Intracerebroventricular Injection Research Articles

New MiniPromoter Ple389 (ADORA2A) drives selective expression in medium spiny neurons in mice and non-human primates.

Compact cell type-specific promoters are important tools for basic and preclinical research and clinical delivery of gene therapy. In this work, we designed novel MiniPromoters to target D1 and D2 type dopaminoceptive medium spiny neurons in the striatum by manually identifying candidate regulatory regions or employing the OnTarget webserver. We then empirically tested the designs in rAAV-PHP.B for specificity and robustness in three systems: intravenous injection in mice, intracerebroventricular injection in mice, and intracerebroventricular injection in non-human primates. Twelve MiniPromoters were designed from eight genes: seven manually and five using OnTarget. When delivered intravenously in mice, three MiniPromoters demonstrated highly selective expression in the striatum, with Ple389 (ADORA2A) showing high levels of dopamine D2-receptor cell co-localization. The same three MiniPromoters also displayed enriched expression in the striatum when delivered intracerebroventricularly in mice with high levels of DARPP32 co-localization. Finally, Ple389 (ADORA2A) was intracerebroventricularly injected in non-human primates and showed enriched expression in the striatum as in the mouse. Ple389 (ADORA2A) demonstrated expression in the medium spiny neurons in all three systems tested and exhibited the highest level of D2-MSNs and DARPP32 co-labeling in mice, demonstrating its potential as a tool for gene therapy approaches for Parkinson and Huntington disease treatment.

Cannabidiol Plays a Modulatory Function on the Methamphetamine-Induced Reward Through Hippocampal D2-Like Dopamine Receptors.

Methamphetamine (METH), a stimulant that is extremely addictive, directly affects the central nervous system. METH's abuse and consumption are directly linked to mental illnesses, psychosis, and behavioral and cognitive impairments. It may disrupt the reward system and dopaminergic transmission. METH's rewarding qualities are associated with a rise in dopamine. Additionally, cannabidiol (CBD), one of the primary cannabinoid components of the cannabis plant, significantly affects dopaminergic transmission and may aid in reward- and addiction-related behaviors. To shed light on the role of the D2-like dopamine receptor (D2R) in the hippocampal dentate gyrus (DG), the present study examined the effects of CBD on the acquisition and expression of the conditioned place preference (CPP) induced by METH. The function of D2R was ascertained by delivering Sulpiride microinjections, as a D2R antagonist Sulpiride (0.25, 1, and 4μg/0.5 μL DMSO12%) into the DG. Moreover, an intracerebroventricular injection of CBD at a dose of 10μg/5 μL for CPP acquisition and 50μg/5 μL for CPP expression was given to rats. According to the current research, CBD dramatically reduced the acquisition and expression of CPP resulting from METH. However, Sulpiride suppressed the effect of CBD on METH-induced CPP acquisition and expression, with a greater impact on expression experiments. Ultimately, this study proposed that the expression experiment of METH-induced CPP appears to be heavily dependent on D2R in the DG.

Cannula Implantation Reduces the Severity of the Beta Amyloid Effect on Peroxidized Lipids and Glutathione Levels in the Brain of BALB/c Mice

Sporadic Alzheimer’s disease (sAD) is the most common of neurodegenerative disorders. The lack of effective therapy indicates that the mechanisms of sAD development remain poorly understood. To investigate this pathology in animals, intracerebroventricular injection of β-amyloid peptide (Aβ) using a Hamilton syringe, either during stereotactic surgery or through a pre-implanted cannula, is used. In this study, we analyzed the effect of chronic cannula implantation on the severity of Aβ effects at the behavioral, histological, and biochemical levels. The results showed that the local damage to neural tissue caused by cannulation has no bearing on the effect of Aβ on animal behavior and the microglial parameters of the unilateral hippocampus two weeks after the Aβ administration. However, cannula implantation fundamentally modifies some biochemical markers of the oxidative stress that occurs in the brain tissue in response to Aβ administration. Thus, the presence of a cannula reduces the severity of the Aβ impact on the levels of peroxidized lipids and glutathione two- and 10-fold, respectively. It is important to note that the detected changes are chronic and systemic. This is known because the homogenate of the entire contralateral (in relation to the cannula implantation site) hemisphere was analyzed, and the analysis was performed two weeks after implantation. At the same time, cannulation does not affect the rate of reactive oxygen species production. The obtained data indicate that chronic implantation of a cannula into the brain of experimental animals fundamentally distorts some parameters of oxidative stress in the neural tissue, which are widely used to assess the severity of experimental Alzheimer’s-type diseases.

