Healthy Mouse Brain Research Articles

DNA methylation controls stemness of astrocytes in health and ischaemia

Astrocytes are the most abundant cell type in the mammalian brain and provide structural and metabolic support to neurons, regulate synapses and become reactive after injury and disease. However, a small subset of astrocytes settles in specialized areas of the adult brain where these astrocytes instead actively generate differentiated neuronal and glial progeny and are therefore referred to as neural stem cells1–3. Common parenchymal astrocytes and quiescent neural stem cells share similar transcriptomes despite their very distinct functions4–6. Thus, how stem cell activity is molecularly encoded remains unknown. Here we examine the transcriptome, chromatin accessibility and methylome of neural stem cells and their progeny, and of astrocytes from the striatum and cortex in the healthy and ischaemic adult mouse brain. We identify distinct methylation profiles associated with either astrocyte or stem cell function. Stem cell function is mediated by methylation of astrocyte genes and demethylation of stem cell genes that are expressed later. Ischaemic injury to the brain induces gain of stemness in striatal astrocytes7. We show that this response involves reprogramming the astrocyte methylome to a stem cell methylome and is absent if the de novo methyltransferase DNMT3A is missing. Overall, we unveil DNA methylation as a promising target for regenerative medicine.

The impact of astrocytic NF-κB on healthy and Alzheimer’s disease brains

Astrocytes play a role in healthy cognitive function and Alzheimer’s disease (AD). The transcriptional factor nuclear factor-κB (NF-κB) drives astrocyte diversity, but the mechanisms are not fully understood. By combining studies in human brains and animal models and selectively manipulating NF-κB function in astrocytes, we deepened the understanding of the role of astrocytic NF-κB in brain health and AD. In silico analysis of bulk and cell-specific transcriptomic data revealed the association of NF-κB and astrocytes in AD. Confocal studies validated the higher level of p50 NF-κB and phosphorylated-p65 NF-κB in glial fibrillary acidic protein (GFAP)+-astrocytes in AD versus non-AD subjects. In the healthy mouse brain, chronic activation of astrocytic NF-κB disturbed the proteomic milieu, causing a loss of mitochondrial-associated proteins and the rise of inflammatory-related proteins. Sustained NF-κB signaling also led to microglial reactivity, production of pro-inflammatory mediators, and buildup of senescence-related protein p16INK4A in neurons. However, in an AD mouse model, NF-κB inhibition accelerated β-amyloid and tau accumulation. Molecular biology studies revealed that astrocytic NF-κB activation drives the increase in GFAP and inflammatory proteins and aquaporin-4, a glymphatic system protein that assists in mitigating AD. Our investigation uncovered fundamental mechanisms by which NF-κB enables astrocytes' neuroprotective and neurotoxic responses in the brain.

Effect of reducing isoflurane level on glucosamine uptake in the mouse brain during magnetic resonance imaging studies

Anesthesia is often required during magnetic resonance imaging (MRI) examinations in animal studies. Anesthetic drugs differ in their capacity to interfere with homeostatic mechanisms responsible for glucose metabolism in the brain, which may create a constraint in the study design. Recent studies suggest that the chemical exchange saturation transfer (CEST) MRI scanning technique can detect localized metabolic changes in rodent brains induced by the uptake of glucose or its analogs; however, most of these studies do not account for the impact of anesthesia type on the brain metabolism. Herein, we aimed to evaluate the effect of reduced isoflurane levels on the preclinical imaging of glucosamine (GlcN) uptake in healthy mouse brains to establish optimal conditions for future brain imaging studies using the CEST MRI technique. The commonly used anesthesia protocol for longitudinal MRI examinations using 1.5% isoflurane level was compared to that using a mixture of low isoflurane (0.8%) level combined with midazolam (2 mg/kg, SC). Magnetization transfer ratio asymmetry (MTRasym) and area under the curve (AUC) analyses were used to characterize GlcN signals in the brain. The results indicated that mice injected with GlcN and anesthetized with 1.5% isoflurane exhibited low and insignificant changes in the MTRasym and AUC signals in the frontal cortex, whereas mice administered with 0.8% isoflurane combined with midazolam demonstrated a significant increase in these signals in the frontal cortex. This study highlights the diverse GlcN metabolic changes observed in mouse brains under variable levels of isoflurane anesthesia using the CEST MRI method. The results suggest that it is feasible to maintain anesthesia with low-dose isoflurane by integrating midazolam, which may enable the investigation of GlcN uptake in the brain. Thus, reducing isoflurane levels may support studies into mouse brain metabolism using the CEST MRI method and should be considered in future studies.

