Sex-Specific Anxiety and Prefrontal Cortex Glutamatergic Dysregulation Are Long-Term Consequences of Pre-and Postnatal Exposure to Hypercaloric Diet in a Rat Model

Both maternal and early life malnutrition can cause long-term behavioral changes in the offspring, which depends on the caloric availability and the timing of the exposure. Here we investigated in a rat model whether a high-caloric palatable diet given to the mother and/or to the offspring during the perinatal and/or postnatal period might dysregulate emotional behavior and prefrontal cortex function in the offspring at adult age. To this end, we examined both anxiety responses and the mRNA/protein expression of glutamatergic, GABAergic and endocannabinoid signaling pathways in the prefrontal cortex of adult offspring. Male animals born from mothers fed the palatable diet, and who continued with this diet after weaning, exhibited anxiety associated with an overexpression of the mRNA of Grin1, Gria1 and Grm5 glutamate receptors in the prefrontal cortex. In addition, these animals had a reduced expression of the endocannabinoid system, the main inhibitory retrograde input to glutamate synapses, reflected in a decrease of the Cnr1 receptor and the Nape-pld enzyme. In conclusion, a hypercaloric maternal diet induces sex-dependent anxiety, associated with alterations in both glutamatergic and cannabinoid signaling in the prefrontal cortex, which are accentuated with the continuation of the palatable diet during the life of the offspring.


Introduction
Maternal nutrition plays a crucial role in the offspring's brain development during pregnancy and during a critical period after birth. Initial studies on perinatal programming have shown that malnutrition during pregnancy and/or lactation induces metabolic disorders such as hyperphagia, adiposity, hyperlipidemia and glucose intolerance in offspring, even into adulthood [1][2][3][4]. Moreover, growing evidence has revealed a modulation of the offspring behavior related to depression, anxiety-like behaviors and addiction (preference of hypercaloric foods and drugs) due to the consumption of a hypercaloric diet in the perinatal period, being associated with long-term structural and functional alterations in the neurodevelopment of the prefrontal cortex (PFC) [5][6][7][8].

Experimental Design
After 8 weeks of pregestation with the assigned diets, the females were mated for 24 h from the start of the proestrus. Gestational day 0 (GD0) was determined by the presence of sperm. The dietary paradigm was maintained during pregnancy and lactation. At 14 h after birth (PND0), pups were weighed and sexed. The litter size was arranged to comprise 8 pups consisting of 4 males and 4 females where possible. The remaining pups were quickly sacrificed by decapitation. Mating was successful in 9 female rats exposed to the C diet, resulting in 9 C litters, and in 11 mothers exposed to the free-choice P diet, resulting in 11 P litters. All litters were equally represented in each offspring group. Initially, we randomly distributed 3-4 pups per litter, resulting in 35-41 pups per experimental group (35 C males, 36 C females, 38 P males and 41 P females). At PND 22-23, all offspring were weaned and randomly assigned to a C diet or free-choice P diet, resulting in eight experimental groups with 1-2 pups per litter (n = 15-20): C diet-fed male and female offspring born to C diet-fed dams (15 CC males and 16 CC females); C diet-fed male and female offspring born to free-choice P diet-exposed dams (17 PC males and 21 PC females); free-choice P diet-exposed male and female offspring born to C diet-fed dams (16 CP males and 16 CP females); and free-choice P diet-exposed male and female offspring born to free-choice P diet-exposed dams (17 PP males and 20 PP females). To minimize estrous cycle-related variability, the female offspring were closely housed in adjacent cages and were separated from males [26]. Food intake and body weight were measured daily during pregnancy and lactation and weekly after weaning. At postnatal weeks 21-23, the adult offspring were sacrificed.

Sample Collection
The adult offspring were sacrificed at postnatal weeks 21-23, by decapitation after the administration of Equitesin ® (3 mg kg −1 ). Sacrifices were made in an isolated room two hours after the start of the dark phase. Brains were frozen and stored at -80 • C (n = 5-6; 1 rat per litter was randomly selected). The prefrontal cortex (PFC) was dissected from Bregma 4.70 mm to 2.20 mm with fine surgical instruments [27]. Brain samples were weighed and stored at −80 • C until they were used for mRNA and protein analysis.

