Macrophages mobilized by the overexpression of the macrophage-colony stimulating factor promote efficient recovery of the ischemic muscle functionality.

AIMS: Narrowing or occlusion of arteries that supply the limbs can evolve to critical limb ischemia. M-CSF promotes proliferation, differentiation and survival of monocytes and macrophages, and polarization of macrophages to M2-subtype, which are essential elements for vessel formation and tissue repair. Based on these properties of M-CSF, we hypothesize that transfection of M-CSF into ischemic limbs may promote vessel formation and repair of ischemic limbs. MAIN METHODS: Hindlimb ischemia was surgically induced in 10-12 weeks old Balb/c and gene therapy was performed with intramuscular application of either uP-MCSF or uP plasmids (100 µg). Macrophage and monocyte subpopulations were assessed by flow cytometry and blood flow was monitored by Laser Doppler Perfusion Imaging (LDPI). Thirty days after transfection, we assessed gastrocnemius mass and muscle force, subsequently collecting the muscle for histology. KEY FINDINGS: We successfully developed the uP-MCSF plasmid, which increases M-CSF expression in the muscle transiently. Thirty days after uP-MCSF gene therapy in ischemic muscles, the treated group presented: improved muscle force, reduced fibrosis and increased arteriogenesis, although LDPI analysis did not show any significant difference in blood flow among groups. Noteworthy, we observed a temporary increase in MHCIIhighCD206high macrophages after uP-MCSF transfection. SIGNIFICANCE: M-CSF gene therapy improved ischemic muscle functionality by promoting arteriogenesis and decreasing fibrosis, likely through increased MHCIIhighCD206high macrophages and not via classically known M2-macrophages.

Pinealectomy in Rats.

The pinealectomy technique consists of the surgical removal of the superficial pineal gland. This procedure allows the ablation of circulating indoles produced by this gland. Withdrawal of systemic melatonin, a pineal hormone, affects animal circadian rhythms and induces several physiological changes that are the subject of many investigations. In this chapter, we describe the pinealectomy protocol adapted to rats. We describe the animal placement on the stereotaxic fixation system, and the procedure for the pineal gland removal and animal recovery from surgery.

The Crosstalk between Melatonin and Sex Steroid Hormones.

Melatonin, an indolamine mainly released from the pineal gland, is associated with many biological functions, namely, the modulation of circadian and seasonal rhythms, sleep inducer, regulator of energy metabolism, antioxidant, and anticarcinogenic. Although several pieces of evidence also recognize the influence of melatonin in the reproductive physiology, the crosstalk between melatonin and sex hormones is not clear. Here, we review the effects of sex differences in the circulating levels of melatonin and update the current knowledge on the link between sex hormones and melatonin. Furthermore, we explore the effects of melatonin on gonadal steroidogenesis and hormonal control in females. The literature review shows that despite the strong evidence that sex differences impact on the circadian profiles of melatonin, reports are still considerably ambiguous, and these differences may arise from several factors, like the use of contraceptive pills, hormonal status, and sleep deprivation. Furthermore, there has been an inconclusive debate about the characteristics of the reciprocal relationship between melatonin and reproductive hormones. In this regard, there is evidence for the role of melatonin in gonadal steroidogenesis brought about by research that shows that melatonin affects multiple transduction pathways that modulate Sertoli cell physiology and consequently spermatogenesis, and also estrogen and progesterone production. From the outcome of our research, it is possible to conclude that understanding the correlation between melatonin and reproductive hormones is crucial for the correction of several complications occurring during pregnancy, like preeclampsia, and for the control of climacteric symptoms.

Regenerative effect of platelet-rich plasma in the murine ischemic limbs.

