Traumatic brain injury (TBI) is a significant public health concern in military and civilian populations (Chiu and LaPorte, 1993). The estimated number of TBI cases occurring each year is 10 million globally and 1.7–3.8 million in the United States alone. 75–90% of those are defined as mild TBI (mTBI) (Selassie et al., 2013; Marin et al., 2014).

The post-concussion syndrome (PCS) is a complex of symptoms consisting of headaches, dizziness, imbalance, vertigo, fatigue, changes in sleep pattern, neuropsychiatric symptoms (e.g., behavioral and mood changes, confusion), and cognitive impairments (in memory, attention, concentration and executive functions) (McCauley et al., 2005). PCS is most often described in the setting of mTBI, but may also occur after moderate and severe TBI. In Eight to Ninty percent of mTBI cases, the symptoms fade away in 7–10 days (McCrory et al., 2005; Hadanny and Efrati, 2016). Still, in 10–20 percent, PCS may persist for weeks or months due to structural and/or metabolic brain damage. Twenty-five to thirty-three percent of those retain a permanent brain injury and experience persistent PCS; the symptoms turn chronic and endure more than 6 months (Kashluba et al., 2004; Bazarian et al., 2005; Iverson, 2005; Sterr et al., 2006; King and Kirwilliam, 2011).

The sensitivity of classic anatomical brain imaging techniques, such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), is generally not sufficient for detection of the pathophysiologic effects of mTBI. New techniques are increasingly utilized for objective evaluation of brain damage. Diffuse Tensor Imaging (DTI) can reveal the combination of axonal injury and secondary gliosis with local microvascular injury (Niogi and Mukherjee, 2010). Dynamic susceptibility contrast MR perfusion can demonstrate reduced cerebral blood flow (CBF), global and regional, as well as cerebral blood volume (CBV) (Tal et al., 2015).

The existing pharmacologic and non-pharmacologic treatments have mostly failed to elicit efficacious results in both the clinical symptoms and the pathophysiologic cascade leading to permanent brain injury (Hadanny and Efrati, 2016). In recent years, both basic (animal models) (Neubauer and James, 1998; Zhang et al., 2005; Vlodavsky et al., 2006; Chen et al., 2010; Huang and Obenaus, 2011; Lin et al., 2012; Efrati and Ben-Jacob, 2014) and clinical studies (Shi et al., 2003, 2006; Barrett et al., 2004; Golden et al., 2006; Wright et al., 2009; Harch et al., 2012; Boussi-Gross et al., 2013; Tal et al., 2015) have shown that hyperbaric oxygen therapy (HBOT) can improve PCS by targeting the basic pathological processes responsible for post-concussion symptoms (Hadanny and Efrati, 2016). In our previous study, it was evident that HBOT can induce brain angiogenesis, demonstrated by perfusion MRI with significant increase in CBF and CBV following HBOT along with significant cognitive improvement in patients post TBI (Tal et al., 2015).

The current study was aimed at evaluating the effects of HBOT on brain microstructure in chronic neurological deficiencies stemming from TBI. This has not been studied in humans so far.

A retrospective analysis of patients suffering from chronic neurocognitive impairment due to TBI, treated at Sagol Center for Hyperbaric Medicine and Research at Assaf Harofeh Medical Center, Israel, between September 2013 and December 2015. The study was approved by the Institutional Review Board of the hospital (0030-15-ASF) and registered in the US National Institute of Health Clinical Trials registry (NCT02452619).

Inclusion criteria: Patients with chronic neurocognitive impairment started only after TBI, persisting over 6 months who underwent two MRI brain imaging (including DTI and DSC sequences) and two neurocognitive tests, pre- and post- hyperbaric oxygen therapy (HBOT). All patients applied for HBOT of their own volition. Patients with other neurological conditions were excluded from the study's cohort.

The classification of TBI was based on the American Congress of Rehabilitation Medicine (ACRM) and the Centers of Disease Control (CDC), where mTBI is defined as altered brain function engendered by external forces with one or more of the following: loss of consciousness with duration of 0–30 min, post-traumatic amnesia with duration of less than 24 h, and Glasgow Coma Scale grade of 13–15 (Malec et al., 2007). GCS score of 3–8, or post-traumatic amnesia of more than 7 days, or loss of consciousness for more than 24 h is classified as severe TBI; GCS score of 9–12, or post-traumatic amnesia of 1–7 days, or loss of consciousness between 30 min and 24 h, is classified as moderate TBI (Malec et al., 2007).