40 Hz light preserves synaptic plasticity and mitochondrial function in Alzheimer’s disease model

Alzheimer’s disease (AD) is the most prevalent type of dementia. Its causes are not fully understood, but it is now known that factors like mitochondrial dysfunction, oxidative stress, and compromised ion channels contribute to its onset and progression. Flickering light therapy has shown promise in AD treatment, though its mechanisms remain unclear. In this study, we used a rat model of streptozotocin (STZ)-induced AD to evaluate the effects of 40 Hz flickering light therapy. Rats received intracerebroventricular (ICV) STZ injections, and 7 days after, they were exposed to 40 Hz flickering light for 15 min daily over seven days. Cognitive and memory functions were assessed using Morris water maze, novel object recognition, and passive avoidance tests. STZ-induced AD rats exhibited cognitive decline, elevated reactive oxygen species, amyloid beta accumulation, decreased serotonin and dopamine levels, and impaired mitochondrial function. However, light therapy prevented these effects, preserving cognitive function and synaptic plasticity. Additionally, flickering light restored mitochondrial metabolites and normalized ATP-insensitive mitochondrial calcium-sensitive potassium (mitoBKCa) channel activity, which was otherwise downregulated in AD rats. Our findings suggest that 40 Hz flickering light therapy could be a promising treatment for neurodegenerative disorders like AD by preserving synaptic and mitochondrial function.

In silico and in vivo evaluations of fisetin and fisetin-loaded nanosuspension on monoamine oxidase inhibition in Aβ(25-35) induced dementia in mice model

BackgroundAmyloid beta (Aβ) plaques on the extracellular matrix and intracellular neurofibrillary tangles comprise the key indicative pathology of Alzheimer's disease (AD). Fisetin, an antioxidant bioactive compound having pharmacotherapeutic effects is applied by traditional Chinese medicine (TCM) such as Toxicodendron vernicifum (Chinese lacquer tree), Cotinus coggygria Scop, Gan Shuang granulates and formula of Acacia catechu-Scutellariae Radix. Nevertheless, fisetin has constraints such as low oral bioavailability, insignificant aqueous solubility and high hepatic metabolisms. ObjectiveThis investigation aimed to envisage the effects of fisetin and its optimised nanoformulation on Aβ(25-35) desirous neurotoxicity in mice through deciphering inhibitory actions against monoamine oxidase A and B (MAO-A and B) enzymes following molecular docking. MethodsMolecular docking with MAO-A and B enzymes were accomplished by AMDock's integrated AutoDock Tools (ADT) scripts. For in vivo studies, fisetin nanosuspension was prepared by nanoprecipitation method and evaluated for standard characterization. 10 and 20 mg/kg of fisetin and 10 mg/kg of fisetin nanosuspension were given once daily to mice for 21 days. On the 15th day, the mice were challenged with Aβ(25–35) by intracerebroventricular injection (ICV) and behavioural tests (open field and elevated plus maze) were performed on the 20th day. Biochemical and histology were examined in brain tissues. ResultsFisetin docked to the catalytic positions of MAO-A and B, unveiling good binding scores and molecular interactions with amino acid residues for inhibition activities. Fisetin nanosuspension has average particle size (225.4 ± 2.95 nm) with low polydispersity index (0.19) and standard zeta potential (-19.13 ± 1.17 mV). Findings showed that fisetin increased locomotor activity and reduced anxiety-like behaviour. Fisetin and its nanosuspension significantly reduced the concentration of MAO-A (P < 0.01) and MAO-B enzymes suggesting a potential neuroprotection effect in Aβ peptide-induced amnesia in mice. ConclusionFisetin with optimized bioavailability, effectively exhibits neuroprotection through molecular interactions of MAO enzymes. Further investigations affidavits the neuroprotection through bidirectional pathways related to biogenic amines and their deamination on Aβ stress conditions.

