Traumatic brain injury (TBI) is the consequence of an external mechanical force applied to the cranium and its content. This event may cause temporary or permanent disabilities on TBI survivors because of its consequences at physical, cognitive or psychological levels (Niedzwecki et al., 2008; Steyerberg et al., 2008; Stocchetti and Zanier, 2016; Wolfe et al., 2018). The TBI’s severity can be classified as mild (mTBI), moderate or severe taking into account the clinical presentation of a patient’s neurologic signs and symptoms (Kay et al., 1993; Katz and Alexander, 1994; Ponsford et al., 2016). Furthermore, TBI is considered a worldwide increasingly critical health problem, according to recent studies in the United States, revealing that the number of “TBI-related emergency department visits, hospitalizations, and deaths increased by 53%” from 2006 to 2014 (Centers for Disease Contr, 2019).

Injuries related to TBI can be classified into primary and secondary. The first ones, occur at the moment of trauma and can be manifested as focal injuries (e.g. skull fractures, intracranial hematomas, lacerations, concussions, penetrating wounds), or diffuse injuries (as in diffuse axonal injury) (Alain and Wang, 2008; Katerji et al., 2019). The secondary injuries appear immediately after trauma and may last for long periods (Nortje and Menon, 2004; Pearn et al., 2017). They are frequently associated with ischemia, brain edema, and neuroinflammation, and may take place in a period from days to weeks, or even months, triggering a complex cascade of intracellular signaling, which include ATP depletion, mitochondrial dysfunction, oxidative stress (OS), and cytoskeleton damage (Montivero et al., 2021). In turn, those events induce the production of toxic and pro-inflammatory mediators, such as prostaglandins, oxidative metabolites, chemokines, and proinflammatory cytokines, which can lead to lipid peroxidation, protein oxidation, enhanced blood brain barrier (BBB) disruption and brain edema to finally induce neuronal dysfunction or death (Krishnamurthy et al., 2016; Pearn et al., 2017).

It has been described that secondary injury mechanisms, as outlined above, may contribute to the cognitive deficits observed even long-term after injury. Individuals suffering from TBI of any severity could experience a cluster of symptoms for a prolonged period of time (Kushner, 1998; Ryan and Warden, 2003; McMahon et al., 2014; Stocchetti and Zanier, 2016), commonly recognized as post-concussive syndrome (PCS) (Alexander, 1995; Ryan and Warden, 2003; Stocchetti and Zanier, 2016; Montivero et al., 2021). The PCS includes a variety of symptoms ranging from cognitive symptoms (speech changes, attention loss, dysfunction in executive function and memory, or mental slowing, among others) Kaplan et al. (2018); physical and somatic symptoms (hearing and visual disturbances, sensitivity to light or sound, pain, headaches, dizziness, nausea, fatigue, sleep disruption, and even seizures) (Lozano et al., 2015; Webster et al., 2017; Kaplan et al., 2018; Wolfe et al., 2018) and emotional/behavioral symptoms (executive dysfunction, anxiety, irritability, depression, and attention deficit) (Arciniegas et al., 2005; Bryan, 2013; Paterno et al., 2017). Regretfully, it is difficult to identify those individuals at risk for PCS with the standard clinical assessment used. Nevertheless, since the working memory is one of the cognitive functions primarily affected by TBI, its clinical evaluation, including functional magnetic resonance imaging performed within the first week of injury, may contribute to predicting the patients´ outcome (Christodoulou et al., 2001; Ricker et al., 2001; Hillary et al., 2011; Wylie et al., 2015; Montivero et al., 2021).

