Discussion

The present study has shown an early weaning model achieved by maternal deprivation that combines nutritional insult and emotional stress. It was capable of raising cardiometabolic risk since youth, allowing cardiovascular disease development in adulthood/elderly. We found that early-weaned male rats presented higher body mass, SBP, and impaired serum lipid profile than controls. Besides, diastolic dysfunction associated with a suggestive pattern of cardiac hypertrophy, as well as exercise intolerance, was also observed in these animals.

Soon after early weaning, 30-day-old rats have presented lower body weight and BMI than controls, as reported in previous studies involving mother–offspring separation^{Reference McIntosh, Anisman and Merali26}, bromocriptine administration,^{Reference Bonomo, Lisboa, Pereira, Passos and de Moura9} and bandages.^{Reference Lima, Moura and Passos12} Thus, these data validate the proposed experimental model of malnutrition during lactation.

Upward postnatal day 150, early-weaned rats have presented a slightly higher body weight than controls, accompanied by hyperphagia, since postnatal day 90. This observation may be due to ‘catch-up growth’ defined by Wit and Boersma^{Reference Wit and Boersma27} as an accelerated growth after a period of its inhibition determined by early undernutrition. In elderly animals, there was an increased abdominal/thoracic circumferences ratio. According to literature, this profile is related to a higher cardiometabolic risk.^{Reference Huxley, Mendis, Zheleznyakov, Reddy and Chan28}

Dyslipidemic profile corroborates the increase in cardiovascular disease risk, worsened with aging. There were higher plasma levels of TG and VLDL-c, as well as increased TG/HDL-c ratio since postnatal day 30, followed by the rise of the Castelli index and the decrease in HDL-c levels. Dyslipidemic profile was also observed in different models of early weaning.^{Reference Moura, Bonomo and Neto10,Reference Lima, Moura and Passos12,Reference Franco, Lisboa and Lima29} Experimental studies have shown that hypertriglyceridemia is a risk factor for coronary heart disease, regardless of overweight. TG-rich lipoproteins, particularly VLDLs, have a direct atherogenic effect.^{Reference Murad, Hazem and Coto-Yglesias30-Reference Havel and Hamilton32} VLDLs are hydrolyzed in the liver by lipase and converted to LDL particles, a crucial trigger of atherosclerosis genesis that favors lipid deposition in vessels.^{Reference Brown and Goldstein33-Reference Klop, Elte and Cabezas35} Besides, HDL-c has a protective role partly due to its ability to remove intracellular cholesterol and perform reverse transport, allowing its redistribution in the body and biliary excretion.^{Reference Rizzo, Otvos, Nikolic, Montalto, Toth and Banach36} HDL-c presents antioxidant and anti-inflammatory properties, inactivating or preventing oxidation of phospholipids from LDL-c.^{Reference Gogonea37,Reference Dobiásová38} HDL-c also inhibits apoptosis of endothelial cells and is associated with a relevant antiatherogenic action.^{Reference Klop, Elte and Cabezas35-Reference Gogonea37}. According to literature, the Castelli I index and TG/HDL ratio are atherogenic markers. They are useful instruments to predict coronary artery disease risk in daily practice, especially in people with other risk factors.^{Reference Marques, Motta and Kummerle21,Reference Reaven24,Reference Sayın, Kutlu and Koçak39}

Leptin serum levels have also increased in early-weaned male rats upward postnatal day 150. Usually, leptin increases the expression of neuropeptides associated with food intake inhibition. Proopimelanocortin, as well as cocaine and amphetamine-regulated transcripts, stimulates total energy expenditure via sympathetic activation. Besides, it decreases the expression of neuropeptide Y and agouti-related protein, associated with increased food intake and reduced energy expenditure.^{Reference Sayın, Kutlu and Koçak39} Leptin plays an essential role in regulating energy homeostasis, decreasing appetite and increasing energy expenditure. Thus, the hyperphagia and the higher body weight suggest leptin hypothalamic resistance. Hyperphagia, higher body mass, and TG/leptin serum levels as well as leptin resistance outcomes were also observed in previous studies encompassing other animal models of early weaning.^{Reference Moura, Bonomo and Neto10,Reference Bonomo, Lisboa, Passos, Alves, Reis and de Moura11,Reference Franco, Lisboa and Lima29,Reference Pietrobon, Bertasso and Silva41,Reference Lima, Moura and Franco42}