A glial circadian gene expression atlas reveals cell type and disease-specific reprogramming in response to amyloid pathology or aging.

While circadian rhythm disruption may promote neurodegenerative disease, how aging and neurodegenerative pathology impact circadian gene expression patterns in different brain cell types is unknown. Here, we used translating ribosome affinity purification methods to define the circadian translatomes of astrocytes, microglia, and bulk cerebral cortex, in healthy mouse brain and in the settings of amyloid-beta plaque pathology or aging. Our data reveal that glial circadian translatomes are highly cell type-specific and exhibit profound, context-dependent reprogramming of rhythmic transcripts in response to amyloid pathology or aging. Transcripts involved in glial activation, immunometabolism, and proteostasis, as well as nearly half of all Alzheimer Disease (AD)-associated risk genes, displayed circadian oscillations, many of which were altered by pathology. Amyloid-related differential gene expression was also dependent on time of day. Thus, circadian rhythms in gene expression are cell- and context dependent and provide important insights into glial gene regulation in health, AD, and aging.

High-resolution 1H-MRSI at 9.4 T by integrating relaxation enhancement and subspace imaging.

Achieving high-resolution and high signal-to-noise ratio (SNR) in vivo metabolic imaging via fast magnetic resonance spectroscopic imaging (MRSI) has been a longstanding challenge. This study combines the methods of relaxation enhancement (RE) and subspace imaging for the first time, enabling high-resolution and high-SNR in vivo MRSI of rodent brains at 9.4 T. Specifically, an RE-based chemical shift imaging sequence, which combines a frequency-selective pulse to excite only the metabolite frequencies with minimum perturbation of the water spins and a pair of adiabatic pulses to spatially localize the slice of interest, is designed and evaluated in vivo. This strategy effectively shortens the apparent T1 of metabolites, thereby increasing the SNR during relatively short repetition time ((TR) compared with acquisitions with only spatially selective wideband excitations, and does not require water suppression. The SNR was further enhanced via a state-of-the-art subspace reconstruction method. A novel subspace learning strategy tailored for 9.4 T and RE acquisitions is developed. In vivo, high-resolution (e.g., voxel size of 0.6 × 0.6 × 1.5 mm3) MRSI of both healthy mouse brains and a glioma-bearing mouse brain in 12.5 min has been demonstrated.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Liposome-based loading enhances the distribution of nicotinamide riboside chloride into the brain and its neuroprotective effects in cerebral ischemic mice

ObjectiveNicotinamide riboside (NR) is neuroprotective; however, its low permeability through the blood‒brain barrier restricts its therapeutic efficacy in central nervous system diseases. Compared with oral NR administration, liposome-based NR loading is hypothesized to improve its pharmacological properties during cerebral ischemia, especially when administered intravenously. MethodsNR chloride (NRC) was encapsulated in an optimized liposome composition and administered by bolus intravenous injection. This was followed by examination of its pharmacokinetics, organ distribution, and effects on cerebral ischemia in mice. ResultsCompared with conventional NRC solution, the liposome form led to a 2.76-fold higher Cmax and a 5.32-fold higher AUC0–24h in plasma after a bolus injection of 40 mg/kg. In healthy mouse brain, it caused a significant elevation of Cmax (2.93-fold) and AUC0.25–24h (2.68-fold). In cerebral ischemia model mice, NRC liposomes increased the drug concentration at 1 and 6 h post-ischemia, increased tissue NAD+ and ATP levels, reduced infarct volume (by a further decrease of 35.4%), ensured neuronal survival, attenuated glial activation, and significantly improved behavioral recovery compared with conventional NRC treatment. ConclusionLiposome loading enhances the brain distribution and therapeutic effects of NRC, which strengthens its possibility for clinical translation and neurorestoration in stroke.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

