Complement Drives Synaptic Degeneration and Progressive Cognitive Decline in the Chronic Phase after Traumatic Brain Injury

Cognitive deficits following traumatic brain injury (TBI) remain a major cause of disability and early-onset dementia, and there is increasing evidence that chronic neuroinflammation occurring after TBI plays an important role in this process. However, little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation after TBI. Here, we identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response that is associated with cognitive decline. Three months after an initial insult, there is ongoing complement activation in the injured brain of male C57BL/6 mice, which drives a robust chronic neuroinflammatory response extending to both hemispheres. This chronic neuroinflammatory response promotes synaptic degeneration and predicts progressive cognitive decline. Synaptic degeneration was driven by microglial phagocytosis of complement-opsonized synapses in both the ipsilateral and contralateral brain, and complement inhibition interrupted the degenerative neuroinflammatory response and reversed cognitive decline, even when therapy was delayed until 2 months after TBI. These findings provide new insight into our understanding of TBI pathology and its management; and whereas previous therapeutic investigations have focused almost exclusively on acute treatments, we show that all phases of TBI, including at chronic time points after TBI, may be amenable to therapeutic interventions, and specifically to complement inhibition.SIGNIFICANCE STATEMENT There is increasing evidence of a chronic neuroinflammatory response after traumatic brain injury (TBI), but little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation. We identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response, and further that this response is associated with cognitive decline. Complement inhibition interrupted this response and reversed cognitive decline, even when therapy was delayed until 2 months after injury. The data further support the concept that TBI should be considered a chronic rather than an acute disease condition, and have implications for the management of TBI in the chronic phase of injury, specifically with regard to the therapeutic application of complement inhibition.     

The NLRP3 inflammasome: a potential therapeutic target for traumatic brain injury

Although the precise mechanisms contributing to secondary brain injury following traumatic brain injury are complex and obscure, a number of studies have demonstrated that inflammatory responses are an obvious and early feature in the pathogenesis of traumatic brain injury. Inflammasomes are multiprotein complexes that prompt the stimulation of caspase-1 and subsequently induce the maturation and secretion of proinflammatory cytokines, such as interleukin-1β and interleukin-18. These cytokines play a pivotal role in facilitating innate immune responses and inflammation. Among various inflammasome complexes, the NOD-like receptor family pyrin domain-containing 3(NLRP3) inflammasome is the best characterized, a crucial role for NLRP3 has been demonstrated in various brain diseases, including traumatic brain injury. Several recent studies have revealed the contribution of NLRP3 inflammasome in identifying cellular damage and stimulating inflammatory responses to aseptic tissue injury after traumatic brain injury. Even more important, blocking or inhibiting the activation of the NLRP3 inflammasome may have substantial potential to salvage tissue damage during traumatic brain injury. In this review, we summarize recently described mechanisms that are involved in the activation and regulation of the NLRP3 inflammasome. Moreover, we review the recent investigations on the contribution of the NLRP3 inflammasome in the pathophysiology of TBI, and current advances and challenges in potential NLRP3-targeted therapies. A significant contribution of NLRP3 inflammasome activation to traumatic brain injury implies that therapeutic approaches focused on targeting specific inflammasome components could significantly improve the traumatic brain injury outcomes.     

Early metabolic crisis-related brain atrophy and cognition in traumatic brain injury

Traumatic brain injury often results in acute metabolic crisis. We recently demonstrated that this is associated with chronic brain atrophy, which is most prominent in the frontal and temporal lobes. Interestingly, the neuropsychological profile of traumatic brain injury is often characterized as ‘frontal-temporal’ in nature, suggesting a possible link between acute metabolic crisis-related brain atrophy and neurocognitive impairment in this population. While focal lesions and diffuse axonal injury have a well-established role in the neuropsychological deficits observed following traumatic brain injury, no studies to date have examined the possible contribution of acute metabolic crisis-related atrophy in the neuropsychological sequelae of traumatic brain injury. In the current study we employed positron emission tomography, magnetic resonance imaging, and neuropsychological assessments to ascertain the relationship between acute metabolic crisis-related brain atrophy and neurocognitive outcome in a sample of 14 right-handed traumatic brain injury survivors. We found that acute metabolic crisis‐related atrophy in the frontal and temporal lobes was associated with poorer attention, executive functioning, and psychomotor abilities at 12 months post-injury. Furthermore, participants with gross frontal and/or temporal lobe atrophy exhibited numerous clinically significant neuropsychological deficits in contrast to participants with other patterns of brain atrophy. Our findings suggest that interventions that reduce acute metabolic crisis may lead to improved functional outcomes for traumatic brain injury survivors.     

