BLOOD–BRAIN BARRIER: THE NEUROVASCULAR UNIT

The NVU is defined in the literature as “a physiologic entity that is structurally defined by interactions occurring between endothelial cells, pericytes, smooth muscle cells, astrocytes, and neurons.”² Its foundation is composed of the basal lamina deep to the endothelial cell monolayer; the latter cells are interspersed with tight junction protein complexes that regulate paracellular transport. Transporters and receptors on the luminal and basolateral surfaces of these endothelial cells are also important to the NVU as they mediate solute traffic and aid in the communication between various cells. Other cells included in the neurovascular unit are pericytes, smooth muscle cells, circulating white blood cells, microglia, and astrocytes, whose end-foot processes line most of the vascular walls and some synapses.³

Recent reviews in the literature have divided the BBB into three functional barriers based on their major functions: physical, transport, and metabolic.⁴ Protein complexes between cells called adherens junctions and tight junctions compose the “physical” barrier that prevents paracellular diffusion. This ensures that substances either remain in the bloodstream to exit the central nervous system (CNS) or diffuse transcellularly into the CNS. Adherens junctions not only join endothelial cells but also contribute to the proper formation of tight junctions. Tight junctions are composed of claudins, occludins, and other transmembrane proteins held together by zona occludens proteins that scaffold these complexes to the cytoskeleton. The “transport” barrier is composed of transport proteins, receptors for endocytosis, and ATP-binding proteins on the endothelial cells that promote the influx of nutrients and ions and the efflux of toxins. Finally, the “metabolic” barrier encompasses enzymes such as P450 and monoamine oxidase in order to metabolize molecules that enter endothelial cells.^4,5 These three major functions of the blood-brain barrier demonstrate that the NVU is both structurally and physiologically dynamic.

TRAUMATIC BRAIN INJURY: PRIMARY AND SECONDARY INJURY

There are two general stages in the development of brain injury: primary damage and secondary damage. These stages are also known as immediate and delayed dysfunction. Traumatic brain injury (TBI) is a direct result of direct or indirect mechanical forces on the brain that results in acute changes such as shearing injuries, contusions, and hematomas. These forces on the brain all encompass the primary injury. Vascular and parenchymal damage in the brain contribute to what is known in the literature as BBB breakdown. Once this barrier is disrupted, secondary changes such as edema, inflammation, and hyperexcitability often occur. Several negative effects occur, setting off a cascade of additional complications that begin hours to days after injury. Some of the delayed responses to TBI include neovascularization, impaired cerebral blood flow, glial cell dysfunction, and cell degeneration, and all of these pathophysiological events are vulnerable to secondary injury (i.e., hypoxia, hypotension). A TBI in a patient may result in coma or death, seizures, or cognitive and behavioral disabilities. This secondary damage is the focus of current TBI research.⁶ BBB breakdown and its downstream effects, namely edema, will be the focus of this chapter.

TBI AND THE BBB

What do the BBB and the NVU have to do with TBI? There is a consensus in the literature that the BBB is an integral part of the neurovascular unit. It maintains homeostasis in the brain by regulating uptake of molecules and helps regulate cerebral blood flow. Traumatic brain injury causes primary damage to the brain parenchyma, damaging cerebral microvessels and leads to NVU pathophysiology.

The secondary damage from TBI begins with and revolves around the cerebral vasculature. From animal TBI models, we know that trauma disrupts the structural and physiological integrity of vessels. Disruption of the walls of microvessels in the BBB activates the coagulation cascade. Intravascular coagulation leads to ischemia in the areas surrounding the impact site resulting in severely decreased blood flow; this is known as the “no-reflow” phenomenon.^7–10 Since the integrity of the BBB is compromised after injury, blood-borne factors such as thrombin, albumin, and fibrinogen, can now enter the brain. These factors are thought by some to cause microglia activation, proliferation, and its production of pro-inflammatory factors.¹¹