HULL’S MAGIC BOX: BUILDING THE CASE FOR CLINICAL TRIALS USING ARGININE METHYLATION INHIBITORS AGAINST GBM

Abstract AIMS • Assess/evaluate transcriptomic changes in GBM, maintained on a microfluidics system, in response to treatment with arginine methylation inhibitor GSK3368715, currently in clinical trials, to identify novel therapeutic strategies, synergising with personalised patient care and precision medicine • Investigate molecular differences between healthy brain and GBM maintained on-chip in response to GSK3368715, including proliferation, apoptosis and splicing. METHOD GBM biopsies from Hull Royal Infirmary, or healthy mouse brain tissues, were maintained on-chip for 8- days and perfused with GSK3368715-treated media, at 3 μl/min, mimicking the in vivo environment. RNA- sequencing determined thousands of differentially expressed genes and alternative splicing (AS) events resulting from GSK3368715 treatment. Immunohistochemistry displayed the effect of GSK3368715 on healthy mouse brain tissue to ascertain the specificity of the arginine methylation inhibitor in leading to GBM apoptosis. RESULTS RNA-sequencing showed highly significant GO-term-enrichment in ribosome and translation pathways, suggesting decreased protein synthesis capacity after GSK3368715 treatment. Additionally, several hundreds of genes were found to be undergoing AS, compatible with a mechanism where changes in the arginine methylation pattern of spliceosome member FUS, upon GSK3368715 treatment, contribute to AS. Of these events, 136 (more than expected by chance, χ2 = 7.16, p = 0.007) were known FUS splicing targets. GO-term-enrichment of these AS events was found in DNA damage and cell death . Together these results are consistent with immunohistological findings that GSK3368715 causes apoptosis in GBM-on-chip tissue, in a specific manner. CONCLUSIONS Our results highlight an exciting potential therapeutic target for GBM, via induction of alternative splicing path- ways, through arginine methylation inhibition with GSK3368715.

Immunohistochemical distribution of secretagogin in the mouse brain.

Calcium is essential for the correct functioning of the central nervous system, and calcium-binding proteins help to finely regulate its concentration. Whereas some calcium-binding proteins such as calmodulin are ubiquitous and are present in many cell types, others such as calbindin, calretinin, and parvalbumin are expressed in specific neuronal populations. Secretagogin belongs to this latter group and its distribution throughout the brain is only partially known. In the present work, the distribution of secretagogin-immunopositive cells was studied in the entire brain of healthy adult mice. Adult male C57BL/DBA mice aged between 5 and 7 months were used. Their whole brain was sectioned and used for immunohistochemistry. Specific neural populations were observed in different zones and nuclei identified according to Paxinos mouse brain atlas. Labelled cells were found with a Golgi-like staining, allowing an excellent characterization of their dendritic and axonal arborizations. Many secretagogin-positive cells were observed along different encephalic regions, especially in the olfactory bulb, basal ganglia, and hypothalamus. Immunostained populations were very heterogenous in both size and distribution, as some nuclei presented labelling in their entire extension, but in others, only scattered cells were present. Secretagogin can provide a more complete vision of calcium-buffering mechanisms in the brain, and can be a useful neuronal marker in different brain areas for specific populations.

β-Amyloid targeting nanodrug for neuron-specific delivery of nucleic acids in Alzheimer's disease mouse models

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood–brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aβ) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Localized Increased Permeability of Blood-Brain Barrier for Antibody Conjugates in the Cuprizone Model of Demyelination.

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Trehalose-Guanosine Glycopolymer Hydrogels Direct Adaptive Glia Responses in CNS Injury.

