The microarchitecture and chemical composition of the femur neck of senescent female rats after different physical training protocols.

A sedentary lifestyle, coupled with a decrease in estrogen, impairs bone homeostasis, favoring to the development of osteopenia and osteoporosis, both recognized as risk factors for fractures. Here, we investigated the quality of the femur, particularly the femur neck region, and the ambulation performance of senescent rats subjected to three different physical training protocols during the periestropause period. Forty-eight female rats, 18 months of age, were subjected to a 120-day training period, three times a week. The rats were distributed into four groups: aerobic training (AT), strength training (ST), concurrent training (CT), or no training (NT). After the experimental period, at 21 months of age, ambulation performance and femur were analyzed using microtomography, Raman stereology, densitometry, and mechanical strength tests. The results demonstrated greater remodeling activity and improvement in resistance and bone microarchitecture in the femur neck of senescent female rats after undergoing physical training. Our verified higher intensities of bands related to collagen, phosphate, amide III, and amide I. Furthermore, the analysis of the secondary collagen structures indicated alterations in the collagen network due to the exercise, resulting in increased bone strength. Both AT and strength-based training proved beneficial, with AT showing greater adaptations in bone density and stiffness in the femur, while strength-based training greater adaptations in trabecular and cortical structure. These insights contribute to the understanding of the potential interventions for preventing osteopenia and osteoporosis, which are critical risk factors for fractures.

Vasopressinergic Activity of the Suprachiasmatic Nucleus and mRNA Expression of Clock Genes in the Hypothalamus-Pituitary-Gonadal Axis in Female Aging.

The important involvement of the suprachiasmatic nucleus (SCN) and the activity of vasopressinergic neurons in maintaining the rhythmicity of the female reproductive system depends on the mRNA transcription-translation feedback loops. Therefore, circadian clock function, like most physiological processes, is involved in the events that determine reproductive aging. This study describes the change of mRNA expression of clock genes, Per2, Bmal1, and Rev-erbα, in the hypothalamus-pituitary-gonadal axis (HPG) of female rats with regular cycle (RC) and irregular cycle (IC), and the vasopressinergic neurons activity in the SCN and kisspeptin neurons in the arcuate nucleus (ARC) of these animals. Results for gonadotropins and the cFos/AVP-ir neurons in the SCN of IC were higher, but kisspeptin-ir was minor. Change in the temporal synchrony of the clock system in the HPG axis, during the period prior to the cessation of ovulatory cycles, was identified. The analysis of mRNA for Per2, Bmal1, and Rev-erbα in the reproductive axis of adult female rodents shows that the regularity of the estrous cycle is guaranteed by alternation in the amount of expression of Bmal1 and Per2, and Rev-erbα and Bmal1 between light and dark phases, which ceases to occur and contributes to determining reproductive senescence. These results showed that the desynchronization between the central and peripheral circadian clocks contributes to the irregularity of reproductive events. We suggest that the feedback loops of clock genes on the HPG axis modulate the spontaneous transition from regular to irregular cycle and to acyclicity in female rodents.

Oxytocin and bone quality in the femoral neck of rats in periestropause.

The objective of this study is to identify whether oxytocin (OT) contributes to the reduction of osteopenia in the femoral neck of rats in periestropause. Animals in irregular estrous cycles received two NaCl injections (0.15 mol/L) or OT (134 µg/kg) over a 12-h interval, and after thirty-five days without treatments, the biological sample collection was performed. The oxytocin group (Ot) demonstrated the highest enzymatic activity of alkaline phosphatase (p = 0.0138), lowest enzymatic activity of tartrate-resistant acid phosphatase (p = 0.0045), higher percentage of compact bone (p = 0.0359), cortical expression of runt-related transcription factor 2 (p = 0.0101), osterix (p = 0.0101), bone morphogenetic protein-2/4 (p = 0.0101) and periostin (p = 0.0455). Furthermore, the mineral-to-matrix ratio (ν1PO4/Proline) was higher and type-B carbonate substitution (CO3/ν1PO4) was lower (p = 0.0008 and 0.0303) in Ot group. The Ot showed higher areal bone mineral density (p = 0.0050), cortical bone area (p = 0.0416), polar moment of inertia, maximum, minimum (p = 0.0480, 0.0480, 0.0035), bone volume fraction (p = 0.0166), connectivity density (p < 0.0001), maximal load (p = 0.0003) and bone stiffness (p = 0.0145). In Ot percentage of cortical pores (p = 0.0102) and trabecular number (p = 0.0088) was lower. The results evidence action of OT in the reduction of osteopenia, suggesting that it is a promising anabolic strategy for the prevention of primary osteoporosis during the periestropause period.