Early-weaned rats presented higher LVM, IVS thickness and LVPW thickness, both in diastole, compared to respective adult and elderly controls. RWT was higher in this group at postnatal day 90 but not 150 or 365. These data point to concentric remodeling that evolved into eccentric patterns commonly observed in patients with diastolic heart failure.^{Reference Nagueh, Appleton and Gillebert43} The observed increase in LA/Ao diameter ratio suggests pressure raise in LA and consequently dilatation of its cavity, possibly due to ventricular relaxation impairment during diastole.^{Reference Hoss, Petterson and Scherer44,Reference Boon45} MDT, intimately related to left ventricular compliance, was shorter in early-weaned animals characterizing diastolic dysfunction and left atrial dilatation does.^{Reference Nagueh, Appleton and Gillebert43}

The maximal effort ergometer test infers the cardiorespiratory capacity.^{Reference Marques, Barros, Rocha and Scaramello15} There is a linear relationship between maximum speed developed by the animal and its oxygen uptake rate.^{Reference Rodrigues, Figueroa, Mostarda, Heeren, Irigoyen and De Angelis46} Thus, a less lasting test accompanied by a shorter distance traveled and an inferior maximum speed described to elderly early-weaned rats suggest a diminished tolerance to exercise compared to respective controls. Diastolic dysfunction favors exercise intolerance.^{Reference Cohen-Solal, Beauvais and Tabet47} It is noteworthy that maximum cardiac output during exercise is dependent on diastolic filling. If relaxation abnormalities accompany diastole, lower ventricular filling rates are achieved and become insufficient to supply cardiac output required during exercise sessions. Consequently, filling pressure increases and maximum capacity decreases; thus, as diastolic dysfunction progresses, the lower will become exercise capacity.^{Reference Otto, Pereira, Beck and Milani48,Reference Little, Kitzman and Cheng49}

Early-life stress is also a relevant factor involved in metabolic programming.^{Reference Souza, de Moura and Lisboa7} Despite this, biometric, nutritional, and biochemical outcomes here described are similar to those observed in studies using pharmacological and non-pharmacological models of early weaning that avoid emotional stress.^{Reference Bonomo, Lisboa, Passos, Passos-Moura, Reis and Moura8-Reference Lima, Moura and Passos12,Reference Franco, Lisboa and Lima29,Reference Pietrobon, Bertasso and Silva41,Reference Lima, Moura and Franco42}

According to Tractenberg et al.,^{Reference Tractenberg, Levandowski and de Azeredo50} maternal deprivation protocols present methodological pitfalls due to different degrees of stress exposure in maternal separation-reared pups. Nevertheless, here we took care of important methodological issues to avoid bias, such as housing/husbandry conditions, animal/standard facility rearing, and litter controls.

Kikusui, Ichikawa, and Mori^{Reference Kikusui, Ichikawa and Mori51} observed that mice weaned on postnatal day 14 showed a prolonged increase in corticosterone compared to the littermates stayed with dam until postnatal day 21. The last ones have presented a transient increase. Moura et al. ^{Reference Moura, Bonomo and Neto10} also reported hypercorticosteronaemia and higher total catecholamine levels later in life. According to Huang et al.,^{Reference Huang, White, Ahmad and Leenen52} hypercorticosteronemia could explain the higher blood pressure seen because glucocorticoids increase blood pressure. For instance, hypertension is often associated with left ventricular hypertrophy and heart failure with preserved ejection fraction, that impair exercise tolerance, outcomes described in this study.^{Reference Otto, Pereira, Beck and Milani48,Reference Little, Kitzman and Cheng49,Reference Dickstein, Cohen-Solal and Filippatos53,Reference Montera, Almeida and Tinoco54}

Literature report that adaptation mechanisms may be different between sexes in early weaning models.^{Reference Souza, de Moura and Lisboa7} Nevertheless, our main objective was to describe outcomes in male rats submitted to an early weaning model encompassing an emotional stress, as widely performed with pharmacological and non-pharmacological models.

The measurement of corticosterone level would contribute to a better understanding of this model, although not compromise the relevance of our findings. Along with corticosterone measurement, sexual dimorphism also encompasses a perspective for future studies in this experimental model.

In conclusion, the present work demonstrates, for the first time, cardiovascular disease development in later life due to maternal deprivation at postnatal day 18. This early weaning model simulates mother abandoning, a social health challenge. As cardiovascular diseases are the leading death causes worldwide, public policies favoring exclusive breastfeeding until 6 months of age may constitute an important strategy to reduce their incidence, reducing direct and indirect health expenditures.