Prognostic Value of Plasma Biomarkers S100B and Osteopontin in Pediatric TBI: A Prospective Analysis Evaluating Acute and 6-Month Outcomes after Mild to Severe TBI.

Blood based traumatic brain injury (TBI) biomarkers offer additional diagnostic, therapeutic, and prognostic utility. While adult studies are robust, the pediatric population is less well studied. We sought to determine whether plasma osteopontin (OPN) and S100B alone or in combination predict mortality, head Computed tomography (CT) findings, as well as 6-month functional outcomes after TBI in children. This is a prospective, observational study between March 2017 and June 2021 at a tertiary pediatric hospital. The sample included children with a diagnosed head injury of any severity admitted to the Emergency Department. Control patients sustained trauma-related injuries and no known head trauma. Serial blood samples were collected at admission, as well as at 24, 48, and 72 h. Patient demographics, acute clinical symptoms, head CT, and 6-month follow-up using the Glasgow outcome scale, extended for pediatrics (GOSE-Peds), were also obtained. The cohort included 460 children (ages 0 to 21 years) and reflected the race and sex distribution of the population served. Linear mixed effect models and logistic regressions were utilized to evaluate the trajectory of biomarkers over time and predictors of dichotomous outcomes. Both OPN and S100B correlated with injury severity based on GCS. S100B and OPN showed lower AUC values (0.59) in predicting positive head CT. S100B had the largest AUC (0.87) in predicting mortality, as well as 6-month outcomes (0.85). The combination of the two biomarkers did not add meaningfully to the model. Our findings continue to support the utility of OPN as a marker of injury severity in this population. Our findings also show the importance of S100B in predicting mortality and 6-month functional outcomes. Continued work is needed to examine the influence of age-dependent neurodevelopment on TBI biomarker profiles in children.

Manganese Neurotoxicity as a Stroke Mimic: A Case Report.

Manganese (Mn)-induced cerebral toxicity is a rare neurological condition that can present as a stroke mimic in high-risk populations. We present a case of a 40-year-old male with no known comorbidities who was brought to the emergency department with complaints of nonprogressive slurred speech and left facial weakness for eight days. Further history revealed that he had been working as a welder in a steel factory for the past seven years without using proper personal protective equipment (PPE). On physical examination, an upper motor neuron (UMN) type weakness on the left side of his face and spastic dysarthria could be appreciated. Following a brain computed tomography (CT) scan that showed ill-defined hypodensities in the basal ganglia without any signs of a hemorrhage, he was admitted to the stroke unit for conservative management and further investigations. A magnetic resonance imaging (MRI) scan of the brain done later showed features of manganese deposition and absorption in the globus pallidus and corticospinal tracts, indicating a diagnosis of manganese-induced cerebral toxicity. His serum manganese levels obtained during admission were normal. He was managed conservatively with intravenous rehydration and was discharged after symptomatic improvement. He was counseled and educated regarding the importance of wearing protective equipment while at work to reduce further exposure to the metal. During his follow-up visit, his symptoms had considerably improved with proper adherence to workplace safety measures.

Neurotherapeutic Potential of Water-Soluble pH-Responsive Prodrugs of EIDD-036 in Traumatic Brain Injury.

The C-20 oxime of progesterone, EIDD-036 (2), demonstrates neuroprotection and improved outcomes in animal models of traumatic brain injury (TBI). However, 2 suffers from poor solubility, which renders it unsuitable for rapid administration. Previous prodrugs of 2 aimed at improving solubility by incorporating enzymatically labile amino acid and phosphate ester promoieties. These approaches were effective but led to limitations with in vivo administration. Herein, we disclose a pH-responsive water-soluble prodrug strategy to improve exposure to 2 through enzyme-independent activation. Compound 13l was identified as a lead that exhibits water-solubility, stability in acidic solutions, and rapid conversion to 2 at physiological pH. Administration of 13l to rats resulted in a twofold increase in exposure to 2 compared to the previous generation phosphate prodrug, EIDD-1723 (6). In a rat model of TBI, treatment with 13l resulted in a significant decrease in cerebral edema when administered postinjury.