Effect of pre-treatment of strength training and raloxifene in periestropause on bone healing.

BACKGROUND: There is evidence that strength training (ST) and raloxifene (Ral) treatment during periestropause promotes better bone quality. We wanted to determine whether the skeletal benefits of ST or Ral treatment, performed during periestropause, would persist after fracture. Therefore, the present study aimed to analyze the influence of pre-treatment with ST and administration of Ral during periestropause on bone healing after total unilateral osteotomy. METHODS: Senescent female Wistar rats between 18 and 21 months of age, performed ST on a ladder three times per week, were administered Ral by gavage (2.3 mg/kg/day), or an association of both. After 120 days, the treatments were interrupted, and a total osteotomy was performed on the left tibia in all animals. They were euthanized 1 and 8 weeks post-osteotomy. RESULTS: The administration of Ral during periestropause worsened the biochemical and oxidative profile, decreased gene expression of markers related to bone resorption and remodeling, which negatively affected the physicochemical properties; this lead to changes in the bone callus microarchitecture and mass, as well as a decrease in callus resistance to torsional deformation, resulting in lower tissue quality during bone healing. In contrast, ST performed prior to the osteotomy resulted in better bone healing, improvement of the biochemical and oxidative profile, alteration of the genetic profile in favor of bone formation and resorption, as well as the physic-ochemical properties of the callus. These changes led to better microarchitecture and bone mass and increased callus resistance to torsional deformation, confirming its beneficial effect on the quality of bone tissue, providing acceleration of bone consolidation. The combination of therapies at this exercise intensity and drug dosage showed a negative interaction, where the negative effect of Ral overcame the positive effect of ST, leading to decreased tissue quality in the bone healing process. CONCLUSIONS: This study indicates that in addition to excellent non-pharmacological therapy and action in the prevention of osteoporosis, ST performed during the aging period may increase bone quality at the onset of healing and provide improved bone consolidation. Furthermore, the anti-osteoclastogenic effect of Ral shown in this model delayed the bone repair process, resulting in considerable clinical concern.

Atuação das proteínas do relógio na senescência reprodutiva de ratas Wistar / Clock protein action in the senescence reproductive of female Wistar rats

