Assessing Mild Traumatic Brain Injury Associated Blood Brain Barrier (BBB) Damage and Restoration Using Late Phase Perfusion Analysis by 3D ASL MRI- Implications for Predicting Progressive Brain Injury

1. Introduction

Mild traumatic brain injury (mTBI) is a pervasive problem worldwide and typically managed without hospitalization thus unreported. The estimated prevalence rate of 369 per 100,000 individuals underestimates the scope of the problem as a result [1,2,3]. Most head injuries are mild (90%) defined by clinical functionality scales such as the Glasgow Coma scale where a score of 13-15/15 is defined as mild [4]. The scale includes basic tests of level of consciousness as defined by verbal, eye opening and motor responses to commands or noxious stimuli. It is an invaluable tool for assessing aTBI and follow up of moderate or severe TBI. More subtle brain injury however is not captured by this scale, as it is meant as a simple stratification tool for rapid assessment and prognosis of more serious head trauma [4].

The assessment of mTBI is currently the sole prevue of clinical assessment to categorize acute cognitive or other subtle neurologic deficit, along with behavioral/emotional effects [5]. Likewise, return to normal learning or sport activities are based on these measures [6]. Since anatomic imaging studies in mTBI are almost invariable normal, they are often not employed [9,10]. However, developing a noninvasive physiologic measure of acute injury and subsequent recovery would provide a critical objective assessment of safe return to sport/usual activities.

1.1. Background

1.1.1. Minor Closed Head Injury

Mild traumatic brain injury (mTBI) is defined as traumatic brain injury resulting in brief if any loss of consciousness with associated transient cognitive (thinking, memory, general confusion), neurologic (vertigo, delayed reaction times), and/or behavioral (depression, aggression) dysfunctions, and often headache [11]. Most frequent causes include contact sport, blast, and acceleration-deceleration injuries. Sports injuries are acutely assessed on the field acutely or within 72 hours using validated clinical testing instruments such as Sport concussion Assessment Tool (SKAT 6), Glascow coma scale, Vestibular Ocular Motor Screening (VOMS) [5,12,13]. Along with inclusion of the testing measures cognitive function (orientation, immediate and delayed memory recall, concentration, emotionality, depression, anxiety, speech, and task specific processing, as well as coordination, balance, eye movements, and headache [14]. A more comprehensive version for office use, SCOAT 6, has also been validated for use within 72 hours of injury. In addition to testing SKAT 6 items, it catalogues prior head injuries, cognitive and emotional history, current medications, and a detailed neurologic examination. Further it includes recommended strategies for return to learning and return to sport activities [15,16]. Currently corroborating physiologic testing is not commonly employed.

To that end, the physiologic consequences of mTBI will be reviewed, and methods to identify those changes using imaging modalities will be discussed. The potential combined use of both clinical and physiologic testing may improve the accuracy of diagnosis and readiness to return to usual activities and predict persistent physiologic dysfunction. The latter when associated with prior mTBIs may potentially lead to chronic progressive disease -chronic traumatic encephalopathy (CTE) years later, as seen in professional contact sports athletes.

1.1.2. Pathophysiology

Given the brain is a soft delicate organ contained within a closed box, sudden applied blunt trauma and inertial forces of acceleration, deceleration, or rotational trauma, dictates the severity and location of cortical, white matter tract, and vasculature injury. Unlike moderate or severe TBI, mTBI injury is associated with minimal or no disruption of these structures in most cases, but not all [17,18]. That said physiologic consequences of disrupted electrolyte pumps and blood flow acutely post head injury have been well delineated with acute impairment of electrolyte (sodium potassium, calcium, and magnesium) distribution notably mitochondrial calcium sequestration with altered metabolism in neurons and astroglia and reduced local blood flow [19,20,21]. This results in an acute glycolysis hypermetabolism to restore normal electrolyte balance [21]. Disruption of the blood brain barrier (BBB) even in mild Traumatic Brain injury (mTBI) is reflective of this acute phase of injury [22,23]. The duration of this varies, dependent upon location and severity of injury leading to hypometabolism in the affected regions which can persist for weeks or longer [22]. The chronic phase of injury is largely thought to be related to persistent physiologic alterations from persistent activated microglia and its effect on the neurovascular unit [24,25]. The focus of this article principally addresses the BBB dysfunction and identifying imaging techniques. Additionally, techniques demonstrating shear force white matter tract injury will be discussed.