The current study shows, for the first time in humans, that HBOT can induce brain microstructure recovery in TBI patients. Brain recovery encompassed gray and white matter areas, white matter tracts and angiogenesis. Post HBOT, FA increased and MD decreased in the DTI sequence, CBV and CBF increased in the DSC (perfusion) sequence, along with improved cognitive functions. Recovery was induced in the late chronic stage of TBI (6.7 ± 2.1 years post injury).

Previous studies using DTI have reported that patients suffering from TBI may still have microstructural damage months to years after the initial injury (Kraus et al., 2007; Yuan et al., 2007; Lipton et al., 2008; Sugiyama et al., 2009; Hartikainen et al., 2010; Niogi and Mukherjee, 2010; Murugavel et al., 2014; Perez et al., 2014; Li et al., 2016). DTI characterizes the diffusion of water in the tissues, thus it indicates microstructural density, spacing, and orientational organization of cell membranes, including myelin (Assaf and Pasternak, 2008; Alexander et al., 2011). Animal studies of brain plasticity, revealed that decrease in MD and increase in FA correlates with synaptophysin (a marker of synaptic vesicles), glial fibrillary acidic protein (GFAP; a marker of astrocyte activation), and brain-derived neurotrophic factor (BDNF; a marker of neuronal growth that facilitates learning) (Sagi et al., 2012). With regards to white matter, it was found that myelin density estimated by DTI can accurately predict the actual myelin density seen/measured by electron microscopy (Sepehrband et al., 2015). The injury can be demonstrated by increased water diffusion as measured by MD and reduced directionality of diffusion as measured by FA, suggesting that either axonal injury or disruption of myelination could have altered brain connectivity (Kraus et al., 2007; Yuan et al., 2007; Lipton et al., 2008; Sugiyama et al., 2009; Hartikainen et al., 2010; Niogi and Mukherjee, 2010; Murugavel et al., 2014; Perez et al., 2014; Li et al., 2016). Moreover, FA changes appear to correlate with the severity of the clinical presentation (Benson et al., 2007; Yuan et al., 2007). The observed decrease in FA values may reflect the barriers to axoplasmic transport, the local accumulation of apoptosis in organelles, and secondary Wallerian degeneration in the white matter, while the increased MD values may be the result of vasogenic cerebral edema.

Significant injury foci were reported in dedicated pathways involved in the transmission of efferent and afferent information, such as the corpus callosum, internal capsule, SLF, ILF, SFO, superior frontal gyrus, insula, and fornix (Yuan et al., 2007; Caeyenberghs et al., 2010). Importantly, the microstructure injuries depicted by the DTI imaging markers, FA and MD, correlated with objective measures of general and cognitive functioning (Benson et al., 2007; Assaf and Pasternak, 2008; Sugiyama et al., 2009; Caeyenberghs et al., 2010; Hartikainen et al., 2010; Alexander et al., 2011; Sagi et al., 2012; Wada et al., 2012; Arenth et al., 2014; Haberg et al., 2015; Sepehrband et al., 2015; Li et al., 2016). Disorders in reaction time, executive functions, information processing speed, attention and memory were correlated with axonal lesions in different areas. Moreover, in two recent longitudinal studies, FA values increased in patients with favorable outcome within 6–12 months, while no DTI changes registered in patients with unfavorable outcome (Sidaros et al., 2008; Hartikainen et al., 2010).

In the current study, for the first time in humans, DTI changes of chronic TBI were evaluated before and after HBOT. The increase in FA and decrease in MD post HBOT, together with cognitive function improvement of patients in the late chronic stage of TBI, suggest that brain microstructure recovery can be induced by HBOT.

