Treating amyotrophic lateral sclerosis with allogeneic Schwann cell-derived exosomal vesicles: a case report.

Schwann cells are essential for the maintenance and function of motor neurons, axonal networks, and the neuromuscular junction. In amyotrophic lateral sclerosis, where motor neuron function is progressively lost, Schwann cell function may also be impaired. Recently, important signaling and potential trophic activities of Schwann cell-derived exosomal vesicles have been reported. This case report describes the treatment of a patient with advanced amyotrophic lateral sclerosis using serial intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles, marking, to our knowledge, the first instance of such treatment. An 81-year-old male patient presented with a 1.5-year history of rapidly progressive amyotrophic lateral sclerosis. After initial diagnosis, the patient underwent a combination of generic riluzole, sodium phenylbutyrate for the treatment of amyotrophic lateral sclerosis, and taurursodiol. The patient volunteered to participate in an FDA-approved single-patient expanded access treatment and received weekly intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles to potentially restore impaired Schwann cell and motor neuron function. We confirmed that cultured Schwann cells obtained from the amyotrophic lateral sclerosis patient via sural nerve biopsy appeared impaired (senescent) and that exposure of the patient's Schwann cells to allogeneic Schwann cell-derived exosomal vesicles, cultured expanded from a cadaver donor improved their growth capacity in vitro. After a period of observation lasting 10 weeks, during which amyotrophic lateral sclerosis Functional Rating Scale-Revised and pulmonary function were regularly monitored, the patient received weekly consecutive infusions of 1.54 × 10 12 (×2), and then consecutive infusions of 7.5 × 10 12 (×6) allogeneic Schwann cell-derived exosomal vesicles diluted in 40 mL of Dulbecco's phosphate-buffered saline. None of the infusions were associated with adverse events such as infusion reactions (allergic or otherwise) or changes in vital signs. Clinical lab serum neurofilament and cytokine levels measured prior to each infusion varied somewhat without a clear trend. A more sensitive in-house assay suggested possible inflammasome activation during the disease course. A trend for clinical stabilization was observed during the infusion period. Our study provides a novel approach to address impaired Schwann cells and possibly motor neuron function in patients with amyotrophic lateral sclerosis using allogeneic Schwann cell-derived exosomal vesicles. Initial findings suggest that this approach is safe.

Improvement in edema and cognitive recovery after moderate traumatic brain injury with the neurosteroid prodrug NTS-104.

Neuroactive steroids reduce mortality, decrease edema, and improve functional outcomes in preclinical and clinical traumatic brain injury (TBI) studies. In this study, we tested the efficacy of two related novel neuroactive steroids, NTS-104 and NTS-105, in a rat model of TBI. NTS-104 is a water-soluble prodrug of NTS-105, a partial progesterone receptor agonist. To investigate the effects of NTS-104 on TBI recovery, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion injury or sham surgery and were treated with vehicle or NTS-104 (10 â€‹mg/kg, intramuscularly) at 4, 10, 24, and 48 â€‹h post-TBI. The therapeutic time window was also assessed using the neuroactive steroid NTS-105 (3 â€‹mg/kg, intramuscularly). Edema in the parietal cortex and hippocampus, measured at 3 days post-injury (DPI), was reduced by NTS-104 and NTS-105. NTS-105 was effective in reducing edema when given at 4, 10, or 24 â€‹h post-injury. Sensorimotor deficits in the cylinder test at 3 DPI were ameliorated by NTS-104 and NTS-105 treatment. Cognitive recovery, assessed with cue and contextual fear conditioning and retention of the water maze task assessed subacutely 1-3 weeks post-injury, also improved with NTS-104 treatment. Cortical and hippocampal atrophy at 22 DPI did not improve, indicating that NTS-104/NTS-105 may promote post-TBI cognitive recovery by controlling edema and other processes. These results demonstrate that NTS-104/NTS-105 is a promising therapeutic approach to improve motor and cognitive recovery after moderate TBI.