Unfortunately, there are no available pharmacological therapies to prevent or reverse early secondary events in order to reduce long-term disabilities described in TBI patients (Paterno et al., 2017). Several neurotrophins synthesis can be locally stimulated in the brain after TBI, among them, insulin like growth factor-1 (IGF-1) (Madathil et al., 2010; Schober et al., 2010). However, TBI-induced increments in IGF-1 and IGF-1-related signaling molecules seem to be transient and probably are not enough to provide neuroprotection or stimulate sub-acute repair or regenerative mechanisms. Early studies in TBI patients have shown a reduction in serum IGF-1 levels (Agha et al., 2004; Popovic et al., 2004; Wagner et al., 2010; Zgaljardic et al., 2011; Olivecrona et al., 2013), even after mTBI Robles et al. (2009), Wagner et al. (2010), while other investigations have reported that there were either no changes Bondanelli et al. (2002) or a long-term increase in serum IGF-1 levels after injury (Wildburger et al., 2001). These differences can be due to the diverse patient population included in those studies, as well as different trauma types, gender, injury severity, patient age, and brain regions affected (Madathil et al., 2015). Nonetheless, low IGF-1 serum levels have been implicated in the development of cognitive dysfunction Trejo et al. (2004), Koopmans et al. (2006) and were positively correlated with cognitive impairment in TBI survivors tested up to a year post injury (Madathil et al., 2015). Thus, it is plausible to consider that low plasma IGF-1 levels may contribute to the primary causes of cognitive disorders after TBI (Madathil et al., 2015). However, even though early IGF-1 administration promotes regenerative events such as neurogenesis and angiogenesis in a TBI animal model Whitaker-Lea and Valadka (2017), its therapeutic potential after TBI has not yet been assessed. It should be emphasized, that different clinical trials have shown acceptable tolerability of systemic IGF-1 therapy in moderate to severe TBI patients, as well as enhanced metabolic outcome in comparison to placebo-treated patients (Hatton et al., 1997; Madathil et al., 2015). Furthermore, systemic growth hormone administration produced increments in IGF-1 levels, inducing a tendency to improve the functional outcomes in treated patients (Dubiel et al., 2018).

Considering all the evidence aforementioned, and the fact that IGF-1 gene therapy targets brain cells in vivo Hereñú et al. (2009) and can decrease behavioral functional impairments in aged rats (Nishida et al., 2011; Pardo et al., 2017), the aim of the present study is to evaluate the effectiveness of early IGF-1 gene therapy in the treatment of TBI for preventing OS as well as improving cognitive deficits.

In this work, we showed that mTBI increases some OS markers as a result of secondary injury. These markers were observed early after TBI (60 min), reaching their highest levels at 24 h in all brain structures analyzed. In addition, even though AOPP and MDA products were significantly reduced on day 7 after TBI, they did not return to basal levels. These results are in line with studies that point out neuroinflammation as a key player in brain damage severity and patients' outcome after any kind of TBI. For instance, recognized neuroinflammation serum protein markers, such as Glial Fibrillary Acidic Protein (GFAP) and S100B, have demonstrated to have a correlation to both severity and outcome in TBI (Pelinka et al., 2004; Luoto et al., 2017). Furthermore, the more severe the TBI is, the less successful outcome is observed in patients and animal models. Many severe TBI models have shown important brain damage and correlate neuroinflammation with memory disorders (Farbood et al., 2015; Zhang et al., 2017). Thus, with our model, we have shown that even mTBI can lead to memory impairment as a sign of PCS. The observed deficits in working memory 7 days after TBI, related to impaired cognitive function, were probably the result of the early neuroinflammatory cascade triggered by secondary injury mechanisms. Working memory performance depends on the PFC integrity (Riggall and Postle, 2012; Lara and Wallis, 2015). Interestingly, we have observed an increase of OS markers in this brain structure that remains elevated over a 7-days period after TBI, compared to Sham animals. This could be explained as the result of microglia hyperactivation, as it occurs in the pathogenesis of many CNS diseases Pekny and Pekna (2016), and/or changes in cerebral blood perfusion (Babior, 2000; Block et al., 2006; Block and Hong, 2007), which in turn may affect neuronal excitability that finally shape the disrupted working memory performance observed.

