Biology subjects

Fleiss, B.

Publications and source records attributed to Fleiss, B..

9 recordsLinked to original sources

bioRxiv2026

Validated LC-MS/MS quantification of kynurenine pathway metabolites and pathway-balance ratios in brain tissue and microglial culture supernatant: application to neonatal stroke

Kynurenine pathway (KP) metabolite imbalance is implicated in perinatal brain injury, and KP metabolites are measured in human plasma and cerebrospinal fluid as markers of neuroinflammation. Methods validated in neonatal brain tissue or glial culture supernatant to the same standard are scarce, limiting comparisons across preclinical and clinical measurements. We optimised LC-MS/MS detection of tryptophan (TRYP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), anthranilic acid and picolinic acid after protein precipitation and Enhanced Matrix Removal-Lipid clean-up, validated it against Clinical and Laboratory Standards Institute (CLSI) acceptance criteria in brain tissue homogenate and cell culture supernatant, and applied it to a rat model of neonatal stroke and immune-activated BV2 microglia, reporting KYN/TRYP, KYNA/KYN, 3-HK/3-HAA and QA/KYNA ratios. The lower limit of quantification was 6.53-34.17 nM; imprecision was 0.57-11.47%, accuracy was 84.9-108.3%, recovery was 83.2-112.3%, and matrix effect was 87.8-108.8%, with linear calibration (R2 > 0.99) over 6.5-2000 nM. KYN/TRYP and QA/KYNA ratios were higher in the stroke hemisphere than in control brain at 1, 7 and 14 days (p<0.001), and KYNA/KYN was lower; lipopolysaccharide- and cytokine-stimulated BV2 microglia showed the same change in all four ratios (p<0.01). The assay quantifies eight KP metabolites and four pathway-balance ratios in brain tissue and microglial supernatant within clinical acceptance limits, allowing direct comparison with KP ratios reported in human plasma and cerebrospinal fluid. Changes in these ratios after neonatal stroke identify KYN/TRYP and QA/KYNA as candidate targets for neonatal biofluid studies.

biochemistry↗

bioRxiv2025

A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury

BackgroundNeonatal brain injuries such as stroke cause focal ischemic lesions that often result in lifelong neurological disabilities, as treatment options are limited. To speed up the discovery of potential therapies, early-phase screening with models that reliably reproduce brain injury, with scalable injury volume and minimal confounders, such as varying anaesthesia duration and painful procedures, is essential. MethodsPostnatal day 10 Sprague-Dawley rats of both sexes, with four litters per group and timepoint, were randomly allocated to delivery of intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and 10 minutes of light-emitting diode illumination through the intact scalp and skull. Infarct progression and reproducibility were assessed at 24 hours, 7 days, and 14 days post-injury. Outcomes included infarct volume and sensorimotor function, and cleaved caspase-3, glial fibrillary acidic protein (GFAP), and ionised calcium-binding adaptor molecule 1 (Iba1) immunoreactivity, with analysis of sex differences. Data were analysed using one-way or two-way ANOVA with Sidaks post-hoc tests. ResultsThere was no mortality due to the infarct, and procedure time was approximately 19 minutes across all groups; the lesion was consistent and supported scalability. The 25 mg/kg dose produced a reproducible cortical infarct (3.74 {+/-} 0.58 mm3; CV = 31%). Lesion size increased with dose and decreased over time (11.15 {+/-} 0.63 mm3 at 60 mg/kg versus 0.05 {+/-} 0.007 mm3 at 14 days; p < 0.0001). Cleaved caspase-3 and glial activation persisted for 14 days, indicating ongoing apoptosis and gliosis. No sex-dependent effects were observed in lesion volume, behaviour, or gliosis. ConclusionsThis refined neonatal photothrombotic ischaemia model is reproducible, scalable, and ethically improved, requiring no skin incision. Its minimal surgical burden, absence of mortality, consistent histopathology, and measurable functional outcomes make it an ideal platform for preclinical screening of neuroprotective and reparative interventions in the developing brain.

developmental biology↗

bioRxiv2024

Evaluation of the efficacy of a full-spectrum medicinal cannabis plant extract with less than 0.3% Delta9-tetrahydrocannabinol in in vitro models of inflammation and excitotoxicity

