Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and loss-of-function coding variants are major risk factors for late onset Alzheimer’s disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling in microglia, we generated a TREM2 reporter mouse. In APP transgenic animals, bulk RNA-sequencing of isolated microglia sorted based on reporter expression highlighted TREM2 level-related changes in major immunometabolic pathways, and enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. Metabolic and lipidomic profiling of sorted microglia showed that, independent of Aβ pathology, TREM2 expression correlated with signatures consistent with increased cellular redox, energetics, and cholesterol homoeostasis. In accordance, metabolic activity correlated with phagocytic capacity. Finally, we performed chronic treatment with a TREM2 agonist antibody and identified a window of TREM2 expression where microglia are most responsive, thereby informing clinical applications of TREM2 agonists. TREM2 is an important AD risk factor playing essential roles in the microglial response to amyloid pathology. Here, authors show using a TREM2 reporter mouse that TREM2 levels are critical for efficacy of TREM2 agonism informing current clinical efforts.

Due to its selective permeability, the blood-brain barrier presents a major hurdle in brain targeted drug delivery, especially for macromolecules such as proteins or nucleic acids. Nose-to-brain delivery offers a promising opportunity for drug delivery via the olfactory and trigeminal pathway resulting in effective concentrations in the brain tissue and central nervous system when compared to systemic delivery. In this study, polymeric nanoparticles constituted of chitosan, which were further chemically modified and decorated with transferrin as targeting ligand, were utilized to examine the nose-to-brain delivery of proteins. The aim of this study was to evaluate chitosan nanoparticles, with or without transferrin decoration, as an intranasal platform for nose-to-brain protein delivery using β-galactosidase as a model protein. The synthesized chitosan nanoparticles and transferrin decorated chitosan nanoparticles were evaluated for their physicochemical characteristics, release kinetics, cellular uptake and permeability through an epithelial cell layer. β-galactosidase-loaded chitosan nanoparticles and transferrin-decorated chitosan nanoparticles showed particle sizes of 120 ± 16 nm and 142 ± 21 nm, respectively, and encapsulation efficiencies of 61 ± 9% and 53 ± 6%. Transferrin decoration enhanced cellular uptake in vitro and showed a favorable trend in epithelial permeability, with 77 ± 10% of the protein load transported after 24 h compared to 54 ± 24% for non-decorated chitosan nanoparticles. The ability of the nanosystem to reach the central nervous system after intranasal administration was assessed using fluorescently labeled nanoparticles containing the model protein β-galactosidase. After intranasal administration of mucus-stable, non-decorated chitosan nanoparticles in mice, approximately 15% of the administered fluorescently labeled nanoparticles and 5.5% of enzymatically active β-galactosidase were detected in the cerebrospinal fluid after 1 h. The results suggest that chitosan nanoparticles can be used for successful delivery of intact proteins to the brain.

Following ischemic stroke, neutrophil adhesion to activated cerebral endothelium triggers the initial inflammatory cascade. Real-time, quantitative in vivo detection of this early interaction could help identify individuals most likely to benefit from immunomodulatory therapies. To achieve this, we analyzed single-cell RNA sequencing data from brain tissue and identified E-selectin (Sele) as one of the earliest adhesion molecules selectively upregulated in activated endothelial cells with a venous-like transcriptional signature. We then developed iron oxide microparticles targeting E-selectin, designed to mimic neutrophil adhesion and to function as MRI probes for early endothelial activation. Within seconds of injection, these probes adhered to inflamed vessels in models of LPS-induced neuroinflammation and ischemic stroke, allowing rapid emergency imaging. We observed an MRI-detectable signal as early as 4 h following LPS stimulation and 8 h post-stroke. This binding was significantly associated with neutrophil infiltration, but not with lesion volume, blood-brain barrier disruption, or the accumulation of T cells and monocyte-derived cells. These findings demonstrate its specificity for neutrophil-driven inflammation and its relevance as a biomarker of ultra-early immune activation. These results suggest that neutrophil-mimetic MRI probes could represent a promising approach for detecting the initial phase of stroke-induced inflammation and for guiding personalized immunomodulatory strategies.