Osteopontin as a biomarker for COVID-19 severity and multisystem inflammatory syndrome in children: A pilot study.

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0-21 years of age) admitted to Children's Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention's case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.

Prophylactic progesterone prevents adverse behavioural and neurocognitive effects of neonatal anaesthesia exposure in rat.

BACKGROUND: Evidence from animal models and human studies suggests an association between early general anaesthesia exposure and development of long-lasting neurocognitive problems including learning and memory impairments and an anxious phenotype. Because millions of children each year undergo procedures that require anaesthesia, it is important to investigate ways to protect the vulnerable developing brain. We evaluated whether progesterone treatment administered before general anaesthesia exposure could prevent long-term anaesthesia-induced neurocognitive and behavioural changes. METHODS: Female and male Long-Evans rat pups were repeatedly exposed to 2 h of sevoflurane or control procedures at postnatal days 7, 10, and 13. Subcutaneous injections of progesterone or vehicle were administered immediately before general anaesthesia exposure or control procedures. Neurobehavioural and cognitive outcomes were evaluated using elevated plus maze and Morris water maze tests. RESULTS: Prophylactic progesterone treatment attenuated the chemokine (C-X-C motif) ligand 1 (CXCL1) response to sevoflurane exposure. Rats given vehicle treatment with general anaesthesia exposure exhibited increased anxiety on the elevated plus maze and learning and memory impairments on the Morris water maze. However, rats treated with progesterone before general anaesthesia lacked these impairments and performed in a similar manner to controls on both tasks. CONCLUSIONS: Progesterone attenuated the anaesthesia-induced, acute peripheral inflammatory response and prevented cognitive and behavioural alterations associated with early repeated general anaesthesia exposure. Importantly, our results suggest that progesterone treatments given before general anaesthesia may help to protect the developing brain.

Neurocognitive Outcomes in a Cisternal Blood Injection Murine Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) results in neurocognitive dysfunction and anxiety in humans and in animal models. Neurobehavioral tests such as the Morris Water Maze (MWM) and Elevated Plus Maze (EPM) tests are validated in several models of SAH but have not been tested in the murine cisternal blood injection SAH model. METHODS: Adult C57BL/6 mice (n=16) were randomized into two groups. Group 1 (n=8) received sham surgery. Group 2 (n=8) underwent SAH with 60 µL of autologous blood injected into the cisterna magna. Mice were then tested using the Modified Garcia Score on post-operative day 2 (POD2), EPM on POD5 & POD16, and MWM on POD6-16.Brain tissues harvested on POD16 were stained with Fluoro-Jade C to identify neurodegeneration in the hippocampus and cortex and Iba-1 immunofluorescence staining for microglial activation in the dentate gyrus and CA1 region of the hippocampus. RESULTS: SAH mice showed increased escape latency on POD10. Swim distance was significantly increased on POD9-10 and swim speed was significantly decreased on POD6&POD10 in SAH mice. SAH mice exhibited a trend for lowered proportion of covered arm entries in EPM on POD16. Modified Garcia Score was not significantly different between the groups on POD2. The area of microglial activation in the dentate gyrus and CA1 region of the hippocampus was mildly increased but not significantly different at day 16 after SAH. Similarly, no significant differences were noted in the number of Fluoro-Jade C (+) cells in cortex or hippocampus. CONCLUSIONS: Cisternal single blood injection in mice produces mild neurocognitive deficits most pronounced in spatial learning and most evident 10 days after SAH.

Patterns of Osteopontin Expression in Abusive Head Trauma Compared with Other Causes of Pediatric Traumatic Brain Injury.