Inflammasome Proteins Are Reliable Biomarkers of the Inflammatory Response in Aneurysmal Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is caused by abnormal blood vessel dilation and subsequent rupture, resulting in blood pooling in the subarachnoid space. This neurological insult results in the activation of the inflammasome, a multiprotein complex that processes pro-inflammatory interleukin (IL)-1 cytokines leading to morbidity and mortality. Moreover, increases in inflammasome proteins are associated with clinical deterioration in many neurological diseases. Limited studies have investigated inflammasome protein expression following aSAH. Reliable markers of the inflammatory response associated with aSAH may allow for earlier detection of patients at risk for complications and aid in the identification of novel pharmacologic targets. Here, we investigated whether inflammasome signaling proteins may serve as potential biomarkers of the inflammatory response in aSAH. Serum and cerebrospinal fluid (CSF) from fifteen aSAH subjects and healthy age-matched controls and hydrocephalus (CSF) no-aneurysm controls were evaluated for levels of inflammasome signaling proteins and downstream pro-inflammatory cytokines. Protein measurements were carried out using Simple Plex and Single-Molecule Array (Simoa) technology. The area under the curve (AUC) was calculated using receiver operating characteristics (ROCs) to obtain information on biomarker reliability, specificity, sensitivity, cut-off points, and likelihood ratio. In addition, a Spearman r correlation matrix was performed to determine the correlation between inflammasome protein levels and clinical outcome measures. aSAH subjects demonstrated elevated caspase-1, apoptosis-associated speck-like protein with a caspase recruiting domain (ASC), IL-18 and IL-1ß levels in serum, and CSF when compared to controls. Each of these proteins was found to be a promising biomarker of inflammation in aSAH in the CSF. In addition, ASC, caspase-1, and IL-1ß were found to be promising biomarkers of inflammation in aSAH in serum. Furthermore, we found that elevated levels of inflammasome proteins in serum are useful to predict worse functional outcomes following aSAH. Thus, the determination of inflammasome protein levels in CSF and serum in aSAH may be utilized as reliable biomarkers of inflammation in aSAH and used clinically to monitor patient outcomes.

The Inflammasome Adaptor Protein ASC in Plasma as a Biomarker of Early Cognitive Changes.

Dementia is a group of symptoms including memory loss, language difficulties, and other types of cognitive and functional impairments that affects 57 million people worldwide, with the incidence expected to double by 2040. Therefore, there is an unmet need to develop reliable biomarkers to diagnose early brain impairments so that emerging interventions can be applied before brain degeneration. Here, we performed biomarker analyses for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and amyloid-ß 42/40 (Aß42/40) ratio in the plasma of older adults. Participants had blood drawn at baseline and underwent two annual clinical and cognitive evaluations. The groups tested either cognitively normal on both evaluations (NN), cognitively normal year 1 but cognitively impaired year 2 (NI), or cognitively impaired on both evaluations (II). ASC was elevated in the plasma of the NI group compared to the NN and II groups. Additionally, Aß42 was increased in the plasma in the NI and II groups compared to the NN group. Importantly, the area under the curve (AUC) for ASC in participants older than 70 years old in NN vs. NI groups was 0.81, indicating that ASC is a promising plasma biomarker for early detection of cognitive decline.

Extracellular vesicles mediate inflammasome signaling in the brain and heart of Alzheimer's disease mice.

Introduction: Alzheimer's disease (AD) is an inflammatory neurodegenerative disease characterized by memory loss and cognitive impairment that worsens over time. AD is associated with many comorbidities, including cardiovascular disease that are associated with poorer outcomes. Comorbidities, especially heart disease and stroke, play a significant role in the demise of AD patients. Thus, it is important to understand how comorbidities are linked to AD. We have previously shown that extracellular vesicle (EV)-mediated inflammasome signaling plays an important role in the pathogenesis of brain injury and acute lung injury after traumatic brain injury. Methods: We analyzed the cortical, hippocampal, ventricular, and atrial protein lysates from APP/PS1 mice and their respective controls for inflammasome signaling activation. Additionally, we analyzed serum-derived EV for size, concentration, and content of inflammasome proteins as well as the EV marker CD63. Finally, we performed conditioned media experiments of EV from AD patients and healthy age-matched controls delivered to cardiovascular cells in culture to assess EV-induced inflammation. Results: We show a significant increase in Pyrin, NLRP1, caspase-1, and ASC in the brain cortex whereas caspase-8, ASC, and IL-1ß were significantly elevated in the heart ventricles of AD mice when compared to controls. We did not find significant differences in the size or concentration of EV between groups, but there was a significant increase of caspase-1 and IL-1ß in EV from AD mice compared to controls. In addition, conditioned media experiments of serum-derived EV from AD patients and age-matched controls delivered to cardiovascular cells in culture resulted in inflammasome activation, and significant increases in TNF-α and IL-2. Conclusion: These results indicate that EV-mediated inflammasome signaling in the heart may play a role in the development of cardiovascular diseases in AD patients.

