TBI is a global health issue and one of the major leading cause of death and disability for individuals under the age of 50. According to the statistics, approximately half the global population will experience one or more TBI events over their lifetime (8). Oxidative stress caused by the redox imbalance promoted by highly reactive oxygen species (ROS; including free oxygen radicals and reactive anions) has been proven to play a major role in post-traumatic secondary brain damage (17, 31). Recent studies have shown that TS is highly enriched with ROS and that chronic TS exposure is associated with impairment of the antioxidative response system and dysfunction of normal endothelial physiology, thus promoting the onset of major cerebrovascular and neuroinflammatory/degenerative disorders (20, 24, 25, 32-35). However, there is still considerable controversy regarding the influence of cigarette smoking as a commonly premorbid factor on how it relates to TBI and its impact on post-traumatic secondary brain injury and post-TBI recovery (3).
In the present study, we evaluated the potential influence of chronic tobacco smoking on pathophysiological mechanisms underlying the exacerbation of TBI and retardation of post TBI recovery using a weight-drop mice model. In this model, a fixed weight is released for a free fall based on a defined path and height. The weight and the height from which it is imposed determines the severity of the injury, and it can range from a mild level of injury to severe brain injury. This model was chosen because of its ability to simulate traumatic head injuries comparable to those observed in road accidents or falls (4). Based on our results, both groups of smoked mice demonstrated a loss of body weight when compared to control, confirming the common metabolic stimulatory effect of TS. Longitudinal increase of body weight was also significantly dampened after TBI induction which is consistent with the well-observed reduced appetite after TBI. In line with these findings, the behavioral analysis confirmed similar changes in the state of consciousness and awareness immediately after TBI and during recovery. While TS was consistently associated with an increase in motor activity as a standalone stimulus, when it was combined with TBI (see Fig. 8) is further depressed motor activity when data were compared to TBI mice that were not exposed to TS. The Post-traumatic motor recovery was also significantly reduced when compared against the same group. These data are consistent with increased severity of post-traumatic brain injury promoted by TS and well correlated with the analysis of inflammatory biomarkers as well as BBB integrity.
Nrf2, a basic region-leucine zipper (bZip) redox-sensitive transcription Factor is the master regulator of multiple cytoprotective responses which controls the redox state of cells in harmful stresses (17, 36, 37). Under basal conditions, Nrf2 is localized in the cytoplasm by its inhibitor, Kelch-like ECH-associated protein 1 (Keap1). Nevertheless, under conditions of oxidative or xenobiotic stress, the cysteine residues of Keap1 become oxidized, Nrf2 dissociates from Keap1, translocate into the nucleus, binds to the antioxidant response element (ARE), and promotes the transcription of over 500 genes encompassing phase 1 and 2 enzymes, regulators of redox metabolism; production of ATP and antioxidative agents (including NADH and glutathione), and TJ expression at the BBB (16, 38, 39). Based on valid evidence, Nrf2 also promotes anti-inflammatory mediators, the activity of the proteasome and other transcription factors involved in mitochondrial biogenesis (40). According to recent studies, while suppression of Nrf2 activity and impairments of the Nrf2–ARE pathway exacerbate TBI-induced oxidative damage as well as post-traumatic neurological deficits, NRF2 played a significant neuroprotective role in TBI and neurodegenerative disorders (4, 11). Since the upregulation of Nrf2 activity ameliorates TBI-induced brain injury it could be suggested that positive modulation of Nrf2 could better TBI outcomes through reduction of oxidative stress, inflammation, and protection of BBB integrity (23, 24, 41-45).
In line with these findings, we assessed the impact of TS-exposure and TBI on Nrf2 expression levels, as well as its downstream effector molecules NQO-1 and HO-1, which are known for exerting acute detoxification and cytoprotective functions. Our in vivo data that the Nrf2-ARE system gets activated in response to TBI (see Fig. 6) This effect could be due to a direct modulatory activity toward Nrf2 expression and activation of the Nrf2–ARE pathway in response to trauma and are in line with the results obtained in a previous study by Li et al. indicating significantly enhanced Nrf2, NQO-1 and HO-1 protein expression following TBI (44). However, chronic TS exposure as a stand-alone stimulus has the opposite effect (see Fig. 6A) which is also in line with previous in vitro and in vivo observations recently published by our group (22-24, 46). As a comorbid stimulus, when TS exposure is combined with TBI, it abrogates the post-traumatic activation/upregulation of Nrf2 that follows brain trauma, thus preventing this physiological recovery system from being activated. The overall effect is the impairment of the BBB (not observed in TBI animals not exposed to TS; see also Fig. 7) and an overall increase of post-traumatic inflammatory responses including overexpression of cytokines (see Fig. 4), the pro-inflammatory transcription factor NF-kB and vascular endothelial adhesion molecules (see Fig. 5). These findings are consistent with the analysis of post traumatic motor activity showing that animals chronically exposed to TS prior TBI fared significantly worse than those undergoing the same traumatic injury but were not exposed to TS (see Fig. 8). In this specific case, the overall cerebrovascular/BBB impairment promoted by TS could explain the phenomenon.