RNA Isolation and Real-Time Quantitative PCR Analysis
Real-time PCR (TaqMan, ThermoFisher Scientific, Waltham, MA, USA) was performed as described previously [28] using TaqMan Gene Expression Assays primers shown in Table 2. Prefrontal cortex RNA (n = 5-6; 1 rat per litter was randomly selected) was extracted following the Trizol ® method Nutrients 2020, 12, 1829 4 of 18 (ThermoFisher Scientific). RNA samples were isolated with the RNeasy MinElute cleanup kit, including digestion with a DNase I column (Qiagen, Hilden, Germany). Reverse transcript reaction of 1 µg of RNA and quantitative real-time reverse transcription polymerase chain reaction (qPCR) were performed in a CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the FAM dye-labeled format for the TaqMan ® Gene Expression Assays (ThermoFisher Scientific). We found that a single product was amplified using a melting curve. The results were normalized in relation to Actb after checking their homogeneity between groups.

Western Blot Analysis
Western blotting was performed as described previously [29]. Briefly, prefrontal cortex (n = 4; 1 rat per litter were randomly selected) were homogenized in 500 µL of ice-cold lysis buffer containing Triton X-100, 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 M ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, sodium fluoride (NaF), sodium orthovanadate (NaOV) and protease inhibitors using a tissue-lyser system (Qiagen). After centrifuging at 26,000× g for 30 min at 4 • C, the supernatant was transferred to a new tube. Bradford method was used to measure the protein concentration of the samples. A quantity of 30 µg of each total protein sample was separated on 4-12% polyacrylamide gradient gels. The gels were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) and stained with Ponceau red, Membranes were blocked in TBS-T (50 mM Tris-HCl pH 7.6, 200 mM NaCl, and 0.1% Tween 20) with 2% albumin fraction V from bovine serum (BSA, Roche, Mannheim, Germany) for 1 h at room temperature. The primary antibodies to the proteins of interest (Table 3) were incubated overnight at 4 • C. Mouse βactin was used as the reference protein. After several washes in TBS containing 1% Tween 20, an HRP-conjugated anti-rabbit or anti-mouse IgG (H+L) secondary antibody (Promega, Madison, WI, USA), diluted 1:10,000, was added followed by incubation for 1 h at room temperature. After extensive washing in TBS-T, the membranes were incubated for 1 min with the Western Blotting Luminol Reagent kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the specific protein bands were visualized and quantified by chemiluminescence using a ChemiDocTM MP Imaging System (Bio-Rad, Barcelona, Spain). The results are expressed as the target protein/ βactin ratios.

Behavioral Studies
At week 7-8 postnatal, anxiety-related behaviors were evaluated in the offspring using the open field and elevated plus maze tests.
The elevated plus-maze (Panlab, Barcelona, Spain) is a cross-shaped platform elevated 65 cm from the floor with two opposing open arms (50 × 10 cm) and two closed arms (50-cm high opaque walls). A central area of 10 cm 2 connected four arms. The light intensity was set at 150 lux in the open arms and 80 lux in the closed arms. The test was performed at 5 h after the beginning of the dark phase. The animal was allowed to explore the maze freely for 5 min. A computer-controlled system recorded the number of entries and the time spent in each arm. Data were analyzed by using the MAZEsoft software (Panlab, Barcelona, Spain). Animals that fell off the maze during the test were excluded from the analysis.
Two days after the elevated plus maze test, the open field test was carried out, which isa square arena (80 cm × 80 cm and 40 cm high) virtually divided into a peripheral zone and a central zone (40 cm × 40 cm) located in a room with low light intensity (30 lux). The test was performed 5 h after Nutrients 2020, 12, 1829 6 of 18 the beginning of the dark phase. Rats explored freely for 5 min. A video tracking motion analysis system (Smart, Panlab, Harvard Apparatus, Spain) measured the total distance traveled (cm) and mean speed (cm/s), and calculated the time spent on central area as well as the number of entries to the central zone as an index of anxiety-like behavior.

Statistical Analysis
All data are expressed as the mean ± SEM. Animal model data were analyzed by three-way ANOVA (sex, maternal diet and offspring diet as factors) using IBM SPSS Statistics 23. Subsequent comparisons between two groups were carried out using Student's t-test. Pearson's correlation analysis was performed using IBM SPSS Statistics 23. p < 0.05 was considered statistically significant.