Allele-Specific Editing of a Dominant SCN8A Epilepsy Variant Protects against Seizures and Lethality in a Murine Model.

Developmental and epileptic encephalopathies (DEEs) can result from dominant, gain of function variants of neuronal ion channels. More than 450 de novo missense variants of the sodium channel gene SCN8A have been identified in individuals with DEE. We studied a mouse model carrying the patient Scn8a variant p.Asn1768Asp. An AAV-PHP.eB virus carrying an allele-specific single guide RNA (sgRNA) was administered by intracerebroventricular injection. Cas9 was provided by an inherited transgene. Allele-specific disruption of the reading frame of the pathogenic transcript generated out-of-frame indels in 1/4 to 1/3 of transcripts throughout the brain. This editing efficiency was sufficient to rescue lethality and seizures. Neuronal hyperexcitability was reduced in cells expressing the virus. The data demonstrate efficient allele-specific editing of a dominant missense variant and support the feasibility of allele-specific therapy for DEE epilepsy. ANN NEUROL 2024;96:958-969.

Fructus Arctii Mitigates Depressive Disorder via the Let-7e-Modulated Toll-Like Receptor (TLR) Signaling Pathway.

Depressive disorder is a common and serious public health challenge globally. Fructus arctii is a traditional medicinal plant ingredient with diverse pharmacological effects. This study aimed to investigate the therapeutic potential of Fructus arctii in alleviating depressive-like behaviors. We established a chronic unpredictable mild stress (CUMS)-induced depression mouse model to assess the antidepressant effects of Fructus arctii. BV2 cells treated with lipopolysaccharide (LPS) were used to mimic neuronal damage. Behavioral tests, including the sucrose preference test, tail-suspension test, and forced swim test, were conducted to evaluate the impact of Fructus arctii on depressive-like behaviors. Let-7e expression was detected by RT-qPCR, and TLR4 signaling pathway activation was evaluated by western blot analysis, which also assessed the inflammatory response by measuring levels of IL-6, IL-1β, MCP-1, TNF-α, and iNOS. Immunohistological analysis was conducted to detect the expression of microglia markers. Luciferase reporter assays verified the interaction between let-7e and TLR4. Fructus arctii administration effectively alleviated depressive-like behaviors induced by CUMS in mice, as evidenced by improved sucrose preference and reduced immobility time in behavioral tests. Mechanistically, Fructus arctii reversed the CUMS-induced downregulation of let-7e and upregulation of TLR4 and MyD88 protein levels in mice hippocampus tissues. In addition, Fructus arctii suppressed microglial activation and reduced the levels of inflammatory factors by upregulating let-7e. Let-7e was verified to bind to TLR4, thereby negatively regulating its expression. TLR4 overexpression reversed the suppressive effect of let-7e upregulation on inflammatory reactions and microglial activation. Furthermore, intracerebroventricular injection of let-7e agomiR alleviated depressive-like behavior and inhibited microglial activation in vivo. In summary, Fructus arctii mitigates depression by regulating the let-7e/TLR4/MyD88 pathway, offering new insights into potential depression therapies.

