In the present study, it was evidenced that maternal deprivation protocol induced dysfunction of the BBB (in the prefrontal cortex and hippocampus) in both sexes and all ages evaluated, except in prefrontal cortex of females at postnatal day 41. We analyzed these two brain regions because the prefrontal cortex and hippocampus are two of the brain regions most involved in the pathophysiology of depression and which also seem to influence according to sex (Malhi and Mann 2018; LeGates et al. 2019).
The BBB provides a stable environment for all neural functions, besides being important for brain nutrition, and protecting of the brain from neurotoxins. Thus, BBB dysregulation may be associated with several neurological diseases (Abbott et al. 2010). Reviews of clinical and preclinical studies suggest that there is an association between neurovascular unit dysfunction, BBB hyperpermeability, and major depressive disorder, and that oxidative stress and inflammation is correlated with BBB dysfunction (Najjar et al. 2013; Kealy et al. 2020). It is worth mentioning that individuals with major depressive disorder have an increase in oxidative stress markers (Black et al. 2015) and inflammatory cytokines (Dowlati et al. 2010).
A good deal of data has established that early life stress is associated with depressive symptoms in humans (Chapman et al. 2004; Heim and Binder 2012) and depressive-like behavior in animals (Réus et al. 2011, 2017). There is also evidence to demonstrate that the maternal deprivation protocol is capable of inducing an increase in inflammatory cytokines in brain tissues and serum (Réus et al. 2017), and oxidative stress markers (Réus et al. 2015a), besides increase microglial activation and generate atrophy of astrocytes (Réus et al. 2019). These changes could justify the increase in BBB permeability observed in the present study. This is in line with another preclinical study that showed the relationship between BBB and early life stress (Gómez-González and Escobar 2009). In this study, maternal deprivation protocol increased Evans blue entry to the neocortex, hippocampus, diencephalon, basal ganglia, olfactory bulb, brain stem, cerebellum, and spinal cord at postnatal day 10, but not at postnatal day 20 or postnatal day 30. It is worth noting that in this study, males and females were evaluated together (Gómez-González and Escobar 2009). It is worth mentioning that others stress protocols can change BBB integrity (Sántha et al. 2016; Menard et al. 2017; Caron et al. 2018). Menard et al. (2017) evidenced that chronic social stress altered BBB integrity through downregulation of the tight junction protein claudin-5 in the nucleus accumbens, which, combined with stress-induced recruitment of peripheral immune signals, resulted in increased BBB permeability, the passage of blood circulating proteins such as IL-6, and the development of depression-like behaviors (Menard et al. 2017).
In the present study, the impact of three types of treatments on BBB integrity was also assessed. Regarding escitalopram, it was observed that in females, whenever the maternal deprivation protocol increased BBB permeability, the antidepressant was able to reverse this change (at all ages evaluated and in both brain regions). However, in males, escitalopram only reversed the increase in BBB permeability (in the prefrontal cortex and hippocampus) at postnatal day 21. This suggests that escitalopram was more effective in females than in male rats.
As far as we know, this was the first study that investigated the effect of escitalopram on BBB integrity. On the other hand, there is a study that evidenced that BBB dysfunction results in poorer less response to escitalopram treatment (Jha et al. 2019). It is worth mentioning that escitalopram was able to reverse the depressive-like behavior and the gut inflammation induced by a colitis protocol in ovariectomized rats, suggesting a potential anti-inflammatory effect of escitalopram (Abdo et al. 2019). Besides, a clinical study demonstrated that the treatment with SSRIs, including escitalopram, was able to decrease oxidative stress index, total oxidant status, and increase total antioxidant capacity in patients with depression (Cumurcu et al. 2009). In line with this, a recent preclinical study evidenced that escitalopram can change the expression and methylation level of genes involved in the oxidative and nitrosative stress in the hippocampus, amygdala, cerebral cortex, and blood of rats exposed to chronic mild stress (Wigner et al. 2021). This antioxidant and anti-inflammatory action could, at least in part, justify the results found in the present study. Interestingly, a clinical study with individual with post-stroke depressive symptoms evidenced that treatment responses of escitalopram tended to be more pronounced in the female group, suggesting different responses according to sex, going according to the data observed in the present study (Lee et al. 2020). Moreover, after review 15 randomized, placebo-controlled trials, Khan et al. (2005) observed that women had a significantly greater response than men to SSRI antidepressants (Khan et al. 2005). It is worth noting that there are sex differences in the pharmacokinetics and pharmacodynamics of antidepressants. For instance, women can absorb more efficiently SSRI and have a greater lipophilic antidepressant (e.g., escitalopram) distribution (Bigos et al. 2009). These differences can occur due to physiological changes, as well as hormonal changes, and differences in synaptic transmission between the sexes (Bigos et al. 2009; LeGates et al. 2019).