Although increasing knowledge about injury mechanisms of TBI has led to a better understanding of its complex pathogenesis, pharmacological strategies focused on ameliorating them are yet missing. To date in clinical practice, medical management of TBI is tailored according to the severity of each case considering the Glasgow scale. In moderate to severe cases the treatment aim is to keep a normal intracranial pressure (ICP) in order to maintain an adequate cerebral blood pressure (CCP), which is an indicator of brain perfusion. The pharmacological armamentarium consists in sedation, neuromuscular blocking, and hyperosmolar agents that contribute to decreasing ICP (Carney et al., 2017). On the other hand, mTBI has a different approach because most of the time the ICP remains within normal ranges, thus the treatment is mainly focused on headaches management and the control of PCS- related symptoms. In these cases, painkillers are the most used medication with only evidence of improving pain, but not decreasing the symptoms duration nor improving cognitive deficits. TBI-related mood disorders can be improved by using antidepressants and psychostimulants, and yet not dealing adequately with memory deficits. Unfortunately, none of them can interfere with the neuroinflammatory cascade that could be the main cause of the sequelae after severe or mTBI (Whyte et al., 2002; Comper et al., 2005; Wheaton et al., 2011; Huang et al., 2016). In the present study we propose an early IGF-1 gene therapy, considering previous reports regarding its neuroprotective and anti-inflammatory effects (Zheng et al., 2000; Carlson and Saatman, 2018; Serhan et al., 2019). In fact, a small clinical trial showed improvement in neurological outcome in severe TBI patients, when intravenous IGF-1 was administered for several days, without effect in those with mTBI (Hatton et al., 1997). Our results indicate that locally administered IGF-1 gene therapy a few minutes after TBI, significantly reduces recognized OS markers, such as the protein and lipid (AOPP and MDA concentrations respectively) peroxidation increments observed at 24 h and impairs its long-term maintenance, also by reducing their levels 7 days after TBI. It is worth noting that this effect is specifically due to IGF-1 over-expression and not only by viruses’ administration since control viruses (RadDsRed) did not reduce AOPP nor MDA levels 7 days after TBI. With this experimental approach, we were able to express the Red protein in brain areas neighboring to the virus administration site (see Figure 4A), and since the effects were observed in frontal brain areas such as PFC and MC, the newly produced IGF-1 could be actively transported through the choroid plexus and translocated to the cerebrospinal fluid to finally reach different areas of the nervous tissue, as it was recently reported (Nishida et al., 2020). Nevertheless, further studies need to be performed to reveal changes in other players involved in the OS balance: antioxidants enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, as well as accumulation of oxidation products of the proteins and lipids, like protein carbonyl and 4-hydroxynonenal (Impellizzeri et al., 2019; Siracusa et al., 2020).

The PFC plays an important role in execution of behavioral tasks that require spatial working memory, and a direct PFC-HIP pathway allows the encoding of salient spatial signals during task execution (Deakin et al., 2012). In fact, lesion- or drug-induced disturbances in HIP function certainly affect spatial working memory performance (Dillon et al., 2008; Tian et al., 2017; Ayabe et al., 2019). In the present study, we found persistent increases in OS markers in these two relevant brain structures that may affect the PFC-HIP bidirectional control required to observe spontaneous alternations. Early IGF-1 gene therapy was able to prevent the behavioral deficits observed in TBI rats after 7 days of injury. Only the group treated with vectors carrying the cDNA of IGF-1 gene showed a percentage of spontaneous arm alternations similar to the control group (Sham), while the TBI or TBI expressing Red protein groups did not show differences from each other and had lower performance rate than the controls in Y-maze spontaneous alternations. All together, these results could indicate that normal working memory can be preserved long-term after injury if critical initial secondary injury mechanisms were prevented in brain structures critical to this cognitive function. This novel therapeutic approach could be beneficial from IGF-1 peripheral administration for many reasons: first, local over-expression can be maintained even after a month from a single administration (Falomir-Lockhart et al., 2019); second, local over-expression may reduce systemic effects compared to peripheral administrations which may need massive doses in order to reach therapeutic CNS IGF-1 concentrations and lastly no doubts about treatment patient’s adherence, since administration must be performed during hospitalization, in a temporal window that guarantees the pharmacological effect.

In conclusion, results presented in this work indicate that brain focalized IGF-1 over-expression could be an effective therapeutic approach targeting neuroinflammation and also improving the cognitive deficits observed after TBI.