Successful Treatment of MDRAcinetobacter baumannii Meningitis in a Young Adult Patient With Intraventricular and Intravenous Polymyxin B‐Tigecycline Based Combinations: A Case Report

ABSTRACTMultiple drug resistance to Acinetobacter baumannii infection treatment is a great challenge for neuro‐intensivists due to poor drug penetration through the blood–brain barrier (BBB). Fortunately, the intraventricular administration of polymyxin‐B and tigecycline seems to be effective; there are few case reports demonstrating the effectiveness of such treatments. Here, we report the case of a 24‐year‐old male who presented with fever and neck rigidity after intracranial drainage following lung infection caused by MDR Acinetobacter baumannii. Due to the presence of turbid CSF, the administration of the intrathecal (ITH) route polymyxin‐B and tigecycline is not possible. In this situation, the neuro‐intensivist decided to start intraventricular tigecycline and polymyxin‐B administration along with IV tigecycline and polymyxin‐B via the intraventricular route, which was feasible because the patient had an external ventricular drain (EVD) due to obstructive hydrocephalus caused by the neurosurgeon after excision of the tumor.

Thyroid Hormone Supplementation Restores Cognitive Deficit, Insulin Signaling, and Neuroinflammation in the Hippocampus of a Sporadic Alzheimer's-like Disease Rat Model.

Thyroid hormones play a crucial role in the development of the central nervous system and are considered pivotal to cognitive functions in the adult brain. Recently, thyroid dysfunction has been associated with Alzheimer's disease. The aim of this study was to assess the neuroprotective effects of triiodothyronine (T3) on insulin signaling, neuroinflammation, apoptosis, and cognitive function in a streptozotocin (STZ)-induced sporadic Alzheimer's disease-like model. Male Wistar rats underwent stereotaxic surgery for intracerebroventricular injections of streptozotocin (STZ; 2 mg/kg) or vehicle in the lateral ventricles to induce an AD-like model. The animals received a daily dose of 1.5 μg of T3/100 g body weight or the same volume of vehicle for 30 days and were subdivided into four experimental groups: (1) animals receiving citrate treated with saline (Control = CTL); (2) animals receiving citrate treated with T3 (T3); (3) animals receiving STZ treated with saline (STZ); and (4) animals receiving STZ treated with T3 (STZ + T3). The novel object recognition test was used to measure cognitive function. Serum analysis, real-time RT-PCR, immunohistochemistry, and immunoblotting analyses were also carried out. Our results demonstrated that T3 treatment reversed cognitive impairment and increased Akt and GSK3 phosphorylation in the treated group, while also reducing microglial activation (Iba-1) and GFAP expression (reactive astrocytes), along with TNF-α, IL-6, and IL-1β levels in the hippocampus. Additionally, T3 treatment increased levels of the anti-apoptotic protein Bcl-2 and reduced the expression of the pro-apoptotic protein BAX in the hippocampus. Our study demonstrated that T3 could potentially protect neurons in an AD model induced by STZ.

Oxytocin alleviates high-fat diet-induced anxiety by decreasing glutamatergic synaptic transmission in the ventral dentate gyrus in adolescent mice

A high-fat diet (HFD)-induced obesity is associated with mental disorders in adolescence. However, the mechanisms underlying these associations remain unclear. In this study, we hypothesized that synaptic remodeling occurs in the ventral hippocampus (vHP) of obese mice. To investigate this, we established a postnatal model of HFD-induced obesity in mice and observed increased body weight, elevated plasma luteinizing hormone and testosterone levels, premature puberty, and enhanced anxiety-like behavior in male subjects. We also examined the effect of HFD on the c-Fos protein expression in the ventral dentate gyrus (vDG) and explored the influence of intracerebroventricular (i.c.v) oxytocin injections on HFD-induced anxiety. Our results indicated an increase in c-Fos-positive cells in the vDG following HFD consumption. Additionally, we recorded the spontaneous synaptic activity of miniature excitatory postsynaptic currents (mEPSCs) in the vDG. Notably, HFD resulted in an elevated mEPSC frequency without affecting mEPSC amplitude. Subsequently, investigations demonstrated that i.c.v oxytocin injections reversed anxiety-like behavior induced by HFD. Moreover, the application of oxytocin in a bath solution reduced the mEPSC frequency in the vDG. These findings suggest that postnatal HFD intake induces synaptic dysfunction in the vDG, associated with the hyperactivity of vDG neurons, potentially contributing to the anxiety-like behavior in juvenile obesity.