The BBB is a dynamic and complex interface between the blood and the central nervous system (CNS)which strictly maintains the brain homeostasis and controls the passage of substances in and out of the brain environment. Among the various control functions of the BBB, the inter-endothelial TJs rigidly control the paracellular pathways blocking the passage of polar molecules (including ions) from moving between adjacent endothelial cells (47). The most important TJ proteins modulating the extremely low BBB permeability to polar molecules are Occludin and Claudins (more specifically claudin-5) forming homotypic bonding with their corresponding counterparts on adjacent endothelial cells. ZO-1 plays the critical function of anchoring this TJ protein to the cell cytoskeleton, thus allowing the cell to direct the distribution of these TJ proteins around the membrane (48). Recent findings have demonstrated that BBB impairment is a key component of post-TBI secondary brain injury and can significantly affect the outcome (49).
Additional evidence also indicate that inflammation is an important contributor to the TBI pathophysiology and exacerbates neuronal damage during post-traumatic brain injury so that sustained and excessive inflammation through secretion of proinflammatory mediators can exacerbate subsequent neurological impairment (50-52). This process involves resident microglia and astrocytes, peripheral leukocytes penetrating through the leaking BBB and inflammatory mediators including cytokines that interfere with normal restorative processes of the brain, thus promoting neuronal cell death (31, 53, 54). Proinflammatory cytokines like IL6, IL-10, and TNF-α, are increased in posttraumatic brain tissue, followed by synthesis of chemokines, prostaglandins and expression of cell adhesion molecules like VCAM-1 and PECAM-1 on the surface of the cerebrovascular endothelium. This latter process can favor the extravasation of inflammatory cells from the blood into the brain (31, 50). There is also a growing consensus that all these processes are the key promoters of the secondary brain damage associated with TBI including dysfunction of astrocytes and microglia, as well as BBB impairment contributing to the increased paracellular permeability and the loss of neurons (50, 55, 56). In fact, proinflammatory molecules play a supplementary role in increased BBB permeability related to loss of Occludin/ZO-1 as well as other tight junction (57, 58). It is also well described that BBB integrity is deeply affected by oxidative stress, so that enhanced ROS production leads to redistribution and/or altered expression of tight-junction proteins, endothelium dysfunction and increased BBB permeability (59-61). Inflammation is also linked to oxidative stress, whereas ROS (such as those released within TS) are considered among the most potent inflammatory mediators (62). In fact, oxidative stress caused by TS is increasingly recognized as a negative contributing factor for neurological outcomes following brain injury (41) whereas TS modulates a cascade of events leading to the activation of NF-κB and the expression of pro-inflammatory cytokines and vascular adhesion molecules (46). This has been observed in glial cells and neurons following TBI and is associated with long-term inflammatory processes (10). Mettang et al., using an experimental model of closed-head injury promoted neuronal cell death, demonstrated the repression of the NF-κB inhibitor system exacerbating the neurological outcome and increasing posttraumatic mortality rate (63). In the contest of Nrf2 - NF-κB interplay, a recent study confirms the cytoprotective mechanisms of action associated with Nrf2 which leads to the downregulation of proapoptotic mediators such as Bax, BAD, and other pro-apoptotic factors which are instead promoted by the activity of NF-κB (64, 65). Nrf2 reduces ROS levels and affects the redox-sensitive NF-κB signaling pathway involved in neuroinflammation. Moreover, in a recent study, it has been reported that Nrf2-/- mice have greater NF-κB activation and generation of pro-inflammatory cytokines in the brain and spinal cord injury compared to their wild-type Nrf2+/+counterparts (66). Relevant to our study is the fact that chronic TS exposure dampened Nrf2 activity in TBI mice. Thus, the cascading effect of Nrf2 downregulation well fit the slowed recovery and overall, worse outcome observed in TBI animals chronically exposed to TS when compared to TBI mice that were not exposed to smoke.
An additional risk factor for TBI patients that is associated with chronic smoking may be derived from the impact of smoking on blood hemostasis. Thrombomodulin is a key component of the anticoagulant protein C pathway and is tightly regulated to maintain blood homeostasis and to ensure control over the process of blood-coagulation (by blocking the activity of the prothrombinase complex and promoting fibrinolysis) following activation of the coagulation cascade in response to vessel injury and/or inflammation (67). Although the mechanism is largely unknown, thrombomodulin is potently inhibited by inflammatory cytokines, thus blocking NF-kB activation effectively prevents cytokine-induced downregulation of thrombomodulin. TS-exposure has been previously observed to promote the downregulation of thrombomodulin at the vascular endothelial level (through its pro-inflammatory activity), thus increasing the risk of blood clot formation and stroke (24). Therefore, the downregulation of thrombomodulin by TS exposure can provide an additional risk for TBI patients were traumatic injury is likely to impact the integrity of the blood vessel and trigger the activation (now poorly controlled) of the coagulation cascade.
Although premature at this stage, we plan to revolve the focus on potential treatment intervention to reduce or eliminate the add-on risk of TS exposure on post-TBI injuries and improve outcomes. This is mostly relevant for patients that recently quit smoking since many of these TS-related harmful effects persist for months or years after quitting, thus remaining at risk of smoking-related comorbidities which may still aggravate the outcome of neurological disorders associated with cerebrovascular impairments (e.g., stroke) and TBI.