Involvement of pro- and anti-inflammatory cytokines and chemokines in the pathophysiology of traumatic brain injury

Despite dramatic improvements in the management of traumatic brain injury (TBI), to date there is no effective treatment available to patients, and morbidity and mortality remain high. The damage to the brain occurs in two phases, the initial primary phase being the injury itself, which is irreversible and amenable only to preventive measures to minimize the extent of damage, followed by an ongoing secondary phase, which begins at the time of injury and continues in the ensuing days to weeks. This delayed phase leads to a variety of physiological, cellular, and molecular responses aimed at restoring the homeostasis of the damaged tissue, which, if not controlled, will lead to secondary insults. The development of secondary brain injury represents a window of opportunity in which pharmaceutical compounds with neuroprotective properties could be administered. To establish effective treatments for TBI victims, it is imperative that the complex molecular cascades contributing to secondary injury be fully elucidated. One pathway known to be activated in response to TBI is cellular and humoral inflammation. Neuroinflammation within the injured brain has long been considered to intensify the damage sustained following TBI. However, the accumulated findings from years of clinical and experimental research support the notion that the action of inflammation may differ in the acute and delayed phase after TBI, and that maintaining limited inflammation is essential for repair. This review addresses the role of several cytokines and chemokines following focal and diffuse TBI, as well as the controversies around the use of therapeutic anti-inflammatory treatments versus genetic deletion of cytokine expression.     

Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury.

Matrix metalloproteinases (MMPs) belong to a class of extracellular proteinases responsible for maintaining and remodeling the extracellular matrix. In addition to multiple functions in normal physiology, abnormal MMP expression and activity may also participate in the pathophysiology of cerebral disease. Here, we show that MMP-9 (gelatinase B; EC.3.4.24.35) contributes to the pathophysiology of traumatic brain injury. After controlled cortical impact in mice, MMP-9 was increased in traumatized brain. Total MMP-9 levels at 24 hr were significantly increased as measured by a substrate cleavage assay. Zymograms showed that MMP-9 was elevated as early as 3 hr after traumatic brain injury, reaching a maximum at approximately 24 hr. Increased MMP-9 levels persisted for up to 1 week. Western blot analysis indicated increased profiles of MMP-9 expression that corresponded with the zymographic data. Knock-out mice deficient in MMP-9 gene expression were compared with wild-type littermates in terms of morphological and motor outcomes after trauma. Motor outcomes were measured at 1, 2, and 7 d after traumatic brain injury by the use of a rotarod device. MMP-9 knock-out mice had less motor deficits than wild-type mice. At 7 d, traumatic brain lesion volumes on Nissl-stained histological sections were significantly smaller in MMP-9 knock-out mice. These data demonstrate that MMP-9 contributes to the pathophysiology of traumatic brain injury and suggest that interruption of the MMP proteolytic cascade may be a possible therapeutic approach for preventing the secondary progression of damage after brain trauma.     