Purpose: Post-traumatic epilepsy (PTE) is one of the most common life-quality reducing consequences of traumatic brain injury (TBI). However, to date there are no pharmacological approaches to predict or to prevent the development of PTE. The P2X7 receptor (P2X7R) is a cationic ATP-dependent membrane channel that is expressed throughout the brain. While increasing evidence suggests a role for the P2X7R during seizures and epilepsy, it is unclear if changes in P2X7R expression can predict TBI-induced epilepsy development, and whether P2X7R antagonism can protect against long-lasting brain hyperexcitability caused by TBI. Methods: TBI was induced in adult male mice using the controlled cortical impact model (CCI). To test the anti-epileptogenic effects of P2X7R antagonism, mice were treated with brain-penetrant P2X7R antagonists JNJ-54175446 (30 mg/kg) or AFC-5128 (30 mg/kg) for 7 days post-CCI. The cell-type specific effects of P2X7Rs on TBI-induced hyperexcitability were analyzed in mice lacking exon 2 of the P2rx7 gene selectively in microglia (P2rx7:Cx3cr1-Cre). Static positron emission tomography (PET) via an intravenous injection of the P2X7R radioligand 18F-JNJ-64413739 and magnetic resonance imaging (MRI) were conducted twice during the first- and third-week post-injury. Results: Following TBI, while there were no obvious changes in P2X7R protein levels in the ipsilateral hippocampus post-injury, there was a delayed increase in P2X7R protein levels in the ipsilateral cortex at 3 months post-injury. Treatment with P2X7R antagonists shortly after TBI reduced long-lasting brain hyperexcitability, reduced cortical contusion volume, and normalized injury-induced hyperactivity to control sham-levels at 3 weeks post-TBI. Notably, mice lacking P2rx7 in microglia had an increased seizure threshold after TBI, suggesting that P2X7R contributed to brain hyperexcitability via its effects on microglia. Finally, P2X7R radioligand uptake after TBI correlated with seizure threshold at 3 weeks post-injury. Conclusions: Our results demonstrate the antiepileptogenic potential of P2X7R antagonism to prevent TBI-induced epilepsy and indicate that P2X7R-based PET imaging may be a useful diagnostic tool to identify people at risk of developing PTE.

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 behavior at the subcellular level while also carrying a Förster 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 naïve mouse brain vasculature exhibit 3D real-time FRET changes, showing size-dependent stability over 2 h in blood circulation. Further, in the Nanostroke model, dynamic intravital two-photon imaging revealed that LNDs accumulated within cerebral postischemic 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, FedEcs represents an advanced 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 poststroke, specifically, accumulation, degradation, and extravasation via poststroke microthrombi, this system could significantly enhance the translational validation of nanocarriers for future treatments of brain diseases.

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. ### Competing Interest Statement K.Kadletz, C.Kimna and A.Erturk have filed for intellectual property on the hybrid nanoparticles described herein. A.Erturk. is a co-founder of Deep Piction. The other authors declare no competing interests. This work was supported by the European Research Council Consolidator grant (no. GA 865323), Nomis Heart Atlas project grant (Nomis Foundation) and Helmholtz Association Program Helmholtz IVF, ZT-I-PF-4-091, FOMIA.