The rapid development of research on the therapeutic benefits of medicinal cannabis, in parallel with an increased understanding of the endocannabinoid system, has driven research of Cannabis sativa constituents for managing neurological conditions. While most studies have focused on the therapeutic potential of the major components of cannabis plant extract isolated or combined, limited research has explored the pharmacological benefits of whole cannabis plant extract. In this study, we investigated the potential anti-inflammatory and neuroprotective effects of NTI-164, a novel full-spectrum cannabis extract with negligible {Delta}9-tetrahydrocannabinol (THC), compared with cannabidiol (CBD) alone in BV-2 microglial and SHSY-5Y neuronal cells. The inflammation-induced upregulation of microglial inflammatory mediators, being tumour necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), inducible nitric oxide synthase (iNOS), and Arginase-1 (Arg-1), were significantly attenuated by NTI-164. This immunomodulatory effect was not observed upon treatment with isolated CBD. Compared to CBD alone, NTI-164 prevented elevated mitochondrial activity while normalising cell numbers in immune-activated microglia cells. NTI-164 also promoted the proliferation of undifferentiated neurons and the survival of differentiated neurons under excitotoxic conditions. Overall, our work shows that the anti-inflammatory and neuroprotective effects of NTI-164 as a full-spectrum cannabis extract are enhanced relative to that of CBD alone, highlighting the potential therapeutic efficacy of NTI-164 for the treatment of neuropathologies such as autism spectrum disorder (ASD) and related neuropathologies. This study has further shown that understanding the synergistic effect of phytocannabinoids is integral to realising the therapeutic potential of full-spectrum cannabis extract to inform the design of botanical-derived treatments for managing neurological disorders.

cell biology↗

bioRxiv2023

C-section and systemic inflammation synergize to disrupt the neonatal gut microbiota and brain development in a model of prematurity

Infants born very preterm (below 28 weeks of gestation) are at high risk of developing neurodevelopmental disorders, such as intellectual deficiency, autism spectrum disorders, and attention deficit. Preterm birth often occurs in the context of perinatal systemic inflammation due to chorioamnionitis and postnatal sepsis (Dammann, O. and Leviton, A., Intermittent or sustained systemic inflammation and the preterm brain. Pediatr Res, 2014. 75(3): p. 376-80). In addition, C-section is often performed for very preterm neonates to avoid hypoxia during a vaginal delivery (Luca, A.,et al., Birth trauma in preterm spontaneous vaginal and cesarean section deliveries: A 10-years retrospective study. PloS one,2022, 17(10), e0275726.) We have developed and characterized a mouse model based on intraperitoneal injections of IL-1{beta} between postnatal days one and five to reproduce perinatal systemic inflammation (Favrais, G.,et al., Systemic inflammation disrupts the developmental program of white matter. Ann Neurol,2011. 70(4): p. 550-65). This model replicates several neuropathological, brain imaging, and behavioral deficits observed in preterm infants. We hypothesized that C-sections could synergize with systemic inflammation to induce more severe brain abnormalities. We observed that C-sections significantly exacerbated the deleterious effects of IL-1{beta} on reduced gut microbial diversity, increased levels of circulating peptidoglycans, abnormal microglia/macrophage reactivity, impaired myelination, and reduced functional connectivity in the brain relative to vaginal delivery plus intraperitoneal saline. These data demonstrate the deleterious synergistic effects of C-section and neonatal systemic inflammation on brain maldevelopment and malfunction, two conditions frequently observed in very preterm infants, who are at high risk of developing neurodevelopmental disorders. Significance StatementIn a well-established mouse model of the encephalopathy of prematurity, we observed that C-section exacerbates the deleterious effects of neonatal systemic inflammation (intraperitoneal injections of IL-1{beta} between postnatal days one and five) on reduced gut microbial diversity, increased levels of circulating peptidoglycans, abnormal microglia/macrophage reactivity, impaired myelination, and reduced brain functional connectivity. These data demonstrate the deleterious synergistic effects of C-section and neonatal systemic inflammation, two conditions frequently observed in very preterm infants, who are at high risk of developing neurodevelopmental disorders.

