Biology subjects

Khalin, I.

Publications and source records attributed to Khalin, I..

12 recordsLinked to original sources

bioRxiv2026

Cerebral microthrombi promote focal Cav-1-dependent blood-brain barrier impairment, triggering neuroinflammation and neuronal damage after traumatic brain injury

Traumatic brain injury (TBI) is frequently accompanied by blood-brain barrier (BBB) dysfunction, yet the microvascular events that initiate barrier failure and secondary neural injury remain poorly understood. Using highly sensitive fluorescent nanoscale tracers, correlative light and electron microscopy, single-cell transcriptomics, and genetic manipulation of Caveolin-1 (Cav-1), we show that BBB leakage after TBI in mice occurs focally at sites of cerebral microthrombus formation. Endothelial cells adjacent to microthrombi remain structurally intact, but exhibit a distinct transcriptional program enriched in both thrombosis- and transcytosis-related genes, including Cav-1. Microthrombi promote Cav-1-dependent transcytosis and size-selective extravasation of blood-borne proteins, exposing the surrounding parenchyma to circulating factors and inducing focal microglia activation and neuronal damage in vivo. In human induced pluripotent stem cell-derived neurons, these proteins directly impaired neuronal structure and network function. Genetic deletion of Cav-1 markedly reduces microthrombus-associated BBB leakage, immune-cell diapedesis, microglial activation and neuronal loss, whereas endothelial re-expression of Cav-1 using AAVs restores leakage. Human TBI data reveal early dynamic coagulation dysregulation associated with adverse clinical outcome, focal cerebral vascular injury and endothelial transcriptional signatures consistent with reduced barrier integrity and increased caveolae-associated transport. Together, these findings identify cerebral microthrombi as focal sites of Cav-1-dependent BBB dysfunction and establish pathological endothelial transcytosis as a mechanistic link between post-traumatic microvascular thrombosis, blood protein extravasation and secondary neuronal injury. Targeting Cav-1-dependent transcytosis may therefore provide a strategy to limit pathological BBB permeability and secondary injury after TBI.

neuroscience↗

bioRxiv2026

Dual-action nanoconjugate for overcoming r-tPA -resistant clots

Clot resistance to pharmacological thrombolysis remains a critical challenge in ischemic stroke (IS) management. Thrombus heterogeneity, particularly the presence of thrombolysis-resistant domains composed of dense fibrin and non-fibrin components, including neutrophil extracellular traps (NETs), significantly limits the efficacy of recombinant tissue-type plasminogen activator (r-tPA) and its variant, Tenecteplase (TNK). Consequently, novel therapeutic strategies are urgently required. Emerging evidence suggests that co-administration of deoxyribonuclease I (DNase I) with r-tPA can degrade DNA fibers and enhance clot lysis. In this study, we optimized a previously developed theranostic agent--iron oxide microparticles coated with polydopamine--by dual-grafting both r-tPA and DNase to target resistant thrombi. Using functional ultrasound imaging (fUS) during the acute phase of IS, we demonstrated accelerated reperfusion with this dual-functionalized platform in a r-tPA resistant IS model. Furthermore, MRI analysis confirmed a significant reduction in lesion volume at 24 hours, correlating with improved functional recovery five days post-ischemia.