It is important to note that although the BBB is often referred to as open or closed, it is a dynamic structure and it can change between these states depending on what proteins are expressed. Permeability of the BBB is largely regulated by the expression of certain tight junction proteins on microvessel endothelial cells. TBI disrupts expression of tight junction proteins as well as interactions between the NVU components. BBB opening can be assessed via IgG or Evans blue extravasation from the vessels. One study showed that TBI promotes BBB opening as early as the day after injury, while others show that the BBB can remain open for as long as 30 days.^12,13 Another study showed high levels of IgG extravasation near the injury site at 1 and 3 days postinjury but much lower levels at 7 days in a controlled cortical impact TBI model with juvenile rats. No extravasation was seen at 60 and 180 days from injury.⁵ MRI studies using a diffuse closed head injury model have shown that the initial BBB opening occurs as early 30 minutes postinjury.¹⁴

BBB permeability can also be assessed by measuring the expression of tight junction proteins such as claudin-5. Claudin-5 plays an important role in regulating paracellular transport in the BBB and is highly expressed on the brain microvascular endothelium. Its level of expression on the BBB is known to fluctuate postinjury.¹⁵ It is also well known from rat TBI models that BBB permeability to albumin and other high molecular weight molecules are biphasic, peaking at 4 to 6 hours and 2 to 3 days after injury.^16–19 It is thought that the first peak in permeability across the BBB is due to an influx of neutrophils and an increase in factors/molecules that lead to BBB dysfunction. The cause of the second peak in BBB permeability is currently unknown but is hypothesized to be a component of the brain’s response to injury.⁶ Experiments repeatedly demonstrate that claudin-5 expression then increases 1 to 2 weeks after injury and can remain elevated as much as 4 to 8 weeks after initial injury.^15,20

ROLE OF BLOOD-BORNE FACTORS THAT AFFECT BBB FUNCTION FOLLOWING TBI

In the normal brain, the neurovascular unit interacts with glial cells and vascular endothelial cells via paracrine signaling. After injury, astrocytes and microglial quickly react by producing and releasing a myriad of molecules that affect the communication between the different components of the BBB. Some of the molecules that contribute to the dysfunction of the BBB include TGF-beta, reactive oxygen species (ROS), VEGF, MMPs, and glutamate. Their presence and abnormally high levels in the brain lead to the disruption of BBB integrity primarily by destroying tight junction proteins or decreasing their expression. This then leads to increased BBB permeability, facilitating the development of cerebral edema and other secondary changes. We will discuss each of these factors individually, presenting evidence for their role in the increased BBB permeability we observe after TBI.

There is evidence that matrix metalloproteinases (MMPs) and VEGF lead to increased BBB permeability via the destruction of tight junction proteins. MMPs are produced by many cerebrovascular endothelial cells, astrocytes, microglia, neurons, and leukocytes especially isoforms 2, 3, and 9. They directly target and degrade basal lamina and tight junction proteins on the BBB, promoting vasogenic edema.^21–23 Brain tissue from TBI patients show increased synthesis and elevated levels of MMP 2 and 9 in plasma and cerebrospinal fluid (CSF).^24–26 Furthermore, deletion of the mmp9 gene in TBI mice resulted in a decrease in brain damage and improved function.^27,28 Tissue inhibitors of MMPs, endogenous regulators of MMPs, have been found to be decreased in animal models of TBI. VEGFA is a member of the VEGF family that has been associated with increased vascular permeability to low molecular weight molecules. It is also an important mitogen for vascular endothelial cells. Astrocyte upregulation of VEGFA synthesis only a few hours after injury was observed in brain tissue from TBI patients and animals. In turn, VEGFA downregulates claudin-5 expression, resulting in BBB “opening.”^29–32

