Biology subjects

Robel, S.

Publications and source records attributed to Robel, S..

6 recordsLinked to original sources

bioRxiv2025

Fast Ripple-Delta Coupling as Early Biomarker for Post-Traumatic Epileptogenesis in Repetitive Brain Injury

Traumatic brain injury (TBI) can induce post-traumatic epilepsy (PTE), but early biomarkers for epileptogenesis are lacking. We used a repetitive diffuse TBI (rdTBI) model in mice with continuous video-EEG monitoring up to 4[1/2] months post-injury to investigate electrographic biomarkers before and during post-traumatic seizure development. 25% of mice developed post-traumatic seizures with highly variable latency (5-126 days post-injury). Most significantly, we identified fast ripple-delta DOWN state coupling as an early biomarker that was detectable at 4 days post-TBI and appeared before seizure onset in all seizure-experiencing mice. This EEG signature distinguished seizure-experiencing from seizure-free TBI mice with high specificity. Power spectrum analysis revealed elevated delta and theta power, reduced physiological fast oscillations (alpha, beta, gamma) and increased pathological high-frequency oscillations (fast ripples) in seizure-experiencing animals, indicating network hyperexcitability. Spike analysis showed that while TBI itself increased cortical excitability, seizure onset triggered a dramatic further escalation in interictal activity. These electrographic signatures were remarkably consistent across all seizure-experiencing animals regardless of single or recurrent seizure pattern. Our results demonstrate that fast ripple-delta coupling represents a promising early biomarker detectable at 4 days post-TBI, before seizure onset, offering potential for early identification of post-traumatic seizure susceptibility. Importantly, this biomarker identified all seizure-prone animals regardless of whether they developed single or recurrent seizures, suggesting shared underlying mechanisms and clinical relevance for any post-traumatic seizure occurrence. These findings emphasize the utility of temporal EEG analysis for detecting early electrographic changes in post-traumatic epileptogenesis and may inform future intervention strategies. Key PointsO_LIFast ripple-delta DOWN state coupling was detectable as early as 4 days post-TBI and appeared before seizure onset in seizure-experiencing mice, representing the first early biomarker that can stratify animals for epileptogenesis risk during the critical latent period. C_LIO_LIDelta and theta power increased while alpha, beta and gamma power decreased in all seizure-experiencing mice post-TBI, creating a consistent electrographic signature regardless of whether animals developed single or recurrent seizures. C_LIO_LIFast ripples were elevated and gamma-to-HFO ratios were reduced in seizure-experiencing mice, reflecting network hyperexcitability shift and potential inhibitory dysfunction that preceded seizure onset. C_LIO_LISeizure onset triggered a 3-fold escalation in spike activity, while baseline spike differences between TBI and pre-seizure mice were not significant, highlighting the limitation of spike counts alone as predictive biomarkers during the latent period. C_LIO_LIElectrographic signatures were almost similar across all seizure patterns (single and recurrent), suggesting shared underlying mechanisms of network dysfunction, though larger studies are needed to determine if biomarkers can predict seizure frequency in addition to seizure susceptibility. C_LI

neuroscience↗

bioRxiv2025

Evidence for the Transient Presence of Atypical Astrocytes in Mice Following a Single, Closed-Head Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) affects roughly 42 million people each year, causes a variety of physical, behavioral, and cognitive symptoms, and increases the risk for developing neurological disorders, including post-traumatic headache (PTH) and Alzheimers disease (AD). Multiple molecular and cellular changes occur following mTBI; here we focus on astrocytes - cells that respond to brain injury and are critical to maintaining neuronal and circuit homeostasis. While some astrocytes become reactive after mTBI, others adopt an atypical state characterized by the loss of multiple functional astrocyte proteins, including glutamate transporters (GLT-1, GLAST) and ion channels (Kir4.1), without upregulation of prototypical reactive astrocyte markers (glial fibrillary acidic protein [GFAP]). Previous studies have shown that repeated mTBI causes atypical astrocytes (AtAs) that can persist for months, but we know much less about whether a single mTBI causes similar astrocyte phenotypes. To address this, we employed a closed-head mild traumatic brain injury (chmTBI) model in male and female mice and quantified the abundance of AtAs both acutely (3-days) and chronically (1-month) after a single injury. We found that 3-days after chmTBI, AtAs were present in areas subject to blunt force trauma (BFT), consistent with previous reports, as well as in other brain regions presumably affected by diffuse injury. One month after chmTBI, however, the proportion of AtAs was similar between chmTBI and sham injured mice, thereby suggesting AtAs do not persist long term in this model. Consistent with previous studies, this chmTBI model did not induce significant GFAP-positive reactive astrocytes as assayed using immunohistochemistry, at either timepoint. Overall, we show an increase in AtAs 3-days after a single chmTBI that returns to sham levels when examined 1-month after injury. This suggests that after a single mTBI, AtAs are present but do not persist long term, unlike in repeated mTBI where AtAs persist for months after injury.