OBJECTIVE: To examine levels of plasma osteopontin (OPN), a recently described neuroinflammatory biomarker, in children with abusive head trauma (AHT) compared with children with other types of traumatic brain injury (TBI). STUDY DESIGN: The study cohort comprised children aged <4 years diagnosed with TBI and seen in the intensive care unit in a tertiary children's hospital. Patients were classified as having confirmed or suspected AHT or TBI by other mechanisms (eg, motor vehicle accidents), as identified by a Child Protection Team clinician. Serial blood samples were collected at admission and at 24, 48, and 72 hours after admission. Levels of OPN were compared across groups. RESULTS: Of 77 patients identified, 24 had confirmed AHT, 12 had suspected AHT, and 41 had TBI. There were no differences in the Glasgow Coma Scale score between the patients with confirmed AHT and those with suspected AHT and those with TBI (median score, 4.5 vs 4 and 7; P = .39). At admission to the emergency department, OPN levels were significantly higher in children with confirmed AHT compared with the other 2 groups (mean confirmed AHT, 471.5 ng/mL; median suspected AHT, 322.3 ng/mL; mean TBI, 278.0 ng/mL; P = .03). Furthermore, the adjusted mean trajectory levels of OPN were significantly higher in the confirmed AHT group compared with the other 2 groups across all subsequent time points (P = <.01). CONCLUSIONS: OPN is significantly elevated in children with confirmed AHT compared with those with suspected AHT and those with other types of TBI. OPN expression may help identify children with suspected AHT to aid resource stratification and triage of appropriate interventions for children who are potential victims of abuse.

Progesterone Attenuates Stress-Induced NLRP3 Inflammasome Activation and Enhances Autophagy following Ischemic Brain Injury.

NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome inhibition and autophagy induction attenuate inflammation and improve outcome in rodent models of cerebral ischemia. However, the impact of chronic stress on NLRP3 inflammasome and autophagic response to ischemia remains unknown. Progesterone (PROG), a neuroprotective steroid, shows promise in reducing excessive inflammation associated with poor outcome in ischemic brain injury patients with comorbid conditions, including elevated stress. Stress primes microglia, mainly by the release of alarmins such as high-mobility group box-1 (HMGB1). HMGB1 activates the NLRP3 inflammasome, resulting in pro-inflammatory interleukin (IL)-1ß production. In experiment 1, adult male Sprague-Dawley rats were exposed to social defeat stress for 8 days and then subjected to global ischemia by the 4-vessel occlusion model, a clinically relevant brain injury associated with cardiac arrest. PROG was administered 2 and 6 h after occlusion and then daily for 7 days. Animals were killed at 7 or 14 days post-ischemia. Here, we show that stress and global ischemia exert a synergistic effect in HMGB1 release, resulting in exacerbation of NLRP3 inflammasome activation and autophagy impairment in the hippocampus of ischemic animals. In experiment 2, an in vitro inflammasome assay, primary microglia isolated from neonatal brain tissue, were primed with lipopolysaccharide (LPS) and stimulated with adenosine triphosphate (ATP), displaying impaired autophagy and increased IL-1ß production. In experiment 3, hippocampal microglia isolated from stressed and unstressed animals, were stimulated ex vivo with LPS, exhibiting similar changes than primary microglia. Treatment with PROG reduced HMGB1 release and NLRP3 inflammasome activation, and enhanced autophagy in stressed and unstressed ischemic animals. Pre-treatment with an autophagy inhibitor blocked Progesterone's (PROG's) beneficial effects in microglia. Our data suggest that modulation of microglial priming is one of the molecular mechanisms by which PROG ameliorates ischemic brain injury under stressful conditions.