Effect of Maternal and/or Postnatal Hypercaloric Diet on Offspring Behavior
The  Figure 1D).
Regarding the locomotor activity, a sex effect was found in the total distance travelled in the open field test [F (1,124) = 50.772; p < 0.001], with an overall increase in the distance traveled by PP females (* p < 0.05; # p < 0.05; & p < 0.05; Figure 1E).
A sex effect was also found in the total entries in the elevated plus maze test [F (1,124) = 23.678; p < 0.001], with an overall decrease in the number of entries made by the PP male group (** p < 0.01; # p < 0.05; & p < 0.05; Figure 1F).

Effects of Maternal and/or Postnatal Hypercaloric Diet on Glutamatergic Signaling in Adult Offspring Prefrontal Cortex
Regarding glutamate synthesis and transport, an interaction between maternal and postnatal diets was found on the mRNA levels of the glutamate synthesizing enzyme Gls [F (1,41) = 8.399; p < 0.01], with an overall increase in Gls mRNA in PP male animals compared to CP and PC groups ( # p < 0.05; & p < 0.05; Figure 2A

Effects of Maternal and/or Postnatal Hypercaloric Diet on Glutamatergic Signaling in Adult Offspring Prefrontal Cortex
Regarding glutamate synthesis and transport, an interaction between maternal and postnatal diets was found on the mRNA levels of the glutamate synthesizing enzyme Gls [F (1,41) = 8.399; p < 0.01], with an overall increase in Gls mRNA in PP male animals compared to CP and PC groups ( # p < 0.05; & p < 0.05; Figure 2A). A significant interaction between postnatal diet and sex was also found on the mRNA levels of glutamate uptake protein Slc1a1 [F (1,41) = 12.359; p < 0.01], with the hypercaloric postnatal diet increasing Slc1a1 mRNA levels in CP and PP male offspring (* / ** p < 0.05/0.01; & p < 0.05; Figure 2C).    response to maternal and postnatal hypercaloric diets in the male offspring (PP group) (** p < 0.01; # p < 0.05; Figure 2H).
Regarding metabotropic glutamate receptors, there was a significant interaction between postnatal diet and sex on mRNA levels of Grm5 [F (1,41) = 8.199; p < 0.01], with an increase in Grm5 mRNA levels in CP, PC and PP males. compared to the CC group ( */** p < 0.05/0.01; Figure 2M). No main effects or interactions between factors were found in Grm3 expression, however Student's t test showed an increase in Grm3 mRNA levels in the CP and PP male groups, compared to the control group (* p < 0.05; Figure 2L).
In order to establish the relationship between the main changes found in anxiety-related behavioral tests and those found in the mRNA expression of glutamatergic signaling in males, we performed correlation studies.
Our results showed a negative correlation between the time spent in the central area of the open field test and the mRNA expression of Gls and Gria2 (p < 0.01).
The time spent in open arms of the elevated plus maze test also correlated negatively with gene expression of the glutamatergic receptors Gria1 and Grm5 (p < 0.05).
Finally, the number of entries that the animals made into the open arms of the elevated plus maze test showed a negative correlation with the mRNA levels of Slc1a1 (p < 0.05), Grin1 (p < 0.01), Gria1 (p < 0.05), Grm3 (p < 0.05) and Grm5 (p < 0.01). The correlation coefficients are shown in Table 4. Table 4. Pearson's correlation coefficients between behavioral parameters related to anxiety and elements of the glutamatergic system. No correlation was found between the number of entries into the central area of the open field test and the glutamatergic system genes included in this part of the study.