Oxidative stress is now known to be a post-TBI event that produces molecules that can lead to BBB dysfunction. Traumatic injury to the brain promotes lipid peroxidation of cell membranes through which certain reactive oxygen species (ROS) are produced. There is evidence that an ROS called 4-HNE significantly increases BBB permeability.³³ Normal cells use endogenous molecules like glutathione to protect themselves from ROS. One study showed that glutathione depletion leads to a significant increase in paracellular BBB permeability to low molecular weight markers.³⁴ Another study demonstrated that claudin-5 and occludin actually get redistributed and degraded when brain endothelial monolayers were exposed to a mixture of ROS.³⁵ Adding to the list of molecules that contribute to BBB permeability after TBI is glutamate that gets released from parenchymal brain cells in large amounts. By binding to its mGluR receptor, it increases endothelial monolayer permeability in vitro. Interestingly, these two molecules’ actions may be linked as there is some evidence that glutamate increases ROS production to promote apoptosis.³⁶

TGF-beta is a molecule with very important cell functions such as proliferation and differentiation and it is produced in many cell types, including platelets, astrocytes, and microglia. Large amounts of TGF-beta get released from platelets in the latent form after vascular wall damage. Cryogenic brain injury studies have shown that there is an increase in both TGF-beta levels after injury and in the expression of TGF-beta receptors on vascular endothelium.^37–39 There are conflicting studies about whether TGF-beta really increases BBB permeability. Some believe that TGF-beta increases tyrosine phosphorylation to reduce claudin-5 and VE-cadherin expression; others believe that it plays a role in maintaining the barrier as it helps stabilize endothelial cell and pericyte interaction via N-cadherin.^40–42

Paradoxically, many of these molecules initially destroy the BBB after TBI but are later involved in repair processes. Although they seem like good therapeutic targets, blocking their action in the acute phase could lead to problems at a later time. For example, experiments show that glutamate stimulates the activity of heme oxygenase, a protectant against glutamate toxicity, in endothelial cells.^36,43

IMPORTANCE IN DEVELOPMENT OF CEREBRAL EDEMA FOLLOWING TBI

Cerebral edema is a very important secondary consequence of TBI. It is crucial to understand the development of cerebral edema because it is one of the main factors affecting the morbidity and mortality of TBI patients. Post-traumatic cerebral edema leads to the expansion of brain volume against an enclosed skull and an increase in intracranial pressure (ICP). Not only can increased ICP cause herniation, but it also decreases cerebral perfusion pressure, promoting cerebral ischemia in a brain with already tenuous blood supply.^6,44–47

As outlined earlier, we know that BBB permeability changes occur after TBI. Cerebral edema results from a combination of endothelial cell damage, tight junction disruption, and abnormal transcellular transport.⁴⁸ It is important to note that there are two types of edema that can come about, depending on the type of injury. The majority of TBI and blast injuries result in predominantly vasogenic edema—a pathological increase in vascular permeability, in this case, due to vessel damage. This leads to interstitial accumulation of plasma-derived, osmotically active molecules/solutes like plasma proteins followed by water.¹ Ischemic brain injuries tend to result in cytotoxic edema, which is caused by changes in cell metabolism and the failure of membrane associated pumps and ion transporters. This results in cellular accumulation of osmotically active molecules followed by water.¹

There is some evidence that aquaporins may also play a role in the development of post-CNS injury edema, but results from studies have been conflicting. There are many isoforms of this special water transporter but AQP4 is the one most studied. It is located on astrocyte end-foot processes and is believed to play a role in water homeostasis in the NVU.⁴⁹ There have not been many studies with TBI animal models so the role of AQP4 remains unclear. However, in summary, AQP4 seems to aid in cytotoxic edema but helps resolve vasogenic edema after the initial opening of the BBB after injury.⁴⁴

STRATEGIES TO REDUCE CEREBRAL EDEMA AFTER TBI

The traditional approach/first-line treatment to treating cerebral edema is to use mannitol, an osmotic diuretic that draws water out of cells and into the vasculature to be excreted by the body. Neurologic and renal side effects limit its repeated administration. Moreover, it is only effective for short periods of time and mortality can continue to increase if brain swelling continues past 24 hours. Hypertonic saline, decompressive craniotomy, and external ventricular drainage are the only other options for decreasing cerebral edema.⁴⁸ Steroids, despite their anti-inflammatory effects, have not been shown to be effective in reducing cerebral edema due to TBI. Thus, more effective and safe methods for decreasing edema are needed in order to prevent adverse outcomes from TBI.