Plasma osteopontin may predict neuroinflammation and the severity of pediatric traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death in children and adolescents in developed countries, but there are no blood-based biomarkers to support the diagnosis or prognosis of pediatric TBI to-date. Here we report that the plasma levels of osteopontin (OPN), a phosphoprotein chiefly secreted by macrophages and/or activated microglia, may contribute to this goal. In animal models of TBI, while OPN, fibrillary acidic protein (GFAP), and matrix metalloproteinase 9 (MMP-9) were all readily induced by controlled cortical impact in the brains of one-month-old mice, only OPN and GFAP ascended in the blood in correlation with high neurological severity scores (NSS). In children with TBI (three to nine years of age, n = 66), the plasma levels of OPN, but not GFAP, correlated with severe TBI (Glasgow Coma Score ≤ 8) and intracranial lesions at emergency department. In addition, the plasma OPN levels in severe pediatric TBI patients continued to ascend for 72 h and correlated with mortality and the days requiring ventilator or intensive care unit support, whereas the plasma GFAP levels lacked these properties. Together, these results suggest that plasma OPN outperforms GFAP and may be a neuroinflammation-based diagnostic and prognostic biomarker in pediatric TBI.

Is a combination of progesterone and chloroquine more effective than either alone in the treatment of cerebral ischemic injury?

BACKGROUND: In this proof-of-concept paper, we investigated whether combination treatment with progesterone (P4) and chloroquine (CQ) would reduce ischemic injury more effectively than either agent alone in a transient middle cerebral artery occlusion (tMCAO) model in male rats. METHODS: P4 (8 mg/kg) and CQ (25 mg/kg) were given alone or in combination beginning at different times during surgery and for 3 days post-occlusion. Locomotor activity and grip strength were evaluated as measures of impairment and recovery. Infarct size was assessed by TTC staining. Markers of autophagy (LC3 and SQSTM1/p62) and apoptosis (Bcl-2 and Bax) were evaluated with western blotting. RESULTS: At the doses we employed, the combination was not more effective than either drug given separately on measures of grip strength or locomotor activity. However, combination therapy substantially reduced infarct size, and significantly increased Bcl-2 protein levels and suppressed Bax expression. Progesterone decreased the expression of LC3-II 24 h and SQSTM1/p62 after ischemia. CONCLUSIONS: Our findings suggest that combination therapy with P4 and CQ is not detrimental and has a small-to-moderate additive neuroprotective effect on ischemic injury in rats without substantively affecting behavioral outcomes. CQ and P4 may help to regulate the expression of both autophagy-related and apoptosis-related proteins.

Vitamin D deficiency increases blood-brain barrier dysfunction after ischemic stroke in male rats.

Because vitamin D hormone deficiency (VDHdef) can worsen severity and outcome for ischemic stroke, we examined the role of VDH in maintaining blood-brain-barrier (BBB integrity) in a rat model of stroke. In most types of stroke, the BBB is markedly compromised, potentially leading to a cascade of injury processes and functional deficits, so we examined a number of biomarkers associated with BBB disruption to determine whether VDH deficiency would further compromise the BBB following a stroke. Male Wistar rats were randomly assigned to one of two diet cohorts, VDH-sufficient (VDHsuf) and VDHdef. The VDHsuf group was fed standard rat chow and the VDHdef group got a VDH-null version of the same diet for 8 weeks. Animals from both cohorts were subjected to transient middle cerebral artery occlusion (tMCAO) surgery, killed at 72 h post-stroke, and their brains evaluated for BBB permeability and injury severity using expression of immunoglobulin (IgG), matrix metalloproteinase-9 (MMP-9) activity and alteration of tight junction (TJ) proteins as markers of BBB disruption. We also evaluated modulation of glucose transporter-1 (GLUT1), osteopontin (OPN), ß-catenin and vitamin D receptor (VDR) expression in VDHsuf and VDHdef subjects. At the time of MCAO, rats on the VDHdef diet had circulating VDH levels one-fourth that of rats fed control chow. IgG extravasation after MCAO, indicating more severe BBB injury, was significantly higher in the MCAO+VDHdef than the MCAO+VDHsuf rats. Following MCAO, expression of MMP-9, GLUT1, VDR and OPN increased and the TJ proteins occludin and claudin-5 decreased significantly in the VDHdef compared to the VDHsuf group. We also observed significantly lower expression of ß-catenin in the MCAO group of both VDHsuf and VDHdef rats. Under these conditions, VDH deficiency itself can compromise the BBB. We think that low serum VDH levels are likely to complicate stroke severity and its chronic consequences.