Effects of Maternal and/or Postnatal Hypercaloric Diet on ECS and Glutamatergic Signaling Protein Levels in Adult Offspring Prefrontal Cortex
Regarding glutamate receptors, no main effects were found in the protein levels of GRIN1. However, Student´s t test showed an increase in GRIN1 in PFC of CP males compared to the control group (* p < 0.05; Figure 4A). The main effects of postnatal diet were found on the protein level of mGLUR5 [F (1,24) = 10.734; p < 0.01], with an increase in the mGLUR5 expression in the male CP group, compared to the control group, and a decrease in the PC males and females compared to the CP groups (* p < 0.05; # p < 0.05; Figure 4C).  Regarding the endocannabinoid system, an effect of the maternal diet was found in the protein level of CB1 [F (1,24) = 7.597; p < 0.05], with the PC male group expressing significantly less CB1 that the control group (* p < 0.05; Figure 4D). An effect of the postnatal diet was found in the protein level of the cannabinoid receptor CB2 [F (1,24) = 9.489; p < 0.01], with an increase in the CB2 expression in the female CP group compared to the control group (* p < 0.05; Figure 4E). No main effects were found in the protein levels of DAGLβ. However, the Student´s t test showed an increase in DAGLβ in PFC of PP females, compared to the control group (* p < 0.05; Figure 4G). A significant interaction between maternal and postnatal diets and sex was found on the protein level of NAPE-PLD [F (1,24) = 4.877; p < 0.05], with an overall decrease in the NAPE-PLD protein level in CP and PC males (** / *** p < 0.01/0.001; Figure 4I). A significant interaction between maternal and postnatal diets was also found in the protein level of endocannabinoid-degrading enzyme FAAH [F (1,24) = 10.000; p < 0.01], with an overall decrease in the FAAH protein level in PC females (* p < 0.05; Figure 4J). Figure S2 shows membranes used in this study stained with Ponceau red stain for locating protein bands on Western blots, and the proteins analyzed in each one of them.

Effects of Maternal and/or Postnatal Hypercaloric Diet on Inflammation in Adult Offspring Prefrontal Cortex
Regarding gliosis, an interaction between sex and maternal diet was found in the mRNA levels of Gfap [F (1,41) = 4.605; p < 0.05] ( Figure 5D).

Effects of Maternal and/or Postnatal Hypercaloric Diet on Inflammation in Adult Offspring Prefrontal Cortex
Regarding gliosis, an interaction between sex and maternal diet was found in the mRNA levels of Gfap [F (1,41) = 4.605; p < 0.05] ( Figure 5D).
No main effects were found in the mRNA expression of Tlr4. However, the Student´s t test showed an increase in Tlr4 in PFC of the PC male, compared to the CP, group ( # p < 0.05; Figure 5A).

Effects of Maternal and/or Postnatal Hypercaloric Diet on GABAergic Signaling in Adult Offspring Prefrontal Cortex
Regarding GABA receptors, only a sex effect was found on the mRNA levels of Gabbr2 [F (1,41) = 4.558; p < 0.05], with an increase in Gabbr2 mRNA levels in PP males compared to the CC group (* p < 0.05; Figure S1H).
No more main effects were found, however the Student's test showed an increase in the mRNA expression of Gabrb2 and Gabbr1 in PP males (** p < 0.01; # p < 0.05; Figure S1D,G).

Discussion
The present study indicates that hypercaloric diet consumption during the perinatal period No main effects were found in the mRNA expression of Tlr4. However, the Student´s t test showed an increase in Tlr4 in PFC of the PC male, compared to the CP, group ( # p < 0.05; Figure 5A).

Effects of Maternal and/or Postnatal Hypercaloric Diet on GABAergic Signaling in Adult Offspring Prefrontal Cortex
Regarding GABA receptors, only a sex effect was found on the mRNA levels of Gabbr2 [F (1,41) = 4.558; p < 0.05], with an increase in Gabbr2 mRNA levels in PP males compared to the CC group (* p < 0.05; Figure S1H).
No more main effects were found, however the Student's test showed an increase in the mRNA expression of Gabrb2 and Gabbr1 in PP males (** p < 0.01; # p < 0.05; Figure S1D,G).

