littermates after thoracic Spinal Cord Injury (SCI). This observation was surprising, because CHL1 increases neurite outgrowth in vitro. Injury of adult mouse central and peripheral nervous systems upregulate CHL1 expression in neurons and astrocytes, consistent with CHL1's pro-active, homophilic interaction between CHL1 surface molecules in wild-type mice. After SCI, CHL1 expression was observed to increase in the glial scar, areas of axonal regrowth and remodeling of neural circuits. These observations were made only in females, and we therefore sought to analyze SCI in CHL1-deficient male mice. We now show that CHL1-deficient males did not recover better or worse than their male wild-type littermates. Primary and secondary lesion volumes were similar in the two genotypes, as seen in female mice which were studied in parallel with male mice. Assessment of peripheral leukocytes showed a significant increase in numbers of blood neutrophils at 24 h after SCI in males, but not in females. Lymphocyte numbers in mutant males increased slightly, but numbers of lymphocytes or monocytes did not differ significantly between males or females. These results indicate that CHL1-deficient males and females differ in the number of neutrophils but not lymphocytes or monocytes, suggesting that the difference between males and females is unlikely due to differences in leukocytes.