Development of a novel progesterone analog in the treatment of traumatic brain injury.

Although systemic progesterone (PROG) treatment has been shown to be neuroprotective by many laboratories and in multiple animal models of brain injury including traumatic brain injury (TBI), PROG's poor aqueous solubility limits its potential for use as a therapeutic agent. The problem of solubility presents challenges for an acute intervention for neural injury, when getting a neuroprotectant to the brain quickly is crucial. Native PROG (nPROG) is hydrophobic and does not readily dissolve in an aqueous-based medium, so this makes it harder to give under emergency field conditions. An agent with properties similar to those of PROG but easier to store, transport, formulate, and administer early in emergency trauma situations could lead to better and more consistent clinical outcomes following TBI. At the same time, the engineering of a new molecule designed to treat a complex systemic injury must anticipate a range of translational issues including solubility and bioavailability. Here we describe the development of EIDD-1723, a novel, highly stable PROG analog with >104-fold higher aqueous solubility than that of nPROG. We think that, with further testing, EIDD-1723 could become an attractive candidate use as a field-ready treatment for TBI patients. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

Repurposing and repositioning neurosteroids in the treatment of traumatic brain injury: A report from the trenches.

The field of neuroprotection after brain injuries has been littered with failed clinical trials. Finding a safe and effective treatment for acute traumatic brain injury remains a serious unmet medical need. Repurposing drugs that have been in use for other disorders is receiving increasing attention as a strategy to move candidate drugs more quickly to trial while reducing the very high cost of new drug development. This paper describes our own serendipitous discovery of progesterone's neuroprotective potential, and the strategies we are using in repurposing and developing this hormone for use in brain injuries-applications very different from its classical uses in treating disorders of the reproductive system. We have been screening and testing a novel analog that maintains progesterone's therapeutic properties while overcoming its physiochemical challenges, and testing progesterone in combination treatment with another pleiotropic hormone, vitamin D. Finally, our paper, in the context of the problems and pitfalls we have encountered, surveys some of the factors we found to be critical in the clinical translation of repurposed drugs. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Effect of Progesterone on Cerebral Vasospasm and Neurobehavioral Outcomes in a Rodent Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) induces widespread inflammation leading to cellular injury, vasospasm, and ischemia. Evidence suggests that progesterone (PROG) can improve functional recovery in acute brain injury owing to its anti-inflammatory and neuroprotective properties, which could also be beneficial in SAH. We hypothesized that PROG treatment attenuates inflammation-mediated cerebral vasospasm and microglial activation, improves synaptic connectivity, and ameliorates functional recovery after SAH. METHODS: We investigated the effect of PROG in a cisternal SAH model in adult male C57BL/6 mice. Neurobehavioral outcomes were evaluated using rotarod latency and grip strength tests. Basilar artery perimeter, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor 1 (GluR1)/synaptophysin colocalization, and Iba-1 immunoreactivity were quantified histologically. RESULTS: PROG (8 mg/kg) significantly improved rotarod latency at day 6 and grip strength at day 9. PROG-treated mice had significantly reduced basilar artery vasospasm at 24 hours. GluR1/synaptophysin colocalization, indicative of synaptic GluR1, was significantly reduced in the SAH+Vehicle group at 24 hours, and PROG treatment significantly attenuated this reduction. PROG treatment significantly reduced microglial cell activation and proliferation in cerebellum and cortex but not in the brainstem at 10 days. CONCLUSIONS: PROG treatment ameliorated cerebral vasospasm, reduced microglial activation, restored synaptic GluR1 localization, and improved neurobehavioral performance in a murine model of SAH. These results provide a rationale for further translational testing of PROG therapy in SAH.

