To our knowledge, this is the first study that investigated BBB disruption in an animal model of mania induced by AMPH. Corroborating with previous studies, AMPH-injected rats exhibited hyperactivity, which was determined by an increased frequency of crossings and distance travelled. Although our model showed face validity, no changes in peripheral and CNS levels of TNFα and TBARS were observed following AMPH and lithium injection. Consequently, protein levels of claudin-5, the most enriched tight junction protein in the BBB, also remained unchanged in the brain regions analyzed.
Psychostimulant-induced animal model of mania, such as the AMPH model, is frequently used to investigate biological mechanisms and alterations that have already been described in BD (Kara and Einat, 2013; Sharma et al., 2016). Clinical studies often report an increase in inflammatory and oxidative stress parameters (Rowland et al., 2018), and most preclinical evidence is consistent with such findings. For instance, Valvassori et al. (2015) have shown that AMPH injections resulted in a pro-inflammatory effect. Injected rats presented with an augment of IL-4, IL-6, IL-10, and TNFα in the PFC, ST, and serum, which were restored to control levels following treatment with lithium. However, the authors did not find significant alterations in the HIP and cerebrospinal fluid (CSF). Other psychostimulant drugs, such as methylphenidate and methamphetamine, also seem to increase inflammatory markers in the HIP of rats (Beirami et al., 2017; Motaghinejad et al., 2017). However, no changes in TNFα levels were found in the serum and brain regions analyzed in our model. Still, no differences in inflammatory cytokines levels – such as TNFα, IL-1β, and IL-10 – have been previously described in rodents injected with AMPH or its derivates (Bristot et al., 2019; Gubert et al., 2016).
Classically, AMPH is responsible for enhancing dopamine (DA) release by inhibiting its reuptake, promoting reverse transport of DA into the synaptic cleft independent of stimulus and releasing DA from synaptic vesicles in the cytoplasm (Calipari and Ferris, 2013). These are the primary mechanisms involved in AMPH-induced hyperactivity and its neurotoxic effects (Valvassori et al., 2021). If not stored in synaptic vesicles, cytoplasmatic DA has a highly autoxidative capacity that can impair mitochondrial function and increase oxidative stress resulting in cell death (Brown and Yamamoto, 2003; Yamamoto and Bankson, 2005). However, in the present study, lipid peroxidation was not augmented following AMPH injection, given by TBARS levels. TBARS levels were higher in the ST, a brain region with many dopaminergic projections, but no statistical difference between groups was found. Still, a strong positive correlation between TBARS and TNFα levels was observed in this same brain region in AMPH-injected groups, regardless of treatment with lithium. Overall, findings regarding oxidative stress in AMPH models vary in the literature. More acute protocols or higher doses seem to be more likely to promote such alteration in the CNS (Frey et al., 2006; Gomes et al., 2017; Gubert et al., 2016). Also, higher levels of TBARS were observed in the serum of lithium-treated animals, independent of AMPH injection. Lithium per se is known to be nephrotoxic (Carter et al., 2013), and increased levels of TBARS in the kidney have already been described in rats (Davis et al., 2018; Ossani et al., 2019).
Oxidative stress is an important promoter and product of the inflammatory response (Biswas, 2016), and it is supposed to underly AMPH-induced inflammation. As oxidative stress and inflammation are predictive of promoting BBB disruption and increasing its permeability (Patel and Frey, 2015), we hypothesized that AMPH would exert a deleterious effect in the BBB by downregulating claudin-5 expression. However, no significant changes were observed on claudin-5 levels in the PFC, ST and HIP of rats after AMPH and lithium injections. A previous study showing increased levels of inflammatory cytokines in the brain parenchyma but no alteration in the CSF suggested that AMPH-injected rats might not present with disrupted BBB (Valvassori et al., 2015). However, in vivo and in vitro studies have already reported that methamphetamine and other psychostimulant drugs can disturb BBB integrity (Kousik et al., 2012; Northrop and Yamamoto, 2012).
Besides inflammation and oxidative stress, associated mechanisms have been proposed to underlie BBB disruption. For instance, the activation of inflammatory pathways, such as nuclear factor kappa B (NFκB), can result in the amplification of a large array of genes involved in inflammation, including matrix metalloproteinases (MMPs) (Hurtado-Alvarado et al., 2016). MMPs are enzymes that degrade tight junctions, such as claudin-5, which are essential for maintaining BBB properties (Rempe et al., 2016). Methamphetamine has already been described to upregulate MMPs expression (Mizoguchi et al., 2007). Additionally, NFκB pathway activation following TNFα and IL-15 signalling may also be responsible for the downregulating the expression of tight junction proteins (e.g., claudin-2) in vitro (Stone et al., 2011). Therefore, it is relevant to investigate other pathways in BBB disruption and their potential association with BD and other psychiatric disorders.
Although BBB disruption has been implicated in the pathophysiology of psychiatric disorders (including BD; Greene et al., 2020), clinical and preclinical evidence is still scarce. A recent imaging study has shown that, among individuals with BD, only a sub-group exhibited an extensive BBB leakage that significantly differed from controls (Kamintsky et al., 2019). Interestingly, this sub-group of patients have a more chronic course of BD, with more severe symptoms of depression and anxiety. Other studies have evaluated biomarkers in the CSF and described higher levels of catecholamine and serotonin metabolites and inflammatory markers, such as IL-8, in individuals with BD (Isgren et al., 2015; Knorr et al., 2018). Furthermore, increased IL-8 levels in the CSF were associated with lithium treatment. Since its discovery, lithium has remained the first-line therapeutic choice for BD treatment (Yatham et al., 2018), but its effects on inflammation have yet to be fully elucidated. During euthymia, individuals with BD treated with lithium exhibited increased levels of TNFα and IL-4 compared to unmedicated patients (Guloksuz et al., 2010), which has also been described in vitro (Liu et al., 2011); but there is also evidence to show otherwise (Fernandes et al., 2019; Knijff et al., 2007). It is worth mentioning that augmented peripheral TNFα levels were further associated with poor response to lithium treatment in BD (Guloksuz et al., 2012).
Despite its novelty, some limitations should be addressed in our study. First, we did not explore more dynamic markers of the interface integrity between the blood, brain, and CSF, such as Evans Blue, which could be helpful as a first screening. Second, plasmatic levels of lithium were not assessed, but it has been described that therapeutic levels are reached following this protocol (Frey et al., 2006). Third, while only a few parameters were analyzed, their relevance to BD and the rationale for their role in the BBB disruption have been discussed. Although an ideal animal model for BD has not been developed, AMPH injection in rodents remains an established animal model of mania with good construct, face and predictive validity (Sharma et al., 2016). It should be noted that the latter has been questioned (Lan and Einat, 2019). Although this model may not mimic the vast complexity of BD pathophysiology, it would be advantageous to identify novel animal models that allow the evaluation of BBB disruption.
In summary, in one of the first attempts to investigate the effects of AMPH on BBB integrity, we did not find evidence that AMPH or lithium impact brain levels of claudin-5. It is only recently that clinical research has provided BBB disruption as a marker of progression in BD (Kamintsky et al., 2019). Still, our results provide evidence and rationale for future research to establish the best approach to model and better understand this relatively novel pathophysiological mechanism implicated in BD.