Discussion
The present study indicates that hypercaloric diet consumption during the perinatal period induces anxiety-related behavioral changes in adult offspring that kept consuming the palatable diet after weaning (PP animals). Furthermore, increased anxiety in PP animals is associated with specific changes in the prefrontal cortex's (PFC) mRNA expression of ionotropic and metabotropic glutamate receptors, such as Grin1, Gria1 and Grm5, and endocannabinoid signaling elements, such as the Cnr1 receptor or the endocannabinoid-synthesis/degrading enzymes Diacylglycerol lipase-beta (Daglβ) and N-acyl phosphatidylethanolamine phospholipase D (Nape-pld). The alterations suggest an enhanced glutamatergic activity in the PFC, associated with a reduced inhibitory input into the glutamate synapses through the cannabinoid CB1 receptor, whose expression was decreased. These changes are sex-dependent, being more marked in males.
Traditionally, maternal nutrition studies have focused on offspring metabolic diseases, such as glucose tolerance, insulin resistance, dyslipidaemia, hypertension and obesity, in humans and rodents [4,30,31]. However, it is known that continued stress, such as malnutrition with an excess or deficiency in most nutrients, impacting fetal neurodevelopment, can cause neuropsychiatric disorders, including depression, anxiety and attention-deficit/hyperactivity disorder in offspring, even though to adulthood [32]. Thus, both exposure to a high-fat diet and calorie restriction during the perinatal period can lead to eating disorders in offspring, such as hyperphagia and the preference for hypercaloric food [33][34][35]. In addition to eating disorders, perinatal exposure to a high-fat diet has been shown to increase anxiety-related behaviors in offspring, this effect being independent of the type of diet consumed during postnatal life [36][37][38].
Our results confirm that a free-choice palatable diet (P) during the perinatal period exerts an effect by itself on the offspring's time in, and number of entries into, the central area of the open field test. However, we also observed an interaction between the maternal diet and the postnatal diets, affecting the same parameters mentioned, as well as the time in and number of entries into the open arms of the elevated plus maze test. Thus, the male PP group showed the most anxiety-like behaviors, associated with a decrease in these four behavioral indicators despite normal locomotion. These results also indicate a marked sexual dimorphism in long-term hypercaloric diet-induced anxiety behavior.Earlier studies reported the implications of a hypercaloric diet for brain structures and the neurotransmitter systems important in anxiety. For example, obese animals are susceptible to develop anxiety-like behaviors, probably through changes in the glutamatergic and GABAergic neurotransmission within the ventromedial and dorsomedial hypothalamic nucleus [39][40][41], which demonstrated that a high-fat/high-sugar diet induces alterations in the function of the prefrontal cortex through changes in the expression of GABAergic parvalbumin-expressing inhibitory interneurons, which was associated with increased anxiety-like behaviors.
Maternal malnutrition has also been shown to play an important role in programming behavioral disorders, such as anxiety, associated with alterations in the neurodevelopment of the brain areas involved in behavior. However, most studies are focused on the hypothalamic-pituitary-adrenal (HPA) axis [1,5,42]. The prefrontal cortex (PFC) provides executive control by coordinating cognitive, emotional and behavioral responses to threatening stimuli, however, the precise role of signaling within PFC, with respect to the stress of a maternal hypercaloric diet, has been minimally investigated.
Our results showed an overall activation of PFC glutamate signaling in the PP male group, with an increase in the mRNA levels of the glutamate synthesis enzyme Gls, the glutamate transporter Slc1a1, the ionotropic receptors NMDA type Grin1 and Grin2c, the ionotropic receptors AMPA type Gria1, Gria2 and Gria4, and the metabotropic receptors Grm3 and Grm5. Several studies relate behavioral disorders in offspring, such as schizophrenia and anxiety-like behaviors, to alterations in GABAergic signaling (in involved areas such as HPA axis, hippocampus and cortex) induced by such perinatal insults as lipopolysaccharide (LPS), drugs and malnutrition [1,5,22,43]. Contrarily, our data show no effect of perinatal and/or postnatal free-choice P diet on GABAergic signaling in PFC of adult offspring. Furthermore, our results showed a negative correlation between anxiety-related behavioral tests and the main altered genes of the glutamatergic system. Thus, an increase in the expression of the glutamatergic system is associated with less time spent in the central area of the open field test, and less time in, and number of entries into, the open arms of the elevated plus maze test; that is, greater anxiety.
So, results from the present study suggest that the impact of the maternal and/or postnatal P diet on anxiety levels in adult offspring relate to alterations in PFC glutamate levels, or its modulatory systems, including the endocannabinoid system (ECS), as we will discuss below.
The ECS plays an important role in various physiological functions, including neuroprotection, synaptic plasticity and energy homeostasis. ECS is a neuromodulator of the major excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitter systems in the brain. In addition, the ECS plays a crucial role during critical periods of brain development, and its disruption by early stressful events, including metabolic disruptions, can lead to significant neuropsychiatric symptoms. [17]. Our results are in agreement with previous studies that demonstrate alterations in cannabinoid signaling in the brain of adult offspring induced by maternal malnutrition [8,24,28]. We found a decrease in the mRNA expression of the cannabinoid receptor Cnr1 in the PFC of adult animals born from mothers fed with a free-choice palatable diet, and who continued eating the P diet after weaning. Furthermore, the protein levels of CB1 are also affected by the maternal diet, with an overall decrease in its expression in males compared to the control group. The palatable diet consumed from the perinatal period to adulthood (PP groups) also induces a decrease in gene levels of Nape-pld, the main synthesis enzyme of anandamide (AEA). The decrease of both Cnr1 and Nape-pld in the PP groups could indicate a decrease in AEA, that would explain the increase in anxiety in these animals, since the anxiolytic role of this endocannabinoid has been demonstrated, and the pharmacological augmentation of central endogenous cannabinoid (eCB) signaling being a promising treatment for anxiety disorders [44].
Our results also discard a potential diet-associated neuroinflammation factor in these observed long-term alterations, despite previous reports suggesting that a hypercaloric diet might result in neuroinflammation, thus contributing to behavioral disorders [40,45]. Our results do not support this hypothesis, since we did not find alterations in the mRNA expression of several genes that would indicate the activation of inflammatory pathways, such as the toll-like receptor related to pathogen recognition and the activation of innate immunity Tlr4, the cyclooxygenase key in prostaglandin biosynthesis Ptgs2, the microglia/macrophage-specific calcium-binding protein Iba1, the major intermediate filament proteins of mature astrocytes Gfap, and the cannabinoid receptor Cnr2, induced by the perinatal and/or postnatal P diet in the adult offspring PFC.
The perinatal and/or postnatal hypercaloric diet can exert a direct effect on cannabinoid signaling, decreasing cannabinoid tone, interrupting the negative feedback that this system exerts on glutamatergic neurotransmission, which is thus increased. There could also be a direct effect of diet on glutamatergic signaling, by increasing it. Both effects could explain the increase in the anxious behavior of PP animals. Although we have not addressed the impact of specific nutrients as potential sources for the effects observed, we must consider that the fatty acid composition of the diet might dysregulate the amount of endocannabinoid precursors [46], and that maternal hypoproteic diets can also disrupt brain glutamatergic systems [47].
In the present study we have found some inconsistencies between the genetic and protein results. We consider these differences to be a limitation of the study, due to the low number of samples analyzed by western blot. So, future extensive protein studies will be necessary to elucidate this controversy.