According to current TBI management guidelines, decreasing intracranial pressure while optimizing cerebral perfusion pressure caused by TBI can be accomplished using mannitol or hypertonic saline (HS). Mannitol works by expanding blood plasma and drawing water into the vessels to improve cerebral blood flow.⁵⁰ HS decreases cerebral water content by creating a gradient across the BBB, shifting water from brain cells to the vasculature.⁵⁰ As a result, intravascular volume increases, improving blood flow. Historically, mannitol has been used more frequently than HS, although more recently 3% or 23% hypertonic saline has been used, particularly in combat casualty care. There have been numerous studies attempting to tease out which therapy is more beneficial, but results thus far are conflicting and inconclusive. Both therapies shift fluid from extravascular compartments into the vasculature but hypertonic saline expands intravascular volume while mannitol decreases it by promoting diuresis. Surprisingly, they both have anti-inflammatory properties such as decreasing neutrophil-endothelial cell interactions. A recent study performed by Marks et al. attempted to find a difference between mannitol and hypertonic saline in their effect on microvascular permeability.⁵¹ Using a simulated TBI model involving exogenous IL-1beta, they did not find a significant difference in the ability of either therapy to reduce neutrophil-endothelial interactions.⁵² Furthermore, when the BBB is compromised following TBI, these agents may not be as effective since the osmotic gradient may no longer exist in severe areas of injury.

Recent studies have shown that by transiently opening the BBB after TBI, it is possible to decrease post-traumatic cerebral edema. Using a cold-induced cortical impact model to produce focal injury, researchers have demonstrated paradoxically that a transient and size-selective modulation in the BBB promotes enhanced movement of water from the brain parenchyma to the blood vessels, decreasing brain swelling.⁴⁸ Campbell et al. have shown that siRNA knockdown of claudin-5 increases the efflux of low molecular weight molecules (less than 1 kDa) across the barrier between 24 and 72 hours after injection of siRNA. It has been postulated that the extracellular water follows these molecules and in turn decreases vasogenic edema.⁴⁸ To evaluate larger molecules, the extravasation of Evan’s blue dye in the parenchyma, which strongly binds to albumin (70 kDa), was grossly observed. There was a significant decrease in the amount of Evan’s blue dye in the peri-injury area in the mice that received claudin-5 targeted siRNA compared to their nontargeted counterparts. The claudin-5 targeted siRNA group also had a significant decrease in their percentage water content as well as the lesion volume 72 hours postinjury.⁴⁸

NOVEL THERAPEUTIC STRATEGIES TO STABILIZE BBB AFTER TBI

Secondary neuronal injury after TBI may be exacerbated by BBB dysfunction. The ideal strategy would be to prevent BBB breakdown and stabilize it, thereby protecting the brain from factors released from damaged blood vessels that cause further damage. Inflammation, excitotoxicity, oxidative stress, edema, and neuronal injury are all components of secondary neuronal injury after the primary damage caused by TBI. Attempts have been made at curtailing the effects of two endogenous mediators in the formation of brain edema: bradykinin and VEGF (vascular endothelial growth factor). Bradykinin is a pro-inflammatory peptide that promotes vasodilation and vascular permeability, and antagonists at its receptor decrease cerebral edema in animals and humans.^53,54 VEGF, a promoter of angiogenesis, increases vascular permeability in hypoxic conditions by modulating tight junction proteins.⁵⁵ Studies have shown that blocking VEGF decreases BBB permeability in vivo and that corticosteroids may regulate VEGF expression in the midst of BBB injury.

Numerous hormones including ghrelin, neural growth factor, and progesterone have recently been found to have neuroprotective effects following traumatic brain injury and all three have an effect on the BBB or water flux across the BBB.