1.1.3. Shear Injury in TBI

The most troublesome and unfortunately permanent sequelae of TBI is the result of shear injury to white matter tracts [18,26,27]. The extent and severity of damage escalates proportional to force and location of impact with compiled additional effects from edema mass effect and inflammatory upregulation. In mTBI, the general assumption is absence of substantive anatomic injury [17,18]. This however belies subtle changes in personality, and emotionality, particularly in adolescence, from damage to developing limbic connections [26,27,28,29].

The underpinnings of shear injury are quite understandable with sudden mechanical deformation of the brain tearing apart connections [26,30,31]. The longer-term damage though is caused by subsequent mass effect from edema, hemorrhage, or later from inflammation [22,32,33,34]. To understand the latter influences, animal models have been employed using a variety of species, most commonly rodents and rarely large animals [35]. Translatability from rodent to human physiology is problematic given vast differences in brain structure and physiologic responses to injury [36,37].

Current medical state of the art possesses no means of restoring shear tract injury. Prevention of delayed injury by reducing mass effect and swelling preserve life and may reduce secondary shear injury [27,29,30,31,32]. But knowledge of how to best limit the secondary tract damage will require additional mechanistic study in higher animals (swine or primates) with closer anatomic affiliation to the human brain [36,37].

Too often the die is cast for potential clinical recovery based on the initial extent of shear force injury. That said identifying the severity of tract injury has potential importance in prognostic discussions with the patient and family.

1.1.4. Blood Brain Barrier Dysfunction in TBI Acute And Chronic

In mTBI, the microcirculation (capillary system) BBB is disrupted acutely causing reduced mean capillary transit time (cMTT) and reduced glymphatic flow (GF) in the region(s) of impact both coup and contrecoup with limited or no white matter tract injury [39,40]. The effect of this disruption is leak and trapping of normally restricted substances into the brain and thus reduced clearance out of the brain. This results in measurable reduction in clearance rate of intraparenchymal fluid which can be identified by imaging techniques (see below). In animal studies, BBB disruption and leak of IGG locally persisted for up to a month following moderate TBI with associated delayed microhemorrhages at those sites [18,32].

A brief summary of the complexity of BBB structure and function is presented here. Details of the cellular interactions, signaling mechanisms, and what is known of the basic biology are beyond the scope of this review but referenced [40,41,42,43,44,45].

The blood brain barrier is dependent on a complex interplay of cells within the neurovascular unit (NVU), including the endothelial cells, pericytes, and astrocyte end feet [33,46,47]. Signalling within this triad in homeostasis allows for transport of needed electrolyte, glucose, and specific lipids and proteins while excluding common blood constituents that are toxic to the complex interstitial environment [48,49,50,51,52,53,54]. The interaction and communication among elements of the NVU, extracellular matrix (ECM) and microglia dictate the presence, expression, and integrity of the various tight junction (TJ) proteins [45,55,56,57]. Presence of specific inflammatory cytokines may result in either a proinflammatory milieux or counterintuitively, a restorative one [24,37,44,45]. For example, Endothelial cell derived IL1 β acutely downregulates TJ protein ZO-1/occludin thus increasing BBB permeability, but by promoting expression of Pentraxin 3 later enhances its restoration [58]. The relative balance of local microglial proinflammatory versus homeostatic regulatory influences also determines the BBB integrity [59,60,61]. That balance is dictated by neuronal and astrocyte signaling which is influenced by presence of cell injury and local inflammation [61].