Stem Cells Treatment for Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) refers to bleeding in the subarachnoid space, which is a serious neurologic emergency. However, the treatment effects of SAH are limited. In recent years, stem cell (SC) therapy has gradually become a very promising therapeutic method and advanced scientific research area for SAH. The SCs used for SAH treatment are mainly bone marrow mesenchymal stem cells (BMSCs), umbilical cord mesenchymal stem cells (hUC-MSCs), dental pulp stem cells (DPSCs), neural stem cells (NSCs)/neural progenitor cell (NPC), and endothelial progenitor cell (EPC). The mechanisms mainly included differentiation and migration of SCs for tissue repair; alleviating neuronal apoptosis; anti-inflammatory effects; and blood-brain barrier (BBB) protection. The dosage of SCs was generally 106 orders of magnitude. The administration methods included intravenous injection, nasal, occipital foramen magnum, and intraventricular administration. The administration time is generally 1 hour after SAH modeling, but it may be as late as 24 hours or 6 days. Existing studies have confirmed the neuroprotective effect of SCs in the treatment of SAH. SC has great potential application value in SAH treatment, a few case reports have provided support for this. However, the relevant research is still insufficient and there is still a lack of clinical research on the SC treatment for SAH to further evaluate the effectiveness and safety before it can go from experiment to clinical application.

Widespread Gene Editing in the Brain via In Utero Delivery of mRNA Using Acid-Degradable Lipid Nanoparticles.

In utero gene editing with mRNA-based therapeutics has the potential to revolutionize the treatment of neurodevelopmental disorders. However, a critical bottleneck in clinical application has been the lack of mRNA delivery vehicles that can efficiently transfect cells in the brain. In this report, we demonstrate that in utero intracerebroventricular (ICV) injection of densely PEGylated lipid nanoparticles (ADP-LNPs) containing an acid-degradable PEG-lipid can safely and effectively deliver mRNA for gene editing enzymes to the fetal mouse brain, resulting in successful transfection and editing of brain cells. ADP-LNPs containing Cre mRNA transfected 30% of the fetal brain cells in Ai9 mice and had no detectable adverse effects on fetal development and postnatal growth. In addition, ADP-LNPs efficiently transfected neural stem and progenitor cells in Ai9 mice with Cre mRNA, which subsequently proliferated and caused over 40% of the cortical neurons and 60% of the hippocampal neurons to be edited in treated mice 10 weeks after birth. Furthermore, using Angelman syndrome, a paradigmatic neurodevelopmental disorder, as a disease model, we demonstrate that ADP-LNPs carrying Cas9 mRNA and gRNA induced indels in 21% of brain cells within 7 days postpartum, underscoring the precision and potential of this approach. These findings demonstrate that LNP/mRNA complexes have the potential to be a transformative tool for in utero treatment of neurodevelopmental disorders and set the stage for a frontier in treating neurodevelopmental disorders that focuses on curing genetic diseases before birth.

Long-term reprogramming of primed microglia after moderate inhibition of CSF1R signaling.