Ghrelin is a hormone known for its role in regulating hunger and satiety, but its newly discovered neuroprotective effects are thought to be mediated by maintaining oxidant/anti-oxidant balance, inhibiting pro-inflammatory mediators, and inhibiting neuronal apoptosis.⁵⁶ Ghrelin is believed to inhibit apoptosis by upregulating a mitochondrial uncoupling protein called UCP-2, stabilizing mitochondria. Experiments with TBI, stroke, and Parkinson’s animal models demonstrate that higher levels of UCP-2 are neuroprotective as this results in decreased activation of caspase-3, an apoptotic protein.⁵⁷ Lopez et al. also showed that treating mice with neuropeptide ghrelin preceding and 1 hour after TBI resulted in decreased BBB permeability 6 hours after TBI. Not only did ghrelin treatment significantly decrease BBB permeability, measured by 70kD FITC-dextran extravasation 24 hours after injury, but it also significantly decreased post-TBI apoptosis of injured tissue. This group also showed that this decrease in vascular permeability was linked to a decrease in perivascular aquaporin-4 (AQP-4) expression after ghrelin treatment.⁵⁸ This is a significant finding because perivascular levels of AQP-4 receptors increase significantly 6 hours after TBI. AQP-4 receptors are preferentially located on astrocyte end-foot processes adjacent to cerebral microvessels and play a role in the formation of cerebral edema.⁵⁹ Their work with ghrelin thus far not only suggests a link between BBB breakdown and apoptosis of injured tissue but also that decreased BBB permeability after TBI limits tissue injury.^57,58

A hormone called nerve growth factor (NGF) is currently being studied for its potential to reduce cellular cerebral edema in mice after TBI via downregulation of AQP-4. NGF was delivered intranasally to bypass the BBB in order to preferentially distribute it in the brain, limiting systemic side effects. Cerebral water content measured at 12, 24, and 72 hours post-TBI coincided with reduced expression of AQP-4.⁶⁰

Preliminary studies involving progesterone demonstrates that it too may have neuroprotective effects.^61,62 Not only does cranial edema result from increased BBB permeability after TBI but also from inflammation and secondary brain injury that persists hours later.¹ The latter process is mediated by neutrophil and endothelial cell activation in the microcirculation, leading to microvascular breakdown. Neutrophils are an important part of the inflammatory host response, exerting cytotoxic and phagocytic effects on tissues. However, excessive or inappropriate activation of these cells can lead to unwanted destruction of the microvasculature and surrounding tissue, and in turn exacerbating cerebral edema.^64,65 Progesterone, an important steroid hormone, has been found to inhibit neuronal apoptosis and decrease free radical and cytokine production.⁶⁵ Pascaul et al. found that treating TBI rats with progesterone decreased neutrophil rolling and adhesion to the endothelium of the BBB, and decreased cerebral vascular leakage and edema 36 hours after trauma.⁶⁵ Data from other studies suggests that neutrophils are recruited to the BBB 12 to 36 hours after TBI, creating a window of opportunity to intervene and attenuate this aspect of secondary brain injury.^66,67 Progesterone’s effect on neutrophils is thought to decrease polymorphonuclear (PMN) respiratory burst and oxygen radials as well as decreased PMN adhesion receptor expression. Its effect on the endothelial cells has not yet been studied. A large multicenter RTCs Phase III clinical trial, ProTECT III, just completed investigated progesterone as a potential treatment to improve survival and cognitive recovery post-TBI.⁶⁵ Unfortunately the trial was negative.