neuroscience↗

bioRxiv2024

Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury

Astrocytes play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily formed by connexin43 (Cx43) in the cortical gray matter. These GJs facilitate electrical and metabolic coupling between astrocytes, allowing the passage of ions, glucose, and metabolites. Dysregulation of Cx43 has been implicated in various pathologies, including traumatic brain injury (TBI) and acquired epilepsy. After mild TBI/concussion, we previously identified a subset of atypical astrocytes, which are correlated with the development of spontaneous seizures. These astrocytes exhibit reduced Cx43 expression and coupling. However, atypical astrocytes represent a relatively small subset of astrocytes within the cortical gray matter and previous studies suggest an overall increase of Cx43 protein after TBI. Additionally, Cx43 also has non-junctional and channel-independent functions, which include hemichannel communication with the extracellular milieu, cell adhesion, protein trafficking, protein-protein interactions, and intracellular signaling. In the present study, we set out to determine how mild TBI initiates alterations to Cx43 protein expression and localization, how they may be regulated, and whether they contribute to seizure susceptibility. We demonstrate remarkable heterogeneity of Cx43 protein levels from astrocyte to astrocyte. In accordance with our previous findings, a subset of astrocytes lost Cx43 expression, yet total cortical Cx43 protein increased. At the subcellular level, junctional Cx43 protein levels remained stable, while hemichannels and/or cytoplasmic Cx43 were increased. Phosphorylation of Cx43 at serine 368, a key regulatory site influencing GJ assembly and function, increased after mild TBI. Critically, Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to pentylenetetrazol-induced seizures. These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility, while inhibiting this modification presents a potential therapeutic avenue for mitigating neuronal hyperexcitability and seizure development. Significance statementConnexin43 (Cx43) is the main protein comprising astrocyte gap junctions which mediate astrocyte coupling into cellular networks, but it also has other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we assessed Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocytes lost Cx43 expression, other astrocytes had increased cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility.

neuroscience↗

bioRxiv2024

Optimized Enrichment of Murine Blood-Brain Barrier Vessels with a Critical Focus on Network Hierarchy in Post-Collection Analysis

Cerebrovascular networks contain a unique region of interconnected capillaries known as the blood-brain barrier (BBB). Positioned between upstream arteries and downstream veins, these microvessels have unique structural features, such as the absence of vascular smooth muscle cells (vSMCs) and a relatively thin basement membrane, to facilitate highly efficient yet selective exchange between the circulation and the brain interstitium. This vital role in neurological health and function has garnered significant attention from the scientific community and inspired methodology for enriching BBB capillaries. Extensive characterization of the isolates from such protocols is essential for framing the results of follow-on experiments and analyses, providing the most accurate interpretation and assignment of BBB properties. Seeking to aid in these efforts, here we visually screened output samples using fluorescent labels and found considerable reduction of non-vascular cells following density gradient centrifugation (DGC) and subsequent filtration. Comparatively, this protocol enriched brain capillaries, though larger diameter vessels associated with vSMCs could not be fully excluded. Protein analysis further underscored the enrichment of vascular markers following DGC, with filtration preserving BBB-associated markers and reducing - though not fully removing - arterial/venous contributions. Transcriptional profiling followed similar trends of DGC plus filtration generating isolates with less non-vascular and non- capillary material included. Considering vascular network hierarchy inspired a more comprehensive assessment of the material yielded from brain microvasculature isolation protocols. This approach is important for providing an accurate representation of the cerebrovascular segments being used for data collection and assigning BBB properties specifically to capillaries relative to other regions of the brain vasculature. HIGHLIGHTS- We optimized a protocol for the enrichment of murine capillaries using density gradient centrifugation and follow-on filtration. - We offer an approach to analyzing post-collection cerebrovascular fragments and cells with respect to vascular network hierarchy. - Assessing arterial and venous markers alongside those associated with the BBB provides a more comprehensive view of material collected. - Enhanced insight into isolate composition is critical for a more accurate view of BBB biology relative to larger diameter cerebrovasculature. MOTIVATIONThe recent surge in studies investigating the cerebrovasculature, and the blood-brain barrier (BBB) in particular, has inspired a broad range of approaches to target and observe these specialized blood vessels within murine models. To capture transcriptional and molecular changes during a specific intervention or disease model, techniques have been developed to isolate brain capillary networks and collect their cellular constituents for downstream analysis. Here, we sought to highlight the benefits and cautions of isolating and enriching microvessels from murine brain tissue. Specifically, through rigorous assessment of the output material following application of specific protocols, we presented the benefits of specific approaches to reducing the inclusion of non-vascular cells and non-capillary vessel segments, verified by analysis of vascular-related proteins and transcripts. We also emphasized the levels of larger- caliber vessels (i.e. arteries/arterioles and veins/venules) that are collected alongside cerebral capillaries with each method. Distinguishing these vascular regions with greater precision is critical for attributing specific characteristics exclusively to the BBB where metabolic, ion, and waste exchange occurs. While the addition of larger vessels to molecular / transcriptional analyses or follow-on experiments may not be substantial for a given protocol, it is essential to gauge and report their level of inclusion, as their contributions may be inadvertently assigned to the BBB. Therefore, we present this optimized brain microvessel isolation protocol and associated evaluation methods to underscore the need for increased rigor in characterizing vascular regions that are collected and analyzed within a given study. Graphical AbstractConstructed using resources from BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/613898v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@7b4448org.highwire.dtl.DTLVardef@bff92borg.highwire.dtl.DTLVardef@17fa8b3org.highwire.dtl.DTLVardef@1c950d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Mild traumatic brain injury/ concussion initiates an atypical astrocyte response caused by blood-brain barrier dysfunction