AIMS: The purpose of this study was to investigate the effect of PPRP (pure PRP) and LPRP (PRP with leukocytes) on recovery from limb ischemia and on expression of growth factors involved in angiogenesis, myogenesis and fibrogenesis. MATERIAL AND METHODS: PPRP and LPRP prepared by centrifugation were added to cultures of C2C12 and NIH3T3 cells (1 or 10% PRPs) to evaluate alterations in cell metabolism and expression of growth factors by MTT, ELISA and RT-qPCR, respectively. To evaluate in vivo regenerative effects, PRPs were injected into the ischemic limbs of BALB/c mice and muscle mass/strength and histomorphometry were evaluated after 30 days. KEY FINDINGS: Mice treated with PRPs after limb ischemia showed an increase in the size of myofibers and muscle strength, reduced fibrosis and adipocytes, and decreased capillary density and necrosis scores compared to untreated mice. In cell culture, serum deprivation reduced the viability of C2C12 and NIH3T3 cells to about 50%, but the addition of 1% PRPs completely recovered this loss. Both PRPs, downregulated most of the tested genes; however, angiogenic gene Vegfa in C2C12 and the fibrogenic genes Col1a1 and Col3a1 in NIH3T3 cells were upregulated by LPRP. SIGNIFICANCE: PPRP and LPRP had similar effects in regulation of genes involved in angiogenesis, myogenesis and fibrogenesis. However, the presence of leucocytes did not significantly affect regenerative activities of PRP in the ischemic limb.

Maternal pineal melatonin in gestation and lactation physiology, and in fetal development and programming.

Pregnancy and lactation are reproductive processes that rely on physiological adaptations that should be timely and adequately triggered to guarantee both maternal and fetal health. Pineal melatonin is a hormone that presents daily and seasonal variations that synchronizes the organism's physiology to the different demands across time through its specific mechanisms and ways of action. The reproductive system is a notable target for melatonin as it actively participates on reproductive physiology and regulates the hypothalamus-pituitary-gonads axis, influencing gonadotropins and sexual hormones synthesis and release. For its antioxidant properties, melatonin is also vital for the oocytes and spermatozoa quality and viability, and for blastocyst development. Maternal pineal melatonin blood levels increase during pregnancy and triggers the maternal physiological alterations in energy metabolism both during pregnancy and lactation to cope with the energy demands of both periods and to promote adequate mammary gland development. Moreover, maternal melatonin freely crosses the placenta and is the only source of this hormone to the fetus. It importantly times the conceptus physiology and influences its development and programing of several functions that depend on neural and brain development, ultimately priming adult behavior and energy and glucose metabolism. The present review aims to explain the above listed melatonin functions, including the potential alterations observed in the progeny gestated under maternal chronodisruption and/or hypomelatoninemia.

Skeletal muscle healing by M1-like macrophages produced by transient expression of exogenous GM-CSF.

BACKGROUND: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. METHODS: To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. RESULTS: Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-ß1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. CONCLUSION: Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

Melatonin multiple effects on brown adipose tissue molecular machinery.

Brown adipose tissue (BAT) influences energy balance through nonshivering thermogenesis, and its metabolism daily and seasonal variations are regulated by melatonin through partially known mechanisms. We evaluated the role of melatonin in BAT molecular machinery of male Control, pinealectomized (PINX), and melatonin-treated pinealectomized (PINX/Mel) adult rats. BAT was collected either every 3 hours over 24 hours or after cold or high-fat diet (HFD) acute exposure. HFD PINX animals presented decreased Dio2 expression, while HFD PINX/Mel animals showed increased Dio2, Ucp1, and Cidea expression. Cold-exposed PINX rats showed decreased Dio2 and Lhs expression, and melatonin treatment augmented Adrß3, Dio2, Ucp1, and Cidea expression. Daily profiles analyses showed altered Dio2, Lhs, Ucp1, Pgc1α, and Cidea gene and UCP1 protein expression in PINX animals, leading to altered rhythmicity under sub-thermoneutral conditions, which was partially restored by melatonin treatment. The same was observed for mitochondrial complexes I, II, and IV protein expression and enzyme activity. Melatonin absence seems to impair BAT responses to metabolic challenges, and melatonin replacement reverses this effect, with additional increase in the expression of crucial genes, suggesting that melatonin plays an important role in several key points of the thermogenic activation pathway, influencing both the rhythmic profile of the tissue and its ability to respond to metabolic challenges, which is crucial for the organism homeostasis.