Fundamental questions remain regarding defining the restorative pathways post injury, and long-term consequences of persistent leak [62,63]. In TBI BBB leak is caused by proinflammatory microglial-endothelial signaling and to shear forces disrupting endothelial glycocalyx, the latter allowing leak of IGG and upregulation of inflammatory cytokines causing local conversion of microglia to proinflammatory state [58,59,60,61]. Activated microglia express complement (C3a) fragment which in turn upregulates C3aR in capillary endothelial cells altering its phenotype to a proinflammatory/immune cell attractant state with associated disruption of intercellular tight junctions thus triggering additional BBB leak [64]. In the chronic phase of injury presence of activated microglia induces BBB dysfunction, local chronic inflammatory changes, and neuronal dysfunction [23,39,65]. The persistence of this especially with multiple mTBI result in perivascular accumulation of pTau within astroglia and neurons in the sulcal depths, the hallmark of CTE [65,66,67,68].

Restoration of normal BBB integrity following mTBI generally occurs quickly in youth, however in some persistence of activated microglia can persist for months or longer resulting in reduction in threshold for additional mTBI related pathology with clinical accompaniment [67,68,69]. With advancing age, recovery is less complete and progression of both inflammation with BBB leak with cognitive decline following TBI is more likely [70]. Further, the lower threshold and persistence of mTBI in young women is well recognized and postulated to be influenced by sex hormones, and reduced muscle mass compared to their male counterparts [71]. The mechanisms of the BBB repair process in humans remain incompletely delineated, and in particular the specifics of the pathways active in youth but lost in the circumstances of repetitive TBI or in the normal aging process [72].

The cumulative effects of multiple mTBI events causes ongoing inflammatory upregulation resulting in both permanent microvascular changes and persistent upregulation of inflammation within the neuropil [67]. This induces altered synthesis, degradation, accumulation and transsynaptic spread of toxic misfolded proteins predominantly hyperphosphorylated Tau (pTau), but also Lewey body proteins, TDP-43 and to a delayed and minor degree β amyloid [69,74]. The consequence is the well described high incidence of chronic traumatic encephalopathy (CTE) in professional contact sport athletes [75]. The clinical correlates include aggression, depression, suicide ideation, impulsivity, cognitive decline, parkinsonian features [66,75,76]. To reduce the likelihood of progressive dementia from cumulative brain injury, the addition of objective physiologic testing in mTBI should prove invaluable in conjunction with clinical assessment to identify the early chronic inflammatory conversion.

Given the importance of maintaining BBB integrity, there is likely more than one pathway regulating restoration post injury [62,63]. That said, duration and speed of clinical recovery are correlated with patient’s age, location and magnitude of force, and prior head injuries [60,61]. The inverse correlation of severity of head injury and restoration of BBB integrity are understandable related to anatomic and vascular disruption [77]. The caveat to this general rule in adolescence has shown clinical evidence of emotional dysfunction even in mTBI which can be persistent suggesting limbic system tract injury [66].

Of particular interest is why do these mechanisms rapidly repair the BBB leaks in youth with a single mTBI but flags in the aging brain even without injury? The answer may lie in the long-term presence of proinflammatory microglia and ECM constituent protein alterations which increase local BBB permeability [78]. Alternatively, upregulation of inflammatory cytokines related to other unrelated health issues may contribute [79,80,81,82]. Conversely, is it a failure of normal restorative signaling pathways lost in the aging process that are root cause? Or perhaps a combination all three mechanisms? This question has profound implications for treatment of the underpinnings of other diseases such as neurogenerative diseases where loss of BBB integrity is the nexus of their development [63,79,80,81,82]. Further, of the two pathologic features of traumatic brain injury, restoration of BBB integrity as opposed to shear injury appears to be the most amenable to intervention.