Beneficial Effects of Human Schwann Cell-Derived Exosomes in Mitigating Secondary Damage After Penetrating Ballistic-Like Brain Injury.

There is a growing body of evidence that the delivery of cell-derived exosomes normally involved in intracellular communication can reduce secondary injury mechanisms after brain and spinal cord injury and improve outcomes. Exosomes are nanometer-sized vesicles that are released by Schwann cells and may have neuroprotective effects by reducing post-traumatic inflammatory processes as well as promoting tissue healing and functional recovery. The purpose of this study was to evaluate the beneficial effects of human Schwann-cell exosomes (hSC-Exos) in a severe model of penetrating ballistic-like brain injury (PBBI) in rats and investigate effects on multiple outcomes. Human Schwann cell processing protocols followed Current Good Manufacturing Practices (cGMP) with exosome extraction and purification steps approved by the Food and Drug Administration for an expanded access single ALS patient Investigational New Drug. Anesthetized male Sprague-Dawley rats (280-350g) underwent PBBI surgery or Sham procedures and, starting 30 min after injury, received either a dose of hSC-Exos or phosphate-buffered saline through the jugular vein. At 48h after PBBI, flow cytometry analysis of cortical tissue revealed that hSC-Exos administration reduced the number of activated microglia and levels of caspase-1, a marker of inflammasome activation. Neuropathological analysis at 21 days showed that hSC-Exos treatment after PBBI significantly reduced overall contusion volume and decreased the frequency of Iba-1 positive activated and amoeboid microglia by immunocytochemical analysis. This study revealed that the systemic administration of hSC-Exos is neuroprotective in a model of severe TBI and reduces secondary inflammatory injury mechanisms and histopathological damage. The administration of hSC-Exos represents a clinically relevant cell-based therapy to limit the detrimental effects of neurotrauma or other progressive neurological injuries by impacting multiple pathophysiological events and promoting neurological recovery.

Association of region-specific hippocampal reduction of neurogranin with inflammasome proteins in post mortem brains of Alzheimer's disease.

INTRODUCTION: Neurogranin (Ng) is considered a biomarker for synaptic dysfunction in Alzheimer's disease (AD). In contrast, the inflammasome complex has been shown to exacerbate AD pathology. METHODS: We investigated the protein expression, morphological differences of Ng, and correlated Ng to hyperphosphorylated tau in the post mortem brains of 17 AD cases and 17 age- and sex-matched controls. In addition, we correlated the Ng expression with two different epitopes of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). RESULTS: We show a reduction of Ng immunopositive neurons and morphological differences in AD compared to controls. Ng immunostaining was negatively correlated with neurofibrillary tangles, humanized anti-ASC (IC100) positive neurons and anti-ASC positive microglia, in AD. DISCUSSION: The finding of a negative correlation between Ng and ASC speck protein expression in post mortem brains of AD suggests that the activation of inflammasome/ASC speck pathway may play an important role in synaptic degeneration in AD. Highlights: We show the role that neurogranin plays on post-synaptic signaling in specific hippocampal regions.We demonstrate that there could be clinical implications of using neurogranin as a biomarker for dementia.We describe the loss of plasticity and neuronal scaffolding proteins in the present of AD pathology.We show the response of neuroinflammation when tau proteins phosphorylate in hippocampal neurons.We show that there is a potential therapeutic target for the inflammasome, and future studies may show that IC100, a humanized monoclonal antibody directed against ASC, may slow the progression of neurodegeneration.

Covert Tracking to Immersive Stimuli in Traumatic Brain Injury Subjects With Disorders of Consciousness.