DTI values, FA and MD, were found in our study to correlate with the improvements in cognitive functions in concordance with previous studies (Sugiyama et al., 2009; Wada et al., 2012; Arenth et al., 2014; Haberg et al., 2015). Memory, executive function and information processing speed were all significantly improved. In correlation with these cognitive improvements, MD decreased in most of the frontal lobe white matter, such as the prefrontal cortex that enables executive control (Miller and Cohen, 2001) and the anterior cingulate gyrus involved in error detection, especially in a Stroop task (Bush et al., 2000). Also, FA increased in most of the long association fibers critical for proper cognitive function:

Mechanisms of neuroplasticity and cellular repair by HBOT have been suggested in many animal studies (Hadanny and Efrati, 2016). These include enhanced mitochondrial function and cellular metabolism, improved blood brain barrier and inflammatory reactions, reduced apoptosis, alleviation of oxidative stress, increased levels of neurotrophins and nitric oxide, and up-regulation of axonal guidance agents (Efrati et al., 2013; Efrati and Ben-Jacob, 2014). Moreover, the effects of HBOT on neurons may be mediated indirectly by glial cells. HBOT may also promote neurogenesis of endogenous neural stem cells (Efrati et al., 2013; Efrati and Ben-Jacob, 2014). HBOT may enable the metabolic change simply by supplying the missing oxygen/energy needed for these regeneration processes (Efrati et al., 2013; Efrati and Ben-Jacob, 2014). The ability of HBOT to induce angiogenesis was demonstrated in several different pre-clinical studies (Mu et al., 2011; Lin et al., 2012; Lee et al., 2013; Hu et al., 2014). Hu et al. have demonstrated that HBOT-induced neurogenesis is mediated by ROS/HIF-1α/β-catenin pathway (Hu et al., 2014). In the current study, it is demonstrated that HBOT can induce neuroplasticity in humans even years after the acute insult.

Along with the structural changes, HBOT induces angiogenesis, as shown by the increase of CBF and CBV in this study as well as in our previous study (Tal et al., 2015). The injured areas in the brains post TBI experience hypoxia and hypoperfusion, which serve as a rate-limiting factor for any regenerative process (Graham and Adams, 1971; Graham et al., 1978; Stein et al., 2004; Kim et al., 2010; Ostergaard et al., 2014). HBOT-induced angiogenesis has been amply confirmed in pre-clinical models and can be deduced from brain SPECTs of patients post stroke and post TBI even years after the acute insult (Lin et al., 2012; Boussi-Gross et al., 2013; Efrati et al., 2013; Peng et al., 2014; Duan et al., 2015). The generation of new microvessels renders the local environment non-hypoxic, thus able to induce brain plasticity, enhance neurogenesis and synaptogenesis and foster functional recovery (Chen et al., 2003; Jiang et al., 2005). Unsurprisingly, CBV and CBF increased in the long association fiber tracts discussed above, including corpus callosum, association fibers (SLF, IFOF) and cingulum. Angiogenesis and increased perfusion to the malfunctioning tissue, seen in DSC, serve as infrastructure for the regenerative process and the preservation of newly generated metabolic functioning of the axonal microstructure seen in DTI.

Our study has several limitations. The major one is related to lack of control group. However, one can hardly expect any significant changes in DSC and DTI values or neurocognitive improvement to occur spontaneously years after the acute insult. The cognitive improvement seen here is in line with our earlier randomized controlled trial on patients suffering from mild TBI. In our previous randomized control study it was clearly demonstrated that the control group had no neurocognitive improvement (same cognitive tests used in the current study) or significant change of in brain perfusion measured by SPECT 1-5 years after the acute insult (Boussi-Gross et al., 2013). Nevertheless, one can hardly expect any significant changes in DSC and DTI values or neurocognitive improvement occurring spontaneously years after the acute insult. In addition, a previous randomized controlled trial with a control group showed neurocognitive effects and brain perfusion improvement using SPECT (Boussi-Gross et al., 2013).

HBOT can induce cerebral angiogenesis and recovery of brain microstructure in patients with chronic cognitive impairments due to TBI months to years after the acute injury. The increased integrity of brain fibers correlates with the functional cognitive improvement. The mechanism by which HBOT can induce brain neuroplasticity can be demonstrated by highly sensitive perfusion MRI and DTI. Further studies, using DTI - MRI, are needed in order to gain better understanding of the neuroplasticity effect of HBOT in a larger cohort of patients with different types of brain injuries.

This study was carried out in accordance with the recommendations of Assaf Harfoeh Medical Center Institutional Review Board (0030-15-ASF) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Assaf Harfoeh Medical Center Institutional Review Board (0030-15-ASF).

Conceived and designed the experiments: AH, ST, and SE. Performed the experiments: AH, ST, GS, and SE. Analyzed the data: AH, ES, and SE. Contributed analysis tools: AH, ST, ES, and SE. Wrote the paper: AH, ST, GS, ES, and SE.