neuroscience↗

bioRxiv2021

A unique cerebellar pattern of microglia activation in a mousemodel of encephalopathy of prematurity

Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Because 1 in 10 babies is born preterm cerebellar injury is a significant clinical problem. The causes of cerebellar damage are yet to be fully explained. Herein, we tested the hypothesis that perinatal inflammatory stimuli may play a key role in cerebellar injury of preterm infants. We undertook our studies in an established mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of the prototypic pro-inflammatory cytokine interleukin-1{beta} (IL-1{beta}). Inflammation is induced between postnatal day (P) 1 to day 5, timing equivalent to the last trimester for brain development in humans the period of vulnerability to preterm birth related brain injury. We investigated acute and long-term consequences for the cerebellum on brain volume expansion, oligodendroglial maturation, myelin levels and the microglial transcriptome. Perinatal inflammation induced global mouse brain volume reductions, including specific grey and white matter volume reductions in cerebellar lobules I and II (5% FDR) in IL-1{beta} versus control treated mice from P15 onwards. Oligodendroglia damage preceded the MRI-detectable volume changes, as evidenced by a reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins MOG, MBP and MAG at P10 and P15. Increased density of Iba1+ cerebellar microglia was observed at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebelli and cerebrum revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but in the cerebellum microglia displayed a unique type I interferon signalling dysregulation. Collectively, these data suggest that in our model that systemic inflammation causes chronic activation of microglia and maldevelopment of cerebellum that includes myelin deficits which is driven in the cerebellum by type I interferon signalling. Future protective strategies for preterm infants should consider sustained type I interferon signalling driven cerebellar inflammation as an important target.

neuroscience↗

bioRxiv2020

Perinatal IL-1-induced Inflammation Suppresses Tbr2+ Intermediate Progenitor Cell Proliferation in the Developing Hippocampus accompanied by Long-Term Behavioral Deficits

Meta-analyses have revealed associations between the incidence of maternal infections during pregnancy, premature birth, smaller brain volumes, and subsequent cognitive, motor and behavioral deficits as these children mature. Inflammation during pregnancy in rodents produces cognitive and behavioral deficits in the offspring that are similar to those reported in human studies. These deficits are accompanied by decreased neurogenesis and proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. As systemically administering interleukin-1 {beta} (IL-1{beta}) to neonatal mice recapitulates many of the brain abnormalities seen in premature babies including developmental delays, the goal of this study was to determine whether IL-1-mediated neuroinflammation would affect hippocampal growth during development to produce cognitive and behavioral abnormalities. For these studies, 10 ng/g IL-1{beta} was administered twice daily to Swiss Webster mice during the first 5 days of life, which increased hippocampal levels of IL-1 and acutely reduced the proliferation of Tbr2+ neural progenitors in the DG. In vitro, both IL-1 and IL-1{beta} produced G1/S cell cycle arrest that resulted in reduced progenitor cell proliferation within the transit amplifying progenitor cell cohort. By contrast, IL-1{beta} treatment increased neural stem cell frequency. Upon terminating IL-1{beta} treatment, the progenitor cell pool regained its proliferative capacity. An earlier study that used this in vivo model of perinatal inflammation showed that mice that received IL-1{beta} as neonates displayed memory deficits which suggested abnormal hippocampal function. To evaluate whether other cognitive and behavioral traits associated with hippocampal function would also be altered, mice were tested in tasks designed to assess exploratory and anxiety behavior as well as working and spatial memory. Interestingly, mice that received IL-1{beta} as neonates showed signs of anxiety in several behavioral assays during adolescence that were also evident in adulthood. Additionally, these mice did not display working memory deficits in adulthood, but they did display deficits in long-term spatial memory. Altogether, these data support the view that perinatal inflammation negatively affects the developing hippocampus producing behavioral deficits that persist into adulthood. These data provide a new perspective into the origin of the cognitive and behavioral impairments observed in prematurely-born sick infants. HighlightsO_LISystemic inflammation during the first 5 days of life increases hippocampal pro-inflammatory cytokines and chemokines C_LIO_LINeuroinflammation reduces Tbr2+ neural progenitor cell proliferation in the dentate gyrus. C_LIO_LIIL-1 arrests the cell cycle of Tbr-2+ progenitors and enriches for neural stem cells. C_LIO_LIAdolescent mice that experienced neonatal systemic inflammation have a persistent anxiety disorder. C_LIO_LIMice that experienced neonatal systemic inflammation have deficits in spatial memory as adults. C_LI