In acute neuroinflammation, microglia activate transiently, and return to a resting state later on. However, they may retain immune memory of such event, namely priming. Primed microglia are more sensitive to new stimuli and develop exacerbated responses, representing a risk factor for neurological disorders with an inflammatory component. Strategies to control the hyperactivation of microglia are, hence, of great interest. The receptor for colony stimulating factor 1 (CSF1R), expressed in myeloid cells, is essential for microglia viability, so its blockade with specific inhibitors (e.g. PLX5622) results in significant depletion of microglial population. Interestingly, upon inhibitor withdrawal, new naïve microglia repopulate the brain. Depletion-repopulation has been proposed as a strategy to reprogram microglia. However, substantial elimination of microglia is inadvisable in human therapy. To overcome such drawback, we aimed to reprogram long-term primed microglia by CSF1R partial inhibition. Microglial priming was induced in mice by acute neuroinflammation, provoked by intracerebroventricular injection of neuraminidase. After 3-weeks recovery, low-dose PLX5622 treatment was administrated for 12 days, followed by a withdrawal period of 7 weeks. Twelve hours before euthanasia, mice received a peripheral lipopolysaccharide (LPS) immune challenge, and the subsequent microglial inflammatory response was evaluated. PLX5622 provoked a 40%-50% decrease in microglial population, but basal levels were restored 7 weeks later. In the brain regions studied, hippocampus and hypothalamus, LPS induced enhanced microgliosis and inflammatory activation in neuraminidase-injected mice, while PLX5622 treatment prevented these changes. Our results suggest that PLX5622 used at low doses reverts microglial priming and, remarkably, prevents broad microglial depletion.

WIF-1 contributes to lupus-induced neuropsychological deficits via the CRYAB/STAT4-SHH axis

BackgroundNeuropsychiatric systemic lupus erythematosus (NPSLE) often manifests as cognitive deterioration, with activated microglia and blood-brain barrier (BBB) disruption implicated in these neurological complications. Wnt-inhibitory factor-1 (WIF-1), a secreted protein, has been detected in the cerebrospinal fluid (CSF) of NPSLE patients. However, the contribution of WIF-1 in contributing to lupus cognitive impairment remains poorly understood.MethodsUsing MRL/MpJ-Faslpr (MRL/lpr) lupus-prone mice and TLR7 agonist imiquimod (IMQ)-induced lupus mice, recombinant WIF-1 protein (rWIF-1) and adeno-associated virus (AAV) encoding sh-WIF-1 were administered via intracerebroventricular injection. Behavioral tests, histopathological examinations, flow cytometry, and molecular biology techniques were employed to investigate the underlying mechanisms.ResultsMicroinjection of rWIF-1 exacerbated cognitive deficits and mood abnormalities, increased BBB leakage and neuronal degeneration, and caused aberrant activation of microglia and synaptic pruning in the hippocampus. Conversely, lupus mice injected with AAV-shWIF-1 exhibited significant remission. In vitro, rWIF-1 induced overactivation of microglia with an increased CD86+ pro-inflammatory subpopulation, upregulated phagocytic activity, and excessive synaptic engulfment, contributing to increased BBB permeability. Furthermore, WIF-1 exerted its biological effects through the CRYAB/STAT4 pathway, transcriptionally decreasing SHH production. We also identified that symmetric dimethylarginine (SDMA) could alleviate rWIF-1-induced microglial activation and BBB damage, thereby restoring SHH levels.ConclusionsIn conclusion, WIF-1 exacerbates lupus-induced cognitive dysfunction in mice by triggering aberrant microglial activation and BBB disruption through the CRYAB/STAT4-SHH axis, highlighting the potential therapeutic effects of SDMA for the treatment of NPSLE.

Methionine restriction alleviates diabetes-associated cognitive impairment via activation of FGF21