Efficient and accurate nanocarrier development for targeted drug delivery is hindered by a lack of methods to analyze its cell-level biodistribution across whole organisms. Here we present Single Cell Precision Nanocarrier Identification (SCP-Nano), an integrated experimental and deep learning pipeline to comprehensively quantify the targeting of nanocarriers throughout the whole mouse body at single-cell resolution. SCP-Nano reveals the tissue distribution patterns of lipid nanoparticles (LNPs) after different injection routes at doses as low as 0.0005 mg kg−1—far below the detection limits of conventional whole body imaging techniques. We demonstrate that intramuscularly injected LNPs carrying SARS-CoV-2 spike mRNA reach heart tissue, leading to proteome changes, suggesting immune activation and blood vessel damage. SCP-Nano generalizes to various types of nanocarriers, including liposomes, polyplexes, DNA origami and adeno-associated viruses (AAVs), revealing that an AAV2 variant transduces adipocytes throughout the body. SCP-Nano enables comprehensive three-dimensional mapping of nanocarrier distribution throughout mouse bodies with high sensitivity and should accelerate the development of precise and safe nanocarrier-based therapeutics. An integrated experimental and deep learning pipeline reveals cell-level targeting of nanocarriers in whole bodies.

Molecular probes for cell plasma membranes are 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 to the commonly used MemBright probe 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 accessible 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 for plasma membrane research.

Mild traumatic brain injury (mTBI) accounts for 80% of all TBI, may be associated with chronic impairments, and is difficult to diagnose due to a lack of objective markers. In this study, we investigated whether neurosteroids can serve as blood biomarkers for mTBI. Two cohorts of C57BL/6 mice were subjected to a model of mTBI combining impact with rotational acceleration or sham surgery. The first cohort underwent neurological testing for anxiety, balance, and locomotion before and after mTBI. For the second cohort, brains and plasma were collected 6 or 24 h after mTBI to measure steroid and neurosteroid levels by gas chromatography-tandem mass spectrometry. Traumatized mice exhibited significantly prolonged wake-up time from anesthesia, transiently increased beam-walk time, and mild astrogliosis compared with their control counterparts, but did not suffer from skull fractures, intracranial hemorrhage, or mortality. Isopregnanolone and 3β,5α-tetrahydrodeoxycorticosterone (ISODOC) were significantly decreased by more than 50% in brain parenchyma at 6 and 24 h after mTBI, while ISODOC was also significantly decreased in plasma (-75%). Therefore, ISODOC may be a candidate diagnostic biomarker for mTBI.

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.

Current techniques for inducing intraluminal filamentous middle cerebral artery occlusion (fMCAo) in mice produce highly variable results and often cause additional infarcts in the posterior cerebral artery (PCA) territory. The aim of the current study was to develop a novel procedure to overcome these shortcomings. Male C57BL/6 mice were subjected to 60 min of fMCAo with cerebral blood flow monitored by laser Doppler flowmetry. The influence of the length of the occlusion filament coating and the combination of common carotid artery (CCA) or pterygopalatine artery (PPA) ligation on lesion volume and functional outcome 24 h after reperfusion was evaluated. The use of appropriate filament and PPA ligation while maintaining CCA perfusion prevented the development of infarcts in the PCA area, resulted in pure MCA infarcts (68.3 ± 14.5 mm3) and reduced the variability of infarct volumes by more than half (from 26-38% to 14% standard deviation/mean). Using an improved fMCAo procedure, we were able to produce PCA area-unaffected reproducible (PURE) infarcts exclusively in the MCA territory. Thus PURE-MCAo reduced outcome variability by more than 50%. Our results may thus help to reduce the number of animals in preclinical stroke research and to increase the reproducibility of the fMCAo model.

BACKGROUND In 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.METHODS C57BL/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.RESULT The 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 Swanson’s correction) and reduced the variability of infarct volumes by more than half to 13.9% of the standard deviation divided by mean.CONCLUSIONS Using 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.### Competing Interest StatementThe authors have declared no competing interest.* ANOVA : Analysis of Variance CCA : common carotid artery ECA : external carotid artery fMCAo : filament middle cerebral artery occlusion ICA : internal carotid artery MCA : middle cerebral artery OA : occipital artery PCA : posterior cerebral artery PFA : paraformaldehyde PPA : pterygopalatine artery rCBF : regional cerebral blood flow SD : standard deviation

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 Alzheimer’s 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 Aβ 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. ### Competing Interest Statement JHS, BvL, JWL, GDP and KMM are full-time employees and shareholders of the Denali Therapeutics Inc. CH collaborates with Denali Therapeutics and is a member of the advisory boards of AviadoBio and Cure Ventures.

