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Abstract Background Stimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model. Methods Mice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA. Findings Infusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice. Conclusions Reducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.

The precise neural mechanisms underlying the pathogenesis of depression are largely unknown, though stress-induced brain inflammation and serotonergic plasticity are thought to be centrally involved. Moreover, we previously demonstrated that neuropeptide FF receptor 2 (NPFFR2) overexpression provokes depressive-like behaviors in mice. Here, we assess whether NPFFR2 is involved in priming of depressive-like behaviors and downregulation of serotonergic 1A receptor (5HT1AR) after lipopolysaccharide (LPS) treatment. The forced swimming test (FST) and sucrose preference test (SPT) were used to quantify depressive-like phenotypes in wild-type (WT) and NPFFR2-knockout (KO) mice. A single dose of LPS (i.p. 1 mg/kg) readily caused increases in toll-like receptor 4 and tumor necrosis factor-α along with decreases in 5-HT1AR mRNA in the ventral hippocampus of WT mice. Furthermore, LPS treatment of WT mice increased immobility time in FST and decreased sucrose preference in SPT. In contrast, none of these effects were observed in NPFFR2-KO mice. While WT mice injected with lentiviral 5-HT1AR shRNA in the ventral hippocampus displayed an unaltered response after LPS challenge, LPS-challenged NPFFR2-KO mice displayed a profound decrease in sucrose preference when pretreated with 5-HT1AR shRNA. Taken together, these results suggest that NPFFR2 modulates LPS-induced depressive-like behavioral phenotypes by downregulating 5HT1AR in the ventral hippocampus.

An amendment to this paper has been published and can be accessed via the original article.

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

Introduction There is a need for evidence-based prevention programming that can reduce head impacts and increase reporting and disclosure of concussion. This study assessed an intervention to decrease concussion risk and improve concussion management through improving concussion-related knowledge, attitudes, intentions, and self-efficacy among parents in the middle school (MS) sport setting.Patients and methods This randomized controlled trial (NCT04841473) examined parents of MS-aged children. Participants were randomized into one of two study arms: (1) CDC, which completed an education training module that compiled concussion education from the Centers for Disease Control and Prevention (CDC); and (2) TRAIN + CDC, which completed the CDC training module and an additional TRAIN educational module that provided strategies (originating from the Popular Opinion Leader framework) on communicating such information with one’s personal peer networks and children. Validated measures of concussion-related knowledge, attitudes, intentions, and self-efficacy were collected before completing the training modules and one week following completion. Linear mixed model analyses examined differences in outcomes between study arms.Results Overall, 103 parents completed the training modules and had valid pre- and post-intervention data (TRAIN + CDC n = 49; CDC n = 54). Analyses found that the study arms did not differ in the change scores from pre- to post-intervention across concussion-related outcomes. However, scores from pre- to post-intervention improved across both study arms for knowledge metrics, such as ‘Concussions are less likely to happen when athletes play by the rules of the sport’ (p

Updated recommendations on preseason heat safety in high school (HS) athletics ("2021 Consensus Statements") were published in April 2021. This cross-sectional survey study explored the initial roll-out of the 2021 Consensus Statements, including their visibility among United States HS athletic trainers (ATs) and perceived levels of confidence in implementing them. Recruitment occurred first, from a random selection of ATs from the Board of Certification, Inc., and second, an open invitation via social media. An online cross-sectional questionnaire had participating ATs note whether they had seen the 2021 Consensus Statements. If yes, ATs reported their perceived level of confidence in implementing them (5-point-ordinal scale from "not at all confident" to "very confident); if no, ATs disclosed (open-ended) why they had not yet seen them. Descriptive statistics were calculated for quantitative variables; template analysis identified codes related to visibility of and confidence in implementing 2021 Consensus Statements. Nearly half (45.7%) of 116 responding HS ATs reported having seen at least one 2021 Consensus Statements; 23.3% had reviewed all three. Common reasons among the 63 that had not seen them included: not aware they were published (n = 22), have yet to read them (n = 19), and believed they could not access the journal (n = 10). Of the 53 ATs having seen at least one of the 2021 Consensus Statements, 67.9% (n = 36) were very/fairly confident in implementing them at their HS. Reasons for confidence included their schools ensuring up-to-date EHI prevention and management practices (n = 18) and athletics constituent support (n = 8). This exploratory study observed proportions of surveyed HS ATs that had not seen the 2021 Consensus Statements and were not confident in implementing them. Findings highlight the need to continue improving messaging about access to best-practice recommendations. Further, continued efforts inclusive of active and passive dissemination strategies across all athletics constituents are needed to aid proper implementation.