Glucose metabolism disturbances may result in diabetes-associated cognitive decline (DACI). Methionine restriction (MR) diet has emerged as a potential dietary strategy for managing glucose homeostasis. However, the effects and underlying mechanisms of MR on DACI have not been fully elucidated. Here, we found that a 13-week MR (0.17 % methionine, w/w) intervention starting at 8 weeks of age improved peripheral insulin sensitivity in male db/db mice, a model for type 2 diabetes. Notably, MR significantly improved working as well as long-term memory in db/db mice, accompanied by increased PSD-95 level and reduced neuroinflammatory factors, malondialdehyde (MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG). We speculate that this effect may be mediated by MR activating hepatic fibroblast growth factor 21 (FGF21) and the brain FGFR1/AMPK/GLUT4 signaling pathway to enhance brain glucose metabolism. To further delineate the mechanism, we used intracerebroventricular injection of adeno-associated virus to specifically knock down FGFR1 in the brain to verify the role of FGFR1 in MR-mediated DACI. It was found that the positive effects of MR on DACI were offset, reflected in decreased cognitive function, impaired synaptic plasticity, upregulated neuroinflammation, and balanced enzymes regulating reactive oxygen species (Sod1, Sod2, Nox4). Of note, the FGFR1/AMPK/GLUT4 signaling pathway and brain glucose metabolism were inhibited. In summary, our study demonstrated that MR increased peripheral insulin sensitivity, activated brain FGFR1/AMPK/GLUT4 signaling through FGF21, maintained normal glucose metabolism and redox balance in the brain, and thereby alleviated DACI. These results provide new insights into the effects of MR diet on cognitive dysfunction caused by impaired brain energy metabolism.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling

BackgroundNeuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation.MethodsTo track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation.ResultsWe observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88.ConclusionsCirculating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Intranasal administration of liposomes: Potential brain delivery with prospective ophthalmic reach

The low efficacy of drug transportation from the bloodstream to specific tissues remains a challenge in drug delivery to the central nervous system (CNS), primarily because of the presence of the blood-brain and blood-retinal barriers. There is a need for non-invasive drug delivery techniques to the CNS given the intrusive nature of and potential patient burden associated with intraventricular, intrathecal, and intravitreal administration. Recently, the focus has shifted towards intranasal (i.e., nose-to-brain) drug delivery. Furthermore, there have been reports indicating that the use of drug nanocarriers, such as liposomes, facilitates efficient drug delivery via the intranasal pathway. This study investigates the brain penetration capabilities of intranasally administered liposomes using the hydrophobic fluorescent molecule, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl lindodicarbocyanine, 4-chlorobenzene sulfonate salt (DiD), as a model compound. The primary objective of this study was to elucidate the penetration of intranasally administered liposomes into the CNS, including the eyes. An examination of DiD distribution in the dissected brain and ocular tissues showed that DiD concentrations were elevated specifically at the limbus of the ocular tissue when surface-modified liposomes were employed. These findings suggest that intranasal liposome administration can deliver drugs to the posterior ophthalmic region, specifically the optic nerve, in addition to the brain.

8631 Tanycytic TSHR Activation Regulates Blood-Hypothalamus Barrier Plasticity, Ingestive Behavior, Adipose Remodeling, and Body Weight