Plasma fibronectin is neuroprotective following traumatic brain injury

Promotion of repair and regeneration following traumatic brain injury remains a challenging clinical problem. While significant efforts have been made to reduce inhibitory extracellular matrix expression following central nervous system injury, much less attention has been given to the role of endogenous reparative matrix proteins, such as fibronectin. Traumatic brain injury leads to increased levels of plasma-derived fibronectin in the brain tissue, though the specific function of this protein following neurotrauma was unknown. In this study, we utilized conditional plasma fibronectin (pFN) knockout mice to examine the role of fibronectin following a traumatic insult. Injured mice deficient in pFN performed significantly worse on both motor and cognitive tasks, had significantly increased lesion volume and apoptotic cell death, and had significantly less phagocytic cells in the injured cortex compared to injured mice with normal pFN levels. Moreover, intravenous injections of fibronectin prior to the injury restored the neural deficits seen in the pFN deficient mice to that of wild type injured mice. These results demonstrate that fibronectin is neuroprotective to the traumatically injured brain and identify a novel target for therapeutic interventions.     

Complications of head injury

Management of head injury is based on two concepts, proper treatment of the acute insult and the prevention and treatment of secondary insults. The head injured patient is subject to both intracranial and extracranial secondary insults. This paper will review complications related to the central nervous system as well as the pulmonary, infectious, gastrointestinal, and psychiatric complications frequently seen following traumatic brain injury. Complications following head trauma lead to significant acute and chronic morbidity and mortality. It is essential that clinicians be able to recognize and treat these complications in order to more effectively manage head trauma, improve outcome, and care for patients.     

Traumatic brain injury increases TGF beta RII expression on endothelial cells

Transforming growth factor beta (TGFbeta) modulates a variety of growth related functions following traumatic injury. The cellular response to TGFbeta is predominantly mediated through TGFbeta receptor I (TGFbetaRI) and receptor II (TGFbetaRII) on the cell surface and SMAD proteins intracellularly. We investigated the expression of TGFbeta receptors in the acute and chronic phases of a traumatic cerebral injury (TCI) by immunohistochemistry and in cultures of murine brain microvascular endothelial (EN) cells using cytofluorimetry. Here, we report that TGFbetaRII expression significantly increases on brain endothelial cells in the chronic phase of TCI. SMAD3 and SMAD4 protein expression were also upregulated suggesting the activation of TGFbeta receptor intracellular signaling. When TGFbetaRI and TGFbetaRII expression was studied in in vitro cultures of murine brain microvessel EN cells, TGFbetaRII showed increased expression on proliferating cells that are incorporating BrdU. These data show a differential expression of TGFbetaRI and TGFbetaRII on brain microvessel EN cells in the acute and chronic phases of TCI that might be associated with EN proliferation following injury.     

Potential effects of mesenchymal stem cell derived extracellular vesicles and exosomal miRNAs in neurological disorders

Mesenchymal stem cells are multipotent cells that possess anti-inflammatory, antiapoptotic and immunomodulatory properties. The effects of existing drugs for neurodegenerative disorders such as Alzheimer's disease are limited, thus mesenchymal stem cell therapy has been anticipated as a means of ameliorating neuronal dysfunction. Since mesenchymal stem cells are known to scarcely differentiate into neuronal cells in damaged brain after transplantation, paracrine factors secreted from mesenchymal stem cells have been suggested to exert therapeutic effects. Extracellular vesicles and exosomes are small vesicles released from mesenchymal stem cells that contain various molecules, including proteins, mRNAs and microRNAs. In recent years, administration of exosomes/extracellular vesicles in models of neurological disorders has been shown to improve neuronal dysfunctions, via exosomal transfer into damaged cells. In addition, various microRNAs derived from mesenchymal stem cells that regulate various genes and reduce neuropathological changes in various neurological disorders have been identified. This review summarizes the effects of exosomes/extracellular vesicles and exosomal microRNAs derived from mesenchymal stem cells on models of stroke, subarachnoid and intracerebral hemorrhage, traumatic brain injury, and cognitive impairments, including Alzheimer's disease.     

Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist

The role of complement components in traumatic brain injury is poorly understood. Here we show that secondary damage after acute cryoinjury is significantly reduced in C3-/- or C5-/- mice or in mice treated with C5a receptor antagonist peptides. Injury sizes and neutrophil extravasation were compared. While neutrophil density increased following traumatic brain injury in wild type (C57BL/6) mice, C3-deficient mice demonstrated lower neutrophil extravasation and injury sizes in the brain. RNase protection assay indicated that C3 contributes to the induction of brain inflammatory mediators, MIF, RANTES (CCL5) and MCP-1 (CCL2). Intracranial C3 injection induced neutrophil extravasation in injured brains of C3-/- mice suggesting locally produced C3 is important in brain inflammation. We show that neutrophil extravasation is significantly reduced in both C5-/- mice and C5a receptor antagonist treated cryoinjured mice suggesting that one of the possible mechanisms of C3 effect on neutrophil extravasation is mediated via downstream complement activation products such as C5a. Our data indicates that complement inhibitors may ameliorate traumatic brain injury.     

Astrocyte-targeted expression of IL-6 protects the CNS against a focal brain injury

The effect of CNS-targeted IL-6 gene expression has been thoroughly investigated in the otherwise nonperturbed brain but not following brain injury. Here we examined the impact of astrocyte-targeted IL-6 production in a traumatic brain injury (cryolesion) model using GFAP-IL6 transgenic mice. This study demonstrated that transgenic IL-6 production significantly increased wound healing following the cryolesion. Thus, at 20 days postlesion (dpl) the GFAP-IL6 mice showed almost complete wound healing compared to litter mate nontransgenic controls. It seems likely that a reduced inflammatory response in the long term could be responsible for this IL-6-related effect. Thus, while in the acute phase following cryolesion (1-6 dpl) the recruitment of macrophages and T lymphocytes was higher in GFAP-IL6 mice, at 10-20 dpl it was significantly reduced compared to controls. Reactive astrogliosis was also significantly increased up to but not including 20 dpl in the GFAP-IL6 mice. Oxidative stress as well as apoptotic cell death was significantly decreased throughout the time period studied in the GFAP-IL6 mice compared to controls. This could be linked to the altered inflammatory response as well as to the transgenic IL-6-induced increase of the antioxidant, neuroprotective proteins metallothionein-I + II. These results indicate that although in the brain the chronic astrocyte-targeted expression of IL-6 spontaneously induces an inflammatory response causing significant damage, during an acute neuropathological insult such as following traumatic injury, a clear neuroprotective role is evident.     

Brain to blood glutamate scavenging as a novel therapeutic modality: a review

It is well known that abnormally elevated glutamate levels in the brain are associated with secondary brain injury following acute and chronic brain insults. As such, a tight regulation of brain glutamate concentrations is of utmost importance in preventing the neurodegenerative effects of excess glutamate. There has been much effort in recent years to better understand the mechanisms by which glutamate is reduced in the brain to non-toxic concentrations, and in how to safely accelerate these mechanisms. Blood glutamate scavengers such as oxaloacetate, pyruvate, glutamate–oxaloacetate transaminase, and glutamate-pyruvate transaminase have been shown to reduce blood glutamate concentrations, thereby increasing the driving force of the brain to blood glutamate efflux and subsequently reducing brain glutamate levels. In the past decade, blood glutamate scavengers have gained increasing international interest, and its uses have been applied to a wide range of experimental contexts in animal models of traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, epilepsy, migraine, and malignant gliomas. Although glutamate scavengers have not yet been used in humans, there is increasing evidence that their use may provide effective and exciting new therapeutic modalities. Here, we review the laboratory evidence for the use of blood glutamate scavengers. Other experimental neuroprotective treatments thought to scavenge blood glutamate, including estrogen and progesterone, beta-adrenergic activation, hypothermia, insulin and glucagon, and hemodialysis and peritoneal dialysis are also discussed. The evidence reviewed here will hopefully pave the way for future clinical trials.     