O envelhecimento é considerado processo multidimensional no qual fatores ambientais podem proteger ou, inversamente, agravar seus sinais, de maneira não linear, nos processos fisiológicos e neurocomportamentais. Durante este processo, os ritmos circadianos são interrompidos ou fragmentados com dissociação consequente dos ritmos circadianos do indivíduo e disfunções relacionadas ao relógio circadiano contribuem para o envelhecimento e para patologias a ele relacionadas. O objetivo deste estudo foi averiguar possível alteração temporal do sistema CLOCK no eixo HPG e a relação com às alterações hormonais que caracterizam a periestropausa. Foram utilizadas fêmeas adultas com ciclo estral regular (CD) na fase do diestro e fêmeas senis com ciclo estral irregular e persistência da fase do diestro (IDP). Para análises de expressão gênica dos clock genes Per2, Rev-erbα e Bmal1 no eixo HPG, foram utilizados punchs das regiões do NSQ, onde também foi analisado RNAm de AVP, APO e HMB destes animais, além da adenohipófise e ovários dos quais se extraiu o RNA para confecção do cDNA e realização de qPCR. A determinação da atividade neuronal vasopressinérgica no NSQ foi realizada por imunoistoquíca com dupla marcação para cFos e AVP em tecido previamente fixado com paraformaldeído. A concentração plasmática de gonadotrofinas foi determinada por radioimunoensaio. De modo geral, os animais IDP revelaram alterações no perfil de expressão gênica durante o fotoperíodo, com redução de amplitude, deslocamento/desalinhamento de fase e ausência de antifase. O NSQ de animais IDP apresentou menor expressão de Rev-erbα e maior expressão de RNAm para AVP em relação ao grupo CD. A quantificação relativa de Bmal1 foi semelhante em ambos os grupos e não houve diferenças entre grupos na expressão de Per2. Na APO, animais IDP apresentaram maior expressão de Per2 e menor quantidade de RNAm para Rev-erbα. No HMB observou-se menor expressão para Per2 e Rev-erbα e maior expressão de Bmal1 nas fêmeas IDP. Per2 e Bmal1 na adenohipófise tiveram menor expressão que o gene Rev-erbα no grupo senil e o ovário destes animais revelou maior expressão para Per2 e Rev-erbα, em comparação com os animais CD. As concentrações plasmáticas de FSH foram maiores nas fêmeas com ciclo irregular (2,05 ± 0,44 ng/mL), principalmente durante a fase clara, assim como o LH (0,24 ± 0,07 ng/mL), cujos maiores valores foram encontrados durante a fase escura e com perfil semelhante ao RNAm de AVP. As imunomarcações revelaram alta atividade vasopressinérgica na porção dorsomedial do NSQ das fêmeas IDP. Juntos estes dados permitem concluir que há desarranjo na expressão temporal dos genes Per2, Rev-erbα, Bmal1 que compõem a maquinaria molecular do relógio circadiano, bem como de RNAm para AVP no NSQ, de fêmeas Wistar na periestropausa. Além disso, a maior atividade neuronal vasopressinérgica e a ausência de oscilação de Rev-erbα e Bmal1 no NSQ destes animais, comprometem a correta comunicação do relógio central do NSQ com o eixo HPG, inviabilizando a manutenção da fertilidade feminina e contribuindo para a senescência reprodutiva(AU)

Aging is considered a multidimensional process in which environmental factors can protect or, conversely, aggravate its signals, non-linearly, in physiological and neurobehavioral processes. During this process, circadian rhythms are disrupted or fragmented with consequent dissociation of the individual's circadian rhythms and circadian clock-related dysfunctions contribute to aging and related pathologies. The objective of this study was to investigate possible temporal alteration of the CLOCK system in the HPG axis and the relation with the hormonal changes that characterize periestropause. Adult females with regular estrus cycle in the diestrous phase (RD) and old females with irregular estrous cycle and persistent diestrous phase (IPD). For analyzes of the gene expression of the genes Per2, Rev-erbα and Bmal1 in the HPG axis, punchs from the NSQ regions were used, where AVP, POA and MBH RNAm from these animals were also analyzed, as well as the adenohypophysis and ovaries from which they were extracted the RNA for cDNA production and qPCR performance. The determination of the vasopressinergic neuronal activity in the NSQ was performed by immunohistochemical with double labeling for cFos/AVP in tissue previously fixed with paraformaldehyde. The plasma concentration of gonadotrophins was determined by radioimmunoassay. In general, the IPD animals show alterations in the gene expression profile during the period analyzed, with amplitude reduction, phase shift / misalignment and absence of antiphase. The NSQ of IPD animals presented lower expression of Rev-erbα and higher RNAm expression for AVP than RD group. The relative quantification of Bmal1 was similar in both groups and there were no differences between groups in the expression of Per2. In PAO, IPD animals showed higher expression of Per2 and less amount of RNAm for Rev-erbα. MBH showed lower expression for Per2 and Rev-erbα and higher Bmal1 expression in IPD females. Per2 and Bmal1 in the adenohypophysis had lower expression than the Rev-erbα gene in the old group and the ovary of these animals showed higher expression for Per2 and Rev-erbα, in related to to the RD animals. Plasma concentrations of FSH were higher in females with irregular cycle (2.05 ± 0.44 ng / mL), mainly during the light phase, as well as LH (0.24 ± 0.07 ng / mL) whose values were found during the dark phase and with a profile similar to AVP RNAm. Immunolabeling demonstrated high vasopressinergic activity in the dorsomedial portion of the NSQ of the IPD females. Together these data allow us to conclude that there is a breakdown in the temporal expression of the Per2, Rev-erbα, Bmal1 genes that make up the molecular machinery of the circadian clock, as well as RNAm for AVP in NSQ of Wistar females in peri-masterpause. In addition, the increased vasopressinergic neuronal activity and the absence of Rev-erbα and Bmal1 oscillation in the NSQ of these animals compromise the correct communication of the central clock of the NSQ with the HPG axis, making it impossible to maintain female fertility and contributing to reproductive senescence(AU)