Nanoparticles have a great potential to significantly improve the delivery of therapeutics to the brain and may also be equipped with properties to investigate brain function. The brain, being a highly complex organ shielded by selective barriers, requires its own specialized detection system. However, a significant hurdle to achieve these goals is still the identification of individual nanoparticles within the brain with sufficient cellular, subcellular, and temporal resolution.This review aims to provide a comprehensive summary of the current knowledge on detection systems for tracking nanoparticles across the blood-brain barrier and within the brain. We discuss commonly employed in vivo and ex vivo nanoparticle identification and quantification methods, as well as various imaging modalities able to detect nanoparticles in the brain. Advantages and weaknesses of these modalities as well as the biological factors that must be considered when interpreting results obtained through nanotechnologies are summarized. Finally, we critically evaluate the prevailing limitations of existing technologies and explore potential solutions.

Nanotechnology holds great promise to improve delivery of therapeutics to the brain. Current experimental approaches are, however, hampered by the lack of tools to dynamically monitor cargo delivery in vivo . We developed highly fluorescent lipid nanodroplets (LNDs) that carry a Förster-resonance energy transfer (FRET)-based drug delivery detection system able to monitor cargo release (FedEcs) in vivo . We investigated the distribution, stability, and cargo release of FedEcs-LNDs in the healthy and ischemic mouse brain by intravital multiphoton microscopy. We dynamically observed that LNDs accumulated within cerebral microclots after ischemia, caused by magnetic nanoparticles (Nano-stroke), and released their cargo. Furthermore, the blood-brain barrier (BBB) became permeable at sites of microclots thereby allowing FedEcs-LNDs to cross the BBB and to deliver their cargo to the brain parenchyma. Consequently, FedEcs represents a novel tool to quantitatively investigate the nanocarriers biodistribution and cargo release using intravital microscopy and may thus tremendously ease their translational validation.### Competing Interest StatementThe authors have declared no competing interest.

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. 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. 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.

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. ### Competing Interest Statement The authors have declared no competing interest.

The kallikrein-kinin system is one of the first inflammatory pathways to be activated following traumatic brain injury (TBI) and has been shown to exacerbate brain edema formation in the acute phase through activation of bradykinin 2 receptors (B2R). However, the influence of B2R on chronic post-traumatic damage and outcome is unclear. In the current study, we assessed long-term effects of B2R-knockout (KO) after experimental TBI. B2R KO mice (heterozygous, homozygous) and wild-type (WT) littermates (n = 10/group) were subjected to controlled cortical impact (CCI) TBI. Lesion size was evaluated by magnetic resonance imaging up to 90 days after CCI. Motor and memory function were regularly assessed by Neurological Severity Score, Beam Walk, and Barnes maze test. Ninety days after TBI, brains were harvested for immunohistochemical analysis. There was no difference in cortical lesion size between B2R-deficient and WT animals 3 months after injury; however, hippocampal damage was reduced in B2R KO mice (p = 0.03). Protection of hippocampal tissue was accompanied by a significant improvement of learning and memory function 3 months after TBI (p = 0.02 WT vs. KO), whereas motor function was not influenced. Scar formation and astrogliosis were unaffected, but B2R deficiency led to a gene-dose-dependent attenuation of microglial activation and a reduction of CD45+ cells 3 months after TBI in cortex (p = 0.0003) and hippocampus (p < 0.0001). These results suggest that chronic hippocampal neurodegeneration and subsequent cognitive impairment are mediated by prolonged neuroinflammation and B2R. Inhibition of B2R may therefore represent a novel strategy to reduce long-term neurocognitive deficits after TBI.