Neural tissue damaged after central nervous system (CNS) injury does not naturally regenerate but is instead replaced by non-neural fibrotic scar tissue that serves no neurological function. Scar-free repair to create a more permissive environment for regeneration requires altering the natural injury responses of glial cells. In this work, glycopolymer-based supramolecular hydrogels are synthesized to direct adaptive glia repair after CNS injury. Combining poly(trehalose-co-guanosine) (pTreGuo) glycopolymers with free guanosine (fGuo) generates shear-thinning hydrogels through stabilized formation of long-range G-quadruplex secondary structures. Hydrogels with smooth or granular microstructures and mechanical properties spanning three orders of magnitude are produced through facile control of pTreGuo hydrogel composition. Injection of pTreGuo hydrogels into healthy mouse brains elicits minimal stromal cell infiltration and peripherally derived inflammation that is comparable to a bioinert methyl cellulose benchmarking material. pTreGuo hydrogels alter astrocyte borders and recruit microglia to infiltrate and resorb the hydrogel bulk over 7d. Injections of pTreGuo hydrogels into ischemic stroke alter the natural responses of glial cells after injury to reduce the size of lesions and increase axon regrowth into lesion core environments. These results support the use of pTreGuo hydrogels as part of neural regeneration strategies to activate endogenous glia repair mechanisms.

Functional ultrasound detects frequency-specific acute and delayed S-ketamine effects in the healthy mouse brain.

S-ketamine has received great interest due to both its antidepressant effects and its potential to induce psychosis when administered subchronically. However, no studies have investigated both its acute and delayed effects using in vivo small-animal imaging. Recently, functional ultrasound (fUS) has emerged as a powerful alternative to functional magnetic resonance imaging (fMRI), outperforming it in sensitivity and in spatiotemporal resolution. In this study, we employed fUS to thoroughly characterize acute and delayed S-ketamine effects on functional connectivity (FC) within the same cohort at slow frequency bands ranging from 0.01 to 1.25 Hz, previously reported to exhibit FC. We acquired fUS in a total of 16 healthy C57/Bl6 mice split in two cohorts (n = 8 received saline, n = 8 S-ketamine). One day after the first scans, performed at rest, the mice received the first dose of S-ketamine during the second measurement, followed by four further doses administered every 2 days. First, we assessed FC reproducibility and reliability at baseline in six frequency bands. Then, we investigated the acute and delayed effects at day 1 after the first dose and at day 9, 1 day after the last dose, for all bands, resulting in a total of four fUS measurements for every mouse. We found reproducible (r > 0.9) and reliable (r > 0.9) group-average readouts in all frequency bands, only the 0.01-0.27 Hz band performing slightly worse. Acutely, S-ketamine induced strong FC increases in five of the six bands, peaking in the 0.073-0.2 Hz band. These increases comprised both cortical and subcortical brain areas, yet were of a transient nature, FC almost returning to baseline levels towards the end of the scan. Intriguingly, we observed robust corticostriatal FC decreases in the fastest band acquired (0.75 Hz-1.25 Hz). These changes persisted to a weaker extent after 1 day and at this timepoint they were accompanied by decreases in the other five bands as well. After 9 days, the decreases in the 0.75-1.25 Hz band were maintained, however no changes between cohorts could be detected in any other bands. In summary, the study reports that acute and delayed ketamine effects in mice are not only dissimilar but have different directionalities in most frequency bands. The complementary readouts of the employed frequency bands recommend the use of fUS for frequency-specific investigation of pharmacological effects on FC.

A longitudinal microstructural MRI dataset in healthy C57Bl/6 mice at 9.4 Tesla

Multimodal microstructural MRI has shown increased sensitivity and specificity to changes in various brain disease and injury models in the preclinical setting. Here, we present an in vivo longitudinal dataset, including a subset of ex vivo data, acquired as control data and to investigate microstructural changes in the healthy mouse brain. The dataset consists of structural T2-weighted imaging, magnetization transfer ratio and saturation imaging, and advanced quantitative diffusion MRI (dMRI) methods. The dMRI methods include oscillating gradient spin echo (OGSE) dMRI and microscopic anisotropy (μA) dMRI, which provide additional insight by increasing sensitivity to smaller spatial scales and disentangling fiber orientation dispersion from true microstructural changes, respectively. The technical skills required to analyze microstructural MRI data are complex and include MRI sequence development, acquisition, and computational neuroimaging expertise. Here, we share unprocessed and preprocessed data, and scalar maps of quantitative MRI metrics. We envision utility of this dataset in the microstructural MRI field to develop and test biophysical models, methods that model temporal brain dynamics, and registration and preprocessing pipelines.