The transition to reproductive senescence is characterized by increase in A6 and AVPV neuron activity with attenuation of noradrenaline content.

During the course of life, cyclic females face a state of midlife transition that occurs in a fully functioning neurological system, and results in reproductive senescence. The authors' hypothesis was that changes in the activity noradrenergic neurons may be one of the factors involved in this phenomenon. The aim of this study was to investigate the activity of the neurons in the anteroventral periventricular nucleus (AVPV) and locus coeruleus (LC), to analyze their role in determining reproductive senescence. Adult female Wistar rats in the diestrus phase (4months/cyclic) and old females (18-20months/acyclic) in persistent diestrus, were decapitated or perfused at three different time intervals (10, 14 and 18h) throughout the day. In acyclic rats, the gonadotropin-releasing hormone (GnRH) and noradrenaline (NE) content were reduced; Fos-related antigen (FRA) in AVPV and Fos-related antigen/Tyrosine hydroxylase (FRA/TH) in LC showed immunolabeling of a higher number of neurons in these animals. The 3-methoxy-4-hydroxyphenylglycol/noradrenaline (MHPG/NE) ratio was higher and plasma LH was lower in the acyclic rats. Furthermore, the estradiol level was higher, and the progesterone level was lower after 14h of persistent diestrus. These findings suggested that during the periestropause, there was a higher level of POA/AVPV and NE neuronal activity in the LC of acyclic rats, associated with a lower capacity of synthesis and storage of neurotransmitters and neurohormones contributed to changes in the temporal pattern of neuroendocrine signaling, thereby compromising the accuracy of inhibitory and stimulatory effects, causing irregularity in the estrous cycle and determining reproductive senescence.

Activity of neurons in the preoptic area and their participation in reproductive senescence: Preliminary findings.

Alterations in the hypothalamic-pituitary-gonadal axis in females determine the transition from regular to irregular reproductive cycles, with loss of fertility. Stimulation of noradrenergic neurons of the anteroventral periventricular neurons (AVPV) is essential for regular reproductive cycles. Therefore, we examined the activity of neurons of the AVPV and measure the noradrenaline (NE) of acyclic rats, in constant estrus, and compared it with that of cyclic rats in estrus. Female cyclic (4-5 months) and acyclic (17-18 months) rats were euthanized at 10, 14, and 18 h in estrus. Brains were processed for immunoreactivity to antigens related to Fos (FRA) in AVPV, and the NE was determined by HPLC-ED. Plasma concentrations of LH, FSH, E2 and P4 were determined. In the acyclic animals, plasma LH was higher but the FSH was lower. There was decreasing P4 at different times, while the E2 was constant and lower in acyclic rats. FRA-ir expression in AVPV neurons of acyclic rats as well as turnover of NE was higher when compared with cyclic group. The preliminary findings showed increased activity in AVPV neurons in aging contribute to changes in the temporal pattern of neuroendocrine signaling, compromising the accuracy of inhibitory and stimulatory effects, causing irregularity in the estrous cycle and determining reproductive senescence.