Eye tracking assessments are clinician dependent and can contribute to misclassification of coma. We investigated responsiveness to videos with and without audio in traumatic brain injury (TBI) subjects using video eye-tracking (VET). We recruited 20 healthy volunteers and 10 unresponsive TBI subjects. Clinicians were surveyed whether the subject was tracking on their bedside assessment. The Coma Recovery Scale-Revised (CRS-R) was also performed. Eye movements in response to three different 30-second videos with and without sound were recorded using VET. The videos consisted of moving characters (a dancer, a person skateboarding, and Spiderman). Tracking on VET was defined as visual fixation on the character and gaze movement in the same direction of the character on two separate occasions. Subjects were classified as "covert tracking" (tracking using VET only), "overt tracking" (VET and clinical exam by clinicians), and "no tracking". A k-nearest-neighbors model was also used to identify tracking computationally. Thalamocortical connectivity and structural integrity were evaluated with EEG and MRI. The ability to obey commands was evaluated at 6- and 12-month follow-up. The average age was 29 (± 17) years old. Three subjects demonstrated "covert tracking" (CRS-R of 6, 8, 7), two "overt tracking" (CRS-R 22, 11), and five subjects "no tracking" (CRS-R 8, 6, 5, 6, 7). Among the 84 tested trials in all subjects, 11 trials (13%) met the criteria for "covert tracking". Using the k-nearest approach, 14 trials (17%) were classified as "covert tracking". Subjects with "tracking" had higher thalamocortical connectivity, and had fewer structures injured in the eye-tracking network than those without tracking. At follow-up, 2 out of 3 "covert" and all "overt" subjects recovered consciousness versus only 2 subjects in the "no tracking" group. Immersive stimuli may serve as important objective tools to differentiate subtle tracking using VET.

Prognostic and Diagnostic Utility of Serum Biomarkers in Pediatric Traumatic Brain Injury.

Traumatic brain injury (TBI) remains a major cause of morbidity and death among the pediatric population. Timely diagnosis, however, remains a complex task because of the lack of standardized methods that permit its accurate identification. The aim of this study was to determine whether serum levels of brain injury biomarkers can be used as a diagnostic and prognostic tool in this pathology. This prospective, observational study collected and analyzed the serum concentration of neuronal injury biomarkers at enrollment, 24h and 48h post-injury, in 34 children ages 0-18 with pTBI and 19 healthy controls (HC). Biomarkers included glial fibrillary acidic protein (GFAP), neurofilament protein L (NfL), ubiquitin-C-terminal hydrolase (UCH-L1), S-100B, tau and tau phosphorylated at threonine 181 (p-tau181). Subjects were stratified by admission Glasgow Coma Scale score into two categories: a combined mild/moderate (GCS 9-15) and severe (GCS 3-8). Glasgow Outcome Scale-Extended (GOS-E) Peds was dichotomized into favorable (≤4) and unfavorable (≥5) and outcomes. Data were analyzed utilizing Prism 9 and R statistical software. The findings were as follows: 15 patients were stratified as severe TBI and 19 as mild/moderate per GCS. All biomarkers measured at enrollment were elevated compared with HC. Serum levels for all biomarkers were significantly higher in the severe TBI group compared with HC at 0, 24, and 48h. The GFAP, tau S100B, and p-tau181 had the ability to differentiate TBI severity in the mild/moderate group when measured at 0h post-injury. Tau serum levels were increased in the mild/moderate group at 24h. In addition, NfL and p-tau181 showed increased serum levels at 48h in the aforementioned GCS category. Individual biomarker performance on predicting unfavorable outcomes was measured at 0, 24, and 48h across different GOS-E Peds time points, which was significant for p-tau181 at 0h at all time points, UCH-L1 at 0h at 6-9 months and 12 months, GFAP at 48h at 12 months, NfL at 0h at 12 months, tau at 0h at 12 months and S100B at 0h at 12 months. We concluded that TBI leads to increased serum neuronal injury biomarkers during the first 0-48h post-injury. A biomarker panel measuring these proteins could aid in the early diagnosis of mild to moderate pTBI and may predict neurological outcomes across the injury spectrum.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Multimodal magnetic resonance imaging after experimental moderate and severe traumatic brain injury: A longitudinal correlative assessment of structural and cerebral blood flow changes.