Radiosynthesis and preclinical evaluation of [11C]SNX-ab as an Hsp90α,β isoform-selective PET probe for in vivo brain and tumour imaging

BackgroundThe molecular chaperone, Hsp90, is a key player in the protein quality control system that maintains homeostasis under cellular stress conditions. It is a homodimer with ATP-dependent activity, and is a prominent member of the chaperone machinery that stabilizes, matures and (re)folds an extensive list of client proteins. Hsp90 occurs as four isoforms, cytosolic Hsp90α and Hsp90β, mitochondrial TRAP1 and Grp94 present in the endoplasmic reticulum. An aberrant role of Hsp90 has been attributed to several cancers and neurodegenerative disorders. Consequently, Hsp90 has emerged as an attractive therapeutic target. However, pan-Hsp90 inhibition often leads to detrimental dose-limiting toxicities. Novel strategies for Hsp90-targeted therapy intend to avoid this by using isoform-specific Hsp90 inhibition. In this respect, the radiosynthesis of carbon-11 labeled SNX-ab was developed and [11C]SNX-ab was evaluated as a Hsp90α,β isoform-selective PET probe, which could potentially allow to quantify in vivo Hsp90α,β expression.Results[11C]SNX-ab was synthesized with excellent radiochemical yields of 45% and high radiochemical purity (> 98%). In vitro autoradiography studies on tissue slices of healthy mouse brain, mouse B16.F10 melanoma and U87 glioblastoma using homologous (SNX-ab, SNX-0723) and heterologous (Onalespib and PU-H71) Hsp90 inhibitors demonstrated only limited reduction of tracer binding, indicating that the binding of [11C]SNX-ab was not fully Hsp90-specific. Similarly, [11C]SNX-ab binding to U87 cells was not efficiently inhibited by Hsp90 inhibitors. Ex vivo biodistribution studies in healthy mice revealed limited brain exposure of [11C]SNX-ab and predominantly hepatobiliary clearance, which was confirmed by in vivo full-body dynamic µPET studies.ConclusionOur results suggest that [11C]SNX-ab is not an ideal probe for in vivo visualization and quantification of Hsp90α/β expression levels in tumour and brain. Future research in the development of next-generation Hsp90 isoform-selective PET tracers is warranted to dissect the role played by each isoform towards disease pathology and support the development of subtype-specific Hsp90 therapeutics.

Simultaneous dynamic glucose-enhanced (DGE) MRI and fiber photometry measurements of glucose in the healthy mouse brain

Glucose is the main energy source in the brain and its regulated uptake and utilization are important biomarkers of pathological brain function. Glucose Chemical Exchange Saturation Transfer (GlucoCEST) and its time-resolved version Dynamic Glucose-Enhanced MRI (DGE) are promising approaches to monitor glucose and detect tumors, since they are radioactivity-free, do not require 13C labeling and are is easily translatable to the clinics. The main principle of DGE is clear. However, what remains to be established is to which extent the signal reflects vascular, extracellular or intracellular glucose. To elucidate the compartmental contributions to the DGE signal, we coupled it with FRET-based fiber photometry of genetically encoded sensors, a technique that combines quantitative glucose readout with cellular specificity. The glucose sensor FLIIP was used with fiber photometry to measure astrocytic and neuronal glucose changes upon injection of D-glucose, 3OMG and L-glucose, in the anaesthetized murine brain. By correlating the kinetic profiles of the techniques, we demonstrate the presence of a vascular contribution to the signal, especially at early time points after injection. Furthermore, we show that, in the case of the commonly used contrast agent 3OMG, the DGE signal actually anticorrelates with the glucose concentration in neurons and astrocytes.

Pharmacokinetics and Tissue Distribution of Coumarins from Tagetes lucida in an LPS-Induced Neuroinflammation Model.