There is a growing concern around concussions in rugby union, at all levels of the game. These concerns highlight the need to better manage and care for players. However, consistency around concussion-related responsibilities of stakeholders across the community rugby system remains challenging. Taking a systems thinking approach, this pragmatic, qualitative descriptive study explored key stakeholder groups within New Zealand’s community rugby system’s perceptions of their own and others’ concussion-related responsibilities. Participants included players from schools and clubs, coaches, parents, team leads and representatives from four provincial unions. A total of 155 participants (67 females and 88 males) were included in the study. Focus groups and individual interviews were conducted. Thematic content analysis was used to analyse data. Thirty concussion-related responsibilities were identified. These responsibilities were contained within four themes: (1) policies and support (responsibilities which influence policy, infrastructure, human or financial resources); (2) rugby culture and general management (responsibilities impacting players’ welfare and safety, attitudes and behaviour, including education, injury reporting and communication); (3) individual capabilities (responsibilities demonstrating knowledge and confidence managing concussion, leadership or role/task shifting) and (4) intervention following a suspected concussion (immediate responsibilities as a consequence of a suspected concussion). The need for role clarity was a prominent finding across themes. Additionally, injury management initiatives should prioritise communication between stakeholders and consider task-shifting opportunities for stakeholders with multiple responsibilities. How concussions will realistically be managed in a real-world sports setting and by whom needs to be clearly defined and accepted by each stakeholder group. A ‘framework of responsibilities’ may act as a starting point for discussion within different individual community rugby contexts on how these responsibilities translate to their context and how these responsibilities can be approached and assigned among available stakeholders.

Objective: Parents may use various information sources to obtain information about sport-related concussions (SRC). This study examined SRC-related information sources used by parents of United States middle school children (age: 10–15 years). Methods: A panel of 1083 randomly selected U.S. residents, aged ≥18 years and identifying as parents of middle school children, completed an online questionnaire capturing parental and child characteristics, and utilization and perceived trustworthiness of various sources of SRC-related information. Multivariable logistic regression models identified factors associated with utilizing each source. Adjusted odds ratios (OR) with 95% confidence intervals (95%CIs) excluding 1.00 were deemed significant. Results: Doctors/healthcare providers (49.9%) and other healthcare-related resources (e.g., Centers for Disease Control and Prevention, WebMD) (37.8%) were common SRC-related information sources; 64.0% of parents utilized ≥1 of these sources. Both sources were considered “very” or “extremely” trustworthy for SRC-related information among parents using these sources (doctors/healthcare providers: 89.8%; other healthcare-related resources: 70.9%). A 10-year increase in parental age was associated with higher odds of utilizing doctors/healthcare providers (adjusted odd ratio (ORadjusted) = 1.09, 95%CI: 1.02–1.16) and other healthcare-related resources (ORadjusted = 1.11, 95%CI: 1.03–1.19). The odds of utilizing doctors/healthcare providers (ORadjusted = 0.58, 95%CI: 0.40–0.84) and other healthcare-related resources (ORadjusted = 0.64, 95%CI: 0.44–0.93) were lower among parents whose middle school children had concussion histories versus the parents of children who did not have concussion histories. Conclusion: One-third of parents did not report using doctors/healthcare providers or other healthcare-related resources for SRC-related information. Factors associated with underutilization of these sources may be targets for future intervention. Continuing education for healthcare providers and educational opportunities for parents should highlight accurate and up-to-date SRC-related information.

Middle school (MS) is an intermediary level of education between elementary and secondary school that typically includes students aged 10-15 years. There is limited research within the MS sport setting, particularly related to sport-related injury prevention. This qualitative study aimed to better understand the sport culture within MS sports and the communication strategies used among invested groups (i.e., athletes, parents, staff inclusive of coaches and school nurses). Semi-structured interviews were conducted with 19 athletes, 20 parents, and 18 staff (e.g., coaches, school nurses) from seven MS in two school districts during the 2018/19 and 2019/20 school years. Topics focused on understanding school- and sport-related factors related to education, safety, and communication. Analysis used a consensual qualitative research tradition, in which the research team discussed individually developed themes and categories from transcribed interviews, with the goal of coming to a consensus and creating a codebook. Throughout the coding process, the research team would reconvene to discuss coding decisions until consensus was reached. This study focuses on the themes of sport culture and communication. Dominant categories identified within sport culture related to participants noting why they were interested in MS sports, and their struggles with their perceived roles and engagement (e.g., helping parents stay engaged, finding coaches, oversight of school nurses). Competitiveness and safety could have conflicting roles and priority. Dominant categories identified within communication centered around limited communication between coaches and parents. Technological assistance (e.g., phone apps, websites) was available, but often varied by school and sport. Concussions were seldom discussed unless during the preseason meeting or when one occurred. Findings highlight that the MS sport settings may struggle with incorporating primary prevention into their cultures and ensuring reliable communication among individuals. Novel and tailored approaches to injury prevention are needed to help ensure buy-in and proper implementation.