pathology↗

bioRxiv2025

LipiGo: A Versatile DNA-Lipid Nanoparticle Hybrid for Precision Drug Delivery

Lipid nanoparticles (LNPs) are established carriers for nucleic acid delivery, however, achieving efficient delivery to non-hepatic tissues remains a major challenge. Here, we present LipiGo, a nanocarrier platform engineered by integrating short single stranded DNA molecules into the lipid nanoparticle structure. Using whole body tissue clearing, advanced imaging and AI-based analysis, we show that LipiGo redirects functional mRNA delivery to lymphoid organs, particularly the spleen. Immune cell profiling further reveals enhanced uptake within key immune populations, including antigen-presenting cells, compared to standard LNPs. Beyond passive redistribution, LipiGo leverages DNA hybridization to enable modular attachment of targeting ligands for active targeting as demonstrated by cell-specific delivery to white adipocytes. Overall, the dual-purpose design principle of LipiGo demonstrates high modularity and efficiency, enabling tissue and cell specific delivery beyond hepatic applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/676334v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@9b69eorg.highwire.dtl.DTLVardef@1b45bd0org.highwire.dtl.DTLVardef@1f7b79dorg.highwire.dtl.DTLVardef@37a673_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Fluorescent anionic cyanine plasma membrane probes for live cell and in vivo imaging

Proper staining of cell plasma membrane is indispensable for fluorescence imaging. Herein, we present an array of five anionic cyanine-based turn-on plasma membrane probes with emission spanning from green to near infrared. They are analogous of commonly used MemBright probes family, where two zwitterionic anchor groups are replaced with anionic sulfonates with dodecyl chains. The developed probes provide selective wash-free staining of plasma membranes of live cells in vitro, featuring improved brightness and slower internalization inside the cells. In comparison to protein-based (wheat germ agglutinin) membrane markers, new membrane probes provide better staining in poorly assessable cell-cell contacts. A key challenge is to stain cell plasma membranes directly in vivo. During in vivo brain tissue imaging in living mice by two-photon microscopy, the anionic cyanine probes allowed us to visualize in detail the pyramidal neurons with high image quality, clearly resolving neuron soma, dendrites with dendritic spines and axons with axonal boutons. The developed anionic cyanine-based plasma membrane probes constitute an important extension of the toolbox of fluorescent probes for plasma membrane research.

biochemistry↗

bioRxiv2024

TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and sequence variants are major risk factors for late onset Alzheimers disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling, we generated a TREM2 reporter mouse model and observed a gradual upregulation of reporter expression with increasing plaque proximity. Isolated microglia were sorted based on reporter expression and their transcriptomic profiles acquired in both wildtype and APP transgenic animals, allowing us to disentangle TREM2 versus pathology-specific effects. Bulk RNA-sequencing highlighted TREM2 level-dependent changes in major immunometabolic pathways, with enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. To confirm these findings, we next analysed uptake of fluorodeoxyglucose (FDG) and examined metabolomic and lipidomic profiles. Again, independent of A{beta} pathology, TREM2 expression correlated with uptake of FDG as well as increased cellular redox, energetics, and cholesterol homeostasis. Finally, we performed chronic treatment with a brain penetrant TREM2 agonist and identified a window of TREM2 expression where microglia are most responsive. Thus, our data provide novel insights into TREM2-mediated regulation of microglial metabolic function and informs current efforts to bring TREM2 agonists into clinical application.

neuroscience↗

bioRxiv2024

Using a Foerster-resonance energy transfer (FRET)-based detection system (FedEcs) to monitor nanoparticle cargo delivery to the brain

Nanotechnology holds great promise for improving the delivery of therapeutics to the brain. However, current approaches often operate at the organ or tissue level and are limited by the lack of tools to dynamically monitor cargo delivery in vivo. We have developed highly fluorescent lipid nanodroplets (LNDs) that enable tracking of nanocarrier behaviour at the subcellular level while also carrying a Forster resonance energy transfer (FRET)-based drug delivery detection system (FedEcs) capable of monitoring cargo release in vivo. Using two-photon microscopy, we demonstrate that circulating LNDs in naive mouse brain vasculature exhibit 3D real-time FRET changes, showing size-dependent stability over two hours in blood circulation. Further, in a novel nano-stroke model, dynamic intravital two-photon imaging revealed that LNDs accumulated within cerebral post-ischemic microthrombi, where they released their cargo significantly faster than in normal blood circulation. Furthermore, the blood-brain barrier (BBB) became permeable at the microclot sites thereby allowing accumulated FedEcs-LNDs to cross the BBB and deliver their cargo to the brain parenchyma. This microthrombi-associated translocation was confirmed at the ultrastructural level via volume correlative light-electron microscopy. Consequently, our FedEcs represents a novel tool to quantitatively study the biodistribution and cargo release of nanocarriers at high resolution in real time. By enabling us to resolve passive targeting mechanisms post-stroke, - specifically, accumulation, degradation and extravasation via post-stroke microthrombi - this system could significantly enhance the translational validation of nanocarriers for future treatments of brain diseases.