Regarding ketamine, it was able to reverse the change in BBB in females at postnatal day 21 (only in the prefrontal cortex) and postnatal day 61 (prefrontal cortex and hippocampus). In males, a beneficial effect of ketamine in the prefrontal cortex was observed in the three evaluated moments (postnatal day 21, 41, and 61), and in the hippocampus at postnatal day 41. Consistent with these data, literature findings show that ketamine can protect against bradykinin-induced breakage of the BBB (Chen et al. 2016). Previous data from our group also demonstrated that a single dose of ketamine (administered in postnatal day 46) in male rats submitted to the maternal deprivation protocol was able to decrease the oxidative stress induced by this protocol at postnatal day 60, especially in the hippocampus (Réus et al. 2015a). In the present study, the main results of ketamine in male rats were observed in the prefrontal cortex, however, it is worth mentioning that the treatment protocol with ketamine was different in these two studies. Literature data also show that ketamine has an anti-inflammatory effect (Nowak et al. 2019), and that, at least part of this action, can occur through interaction with gut bacteria (Getachew et al. 2018). In addition, maternal deprivation protocol induced an increase in pro-inflammatory cytokines in serum and cerebrospinal fluid, and on the other hand, ketamine treatment reduced the levels of these cytokines in deprived rats (Réus et al. 2015b).
Concerning the effects of probiotics, it was observed that in females, it reversed the change in the BBB of the hippocampus at all ages evaluated and reversed the change in the BBB of the prefrontal cortex at postnatal day 61. In males, the probiotic reversed the changes in the BBB in the prefrontal cortex at all ages, without any effect on the hippocampus.
A growing body of evidence has suggested that lipopolysaccharide (LPS) induces neuroinflammation and BBB dysfunction (Sumi et al. 2010; Yang et al. 2019). LPS is a structural component of the outer membrane of Gram-negative bacteria and can induce systemic inflammation (Lu et al. 2008). Lactobacillus spp., and Bifidobacterium spp. are the most promising probiotic species that can modulate the gut microbiota, and perform several health benefits through various mechanisms of action (Azad et al. 2018; Khalesi et al. 2019). Here, it is worth noting the probiotics' ability to decrease inflammation. A meta-analysis of randomized clinical trials evidenced that probiotic supplementation reduces serum concentrations of pro-inflammatory cytokines (Milajerdi et al. 2020). Interestingly, chronic treatment with Bifidobacterium infantis reverted the increase in pro-inflammatory cytokine in rats subjected to maternal separation (Desbonnet et al. 2010).
Another important point to be mentioned is that preclinical evidence suggests that gut microbiota influences BBB permeability (Braniste et al. 2014). Probiotic treatment (composed of Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus acidophilus) significantly attenuated BBB injury, inhibited neuroinflammation, reduced oxidative DNA damage in the brain, and decreased plasma LPS in aged mice (Yang et al. 2020). Furthermore, as recently revised, gut microbiota-derived metabolites also influence BBB integrity (Parker et al. 2020). However, as far as we know, this was the first study that investigated the effects of probiotics on the BBB integrity in a maternal deprivation protocol.
In summary, the present study demonstrated that early life stress, induced by maternal deprivation protocol, can lead to long-term BBB integrity changes in male and female rats. Moreover, treatment with escitalopram, ketamine, or probiotics can prevent some of these changes, depending on the age and sex of the animal. Noteworthy female rats at postnatal day 61 were the ones that most responded to all treatments in the two brain regions evaluated, suggesting that a more chronic treatment will bring more effective results. Thus, this study contributes to the understanding of the possible changes that early life stress can cause and possible treatments that could reverse these changes.
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