Conclusions
The conditions of early life contribute greatly to the risk of mental disorders, and therefore, the study of the origins and mechanisms involved in the emergence of these disorders is essential for their prevention and/or treatment.
In this study, we demonstrate that a free-choice palatable diet during the perinatal period induces changes in the glutamatergic signaling of the adult offspring prefrontal cortex, associated with alterations in the endocannabinoid system, a main regulator of the glutamate synapses. Furthermore, these changes are more evident when the palatable diet is maintained after weaning, and its effects are sex-dependent.
Glutamatergic and endocannabinoid disorders induced by maternal diet are related to increased anxiety-like behavior in adulthood.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/12/6/1829/s1, Figure S1: Effect of maternal and/or offspring exposure to a free-choice palatable (P) diet on the relative mRNA levels of GABAergic signaling genes, Figure S2: Ponceau red of the membranes used for western blots and the proteins analyzed in each membrane.
Author Contributions: P.R., M.T.R.-L., J.S. and F.R.d.F. were responsible for the concept and design of the study. P.R., M.T.R.-L., R.T., J.A.N., A.V., J.S. and F.R.d.F. contributed to data acquisition and analysis. P.R., M.T.R.-L., J.S. and F.R.d.F. contributed to data analysis and interpretation. All authors were involved in drafting or revising the manuscript critically for important intellectual content. All authors approved the version to be published.

Conflicts of Interest:
The authors declare no conflict of interest.