Traumatic brain injury (TBI) is a worldwide problem that results in death or disability for millions of people every year. Progressive neurological complications and long-term impairment can significantly disrupt quality of life. We demonstrated the feasibility of multiple magnetic resonance imaging (MRI) modalities to investigate and predict aberrant changes and progressive atrophy of gray and white matter tissue at several acute and chronic time points after moderate and severe parasagittal fluid percussion TBI. T2-weighted imaging, diffusion tensor imaging (DTI), and perfusion weighted imaging (PWI) were performed. Adult Sprague-Dawley rats were imaged sequentially on days 3, 14, and 1, 4, 6, 8, and 12 months following surgery. TBI caused dynamic white and gray matter alterations with significant differences in DTI values and injury-induced alterations in cerebral blood flow (CBF) as measured by PWI. Regional abnormalities after TBI were observed in T2-weighted images that showed hyperintense cortical lesions and significant cerebral atrophy in these hyperintense areas 1 year after TBI. Temporal DTI values indicated significant injury-induced changes in anisotropy in major white matter tracts, the corpus callosum and external capsule, and in gray matter, the hippocampus and cortex, at both early and chronic time points. These alterations were primarily injury-severity dependent with severe TBI exhibiting a greater degree of change relative to uninjured controls. PWI evaluating CBF revealed sustained global reductions in the cortex and in the hippocampus at most time points in an injury-independent manner. We next sought to investigate prognostic correlations across MRI metrics, timepoints, and cerebral pathology, and found that diffusion abnormalities and reductions in CBF significantly correlated with specific vulnerable structures at multiple time points, as well as with the degree of cerebral atrophy observed 1 year after TBI. This study further supports using DTI and PWI as a means of prognostic imaging for progressive structural changes after TBI and emphasizes the progressive nature of TBI damage.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

Background: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found inhibition of GSDMD activation attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. Methods: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal day 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1), a marker of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. Results: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure failed to increase either AIF1+ or TUNEL+ cell numbers, nor decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air- exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, and core development pathways hypoxia-induced factor 1, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. Conclusion: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Increasing Rigor of Preclinical Research to Maximize Opportunities for Translation.

The use of animal models in pre-clinical research has significantly broadened our understanding of the pathologies that underlie traumatic brain injury (TBI)-induced damage and deficits. However, despite numerous pre-clinical studies reporting the identification of promising neurotherapeutics, translation of these therapies to clinical application has so far eluded the TBI research field. A concerted effort to address this lack of translatability is long overdue. Given the inherent heterogeneity of TBI and the replication crisis that continues to plague biomedical research, this is a complex task that will require a multifaceted approach centered around rigor and reproducibility. Here, we discuss the role of three primary focus areas for better aligning pre-clinical research with clinical TBI management. These focus areas are (1) reporting and standardization of protocols, (2) replication of prior knowledge including the confirmation of expected pharmacodynamics, and (3) the broad application of open science through inter-center collaboration and data sharing. We further discuss current efforts that are establishing the core framework needed for successfully addressing the translatability crisis of TBI.

Dose-dependent modulation of microglia activation in rats after penetrating traumatic brain injury (pTBI) by transplanted human neural stem cells.

Traumatic brain injury (TBI) often results in long-lasting patterns of neurological deficits including motor, sensory, and cognitive abnormalities. Cranial gunshot survivors are among the most disabled TBI patients and face a lifetime of disability with no approved strategies to protect or repair the brain after injury. Recent studies using a model of penetrating TBI (pTBI) have reported that human neural stem cells (hNSCs) transplantation can lead to dose and location-dependent neuroprotection. Evidence for regional patterns of microglial activation has also been reported after pTBI with evidence for microglial cell death by pyroptosis. Because of the importance of injury-induced microglial activation in the pathogenesis of TBI, we tested the hypothesis that dose-dependent hNSC mediated neuroprotection after pTBI was associated with reduced microglial activation in pericontusional cortical areas. To test this hypothesis, quantitative microglial/macrophage Iba1 immunohistochemistry and Sholl analysis was conducted to investigate the arborization patterns using four experimental groups including, (i) Sham operated (no injury) + low dose (0.16 million cells/rat), (ii) pTBI + vehicle (no cells), (iii) pTBI + low dose hNSCs (0.16 million/rat), and (iv) pTBI + high dose hNSCs (1.6 million cells/rat). At 3 months post-transplantation (transplants at one week after pTBI), the total number of intersections was significantly reduced in vehicle treated pTBI animals versus sham operated controls indicating increased microglia/macrophage activation. In contrast, hNSC transplantation led to a dose-dependent increase in the number of intersections compared to pTBI vehicle indicating less microglia/macrophage activation. The peak of Sholl intersections at 1 µm from the center of the microglia/macrophages ranged from ~6,500-14,000 intersections for sham operated, ~250-500 intersections for pTBI vehicle, ~550-1,000 intersections for pTBI low dose, and ~2,500-7,500 intersections for pTBI high dose. Plotting data along the rostrocaudal axis also showed that pericontusional cortical areas protected by hNSC transplantation had increased intersections compared to nontreated pTBI animals. These studies using a non-biased Sholl analysis demonstrated a dose-dependent reduction in inflammatory cell activation that may be associated with a neuroprotective effect driven by the cellular transplant in perilesional regions after pTBI.

