In the present work, subcutaneous injection of dexamethasone (10 mg/kg) for 7 days in rats caused significant increases in the molecular markers of neural injury in the cerebral cortex in addition to marked degeneration of the cortical neurons and disruption of its integrity. On the other hand, treating rats with carvedilol, propranolol and doxazosin ameliorated all molecular changes in the cerebral cortex and slightly reduced the histopathological changes elicited by subcutaneous injection of dexamethasone. However, the three treatments used in the current study induced distinct effects on β-arrestin2 expression, a potential neuroprotective protein. Carvedilol significantly increased, while doxazosin significantly decreased β-arrestin2 expression in the cerebral cortex of dexamethasone-treated rats. On the contrary, propranolol did not induce significant changes in β-arrestin2 expression.

Dexamethasone neurotoxicity has been reported in several previous experimental and clinical studies [30,31,32]. Intraperitoneal injection of either 7 or 20 mg/kg dexamethasone caused neurotoxicity in the cerebral cortex after 24 hours by increasing the activity of N-methyl D-aspartate (NMDA) receptors and suppression of neurotrophins [30]. In addition, low dose dexamethasone (0.2 mg/kg) acutely induced c-Fos expression in the hippocampus of neonatal rats and increased apoptotic marker expression specifically in the subiculum [31]. In the same context, a previous study showed that a 10-year-old boy who received a 5-week course of glucocorticoids for an acute asthma attack suffered from progressive decline in academic performance and estimated IQ even after long time of discontinuation of glucocorticoid treatment [32].

In accordance with previous findings [30,31,32], the present study showed that dexamethasone injection for 7 days slightly reduced brain weight and survival rate compared to control group. Also, histopathological examination of the cerebral cortex using H&E and Nissl stains showed marked degeneration in the cortical neurons and disruption of its integrity. In addition, dexamethasone significantly reduced BDNF level and Akt kinase activity in the cerebral cortex. Brain derived neurotrophic factor is a member of neurotrophins that has a vital role in regulating memory in addition to its well-established function in promoting neuronal survival and differentiation [33]. In the same context, Akt is an important survival signal that mediates brain development. Selective downregulation of Akt3, a subtype of Akt proteins, has been found to reduce brain size and weight [34].

On the other hand, dexamethasone significantly increased GFAP and DAG levels in the cerebral cortex compared to control group. GFAP is the main intermediate filament protein in mature astrocytes and a key component of its cytoskeleton during development [35]. Moreover, GFAP expression has been found to increase in the brains of patients suffering from Alzheimer’s disease [36]. In the same context, DAG is an important lipid mediator that can activate several oxidative stress and inflammation signaling. In addition, upregulation of DAG in the cerebral cortex and hippocampus has been found to impair cognitive function [37].

Alpha smooth muscle actin is a subtype of actin proteins that is expressed in vascular smooth muscles and in astrocytes in the central nervous system (CNS) [38]. Expression of α-SMA in astrocytes has been found to be increased in certain types of disorders affecting the CNS such as multiple sclerosis and Alzheimer’s disease [39, 40]. In the latter one α-SMA expression increased in the whole cortex and hippocampus [40]. Like previous findings [39, 40], our results showed significant increases in α-SMA expression in the cerebral cortex of dexamethasone-treated rats compared to control group.

SMAD3 is a component of the transforming growth factor (TGF)-β signaling pathway which is upregulated in response to neural injury as a repairing mechanism. However, SMAD3 can also promote gliosis in addition to being neuroprotective [41]. The present study showed that dexamethasone significantly increased SMAD3 expression in the cerebral cortex compared to control group. In the same context, our results showed that dexamethasone treatment significantly increased the cerebral cortex levels of β-amyloids (1-42) and phospho-Tau protein compared to control group. β-amyloids and phospho-Tau protein are both hallmarks of neurodegenerative disorders such as Alzheimer’s disease. Increased cerebral cortex and hippocampal levels of β-amyloids and phospho-Tau protein were previously recorded in a study investigating the molecular mechanisms of Alzheimer’s disease [42].

Although dexamethasone treatment significantly increased α-SMA level in the cerebral cortex compared to control group, no significant changes were observed in the fibrosis area and collagen deposition. An interpretation of this finding is that secretion of extracellular matrix such as collagen is independent of α-SMA as estimated by a previous study [43]. Furthermore, data available about changes in collagen levels in neurodegenerative disorders is conflicting [44, 45]. A previous study reported a significant increase in collagen deposits in brain tissue of Alzheimer's disease patients [44], in contrast to another study which showed no changes [45].

β-Arrestin2 is an important intracellular protein that mediates desensitization and internalization of GPCRs in addition to initiation of distinct downstream signaling pathways by recruiting and binding intracellular proteins promoting their interaction. Several studies have shown potential neuroprotective effects of β-arrestin2 [11,12,13]. β-Arrestin2 has been found to protect neurons by mediating endogenous opioid arrest of inflammatory microglia [11]. In addition, amisulpride, an atypical antipsychotic drug and a potent dopamine 2 receptor antagonist, can mediate neuroprotection by activating β-arrestin2 signaling [12]. Furthermore, β-arrestin2 has been found to protect against sevoflurane-induced neuronal apoptosis [13].

On the contrary, other studies showed detrimental effects of β-arrestin2 in the development of neurodegenerative disorders [46, 47]. Overexpression of β-arrestin2 has been shown to be associated with increased β-amyloids generation in Alzheimer’s disease [46]. In addition, β-arrestin2 mRNA levels were elevated in postmortem brain tissue from patients with Alzheimer’s disease compared with age-matched controls [47].