To assess presence of BBB leak, physiologic imaging methods are required. Likewise, assessment of future treatment modalities to monitor outcomes of clinical treatment trials addressing BBB repair requires the same [63,83]. In the next sections, current most usable and clinically available methods of anatomic and physiologic imaging will be discussed and their roles in identifying specific pathologic features of head injury.

1.2. Structural Imaging in mTBI

Although anatomic imaging in singular mTBI is infrequently done and if performed is almost invariably normal. It clearly has a place in evaluation of patients with multiple mTBI and of course more severe injury. In moderate or severe acute brain injury, CT head is the study of choice for evaluation of bleeding or mass effect [18,83,84]. Evaluating subacute or chronic injury the benefit of MRI capabilities for defining atrophy with T1sequences, white matter lesions with fluid attenuated inversion recovery (FLAIR), and prior micro or macro hemorrhages with Susceptibility Weighted Images (SWI) are the gold standards [83,84].

1.3. Physiologic Imaging

Assessment of TBI potentially takes three forms: anatomic, clinical cognitive/neurologic examination, and physiologic testing. In mTBI, structural evaluation with CT or MRI is nearly always normal, whereas the clinical cognitive assessments are far more revealing. However subtle changes in processing, memory, personality, and emotional stability may miss subclinical physiologic injuries. Current clinically available methodologies for assessing the physiologic change in BBB function include Dynamic Contrast Enhanced MRI (DCE-MRI) and arterial spin labeling (ASL) MRI [85]. Evaluation of WM tract shear injury can be assessed directly with MRI tractography- Diffusion Tensor Imaging (DTI)., Positron Emission Tomography (PET), assesses reduction in glucose utilization reflective of regional loss of normal metabolic function [85] . Indirect means include Functional MRI imaging (not discussed further as it is not commonly used clinically). Pros and cons of each method will be discussed below.

1.3.1. Diffusion Tensor Imaging

Diffusion Tensor Imaging is a non-invasive method of investigating white matter tract integrity, which leverages imaging of water movement within the brain [86,87]. Water in the absence of obstruction will move randomly in all directions ((isotropy). In normal brain however water is forced to move parallel to white matter tract obstructions (anisotropy). With shearing force injury these neat pathways are interrupted and hence water present in those locations moves more isotopically. Diffusion imaging techniques can identify that disruption (Figure 1). Technical improvement to this methodology is under development. Current methods available lack sensitivity and are hampered by long scan times [85]. Nonetheless, as methods ameliorating persistent BBB leak develop, knowledge of the extent of untreatable shear injury is of value in prognosticating ultimate neurologic recovery.

1.3.2. Dynamic Contrast Enhanced (DCE) MRI

Dynamic Contrast Enhanced (DCE) MRI directly measures leak of contrast agent into the neuropil in excess of its natural infusibility. Given gadolinium contrast agents are mostly excluded from passage through the BBB normally, their passage increases with its disruption. The technique assesses plasma volume, interstitial space and can calculate a volumetric transfer constant from vascular to interstitial space [88,89,90]. Excess accumulation of contrast in the interstitial space beyond the calculated diffusion amount is indicative of BBB leak. This method allows direct visualization of BBB leak but is invasive (requires gadolinium contrast injection) and thus is not suitable for serial measurements over time, given the concern of retained gadolinium contrast within the CNS [91,92,93] (Figure 2). Specific software to sort out normal contrast diffusion through the BBB from that related to excess leak is also required [89]. Depending on patient circumstances though its use as a diagnostic one-off study when coupled with DTI may prove helpful in prognosticating recovery in those slow to do so. The combination would reveal the extent of BBB leak and white matter tract injury.

Figure 2. Individual subject examples from a representative control (A) and subacute TBI subject (B). The TBI nonlesional patient shows similar mild elevations in K^trans and NPI in the anterior frontal white matter (yellow arrows), and additional small areas of K^trans elevation in the occipital lobes (blue arrow).