neuroscience↗

bioRxiv2024

Minimizing variability in the filament middle cerebral artery occlusion model in C57BL/6 mice by surgical optimization - the PURE-MCAo Model

BACKGROUNDIn the intraluminal filament middle cerebral artery occlusion (fMCAo) model, there is considerable variability in infarct volumes, especially in C57BL/6 mice, which often lack the P1 segment of the posterior cerebral artery (PCA) and therefore develop not only MCA but also PCA area infarcts after fMCAo. Another factor contributing to infarct volume variability is collateral flow to the MCA area. The aim of this study was to establish an optimal surgical method to reduce the infarct volume variability in C57BL/6 mice. METHODSC57BL/6 mice were subjected to 60 min of fMCAo with cerebral blood flow monitored by laser Doppler fluxmetry. The influence of the common carotid artery (CCA) ligation, filament morphology, and the pterygopalatine artery (PPA) ligation on lesion volume and neurological severity score 24 hours after reperfusion were assessed. RESULTThe use of filaments with appropriate length of coating and ligation of the PPA while maintaining perfusion of the CCA prevented the development of infarcts in the PCA area, resulted in pure MCA infarcts (68.3{+/-}14.5mm3, 26.1{+/-}3.6% of the hemisphere with Swansons correction) and reduced the variability of infarct volumes by more than half to 13.9% of the standard deviation divided by mean. CONCLUSIONSUsing improved surgical methods with suitable filaments to induce MCA occlusion in mice, we were able to produce PCA area-unaffected reproducible infarcts exclusively in the MCA area with reduced variability (PURE-MCAo). Our results may thus help to increase the reproducibility of the fMCAo model and reduce the number of animals required in preclinical stroke research.

neuroscience↗

bioRxiv2023

Single-cell precision nanotechnology in vivo

Targeting nanoparticle therapeutics with cellular accuracy in whole organisms could open breakthrough opportunities in precision medicine. However, evaluating and fine-tuning the biodistribution of such systems in the whole organism at the cellular level remains a major obstacle. Here, we constructed targetable DNA origami, and analyzed biodistribution in transparent mice, in addition to studying tolerability, clearance kinetics, and immune response parameters. Untargeted DNA origami primarily accumulated in the spleen and the liver, while an immune cell-targeting variant successfully attached to immune cells throughout the body. A cancer cell-targeting mimetic co-localized on solid-tumor metastasis in the liver and the lung. These findings indicate that DNA origami can be directed in vivo, providing an important proof-of-concept and highlights the potential of high-resolution tissue-clearing imaging technologies in their development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/550304v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@ce216borg.highwire.dtl.DTLVardef@f40bb4org.highwire.dtl.DTLVardef@2c6a66org.highwire.dtl.DTLVardef@1fd7fde_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThis study demonstrates the potential of DNA origami-based drug delivery systems as versatile tool for for targeted delivery, which could be used to treat a range of diseases with applications. C_LIO_LIThe immune compatibility, half-life, targeting efficiency, and the biodistribution evaluation of DNA origami indicate its potential for systemic drug delivery. C_LIO_LIOur approach enables the assessment of biodistribution of nanoparticles in the intact body with a sensitivity to the single-cell level, highlighting the high-resolution tissue clearing technoloies in revealing DNA origamis feasibility for drug targeting. C_LI

bioengineering↗

bioRxiv2023

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Like other volume electron microscopy approaches, Automated Tape Collecting Ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. ATUM-SEM of serial semi-thick sections and consecutive ET of selected re-mounted sections combines SEMs fast target recognition and coarse rendering capability with high-resolution volume visualizations, respectively, thus enabling multi-scale interrogation of cellular ultrastructure. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. This workflow is a seamless zoom-in on structural BBB pathology from the micro- to the nanometer scale. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

neuroscience↗

bioRxiv2023

Continued dysfunction of capillary pericytes promotes no-reflow after experimental stroke in vivo