BackgroundRacial and ethnic differences in emergency department (ED) visits have been reported among adolescent patients but are unsubstantiated among adults. Therefore, our purpose in this study was to examine the relationship between race/ethnicity and adult ED visits for concussions, their injury mechanisms, and computed tomography (CT) scan use among a nationally representative sample.MethodsWe used the National Hospital Ambulatory Medical Care Survey database from 2010–2015 to examine 63,725 adult (20–45 years old) patient visits, representing an estimated 310.6 million visits presented to EDs. Of these visits, 884 (4.5 million national estimate) were diagnosed with a concussion. Visit records detailed patient information (age, sex, race/ethnicity, geographic region, primary payment type), ED visit diagnoses, injury mechanism (sport, motor vehicle, fall, struck by or against, “other”), and head CT scan use. The primary independent variable was race/ethnicity (non-Hispanic Asian, non-Hispanic Black or African American, Hispanic/Latinx, non-Hispanic multiracial or another, and non-Hispanic White). We used multivariable logistic and multinomial regression models with complex survey sampling design weighting to examine the relationship between concussion ED visits, injury mechanisms, and CT scan use separately by race/ethnicity while accounting for covariates.ResultsThere were no associations between race/ethnicity and concussion diagnosis among adult ED visits after accounting for covariates. Relative to sports-related injuries, non-Hispanic Black or African American patient visits were associated with a motor vehicle (OR = 2.69, 95% CI: 1.06–6.86) and “other” injury mechanism (OR = 4.58, 95% CI: 1.34–15.69) compared to non-Hispanic White patients. Relative to sports-related injuries, non-Hispanic Asian, multiracial, or patients of another race had decreased odds of falls (OR = 0.20, 95% CI: 0.04–0.91) and “other” injuries (OR = 0.09, 95% CI: 0.01–0.55) compared to non-Hispanic White patients. The odds of a CT scan being performed were significantly lower among Hispanic/Latinx patient visits relative to non-Hispanic White patients (OR = 0.52, 95% CI: 0.30–0.91), while no other race/ethnicity comparisons differed.ConclusionOur findings indicate that the overarching concussion ED visit likelihood may not differ by race/ethnicity in adults, but the underlying mechanism causing the concussion and receiving a CT scan demonstrates considerable differences. Prospective future research is warranted to comprehensively understand and intervene in the complex, multi-level race/ethnicity relationships related to concussion health care to ensure equitable patient treatment.

The objective of this study was to examine associations of lifetime concussion history (CHx) and an advanced metric of lifetime repetitive head impact exposure with resting-state functional connectivity (rsFC) across the whole-brain and among large-scale functional networks (Default Mode; Dorsal Attention; and Frontoparietal Control) in former collegiate football players. Individuals who completed at least one year of varsity collegiate football were eligible to participate in this observational cohort study (n = 48; aged 36-41 years; 79.2% white/Caucasian; 12.5±4.4 years of football played; all men). Individuals were excluded if they reported history/suspicion of psychotic disorder with active symptoms, contraindications to participation in study procedures (e.g., MRI safety concern), or inability to travel. Each participant provided concussion and football playing histories. Self-reported concussion history was analyzed in two different ways based on prior research: dichotomous "High" (≥3 concussions; n = 28) versus "Low" (

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture. [ABSTRACT FROM AUTHOR]

Germ cell development is a fundamental process required to produce offspring. The developmental program of spermatogenesis has been assumed to be similar among mammals. However, recent studies have revealed differences in the molecular properties of primate germ cells compared with the well-characterized mouse germ cells. This may prevent simple application of rodent insights into higher primates. Therefore, thorough investigation of primate germ cells is necessary, as this may lead to the development of more appropriate animal models. The aim of this study is to define molecular signatures of spermatogenic cells in the common marmoset, Callithrix jacchus. Interestingly, NANOG, PRDM1, DPPA3 (STELLA), IFITM3, and ZP1 transcripts, but no POU5F1 (OCT4), were detected in adult marmoset testis. Conversely, mouse testis expressed Pou5f1 but not Nanog, Prdm1, Dppa3, Ifitm3, and Zp1. Other previously described mouse germ cell markers were conserved in marmoset and mouse testes. Intriguingly, marmoset spermatogenic cells underwent dynamic protein expression in a developmental stage-specific manner; DDX4 (VASA) protein was present in gonocytes, diminished in spermatogonial cells, and reexpressed in spermatocytes. To investigate epigenetic differences between adult marmoset and mice, DNA methylation analyses identified unique epigenetic profiles to marmoset and mice. Marmoset NANOG and POU5F1 promoters in spermatogenic cells exhibited a methylation status opposite to that in mice, while the DDX4 and LEFTY1 loci, as well as imprinted genes, displayed an evolutionarily conserved methylation pattern. Marmosets have great advantages as models for human reproductive biology and are also valuable as experimental nonhuman primates; thus, the current study provides an important platform for primate reproductive biology, including possible applications to humans. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.