Stress primes microglial polarization after global ischemia: Therapeutic potential of progesterone.

Despite the fact that stress is associated with increased risk of stroke and worsened outcome, most preclinical studies have ignored this comorbid factor, especially in the context of testing neuroprotective treatments. Preclinical research suggests that stress primes microglia, resulting in an enhanced reactivity to a subsequent insult and potentially increasing vulnerability to stroke. Ischemia-induced activated microglia can be polarized into a harmful phenotype, M1, which produces pro-inflammatory cytokines, or a protective phenotype, M2, which releases anti-inflammatory cytokines and neurotrophic factors. Selective modulation of microglial polarization by inhibiting M1 or stimulating M2 may be a potential therapeutic strategy for treating cerebral ischemia. Our laboratory and others have shown progesterone to be neuroprotective against ischemic stroke in rodents, but it is not known whether it will be as effective under a comorbid condition of chronic stress. Here we evaluated the neuroprotective effect of progesterone on the inflammatory response in the hippocampus after exposure to stress followed by global ischemia. We focused on the effects of microglial M1/M2 polarization and pro- and anti-inflammatory mediators in stressed ischemic animals. Male Sprague-Dawley rats were exposed to 8 consecutive days of social defeat stress and then subjected to global ischemia or sham surgery. The rats received intraperitoneal injections of progesterone (8mg/kg) or vehicle at 2h post-ischemia followed by subcutaneous injections at 6h and once every 24h post-injury for 7days. The animals were killed at 7 and 14days post-ischemia, and brains were removed and processed to assess outcome measures using histological, immunohistochemical and molecular biology techniques. Pre-ischemic stress (1) exacerbated neuronal loss and neurodegeneration as well as microglial activation in the selectively vulnerable CA1 hippocampal region, (2) dysregulated microglial polarization, leading to upregulation of both M1 and M2 phenotype markers, (3) increased pro-inflammatory cytokine expression, and (4) reduced anti-inflammatory cytokine and neurotrophic factor expression in the ischemic hippocampus. Treatment with progesterone significantly attenuated stress-induced microglia priming by modulating polarized microglia and the inflammatory environment in the hippocampus, the area most vulnerable to ischemic injury. Our findings can be taken to suggest that progesterone holds potential as a candidate for clinical testing in ischemic stroke where high stress may be a contributing factor.

Intralipid Vehicle Does Not Interfere with the Efficacy of Progesterone in Attenuating Edema following Traumatic Brain Injury.

The recent disappointing results of phase III trials for progesterone (PROG) in traumatic brain injury (TBI) have triggered speculation about reasons for the negative outcomes. One confounding factor may have been the vehicle used to administer PROG. Virtually all of the many pre-clinical experiments informing the clinical trials and reporting beneficial PROG effects used more soluble 2-hydroxypropyl-b-cyclodextrin as a vehicle given intraperitoneally or subcutaneously rather than a lipid formulation given intravenously (IV). The present investigation compared the effect of PROG infusion with that of lipid emulsion (Intralipid®) as a carrier/vehicle on edema following TBI in rats. Eight-mg/kg doses of PROG with 20% Intralipid were given IV via central venous catheter beginning 1 h post-injury over a 1 h duration (1.2 mL/h). Animals were killed and brains removed at 24 h post-injury. All the brain-injured groups showed more edema compared with the control group. However, PROG+Intralipid significantly attenuated cerebral swelling compared with Intralipid alone. No difference was observed between the TBI-alone and Intralipid groups. Although this study used much a smaller volume and shorter duration of Intralipid infusion than the clinical trials (up to 5 days of continuous infusion), our results suggest that the use of Intralipid in rats did not prevent or mask the beneficial effect of PROG.