Abstract Disclosure: L. Cullen: None. H.S. Kannangara: None. R. Witztum: None. J. Burgess: None. S. Miyashita: None. A. Gumerova: None. F. Korkmaz: None. S.L. Sims: None. U. Cheliadinova: None. G. Pevnev: None. O. Moldavski: None. O. Barak: None. S. Rojekar: None. V. Laurencin: None. T. Frolinger: None. T. Yuen: None. K. Goosens: None. W. Zhou: None. V. Ryu: None. M. Zaidi: None. The notion that TSH acts solely on its primary endocrine target, the thyroid gland, has long been challenged (Abe et al., 2003; PMID: 14567913). More recently, however, we and others have used single transcript technology to document, for the first time, Tshr expression in over 173 brain regions and nuclei, particularly hypothalamic tanycytes (eLife, 2022, PMID: 36052994). The latter cells are known to convey metabolic information across the blood-hypothalamus barrier (BHB) and regulate energy homeostasis. To study whether tanycytic TSHRs have a role in the regulation of BHB permeability, ingestive behavior, adipose tissue remodeling and body weight, we first compared BHB vascular permeability of food-deprived and ad libitum-fed Tshr+/- mice and their wild type (WT) littermates. Transmission electron microscopy and immunostaining was used to study the expression of the constitutive tight junction protein claudin-1 and the glycoprotein MECA-32, which are expressed in tanycytes and endothelial cells, respectively. In loss-of-function studies, fasting of Tshr+/- mice and of WT mice significantly increased the organization of tanycytic honeycomb pattern of claudin-1 and MECA-32-associated fenestral diaphragms in the median eminence and arcuate nucleus, suggesting decreased BHB permeability. As a result, peanut butter consumption following fasting was decreased significantly compared to control mice. In gain-of-function studies, intracerebroventricular (ICV) infusions of a highly specific TSHR agonist, MS438, profoundly increased BHB permeability in fasted mice, as evidenced by decreased claudin-1 and MECA-32 immunostaining and significant increases in peanut butter consumption. This was associated with greater loss of fat mass and body weight following food deprivation, as well as increased influx of fatty acids across the BHB, increased NPY expression (as a measure of increased appetite), and increased secretion of ghrelin from the stomach fundus. We further performed c-Fos immunostaining in brain centers regulating ingestive behavior. ICV MS438 significantly decreased c-Fos immunoreactivity in the hypothalamic paraventricular nucleus, arcuate nucleus and the nucleus of the solitary tract. Early studies using CRSIPR/Cas9 to delete Tshrs in tanycytes via ICV injection resulted in reduced body weight following food deprivation. Finally, using PRV152 (injected into fat tissue) and PRV614 (injected into stomach fundus), we colocalized immunostaining in the hypothalamic paraventricular nucleus, and hindbrain raphe pallidus. Collectively, these data establish a new role for tanycytic TSHRs in regulating BHB plasticity, ingestive behavior, adipose tissue remodeling, and body weight through a novel fast-forward circuit involving the hypothalamus, stomach fundus and fat depots. Presentation: 6/3/2024

AAV8-P301L tau expression confers age-related disruptions in sleep quantity and timing

Sleep timing and quantity disturbances persist in tauopathy patients. This has been studied in transgenic models of primary tau neuropathology using traditional electroencephalograms (EEGs) and more recently, the PiezoSleep Mouse Behavioral Tracking System. Here, we generated a primary tauopathy model using an intracerebroventricular injection of human mutant hSyn-P301L-tau, using adeno-associated virus of serotype 8 (AAV8). We discovered distinctions in sleep architecture with altered quantity and timing in AAV8-P301L tau expressing mice of both sexes using the noninvasive PiezoSleep System. The AAV8-P301L tau mice exhibit striking age-related increases in sleep duration specifically at the active phase onset, suggesting a critical and sensitive time-of-day for tauopathy related sleep disturbances to occur. Since our findings show sleep behavior changes at specific transitional periods of the day, tau neuropathology may impact normal diurnal variation in biological processes, which should be explored using the AAV8-P301L tauopathy model.

Expression-based selection identifies a microglia-tropic AAV capsid for direct and CSF routes of administration in mice.

Microglia are critical innate immune cells of the brain. In vivo targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors. Recently, there has been some progress demonstrating the feasibility and potential of using AAV to transduce microglia after direct intraparenchymal vector injection. Data suggests that combining specific AAV capsids with microglia-specific gene expression cassettes to reduce neuron off-targeting will be key. However, no groups have developed AAV capsids for microglia transduction after intracerebroventricular (ICV) injection. The ICV route of administration has advantages such as increased brain biodistribution while avoiding issues related to systemic injection. Here, we performed an in vivo selection using an AAV peptide display library that enables recovery of capsids that mediate transgene expression in microglia. Using this approach, we identified a capsid, MC5, which mediated enhanced transduction of microglia after ICV injection compared to AAV9. Furthermore, MC5 enhanced both the efficiency (85%) and specificity (93%) of transduction compared to a recently described evolved AAV9 capsid for microglia targeting after direct injection into the brain parenchyma. Exploration of the use of MC5 in a mouse models of Alzheimer's disease revealed transduced microglia surrounding and within plaques. Overall, our results demonstrate that the MC5 capsid is a useful gene transfer tool to target microglia in vivo by direct and ICV routes of administration.