Figure 2. Individual subject examples from a representative control (A) and subacute TBI subject (B). The TBI nonlesional patient shows similar mild elevations in K^trans and NPI in the anterior frontal white matter (yellow arrows), and additional small areas of K^trans elevation in the occipital lobes (blue arrow).

Ware, Jeffrey B., Saurabh Sinha, Justin Morrison, Alexa E. Walter, James J. Gugger, Andrea LC Schneider, Cian Dabrowski et al. "Dynamic contrast enhanced MRI for characterization of blood-brain-barrier dysfunction after traumatic brain injury." NeuroImage: Clinical 36 (2022): 103236. [123]

2213-1582/©2022 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

1.3.3 18-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET)

Utilizing radioactively labeled fluorodeoxyglucose (¹⁸FDG) and measuring cellular uptake thereof via positron emission tomography (PET) indirectly measures metabolic activity [94,95]. With less metabolic activity there is reduced uptake of labeled glucose as occurs in acute mTBI [96,97]. In the acute phase of severe injury there is increased uptake of ¹⁸FDG corresponding to the increased glycolysis required to reestablish electrolyte balance [98,99]. In the chronic phase of injury, there is reduced uptake of glucose corresponding to reduced local activity [99,100,101,102,103]. When overlayed on CT or MRI brain images localization of abnormalities is enhanced (Figure 3). Sensitivity, specificity, cost, and scan times limit its utility.

Morgan, Richard, Jordon Prosapio, Sam Kara, Sreepadma Sonty, Pamela Youssef, and Kester Nedd. "Preliminary clinical diagnostic criteria for chronic traumatic encephalopathy: A case report and literature review." Interdisciplinary Neurosurgery 26 (2021): 101290. [124]

Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

Application of tau PET scan will not be discussed here as they would not be employed in mTBI but in cases of repetitive TBI when the question of CTE arises. The reader is referred to excellent reviews of these techniques [94,104,105,106,107,108,109,110,111,112,113,114].

1.3.4. Arterial Spin Labeling MRI

Another method to indirectly determine BBB leak utilizes the clearance rate of magnetically labelled protons using 3D ASL MRI technology. This technique leverages the physiologic effects of altered perfusion with delayed capillary mean transit time (cMTT) and reduced glymphatic outflow in the late phase of the perfusion cycle, correlating with the location of BBB leak [115] (Figure 4). It is a non-invasive method using blood protons magnetically spin labeled in the neck and after specified post labeling delay (PLD), residual signal averages are sampled in desired cortical regions.

T1 times (63% signal decay) of major signal contributors indicated by the colored dots.

Magenta dot = 800-850 msec T1 of white matter.

Orange dot = 1650 msec T1 of blood.

Green dot = 1700 msec T1 of gray matter (all values are for 3T)

Dk blue dot = 3800 msec T1 of water (CSF fluid); (all values are for 3T)

BAT = bolus arrival time; aaTT =artery-artery arrival time; τ = peak capillary arrival time

Adapted with publisher permission (Wiley) from reference [125].

By using multiple sequential PLD’s in the late phase of the perfusion cycle linear analysis can be employed to determine rate of clearance (slope of the line) [115,116] . In regions of BBB leak there are localized changes in blood flow that correlate with diminished clearance rates. Since the signal measured is largely from trapped labeled water protons in the late phase of perfusion, it is much more sensitive determining minor BBB leaks than gadolinium (large molecule) based DCE [117].