Tchantchou and Zhang found that inhibiting the degradation of the most abundant endocannabinoid in the brain results in decreased BBB dysfunction, brain edema, lesion volume, neuronal death, and better behavioral performance after TBI.^68–72 While these neuroprotective effects of exogenous cannabinoids have been known for some time, psychotropic side effects from global activation of the CB1 receptor make them a poor candidate for treatment in humans. However, new research suggests that it is possible to augment the protective effects of endocannabinoids naturally produced after injury. Endocannabinoids would, instead, activate CB1 and CB2 receptors, expressed on neural cell and inflammatory cells, in specific sites and after a specific event (TBI), minimizing undesirable effects. 2-Arachidonylglycerol (2-AG), the endocannabinoid of interest, increases immediately after TBI.⁶⁸ It is known to be neuroprotective due its anti-oxidant, anti-inflammatory, and anti-excitotoxic effects.^68,73,74 Benefits are shortlived due to 2-AG’s rapid degradation by the enzymes monoacylglycerol (MAGL) and alpha/beta hydroxyl domain 6 (ABHD6).⁷⁵ In this study, mice underwent controlled cortical impact TBI and were given an ABHD6 inhibitor. On a gross level, animals that received the enzyme inhibitor exhibited decreased motor coordination and fine motor deficits. TBI-induced brain lesions were also significantly smaller at 1 and 3 weeks after injury in mice that received the inhibitor via cannabinoid receptior-1 (CB1) blockage. Microscopically, the inhibitor also decreased TBI-induced BBB permeability measured by Evans-blue extravasation and ICAM-1 immunoreactivity. The inhibitor also decreased pro-inflammatory molecules iNOS and COX2 that are usually expressed in glial cells after injury. Additionally, these authors found that chronic administration of the inhibitor reversed the increase in lesional volume that occurs post-TBI. In conclusion, enhancing the action of endocannabinoids in the brain have the potential to decrease BBB breakdown as well as secondary neuronal injury.

STEM CELLS AND BBB RESCUE FOLLOWING TBI

An innovative study by Menge et al. suggests that mesenchymal stem cells (MSCs) may mitigate BBB breakdown following TBI.⁷⁶ Human mesenchymal stem cells produce a molecule called tissue inhibitor of matrix metalloproteinase-3 (TIMP3), an inhibitor of soluble MMP. Using a TBI mouse model, they found that recombinant TIMP3 inhibits BBB permeability in TBI mice by blocking VEGF-A mediated breakdown of endothelial cell adherens and tight junctions in vitro and in vivo. The proposed mechanism involves the fact that 90% of intravenously delivered MSCs travel to the lungs, more specifically the pulmonary endothelium. It is believed that interaction between the stem cells and pulmonary endothelial cells promote production of TIMP3 by both cells. Systemic spread of TIMP3 allows for it to stabilize distant vasculature beds such as the BBB. Experiments by Menge et al. demonstrate that TIMP3 inhibits receptor-mediated VEGF-A binding to the VEGFR2 receptor, preventing vascular permeability. Additionally, recombinant TIMP3 increased adherens junctions in the mouse brain after TBI and downregulation of TIMP3 in TBI mice did not result in this protective phenomenon. This study adds to the growing body of work that human stem cells have therapeutic implications for diseases characterized by vascular instability.⁷⁶

CONCLUSIONS

The pathophysiology of the cascade of events that encompass secondary brain injury following TBI is complex. BBB disruption is a pathological hallmark of severe TBI and is associated with neuroinflammatory events contributing to brain edema and cell death. There is a strong temporal and spatial association between the degree of BBB disruption and the ability of circulating inflammatory cells to quickly migrate to the area of CNS injury. The BBB, however, may also be protective by promoting efflux of water across the BBB to mitigate cerebral edema after severe TBI. The therapeutic window of BBB modulation after TBI remains unknown and is further complicated by the biphasic disruption of the BBB following TBI. Further elucidation of the dynamics of BBB dysfunction after TBI would provide important information to guide the selection of therapeutic agents and timing of treatment. However, since neuroinflammation is a common theme across all severities of TBI, strategies aimed at preserving or repairing the BBB in the early period may be beneficial to improve patient outcome, decrease seizure risk, and promote brain health.

ACKNOWLEDGMENT

Supported by K08 NS075144-06 award to GG from the NIH/NINDS.

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