neuroscience↗

bioRxiv2018

Discordant perturbations of transcriptome and epigenome landscapes highlight dual roles of proinflammatory players in normal and IL1B-compromised OPC maturation trajectory in a prenatal model of diffuse white matter injury

Inflammatory insults accompanying prematurity provokes diffuse white matter injury (DWMI) which is associated with increased risk of neurodevelopmental disorders: pre-term infants have a 10 to 18-fold increased risk of developing autism spectrum disorders, compared to term infants. DWMI is due to maturation arrest in oligodendrocyte precursor cells (OPCs). Using integrated genome-wide approaches in a validated mouse perinatal model of DWMI, induced by systemic- and neuro-inflammation based on repeated interleukin-1B administrations, we show that neuroinflammation induces limited epigenomic disturbances in OPCs. In contrast, we unravel marked transcriptomic alterations of genes of the immune/inflammatory pathways, which are expressed in unstressed OPCs and physiologically downregulated along OPC maturation. Consistently, we observe that transcription factors of the inflammatory pathways occupy DNA both in unstressed and inflamed OPCs. Thus, rather than altering genome-wide chromatin accessibility, neuroinflammation takes advantage of open chromatin regions and deeply counteracts the stage-dependent downregulation of these active transcriptional programs. Therefore, our study opens new avenues for the future development of targeted approaches to protect preterm brains. Highlights{circ} Limited epigenomic impact of inflammation on OPC maturation blockade {circ}Major transcriptomic disturbances take advantage of a primed epigenetic landscape {circ}Proinflammatory genes are active in OPCs and downregulated upon maturation {circ}Neuroinflammation counteracts both this downregulation and maturation in OPCs

developmental biology↗

bioRxiv2018

Loss of the Wnt/β-catenin pathway in microglia of the developing brain drives pro-inflammatory activation leading to white matter injury

Microglia of the developing brain have unique functional properties but how their activation states is regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain with multiple models of neuroinflammation-mediated injury and primary human microglia we found that a reduction in Wnt/{beta}-catenin signalling is necessary and sufficient to drive an oligodendrocyte-injurious microglial phenotype. We validated in a cohort of preterm born infants that genomic variation in the WNT pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a BBB penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

immunology↗

bioRxiv2017

Integrative genomics study of microglial transcriptome reveals effect of DLG4 (PSD95) on white matter in preterm infants.