Tagetes lucida has been widely used as a folk remedy in illnesses associated with the central nervous system and inflammatory ailments. Among the chemical compounds that stand out in the plant against these conditions are coumarins, such as 7-O-prenylscopoletin (PE), scoparone (SC), dimethylfraxetin (DF), herniarin (HR), and 7-O-prenylumbelliferone (PU), considered potential anti-neuroinflammatory compounds. Therefore, the relationship between the therapeutic effect and the dose can be evaluated through pharmacokinetic-pharmacodynamic (PK-PD) studies under a model of neuroinflammation induced by lipopolysaccharide (LPS). Nonetheless, accomplishing those studies requires an accurate and robust analytical method for the detection of these compounds in different biological matrices of interest. Due to the above, in the present study, a bioanalytical method was established by HPLC-DAD-UV for the simultaneous quantification of the coumarins present in the hexane extract of T. lucida, which was able to determine the temporal concentration profiles of each of the coumarins in the plasma, brain, kidney, and spleen samples of healthy and damaged mice. Coumarins showed an increase in plasma concentrations of up to three times in the neuroinflammation model, compared to healthy mice, so it was possible to quantify the therapeutic agents in the main target organ, the brain. The ability of compounds to cross the blood-brain barrier is an advantage in the treatment of diseases associated with neuroinflammation processes that can be studied in future PK-PD evaluations.

Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic review and isolation methods' evaluation.

The aim of our study was to systematically analyze the literature for published flow cytometry protocols for microglia isolation and compare their effectiveness in terms of microglial yield, including our own protocol using sucrose for myelin removal and accutase for enzymatic digestion. For systematic review, the PubMed was searched for the terms "flow cytometry," "microglia," "brain," and "mice." Three different myelin removal methods (Percoll, sucrose, and no removal) and five protocols for enzymatic digestion (accutase, dispase II, papain, trypsin, and no enzymatic digestion) were tested for the effectiveness of microglia (CD11b+CD45int cell population) isolation from the adult mouse brain using flow cytometry. Qualitative analysis of the 32 selected studies identified three most commonly used myelin removal protocols: Percoll, the use of myelin removal kit, and no removal. Nine enzymatic digestion protocols were identified, from which we selected dispase II, papain, trypsin, and no enzymatic digestion. A comparison of these myelin removal methods and digestion protocols showed the Percoll method to be preferable in removal of non-immune cells, and superior to the use of sucrose which was less effective in removal of non-immune cells, but resulted in a comparable microglial yield to Percoll myelin removal. Digestion with accutase resulted in one of the highest microglial yields, all while having the lowest variance among tested protocols. The proposed protocol for microglia isolation uses Percoll for myelin removal and accutase for enzymatic digestion. All tested protocols had different features, and the choice between them can depend on the individual focus of the research.

Development and Validation of a HPLC-MS/MS Method to Measure Nifuroxazide and Its Application in Healthy and Glioblastoma-Bearing Mice.

Nifuroxazide (NAZ), a nitrofuran derivative used to treat diarrhea, has been recently shown to possess anticancer activity. However, its pharmacokinetic profile is poorly known. The pharmacokinetic profile of NAZ was thus investigated in mice using a newly developed method based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). We determined the concentrations of NAZ in the plasma and brain tissue of mice treated with the drug. The method proved to be specific, reproducible, precise, and accurate. It also demonstrated high sensitivity, reaching an LOQ in the order of ppb for both matrices, using samples of 100 µL or 0.2 g. The new HPLC–MS/MS assay was successfully applied to study the pharmacokinetics of NAZ after chronic intraperitoneal administration in mice at a dose of 30 mg/kg. One hour after treatment, plasma concentrations of NAZ were in the range of 336–2640 ng/mL. Moreover, unlike the brains of healthy mice or those with healed mechanical injuries, we found that NAZ was able to cross the injured blood–brain barrier of tumor-infiltrated brains. Thus, following i.p. administration, NAZ reaches systemic levels suitable for testing its efficacy in preclinical models of glioblastoma. Overall, these pharmacokinetic data provide robust evidence supporting the repositioning of NAZ as an antitumor drug.