Association between Cerebrospinal Fluid and Serum Biomarker Levels and Diagnosis, Injury Severity, and Short-Term Outcomes in Patients with Acute Traumatic Spinal Cord Injury.

Acute traumatic spinal cord injury (SCI) is recognized as a global problem that can lead to a range of acute and secondary complications impacting morbidity and mortality. There is still a lack of reliable diagnostic and prognostic biomarkers in patients with SCI that could help guide clinical care and identify novel therapeutic targets for future drug discovery. The aim of this prospective controlled study was to determine the cerebral spinal fluid (CSF) and serum profiles of 10 biomarkers as indicators of SCI diagnosis, severity, and prognosis to aid in assessing appropriate treatment modalities. CSF and serum samples of 15 SCI and ten healthy participants were included in the study. The neurological assessments were scored on admission and at discharge from the hospital using the American Spinal Injury Association Impairment Score (AIS) grades. The CSF and serum concentrations of SBDP150, S100B, GFAP, NF-L, UCHL-1, Tau, and IL-6 were significantly higher in SCI patients when compared with the control group. The CSF GBDP 38/44K, UCHL-L1, S100B, GFAP, and Tau levels were significantly higher in the AIS A patients. This study demonstrated a strong correlation between biomarker levels in the diagnosis and injury severity of SCI but no association with short-term outcomes. Future prospective controlled studies need to be done to support the results of this study.

Activation of inflammasomes and their effects on neuroinflammation at the microelectrode-tissue interface in intracortical implants.

Invasive neuroprosthetics rely on microelectrodes (MEs) to record or stimulate the activity of large neuron assemblies. However, MEs are subjected to tissue reactivity in the central nervous system (CNS) due to the foreign body response (FBR) that contribute to chronic neuroinflammation and ultimately result in ME failure. An endogenous, acute set of mechanisms responsible for the recognition and targeting of foreign objects, called the innate immune response, immediately follows the ME implant-induced trauma. Inflammasomes are multiprotein structures that play a critical role in the initiation of an innate immune response following CNS injuries. The activation of inflammasomes facilitates a range of innate immune response cascades and results in neuroinflammation and programmed cell death. Despite our current understanding of inflammasomes, their roles in the context of neural device implantation remain unknown. In this study, we implanted a non-functional Utah electrode array (UEA) into the rat somatosensory cortex and studied the inflammasome signaling and the corresponding downstream effects on inflammatory cytokine expression and the inflammasome-mediated cell death mechanism of pyroptosis. Our results not only demonstrate the continuous activation of inflammasomes and their contribution to neuroinflammation at the electrode-tissue interface but also reveal the therapeutic potential of targeting inflammasomes to attenuate the FBR in invasive neuroprosthetics.

Covert Tracking to Visual Stimuli in Comatose Patients With Traumatic Brain Injury.

