Robert W. Keane

Pediatric hydrocephalus results from the overproduction, obstruction, or inability to resorb cerebrospinal fluid (CSF), which may lead to ventricular enlargement. Hydrocephalus may be secondary to other pathologies, including intraventricular hemorrhage (IVH). Surgical intervention may include a ventricular access device or a ventriculosubgaleal shunt in premature infants who are too small for permanent CSF diversion. Hydrocephalus, an inflammatory pathology, involves activation of the inflammasome. Elevation in pro-inflammatory proteins is often associated with worsening clinical outcomes in pediatric pathologies. Whether inflammasome proteins are reliable biomarkers for pediatric hydrocephalus has not been established. We analyzed CSF samples via an ELLA assay from pediatric hydrocephalic patients with IVH of prematurity vs. controls and compared changes in inflammasome protein concentrations post-reservoir surgery at different time points. The area under the curve was calculated using receiver operator characteristic curves. The levels of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-18 were significantly elevated in pediatric hydrocephalic patients with IVH vs. controls. Thus, these proteins are reliable biomarkers of the inflammatory response in pediatric hydrocephalus following ROC analysis, with an AUC of 1.0 and high sensitivity and specificity at the corresponding cut-off points. Ongoing work seeks to validate these findings in a larger cohort of pediatric hydrocephalic patients with varying etiologies.

Traumatic brain injury (TBI) is one of the leading causes of death and neurological disability worldwide. The search for biomarkers that indicate TBI severity and prognosis with greater accuracy is ongoing. This study aimed to evaluate the significance of several neuroinflammatory cytokines and chemokines, assessing their potential as biomarkers in pediatric TBI (pTBI). This was an exploratory analysis of inflammatory cytokines and chemokines measured in a subset of 26 children aged 0–18 years with TBI and 21 controls. TBI severity was determined by GCS. The functional outcome was measured via the GOS-E score at 6 weeks and 3, 6, 9, and 12 months post-injury. Serum samples were analyzed for ICAM-1, VCAM-1, SAA, CRP, IFN-g, IL-10, IL-12p70, IL-13, IL-1b, IL-2, IL-4, IL-6, IL-8, TNF-a, TNF-b, eotaxin, eotaxin-3, IP-10, MCP-1, MCP-4, MDC, MIP-1a, MIP-1b and TARC. Levels of IL-6, IL-10, IL-13, IL-16, MDC, and GM-CSF were increased, and IFN-γ, IL-5, IL-8, and eotaxin-3 were decreased at enrollment when compared with controls. Elevated IL-6 and IL-10 at enrollment were associated with severe TBI (AUC of 1, p = 0.0002 and p = 0.8, suggesting biomarker potential. Our data indicate that several cytokines and chemokines measured after TBI may aid in the assessment of pTBI severity and prognosis. IL-6, IL-10, and IL-16 may show potential as biomarkers for pTBI severity and outcomes.

Traumatic brain injury (TBI) pathology is significantly mediated by an inflammatory response involving inflammasome activation, resulting in the release of interleukin (IL)-1β and pyroptotic cell death through gasdermin-D (GSDMD) cleavage. Inflammasome components are transported through extracellular vesicles (EVs) to mediate systemic inflammation in peripheral organs, including the gut. The purpose of this study was to determine the protective effect of GSDMD knockout (KO) on TBI-induced inflammasome activation, EV signaling, and gut function. GSDMD-KO and C57BL6 (WT) mice were subjected to the controlled cortical impact model of TBI. Cytokine expression was assessed with electrochemiluminescent immunoassay and immunoblotting of the cerebral cortex and gut. EVs were examined for pathology-associated markers using flow cytometry, and gut permeability was determined. GSDMD-KO attenuated IL-1β and IL-6 expression in the cerebral cortex and reduced IL-1β and IL-18 in the gut 3 days post-injury. GSDMD-KO mice had decreased neuronal- and gut-derived EVs compared to WT mice post-TBI. GSDMD-KO EVs also had decreased IL-1β and different surface marker expression post-TBI. GSDMD-KO mice had decreased gut permeability after TBI. These data demonstrate that GSDMD ablation improves post-TBI inflammation and gut pathology, suggesting that GSDMD may serve as a potential therapeutic target for the improvement of TBI-associated pathologies.

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.

Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following CNS injury via activation of multiprotein complexes termed inflammasomes that regulate the activation of caspase 1 and the processing of the pro‐inflammatory cytokines IL‐1β and IL‐18. We report here that the expression of components of the nucleotide‐binding and oligomerization domain (NOD)‐like receptor protein‐1 (NLRP‐1) inflammasome, apoptosis speck‐like protein containing a caspase recruitment domain (ASC), and caspase 1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from CSF of SCI and traumatic brain‐injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short‐interfering RNA (siRNA) against ASC and administered to spinal cord‐injured animals. Neuronal‐derived exosomes crossed the injured blood–spinal cord barrier, and delivered their cargo in vivo, resulting in knockdown of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase 1 activation and processing of IL‐1β after SCI. These findings indicate that exosome‐mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury. image We propose the following signaling cascade for inflammasome activation in peripheral tissues after CNS injury: CNS trauma induces inflammasome activation in the nervous system and secretion of exosomes containing inflammasome protein cargo into cerebral spinal fluid. The inflammasome containing exosomes then fuse with target cells to activate the innate immune response in peripheral tissues. We suggest that these findings may be used to develop new therapeutics to treat the devastating inflammation and cell destruction evoked by CNS injuries. IL‐1β and IL‐18 = pro‐inflammatory cytokines.

Background: Members of the mammalian nucleotide binding domain, leucine-rich repeat (LRR)-containing receptor (NLR) family of proteins are key modulators of innate immunity regulating inflammation. Our previous work has shown that among the members of this family, NLRP1/NALP1, present in neurons, plays a crucial role in inflammasome formation and the production of the inflammatory cytokines interleukin (IL) -1β and IL-18 after various types of central nervous system injury.Results: We investigated whether age-related cognitive decline may involve a heightened inflammatory response associated with activation of the NLRP1 inflammasome in the hippocampus. Young (3 months) and aged (18 months) male Fischer 344 rats were tested in a spatial acquisition task via Morris water maze. Following behavioral testing, hippocampal lysates were assayed for expression of NLRP1 inflammasome components and inflammatory cytokines. Hippocampal lysates from aged rats showed significantly higher levels of NLRP1 inflammasome constituents, caspase-1, caspase-11, the purinergic receptor P2X7, pannexin-1 and X-linked inhibitor of apoptosis (XIAP) than lysates from younger animals. Following treatment with probenecid, an inhibitor or pannexin-1, aged animals demonstrated reduction in inflammasome activation and improvement in spatial learning performance.Conclusions: Our behavioral findings are consistent with increases in IL-1β and IL-18 that have been previously shown to correlate with spatial learning deficits. Probenecid reduced activated caspase-1 and ameliorated spatial learning deficits in aged rats. Thus, aging processes stimulate activation of the NLRP1 inflammasome and secretion of IL-1β and IL-18 that may contribute to age-related cognitive decline in the growing elderly population. Moreover, probenecid may be potentially useful as a therapy to improve cognitive outcomes in the aging population.