Since this is a non-invasive technique, it is suitable for sequential studies without risk of exogenous contrast. Currently when combined with FLAIR and Susceptibility Weighted Images (SWI), scan time is about 18 ^1/2 minutes [116]. ASL MRI requires a 3 Tesla magnet and is limited reduced S/N. The latter can be circumvented by measuring a large field of view and obtaining multiple PLDs. Using 3D data acquisition allows for multiple regions of the brain to be investigated in one study. For example, our usage in mTBI acquired clearance rates from 6 brain regions, homologous temporal, frontal, and parietal regions [115]. Newer ASL MRI sequences allow for acquiring multiple PLDs in one sequence, reducing scan times to under 5 minutes [118]. This technique has significant potential in following the progress of BBB repair or alternatively persistent leak. In our pilot study of mTBI in college age athletes, both reduced glymphatic clearance post-acute injury and restoration of BBB integrity as measured by this technique correlated with high sensitivity with clinical recovery [116]. Future studies evaluating more serious head injury sequentially may shed light on those patients with persistent dysfunction which sets the stage for progressive dementia (CTE). In addition, this objective measure could help determine when and if an injured athlete or soldier could safely return to their sport or active duty. Limitations of this technique include the indirect method of determination (results are graphic in nature post analysis as opposed specific images of leak) and low S/N ratio. The latter is compensated for by analyzing multiple data points and acquiring signal from a large FOV. Given the test is noninvasive as well as, time and cost efficient, this method is suitable for serial studies post TBI in the assessment of BBB dysfunction [63,119].

1.3.6 Summary of techniques

Table 1 below summarizes the imaging target, relative advantages, and disadvantages of the four physiologic imaging techniques presented in evaluating mTBI. They provide objective information regarding the state of brain and vascular function at the time they are employed post head injury. The techniques will continue to improve and become more widely available providing both prognostic information. They also will be essential as outcome measures of future treatment trials.

Table 1 summarizes the DTI in identifying shear tract injury, which if present, correlates with persistence of neurologic deficit and recovery depending on its extent. DCE, and ASL MRI identify loss of BBB integrity the former directly and the latter indirectly. ¹⁸FDG-PET measures metabolic activity and thus could be negatively influenced by either shear tract injury or BBB leak.

2. Conclusion

With the mounting evidence of severe late life repercussions (CTE) related to repetitive mTBI among professional athletes in contact sports, there is a need to evaluate the physiology leveraging the loss of BBB integrity post injury, that if persistent, correlates with the progressive changes in CTE [39,60,79,85,120,121]. By adopting physiologic testing measures, safe return to the sport could be more accurately predicted in mTBI. Since the potential of both subtle white matter tact shear injury and persistent BBB leak occur simultaneously assessment of damage requires attention to both which translates into white matter tract imaging as well as evaluation of BBB integrity. Further as we uncover the mechanisms of BBB repair going forward and can leverage that knowledge therapeutically, identifying and assessing outcomes of future treatments will be possible. The benefits of identifying BBB dysfunction spill over from TBI to preclinical stages of neurodegenerative disease (NG), where loss of its integrity is at the nexus of development and prime target for early intervention.

Acronyms Dictionary

TBI- mild traumatic brain injury

TJ protein ZO-1-tight junction protein Zona Occludens 1

C3a- complement C3a protein

C3aR- complement C3a receptor protein

pTau- hyperphosphorylated Tau

TDP-43- Transactive response DNA binding protein- 43

FLAIR- fluid attenuated inversion recovery

SWI- Susceptibility Weighted Images

ASL MRI-Arterial Spin Labeling MRI

PLD- Post labeling delay

DTI MRI- Diffusion Tensor Imaging

DCE MRI- Dynamic Contrast Enhanced MRI

PET- Positron Emission Tomography

S/N- Signal to Noise

SKAT 6- Sport concussion Assessment Tool

VOMS- Vestibular Ocular Motor Screening

SCOAT 6- Sports Concussion Office Assessment Tool

CTE- chronic traumatic encephalopathy

cMTT- mean capillary transit time

GF- glymphatic flow

BBB- Blood brain barrier

IGG- Immunoglobulin G

NVU- the neurovascular unit

ECM- extracellular matrix

IL1 β- interleukin 1 beta