Circulating extracellular vesicles: friends and foes in neurodegeneration

Extracellular vesicles have been identified as pivotal mediators of intercellular communication with critical roles in physiological and pathological conditions.Via this route,several molecules(e.g.,nucleic acids,proteins,metabolites) can be transferred to proximal and distant targets to convey specific information.Extracellular vesicle-associated cargo molecules have been proposed as markers of several disease conditions for their potential of tracking down the generating cell.Indeed,circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems especially in conditions characterized by the accrual of intracellular misfolded proteins.Furthermore,the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo molecules to diseased tissues makes these circulating shuttles possible targets for therapeutic development.The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-associated proteins might reflect molecular events occurring in hardly accessible tissues,such as the brain,thereby serving as a "brain liquid biopsy".The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches.Here,we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases(i.e.,Parkinson's and Alzheimer's diseases).A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.     

Persisting insomnia following traumatic brain injury

Persisting insomnia secondary to traumatic brain injury, rarely reported and documented, is described in an adult male following head injury. The neuronal mechanisms underlying this sleep disorder as well as the neuropsychological concomitants and therapeutic approaches are discussed.     

Elevated intraspinal pressure in traumatic spinal cord injury is a promising therapeutic target

The currently recommended management for acute traumatic spinal cord injury aims to reduce the incidence of secondary injury and promote functional recovery.Elevated intraspinal pressure(ISP)likely plays an important role in the processes involved in secondary spinal cord injury,and should not be overlooked.However,the factors and detailed time course contributing to elevated ISP and its impact on pathophysiology after traumatic spinal cord injury have not been reviewed in the literature.Here,we review the etiology and progression of elevated ISP,as well as potential therapeutic measures that target elevated ISP.Elevated ISP is a time-dependent process that is mainly caused by hemorrhage,edema,and blood-spinal cord barrier destruction and peaks at 3 days after traumatic spinal cord injury.Duraplasty and hypertonic saline may be promising treatments for reducing ISP within this time window.Other potential treatments such as decompression,spinal cord incision,hemostasis,and methylprednisolone treatment require further validation.     

Blockade of brain angiotensin II AT1 receptors ameliorates stress, anxiety, brain inflammation and ischemia: Therapeutic implications

Poor adaptation to stress, alterations in cerebrovascular function and excessive brain inflammation play critical roles in the pathophysiology of many psychiatric and neurological disorders such as major depression, schizophrenia, post traumatic stress disorder, Parkinson's and Alzheimer's diseases and traumatic brain injury. Treatment for these highly prevalent and devastating conditions is at present very limited and many times inefficient, and the search for novel therapeutic options is of major importance. Recently, attention has been focused on the role of a brain regulatory peptide, Angiotensin II, and in the translational value of the blockade of its physiological AT(1) receptors. In addition to its well-known cardiovascular effects, Angiotensin II, through AT(1) receptor stimulation, is a pleiotropic brain modulatory factor involved in the control of the reaction to stress, in the regulation of cerebrovascular flow and the response to inflammation. Excessive brain AT(1) receptor activity is associated with exaggerated sympathetic and hormonal response to stress, vulnerability to cerebrovascular ischemia and brain inflammation, processes leading to neuronal injury. In animal models, inhibition of brain AT(1) receptor activity with systemically administered Angiotensin II receptor blockers is neuroprotective; it reduces exaggerated stress responses and anxiety, prevents stress-induced gastric ulcerations, decreases vulnerability to ischemia and stroke, reverses chronic cerebrovascular inflammation, and reduces acute inflammatory responses produced by bacterial endotoxin. These effects protect neurons from injury and contribute to increase the lifespan. Angiotensin II receptor blockers are compounds with a good margin of safety widely used in the treatment of hypertension and their anti-inflammatory and vascular protective effects contribute to reduce renal and cardiovascular failure. Inhibition of brain AT(1) receptors in humans is also neuroprotective, reducing the incidence of stroke, improving cognition and decreasing the progression of Alzheimer's disease. Blockade of AT(1) receptors offers a novel and safe therapeutic approach for the treatment of illnesses of increasing prevalence and socioeconomic impact, such as mood disorders and neurodegenerative diseases of the brain.     