Preterm birth places newborn infants in an adverse environment that leads to brain injury linked to neuroinflammation. To characterise this pathology, we present a translational bioinformatics investigation, with integration of human and mouse molecular and neuroimaging datasets to provide a deeper understanding of the role of microglia in preterm white matter damage. We examined preterm neuroinflammation in a mouse model of encephalopathy of prematurity induced by IL1B exposure, carrying out a gene network analysis of the cell-specific transcriptomic response to injury, which we extended to analysis of protein-protein interactions, transcription factors, and human brain gene expression, including translation to preterm infants by means of imaging-genetics approaches in the brain. We identified the endogenous synthesis of DLG4 (PSD95) protein by microglia in mouse and human, modulated by inflammation and development. Systemic genetic variation in DLG4 was associated with structural features in the preterm infant brain, suggesting that genetic variation in DLG4 may also impact white matter development and inter-individual susceptibility to injury.\n\nPreterm birth accounts for 11% of all births 1, and is the leading global cause of deaths under 5 years of age 2. Over 30% of survivors experience motor and/or cognitive problems from birth 3, 4, which last into adulthood 5. These problems include a 3-8 fold increased risk of symptoms and disorders associated with anxiety, inattention and social and communication problems compared to term-born infants 6. Prematurity is associated with a 4-12 fold increase in the prevalence of Autism Spectrum Disorders (ASD) compared to the general population 7, as well as a risk ratio of 7.4 for bipolar affective disorder among infants born below 32 weeks of gestation 8.\n\nThe characteristic brain injury observed in contemporary cohorts of preterm born infants includes changes to the grey and white matter tissues, that specifically include oligodendrocyte maturation arrest, hypomyelination and cortical changes visualised as decreases in fractional anisotropy 9-13. Exposure of the fetus and postnatal infant to systemic inflammation is an important contributing factor to brain injury in preterm born infants 12, 14, 15, and the persistence of inflammation is associated with poorer neurological outcome 16. Sources of systemic inflammation include maternal/fetal infections such as chorioamnionitis (which it is estimated affects a large number of women at a sub-clinical level), with the effect of systemic inflammation in the brain being mediated predominantly by the microglial response 17.\n\nMicroglia are unique yolk-sac derived resident phagocytes of the brain 18, 19, found preferentially within the developing white matter as a matter of normal developmental migration 12. Microglial products associated with white matter injury include pro-inflammatory cytokines, such as interleukin-1{beta} (IL1B) and tumour necrosis factor (TNF-)20, which can lead to a sub-clinical inflammatory situation associated with unfavourable outcomes 21. In addition to being key effector cells in brain inflammation, they are critical for normal brain development in processes such as axonal growth and synapse formation 22, 23. The role of microglia in neuroinflammation is dynamic and complex, reflected in their mutable phenotypes including both pro-inflammatory and restorative functions 24. Despite their important neurobiological role, the time course and nature of the microglial responses in preterm birth are currently largely unknown, and the interplay of inflammatory and developmental processes is also unclear. We, and others, believe that a better understanding of the molecular mechanisms underlying microglial function could harness their beneficial effects and mitigate the brain injury of prematurity and other states of brain inflammation25, 26\n\nA clinically relevant experimental mouse model of IL1B-induced systemic inflammation has been developed to study the changes occurring in the preterm human brain 27, 28. This model recapitulates the hallmarks of encephalopathy of prematurity including oligodendrocyte maturation delay with consequent dysmyelination, associated magnetic resonance imaging (MRI) phenotypes and behavioural deficits. Here, we take advantage of this model system to characterise the molecular underpinnings of the microglial response to IL1B-driven systemic inflammation and investigate its role in concurrent development.\n\nIn preterm infants MRI is used extensively to provide in-vivo correlates of white and grey matter pathology, allowing clinical assessment and prognostication. Diffusion MRI (d-MRI) measures the displacement of water molecules in the brain, and provides insight into the underlying tissue structure. Various d-MRI measures of white matter have been associated with developmental outcome in children born preterm 29-32, with up to 60% of inter-individual variability in structural and functional features attributable to genetic factors 33, 34. White matter abnormalities are linked to associated grey matter changes at both the imaging and cellular level 10, 35, 36, with functional and structural consequences lasting into adulthood 37, 38. Tract Based Statistics (TBSS) allows quantitative whole-brain white matter analysis of d-MRI data at the voxel level while avoiding problems due to contamination by signals arising from grey matter 39. This permits voxel-wise statistical testing and inferences to be made about group differences or associations with greater statistical power. TBSS has been shown to be an effective tool for studying white matter development and injury in the preterm brain 40, providing a macroscopic in vivo quantitative measure of white matter integrity that is associated with cognitive, fine motor, and gross motor outcome 11, 41, 42.\n\nIn this work we take a translational systems biology approach to investigate the role of microglia in preterm neuroinflammation and brain injury. We integrate microglial cell-type specific data from a mouse model of perinatal neuroinflammatory brain injury with experimental ex vivo and in vitro validation, translation to the human brain across the lifespan including analysis of human microglia, and assessment of the impact of genetic variation on structure of the preterm brain. We add to the understanding of the neurobiology of prematurity by: a) revealing the endogenous expression of DLG4 (PSD95) by microglia in early development, which is modulated by developmental stage and inflammation; and b) finding an association between systemic genetic variability in DLG4 and white matter structure in the preterm neonatal brain.

genomics↗