CCL2 modulates cytokine production in cultured mouse astrocytes

<p>Abstract</p> <p>Background</p> <p>The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that <it>Ccl2</it>-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, <it>Ccl2</it>-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes <it>in vitro</it>.</p> <p>Methods</p> <p>Primary astrocytes were isolated from neonatal wild-type and <it>Ccl2</it>-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS) and interleukin (IL)-1β for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (r)CCL2 prior to IL-1β stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction.</p> <p>Results</p> <p>LPS (0.1-100 μg/ml; 8 h) induced the significantly greater secretion of five key cytokines and chemokines in <it>Ccl2</it>-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in <it>Ccl2</it>-deficient cells. IL-1β (10 and 50 ng/ml; 2-24 h) also resulted in exacerbated IL-6 production from <it>Ccl2</it>-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml) did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h) prior to stimulation with IL-1β (10 ng/ml, 8 h) significantly reduced IL-6 protein and gene expression.</p> <p>Conclusions</p> <p>Our data indicate that astrocytes are likely responsible for the exacerbated cytokine response seen <it>in vivo </it>post-injury in the absence of CCL2. Furthermore, evidence that CCL2 inhibits cytokine production by astrocytes following IL-1β stimulation, suggests a novel, immunomodulatory role for this chemokine in acute neuroinflammation. Further investigation is required to determine the physiological relevance of this phenomenon, which may have implications for therapeutics targeting CCL2-mediated leukocyte infiltration following TBI.</p

Post-traumatic hypoxia exacerbates neurological deficit, neuroinflammation and cerebral metabolism in rats with diffuse traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>The combination of diffuse brain injury with a hypoxic insult is associated with poor outcomes in patients with traumatic brain injury. In this study, we investigated the impact of post-traumatic hypoxia in amplifying secondary brain damage using a rat model of diffuse traumatic axonal injury (TAI). Rats were examined for behavioral and sensorimotor deficits, increased brain production of inflammatory cytokines, formation of cerebral edema, changes in brain metabolism and enlargement of the lateral ventricles.</p> <p>Methods</p> <p>Adult male Sprague-Dawley rats were subjected to diffuse TAI using the Marmarou impact-acceleration model. Subsequently, rats underwent a 30-minute period of hypoxic (12% O₂/88% N₂) or normoxic (22% O₂/78% N₂) ventilation. Hypoxia-only and sham surgery groups (without TAI) received 30 minutes of hypoxic or normoxic ventilation, respectively. The parameters examined included: 1) behavioural and sensorimotor deficit using the Rotarod, beam walk and adhesive tape removal tests, and voluntary open field exploration behavior; 2) formation of cerebral edema by the wet-dry tissue weight ratio method; 3) enlargement of the lateral ventricles; 4) production of inflammatory cytokines; and 5) real-time brain metabolite changes as assessed by microdialysis technique.</p> <p>Results</p> <p>TAI rats showed significant deficits in sensorimotor function, and developed substantial edema and ventricular enlargement when compared to shams. The additional hypoxic insult significantly exacerbated behavioural deficits and the cortical production of the pro-inflammatory cytokines IL-6, IL-1β and TNF but did not further enhance edema. TAI and particularly TAI+Hx rats experienced a substantial metabolic depression with respect to glucose, lactate, and glutamate levels.</p> <p>Conclusion</p> <p>Altogether, aggravated behavioural deficits observed in rats with diffuse TAI combined with hypoxia may be induced by enhanced neuroinflammation, and a prolonged period of metabolic dysfunction.</p

Tumor necrosis factor-mediated inhibition of interleukin-18 in the brain: a clinical and experimental study in head-injured patients and in a murine model of closed head injury.

Tumor necrosis factor (TNF) and interleukin-(IL)-18 are important mediators of neuroinflammation after closed head injury (CHI). Both mediators have been previously found to be significantly elevated in the intracranial compartment after brain injury, both in patients as well as in experimental model systems. However, the interrelation and regulation of these crucial cytokines within the injured brain has not yet been investigated. The present study was designed to assess a potential regulation of intracranial IL-18 levels by TNF based on a clinical study in head-injured patients and an experimental model in mice. In the first part, we investigated the interrelationship between the daily TNF and IL-18 cerebrospinal fluid levels in 10 patients with severe CHI for up to 14 days after trauma. In the second part of the study, the potential TNF-dependent regulation of intracerebral IL-18 levels was further characterized in an experimental set-up in mice: (1) in a standardized model of CHI in TNF/lymphotoxin-α gene-deficient mice and wild-type (WT) littermates, and (2) by intracerebro-ventricular injection of mouse recombinant TNF in WT C57BL/6 mice. The results demonstrate an inverse correlation of intrathecal TNF and IL-18 levels in head-injured patients and a TNF-dependent inhibition of IL-18 after intracerebral injection in mice. These findings imply a potential new anti-inflammatory mechanism of TNF by attenuation of IL-18, thus confirming the proposed "dual" function of this cytokine in the pathophysiology of traumatic brain injury

The tissue-type plasminogen activator-plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotraum

Anti-Lysophosphatidic Acid Antibodies Improve Traumatic Brain Injury Outcomes

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury. FINDINGS: Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes. CONCLUSIONS: This study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

Variation in neurosurgical management of traumatic brain injury

Background: Neurosurgical management of traumatic brain injury (TBI) is challenging, with only low-quality evidence. We aimed to explore differences in neurosurgical strategies for TBI across Europe. Methods: A survey was sent to 68 centers participating in the Collaborative European Neurotrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. The questionnaire contained 21 questions, including the decision when to operate (or not) on traumatic acute subdural hematoma (ASDH) and intracerebral hematoma (ICH), and when to perform a decompressive craniectomy (DC) in raised intracranial pressure (ICP). Results: The survey was completed by 68 centers (100%). On average, 10 neurosurgeons work in each trauma center. In all centers, a neurosurgeon was available within 30 min. Forty percent of responders reported a thickness or volume threshold for evacuation of an ASDH. Most responders (78%) decide on a primary DC in evacuating an ASDH during the operation, when swelling is present. For ICH, 3% would perform an evacuation directly to prevent secondary deterioration and 66% only in case of clinical deterioration. Most respondents (91%) reported to consider a DC for refractory high ICP. The reported cut-off ICP for DC in refractory high ICP, however, differed: 60% uses 25 mmHg, 18% 30 mmHg, and 17% 20 mmHg. Treatment strategies varied substantially between regions, specifically for the threshold for ASDH surgery and DC for refractory raised ICP. Also within center variation was present: 31% reported variation within the hospital for inserting an ICP monitor and 43% for evacuating mass lesions. Conclusion: Despite a homogeneous organization, considerable practice variation exists of neurosurgical strategies for TBI in Europe. These results provide an incentive for comparative effectiveness research to determine elements of effective neurosurgical care

Guilty Molecules, Guilty Minds? The Conflicting Roles of the Innate Immune Response to Traumatic Brain Injury

Traumatic brain injury (TBI) is a complex disease in the most complex organ of the body, whose victims endure lifelong debilitating physical, emotional, and psychosocial consequences. Despite advances in clinical care, there is no effective neuroprotective therapy for TBI, with almost every compound showing promise experimentally having disappointing results in the clinic. The complex and highly interrelated innate immune responses govern both the beneficial and deleterious molecular consequences of TBI and are present as an attractive therapeutic target. This paper discusses the positive, negative, and often conflicting roles of the innate immune response to TBI in both an experimental and clinical settings and highlights recent advances in the search for therapeutic candidates for the treatment of TBI