Pulsed arterial spin labeling effectively and dynamically observes changes in cerebral blood flow after mild traumatic brain injury

Cerebral blood flow is strongly associated with brain function, and is the main symptom and diagnostic basis for a variety of encephalopathies. However, changes in cerebral blood flow after mild traumatic brain injury remain poorly understood. This study sought to observe changes in cerebral blood flow in different regions after mild traumatic brain injury using pulsed arterial spin labeling. Our results demonstrate maximal cerebral blood flow in gray matter and minimal in the white matter of patients with mild traumatic brain injury. At the acute and subacute stages, cerebral blood flow was reduced in the occipital lobe, parietal lobe, central region, subcutaneous region, and frontal lobe. Cerebral blood flow was restored at the chronic stage. At the acute, subacute, and chronic stages, changes in cerebral blood flow were not apparent in the insula. Cerebral blood flow in the temporal lobe and limbic lobe diminished at the acute and subacute stages, but was restored at the chronic stage. These findings suggest that pulsed arterial spin labeling can precisely measure cerebral blood flow in various brain regions, and may play a reference role in evaluating a patient's condition and judging prognosis after traumatic brain injury.     

Substance P antagonists as a therapeutic approach to improving outcome following traumatic brain injury

Although a number of secondary injury factors are known to contribute to the development of morphological injury and functional deficits following traumatic brain injury, accumulating evidence has suggested that neuropeptides. and in particular substance P, may play a critical role. Substance P is released early following acute injury to the CNS as part of a neurogenic inflammatory response. In so doing, it facilitates an increase in the permeability of the blood—brain barrier and the development of vasogenic edema. At the cellular level, substance P has been shown to directly result in neuronal cell death; functionally, substance P has been implicated in learning and memory, mood and anxiety, stress mechanisms, emotion-processing, migraine, emesis, pain, and seizures, all of which may be adversely affected after brain injury. Inhibition of post-traumatic substance P activity, either by preventing release or by antagonism of the neurokinin-1 receptor, has consistently resulted in a profound decrease in development of edema and marked improvements in functional outcome. This review summarizes the current evidence supporting a role for substance P in acute brain injury.     

Changes in blood-brain barrier permeability to large and small molecules following traumatic brain injury in mice

The entry of therapeutic compounds into the brain and spinal cord is normally restricted by barrier mechanisms in cerebral blood vessels (blood-brain barrier) and choroid plexuses (blood-CSF barrier). In the injured brain, ruptured cerebral blood vessels circumvent these barrier mechanisms by allowing blood contents to escape directly into the brain parenchyma. This process may contribute to the secondary damage that follows the initial primary injury. However, this localized compromise of barrier function in the injured brain may also provide a 'window of opportunity' through which drugs that do not normally cross the blood-brain barriers are able to do so. This paper describes a systematic study of barrier permeability in a mouse model of traumatic brain injury using both small and large inert molecules that can be visualized or quantified. The results show that soon after trauma, both large and small molecules are able to enter the brain in and around the injury site. Barrier restriction to large (protein-sized) molecules is restored by 4-5 h after injury. In contrast, smaller molecules (286-10,000 Da) are still able to enter the brain as long as 4 days postinjury. Thus the period of potential secondary damage from barrier disruption and the period during which therapeutic compounds have direct access to the injured brain may be longer than previously thought.     

Selective CDK inhibitors： promising candidates for future clinical traumatic brain injury trials

Traumatic brain injury induces secondary injury that contributes to neuroinflammation, neuronal loss, and neurological dysfunction. One important injury mechanism is cell cycle activation which causes neuronal apoptosis and glial activation. The neuroprotective effects of both non-selective （Flavopiridol） and selective （Roscovitine and CR-8） cyclin-dependent kinase inhibitors have been shown across mukiple experimental traumatic brain injury models and species. Cyclin-depen- dent kinaseinhibitors, administered as a single systemic dose up to 24 hours after traumatic brain injury, provide strong neuroprotection-reducing neuronal cell death, neuroinflammation and neurological dysfunction. Given their effectiveness and long therapeutic window, cyclin-dependent kinase inhibitors appear to be promising candidates for clinical traumatic brain injury trials.