VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

VEGF-C promotes brain-derived fluid drainage, confers neuroprotection, and improves stroke outcomes.

Meningeal lymphatic vessels promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelium growth factor-C (VEGF-C) is essential for meningeal lymphatic development and maintenance and has therapeutic potential for treating neurological disorders, including ischemic stroke. We have investigated the effects of VEGF-C overexpression on brain fluid drainage, single cell transcriptome in the brain, and stroke outcomes in adult mice. Intra-cerebrospinal fluid administration of an adeno-associated virus expressing VEGF-C (AAV-VEGF-C) increases the CNS lymphatic network. Post-contrast T1 mapping of the head and neck showed that deep cervical lymph node size and drainage of CNS-derived fluids were increased. Single nuclei RNA sequencing revealed a neuro-supportive role of VEGF-C via upregulation of calcium and brain-derived neurotrophic factor (BDNF) signaling pathways in brain cells. In a mouse model of ischemic stroke, AAV-VEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage. AAV-VEGF-C thus promotes CNS-derived fluid and solute drainage, confers neuroprotection, and reduces ischemic stroke damage. Short abstract: Intrathecal delivery of VEGF-C increases the lymphatic drainage of brain-derived fluids confers neuroprotection, and improves neurological outcomes after ischemic stroke.

Imaging Hallmarks of the Tumor Microenvironment in Glioblastoma Progression.

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment.

Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity.

OBJECTIVE: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

Tumor-targeted pH-low insertion peptide delivery of theranostic gadolinium nanoparticles for image-guided nanoparticle-enhanced radiation therapy.

Tumor targeting studies using metallic nanoparticles (NPs) have shown that the enhanced permeability and retention effect may not be sufficient to deliver the amount of intratumoral and intracellular NPs needed for effective in vivo radiosensitization. This work describes a pH-Low Insertion Peptide (pHLIP) targeted theranostic agent to enable image-guided NP-enhanced radiotherapy using a clinically feasible amount of injected NPs. Conventional gadolinium (Gd) NPs were conjugated to pHLIPs and evaluated in vitro for radiosensitivity and in vivo for mouse MRI. Cultured A549 human lung cancer cells were incubated with 0.5 mM of pHLIP-GdNP or conventional GdNP. Mass spectrometry showed 78-fold more cellular Gd uptake with pHLIP-GdNPs, and clonogenic survival assays showed 44% more enhanced radiosensitivity by 5 Gy irradiation with pHLIP-GdNPs at pH 6.2. In contrast to conventional GdNPs, MR imaging of tumor-bearing mice showed pHLIP-GdNPs had a long retention time in the tumor (>9 h), suitable for radiotherapy, and penetrated into the poorly-vascularized tumor core. The Gd-enhanced tumor corresponded with low-pH areas also independently measured by an in vivo molecular MRI technique. pHLIPs actively target cell surface acidity from tumor cell metabolism and deliver GdNPs into cells in solid tumors. Intracellular delivery enhances the effect of short-range radiosensitizing photoelectrons and Auger electrons. Because acidity is a general hallmark of tumor cells, the delivery is more general than antibody targeting. Imaging the in vivo NP biodistribution and more acidic (often more aggressive) tumors has the potential for quantitative radiotherapy treatment planning and pre-selecting patients who will likely benefit more from NP radiation enhancement.

Multimodal Imaging in Rat Model Recapitulates Alzheimer's Disease Biomarkers Abnormalities.

Imaging biomarkers are frequently proposed as endpoints for clinical trials targeting brain amyloidosis in Alzheimer's disease (AD); however, the specific impact of amyloid-ß (Aß) aggregation on biomarker abnormalities remains elusive in AD. Using the McGill-R-Thy1-APP transgenic rat as a model of selective Aß pathology, we characterized the longitudinal progression of abnormalities in biomarkers commonly used in AD research. Middle-aged (9-11 months) transgenic animals (both male and female) displayed mild spatial memory impairments and disrupted cingulate network connectivity measured by resting-state fMRI, even in the absence of hypometabolism (measured with PET [18F]FDG) or detectable fibrillary amyloidosis (measured with PET [18F]NAV4694). At more advanced ages (16-19 months), cognitive deficits progressed in conjunction with resting connectivity abnormalities; furthermore, hypometabolism, Aß plaque accumulation, reduction of CSF Aß1-42 concentrations, and hippocampal atrophy (structural MRI) were detectable at this stage. The present results emphasize the early impact of Aß on brain connectivity and support a framework in which persistent Aß aggregation itself is sufficient to impose memory circuits dysfunction, which propagates to adjacent brain networks at later stages.SIGNIFICANCE STATEMENT The present study proposes a "back translation" of the Alzheimer pathological cascade concept from human to animals. We used the same set of Alzheimer imaging biomarkers typically used in large human cohorts and assessed their progression over time in a transgenic rat model, which allows for a finer spatial resolution not attainable with mice. Using this translational platform, we demonstrated that amyloid-ß pathology recapitulates an Alzheimer-like profile of biomarker abnormalities even in the absence of other hallmarks of the disease such as neurofibrillary tangles and widespread neuronal losses.