OBJECTIVES: This study investigated video eye tracking (VET) in comatose patients with traumatic brain injury (TBI). METHODS: We recruited healthy participants and unresponsive patients with TBI. We surveyed the patients' clinicians on whether the patient was tracking and performed the Coma Recovery Scale-Revised (CRS-R). We recorded eye movements in response to motion of a finger, a face, a mirror, and an optokinetic stimulus using VET glasses. Patients were classified as covert tracking (tracking on VET alone) and overt tracking (VET and clinical examination). The ability to obey commands was evaluated at 6-month follow-up. RESULTS: We recruited 20 healthy participants and 10 patients with TBI. The use of VET was feasible in all participants and patients. Two patients demonstrated covert tracking (CRS-R of 6 and 8), 2 demonstrated overt tracking (CRS-R of 22 and 11), and 6 patients had no tracking (CRS-R of 8, 6, 5, 7, 6, and 7). Five of 56 (9%) tracking assessments were missed on clinical examination. All patients with tracking recovered consciousness at follow-up, whereas only 2 of 6 patients without tracking recovered at follow-up. DISCUSSION: VET is a feasible method to measure covert tracking. Future studies are needed to confirm the prognostic value of covert tracking.

Genetic predisposition to Alzheimer's disease alters inflammasome activity after traumatic brain injury.

Traumatic Brain Injury (TBI) is a major cause of death and disability in the US and a recognized risk factor for the development of Alzheimer's disease (AD). The relationship between these conditions is not completely understood, but the conditions may share additive or synergistic pathological hallmarks that may serve as novel therapeutic targets. Heightened inflammasome signaling plays a critical role in the pathogenesis of central nervous system injury (CNS) and the release of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck from neurons and activated microglia contribute significantly to TBI and AD pathology. This study investigated whether inflammasome signaling after TBI was augmented in AD and whether this signaling pathway impacted biochemical and neuropathological outcomes and overall cognitive function. Five-month-old, 3xTg mice and respective wild type controls were randomized and underwent moderate controlled cortical impact (CCI) injury or served as sham/uninjured controls. Animals were sacrificed at 1 hour, 1 day, or 1 week after TBI to assess acute pathology or at 12 weeks after assessing cognitive function. The ipsilateral cerebral cortex was processed for inflammasome protein expression by immunoblotting. Mice were evaluated for behavior by open field (3 days), novel object recognition (2 weeks), and Morris water maze (6 weeks) testing after TBI. There was a statistically significant increase in the expression of inflammasome signaling proteins Caspase-1, Caspase-8, ASC, and interleukin (IL)-1ß after TBI in both wild type and 3xTg animals. At 1-day post injury, significant increases in ASC and IL-1ß protein expression were measured in AD TBI mice compared to WT TBI. Behavioral testing showed that injured AD mice had altered cognitive function when compared to injured WT mice. Elevated Aß was seen in the ipsilateral cortex and hippocampus of sham and injured AD when compared to respective groups at 12 weeks post injury. Moreover, treatment of injured AD mice with IC100, an anti-ASC monoclonal antibody, inhibited the inflammasome, as evidenced by IL-1ß reduction in the injured cortex at 1-week post injury. These findings show that the inflammasome response is heightened in mice genetically predisposed to AD and suggests that AD may exacerbate TBI pathology. Thus, dampening inflammasome signaling may offer a novel approach for the treatment of AD and TBI.

Inflammasome activation in traumatic brain injury and Alzheimer's disease.

Traumatic brain injury (TBI) and Alzheimer's disease (AD) represent 2 of the largest sources of death and disability in the United States. Recent studies have identified TBI as a potential risk factor for AD development, and numerous reports have shown that TBI is linked with AD associated protein expression during the acute phase of injury, suggesting an interplay between the 2 pathologies. The inflammasome is a multi-protein complex that plays a role in both TBI and AD pathologies, and is characterized by inflammatory cytokine release and pyroptotic cell death. Products of inflammasome signaling pathways activate microglia and astrocytes, which attempt to resolve pathological inflammation caused by inflammatory cytokine release and phagocytosis of cellular debris. Although the initial phase of the inflammatory response in the nervous system is beneficial, recent evidence has emerged that the heightened inflammatory response after trauma is self-perpetuating and results in additional damage in the central nervous system. Inflammasome-induced cytokines and inflammasome signaling proteins released from activated microglia interact with AD associated proteins and exacerbate AD pathological progression and cellular damage. Additionally, multiple genetic mutations associated with AD development alter microglia inflammatory activity, increasing and perpetuating inflammatory cell damage. In this review, we discuss the pathologies of TBI and AD and how they are impacted by and potentially interact through inflammasome activity and signaling proteins. We discuss current clinical trials that target the inflammasome to reduce heightened inflammation associated with these disorders.