Incomplete reperfusion of the microvasculature ("no-reflow") after ischemic stroke damages salvageable brain tissue. Previous ex-vivo studies suggest pericytes are vulnerable to ischemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains underexplored in vivo. Using longitudinal in vivo 2-photon single-cell imaging over seven days we show 87% of pericytes constrict during cerebral ischemia, remain constricted post-reperfusion and 50% of the pericyte population are acutely damaged. Moreover, we reveal ischemic pericytes are fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 hours post-stroke. Despite sustaining acute membrane damage, we observe up to 80% of cortical pericytes survive ischemia, upregulate unique transcriptomic profiles and replicate. Finally, we demonstrate delayed recovery of capillary diameter by ischemic pericytes after reperfusion predicts vessel reconstriction in the sub-acute phase of stroke. Cumulatively, these findings demonstrate surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischemic stroke.

neuroscience↗

bioRxiv2021

Multi-omics and 3D-imaging reveal bone heterogeneity and unique calvaria cells in neuroinflammation

The meninges of the brain are an important component of neuroinflammatory response. Diverse immune cells move from the calvaria marrow into the dura mater via recently discovered skull-meninges connections (SMCs). However, how the calvaria bone marrow is different from the other bones and whether and how it contributes to human diseases remain unknown. Using multi-omics approaches and whole mouse transparency we reveal that bone marrow cells are highly heterogeneous across the mouse body. The calvaria harbors the most distinct molecular signature with hundreds of differentially expressed genes and proteins. Acute brain injury induces skull-specific alterations including increased calvaria cell numbers. Moreover, TSPO-positron-emission-tomography imaging of stroke, multiple sclerosis and neurodegenerative disease patients demonstrate disease-associated uptake patterns in the human skull, mirroring the underlying brain inflammation. Our study indicates that the calvaria is more than a physical barrier, and its immune cells may present new ways to control brain pathologies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/473988v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@978194org.highwire.dtl.DTLVardef@bc45e8org.highwire.dtl.DTLVardef@91afdborg.highwire.dtl.DTLVardef@b06bc6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBone marrow across the mouse body display heterogeneity in their molecular profile C_LIO_LICalvaria cells have a distinct profile that is relevant to brain pathologies C_LIO_LIBrain native proteins are identified in calvaria in pathological states C_LIO_LITSPO-PET imaging of the human skull can be a proxy of neuroinflammation in the brain C_LI Supplementary Videos can be seen at: http://discotechnologies.org/Calvaria/

cell biology↗

bioRxiv2020

Highly fluorescent biodegradable PLGA nano-carriers allows real-time tracking of individual particles in vivo

Poly(lactic-co-glycolic acid) (PLGA)-based drug formulations are approved for the use in humans, however, the potential of PLGA to design nanoparticles (NPs) and target the central nervous system remains to be exploited. The aim of the current study was design PLGA NPs, loading them with bulky fluorophores thereby increasing single particle fluorescence to a level visible by in vivo microscopy, and investigate their brain biodistribution. We developed, highly fluorescent 70 nm PLGA NPs significantly brighter then quantum dots enabling their visualization by intravital real-time 2-photon microscopy. We found that PLGA NPs coated with pluronic F-68 (PF-68) had a substantially longer plasma half-life than uncoated NPs and were taken up by cerebro-vascular endothelial cells. High resolution confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The current data suggest that PF-68 coated PLGA NPs are taken up by mouse cerebral and peripheral endothelial cells in vivo. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

bioengineering↗