Mild traumatic brain injury/ concussion (mTBI) account for 70-90% of all reported TBI cases and cause long lasting neurological consequences in 10 to 40% of patients. Recent clinical studies revealed increased blood-brain barrier (BBB) permeability in mTBI patients, which correlated with secondary damage after mTBI. However, the cascade of cellular events initiated by exposure to blood-borne factors resulting in sustained damage are not fully resolved. We previously reported that astrocytes respond atypically to mTBI rapidly downregulating many proteins essential to their homeostatic function while classic scar formation does not occur. Here, we tested the hypothesis that mTBI -induced BBB damage causes atypical astrocytes through exposure to blood-borne factors. Using a mTBI mouse model, 2-photon imaging, an endothelial cell-specific genetic ablation approach, and serum-free primary astrocyte cultures, we demonstrated that areas with atypical astrocytes coincide with BBB damage and that exposure of astrocytes to plasma proteins is sufficient to initiate downregulation of astrocyte homeostatic proteins. While mTBI resulted in frequent impairment of both physical and metabolic BBB properties and leakage of small-sized blood-borne factors, deposition of the coagulation factor fibrinogen or vessel rupture were rare. Surprisingly, even months after mTBI BBB repair did not occur in areas with atypical astrocytes. Together, these findings implicate that even relatively small BBB disturbances are sustained long-term and render nearby astrocytes dysfunctional, likely at the cost of neuronal health and function.

neuroscience↗

bioRxiv2020

Astrocytes are necessary for blood-brain barrier maintenance in the adult mouse brain

In the adult brain, multiple cell types are known to produce factors that regulate blood-brain barrier properties, including astrocytes. Yet several recent studies disputed a role for mature astrocytes at the blood-brain barrier. To determine if astrocytes contribute a non-redundant and necessary function in maintaining the adult blood-brain barrier, we used a mouse model of tamoxifen-inducible astrocyte ablation. In adult mice, tamoxifen induction caused sparse apoptotic astrocyte cell death within 2 hours. Indicative of BBB damage, leakage of the small molecule Cadaverine and the large plasma protein fibrinogen into the brain parenchyma indicative of BBB damage was detected as early as astrocyte ablation was present. Vessels within and close to regions of astrocyte loss had lower expression of the tight junction protein zonula occludens-1 while endothelial glucose transporter 1 expression was undisturbed. Cadaverine leakage persisted for several weeks suggesting a lack of barrier repair. This is consistent with the finding that ablated astrocytes were not replaced. Adjacent astrocytes responded with partial non-proliferative astrogliosis, characterized by morphological changes and delayed phosphorylation of STAT3, which restricted dye leakage to the brain and vessel surface areas lacking coverage by astrocytes one month after ablation. In conclusion, astrocytes are necessary to maintain blood-brain barrier integrity in the adult brain. Blood-brain barrier-regulating factors secreted by other cell types, such as pericytes, are not sufficient to compensate for astrocyte loss. Main PointsMature astrocytes are necessary for maintenance of endothelial tight junctions in the adult brain. Ablated astrocytes are not replaced by proliferation or process extension of neighboring astrocytes resulting in long-term blood-brain barrier damage.

neuroscience↗