In the same line with the reports that showed β-arrestin2 neuroprotective effects [11,12,13], dexamethasone-induced neurotoxicity was associated with downregulation of β-arrestin2 in the cerebral cortex compared to control group.

Carvedilol is a 3rd generation β-blocker with weak α1-blocking effects. In addition, carvedilol has unique β-arrestin biased agonistic effects, anti-inflammatory, and antioxidant effects [14]. Carvedilol is a moderately lipophilic drug that can cross the blood brain barrier promoting central effects [48]. Previously, carvedilol has been reported to mediate neuroprotective effects in an experimental model of brain ischemia by reducing production of reactive oxygen species (ROS) [17]. In addition, carvedilol showed antioxidant and neuroprotective effects against rat model of diabetic neuropathy [18]. In the same context, carvedilol showed antidepressant effects associated with increased BDNF levels in the cortex and hippocampus of mice exposed to chronic unpredictable stress [49]. Furthermore, carvedilol has shown promising therapeutic effects against Alzheimer’s disease [50]. Chronic oral administration of carvedilol reduced β-amyloids production and cognitive deterioration in two different models of Alzheimer’s disease [51].

In harmony with previous findings [17, 18, 49, 50], the present study showed that carvedilol increased survival rate, slightly reduced the histopathological changes, and significantly increased brain weight, BDNF, Akt kinase activity, and β-arrestin2 levels in the cerebral cortex compared to the dexamethasone group reflecting neuroprotective effects. Moreover, carvedilol significantly decreased GFAP, DAG, α-SMA, SMAD3, β-amyloids (1-42) and phospho-Tau protein levels compared to the dexamethasone group reflecting alleviation of dexamethasone-induced neurotoxicity.

Propranolol is a highly lipophilic non-selective β-blocker that can cross the blood brain barrier and can show weak β-arrestin agonistic effects [51, 52]. Previously, propranolol has shown neuroprotective effect against retinal degeneration in a mouse model of light injury [19]. Moreover, propranolol reduced all behavioral and molecular markers of neural injury in a genetic mice model of Alzheimer's disease [53]. Propranolol treatment for 6 weeks at a dose of 5mg/kg reduced cognitive impairments and hippocampal β-amyloids and phospho-Tau protein levels and increased BDNF and Akt activity in diseased mice [53].

In harmony with previous findings [19, 53], our results showed that propranolol significantly increased BDNF level and Akt kinase activity in the cerebral cortex compared to the dexamethasone group reflecting neuroprotective effects. Also, propranolol slightly reduced the histopathological changes and significantly decreased GFAP, DAG, α-SMA, SMAD3, β-amyloids (1-42) and phospho-Tau protein levels compared to the dexamethasone group reflecting alleviation of dexamethasone-induced neurotoxicity. Notably, propranolol did not change β-arrestin2 level compared to the dexamethasone group. The latter finding shows that propranolol neuroprotective effects may be independent of β-arrestin2 signaling.

Doxazosin is a selective α1-AR blocker that can cross the blood brain barrier mediating central effects [54]. Previously, doxazosin has been reported to protect against neuroblastoma by inhibiting Akt activity leading to apoptosis of undifferentiated neuroblastoma cells [20]. On the contrary, doxazosin increased Akt activity in differentiated nerve cells making it a tool in the management of neurodegenerative disorders [20]. In this context, doxazosin showed neuroprotective effects in an in vitro model of Alzheimer's disease characterized by increased Akt activity, reduced phosphorylation of Tau proteins, and reduced neurotoxic effects of β-amyloids on hippocampal slices [20].

The present study showed that doxazosin significantly increased BDNF level and Akt kinase activity in the cerebral cortex compared to the dexamethasone group reflecting neuroprotective effects. In addition, doxazosin slightly reduced the histopathological changes and significantly decreased GFAP, DAG, α-SMA, SMAD3, β-amyloids (1-42) and phospho-Tau protein levels compared to the dexamethasone group reflecting alleviation of dexamethasone-induced neurotoxicity. Noteworthy, doxazosin significantly decreased β-arrestin2 level in the cerebral cortex compared to the dexamethasone group. Although carvedilol neuroprotective effects were associated with upregulation of β-arrestin2 level in the cerebral cortex, doxazosin showed the reverse. This may reflect multifunction and distinct effects of β-arrestin2 in the nerve cell pathophysiology which may need further investigations to understand the underlying mechanisms.

Considering the changes in the molecular markers of neural injury, both carvedilol and propranolol showed nearly the same neuroprotective effects against dexamethasone-induced neurotoxicity. However, doxazosin was the most potent neuroprotective agent in this model. Notably, the doses of carvedilol and propranolol used in the current study are pharmacologically equivalent based on their anti-hypertensive effects [25].

On the other hand, the dose of doxazosin used in the current study may be much higher than those of carvedilol and propranolol doses regarding the anti-hypertensive effect [27]. However, it is not clear if this variation in neuroprotection is related to the difference in dose levels. This point needs further investigations. Also, it is not clear whether the neuroprotective effects of the used drugs can be achieved by other types of β- and α1-AR blockers or not.

In conclusion, we showed for the first time, to our knowledge, that subcutaneous injection of dexamethasone (10 mg/kg) for 7 days in rats induces neurotoxicity in the cerebral cortex that is similar in its molecular and histopathological patterns to that of neurodegenerative disorders such as Alzheimer’s disease. Furthermore, blocking β- and/or α1ARs by using either carvedilol, propranolol, or doxazosin alleviate dexamethasone-induced neurotoxicity. However, changes in β-arrestin2 level are distinct among different treatments and may reflect multifaceted roles of β-arrestin2 in nerve cell pathophysiology.