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Abstract Background Spinal cord metastatic lesions affect a high number of cancer patients usually resulting in spinal cord compression syndrome. A major obstacle in the research of spinal metastatic disease is the lack of a simple reproducible animal model that mimics the natural course of the disease. In this study, we present a highly reproducible rodent model that can be used for different types of cancers while mimicking the natural course of human metastatic spinal cord compression syndrome. Results All sixteen Fisher 344 rats survived the dorsal approach intraosseous implantation of CRL-1666 adenocarcinoma cells and both rats survived the sham control surgery. By Day 13 functional analysis via the modified Basso-Beattie-Bresnahan (BBB) locomotor rating scale showed significant decrease in motor function; median functional score was 3 for the tumor group (p = 0.0011). Median time to paresis was 8.7 days post-operatively. MR imaging illustrated repeated and consistent tumor formation, furthermore, onset of neurological sequale was the result of tumor formation and cord compression as confirmed by histological examination. Conclusions Analysis of these findings demonstrates a repeatable and consistent tumor growth model for cancer spinal metastases in rats. This novel rat model requires a less intricate surgical procedure, and as a result minimizes procedure time while subsequently increasing consistency. Therefore, this model allows for the preclinical evaluation of therapeutics for spinal metastases that more closely replicates physiological findings.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of β-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1 -/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1 -/- mice die six weeks earlier than male Glb1 -/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1 -/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1 -/- mice as seen in some patients. In addition, Glb1 -/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1 -/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1 -/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Disrupted sleep, including daytime hypersomnolence, is a core symptom reported by primary brain tumor patients and often manifests after radiotherapy. The biological mechanisms driving the onset of sleep disturbances after cranial radiation remains unclear but may result from treatment-induced injury to neural circuits controlling sleep behavior, both circadian and homeostatic. Here, we develop a mouse model of cranial radiation-induced hypersomnolence which recapitulates the human experience. Additionally, we used the model to explore the impact of radiation on the brain. We demonstrated that the DNA damage response following radiation varies across the brain, with homeostatic sleep and cognitive regions expressing higher levels of γH2AX, a marker of DNA damage, than the circadian suprachiasmatic nucleus (SCN). These findings were supported by in vitro studies comparing radiation effects in SCN and cortical astrocytes. Moreover, in our mouse model, MRI identified structural effects in cognitive and homeostatic sleep regions two-months post-treatment. While the findings are preliminary, they suggest that homeostatic sleep and cognitive circuits are vulnerable to radiation and these findings may be relevant to optimizing treatment plans for patients., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Age plays a critical role in disease development and tolerance to cancer treatment, often leading to an increased risk of developing negative symptoms including sleep disturbances. Circadian rhythms and sleep become disrupted as organisms age. In this study, we explored the behavioral alterations in sleep, circadian rhythms, and masking using a novel video system and interrogate the long-term impact of age-based changes in the non-image forming visual pathway on brain anatomy. We demonstrated the feasibility and utility of the novel system and establish that older mice have disruptions in sleep, circadian rhythms, and masking behaviors that were associated with major negative volume alterations in the non-imaging forming visual system, critical for the induction and rhythmic expression of sleep. These results provide important insights into a mechanism, showing brain atrophy is linked to age in distinct non-image forming visual regions, which may predispose older individuals to developing circadian and sleep dysfunction when further challenged by disease or treatment.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome.

Purpose: Abrupt halt of service provision due to pandemic state of COVID-19, significantly affected care of patients with chronic diseases like epilepsy; its impact being greater on caregivers of vulnerable groups such as children with epilepsy. We performed this study to describe difficulties posed by the lockdown to caregivers of children with epilepsy in a low-middle income country and describe their responses and self-management strategies to overcome difficulties and prepare for a recurrence., Method: A cross-sectional all-island survey was carried out at paediatric neurology centers in Sri Lanka. Data was gathered via a face-to-face interview after the lockdown period. Parental stress level was evaluated using a self-rating Stress Assessment Questionnaire., Results: Caregivers of 140 children with epilepsy from seven centers served by paediatric neurologists were interviewed. Mean duration of epilepsy was 7.9 years(SD 4). Majority were on one (52.1 %) or two (20 %) anti-seizure medications regularly. The pandemic did not affect epilepsy control in majority (87.3 %), however, signficant proportion faced difficulties over regular reviews and presecription refills. Despite difficluties, 87.1 % of parents maintained dispensing anti-seizure medications to their child regularly. Caregivers demonstrated healthy self-management strategies such as awareness on medications and access methods to healthcare during lockdown and remained confident of accessability to services. Stress was experienced in < 5%., Conclusion: Lockdown status for COVID-19 did not significantly affect the control of epilepsy in children though it posed difficulties for regular reviews and obtaining medications. Self-management strategies will help caregivers to adopt to new-normal status and potential future outbreaks., (Copyright © 2020 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 10 4 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 10 5 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 10 4 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.

Tumor heterogeneity is one major reason for unpredictable therapeutic outcomes, while stratifying therapeutic responses at an early time may greatly benefit the better control of cancer. Here, we developed a hybrid nanovesicle to stratify radiotherapy response by activatable inflammation magnetic resonance imaging (aiMRI) approach. The high Pearson's correlation coefficient R values are obtained from the correlations between the T 1 relaxation time changes at 24-48 h and the ensuing adaptive immunity (R = 0.9831) at day 5 and the tumor inhibition ratios (R = 0.9308) at day 18 after different treatments, respectively. These results underscore the role of acute inflammatory oxidative response in bridging the innate and adaptive immunity in tumor radiotherapy. Furthermore, the aiMRI approach provides a non-invasive imaging strategy for early prediction of the therapeutic outcomes in cancer radiotherapy, which may contribute to the future of precision medicine in terms of prognostic stratification and therapeutic planning.

Background: Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. Isocitrate dehydrogenase (IDH)-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses toward secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood., Methods: We employed model systems for IDH1 mutant (IDH1mut) gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome, and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic, we analyzed data from The Cancer Genome Atlas., Results: We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially lactate dehydrogenase A (LDHA), and have increased mRNA and metabolite levels compared with the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP)-high molecular subtype in patients and is associated with the worst outcome., Conclusion: We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Metabolic differences among and within tumors can be an important determinant in cancer treatment outcome. However, methods for determining these differences non-invasively in vivo is lacking. Using pancreatic ductal adenocarcinoma as a model, we demonstrate that tumor xenografts with a similar genetic background can be distinguished by their differing rates of the metabolism of 13C labeled glucose tracers, which can be imaged without hyperpolarization by using newly developed techniques for noise suppression. Using this method, cancer subtypes that appeared to have similar metabolic profiles based on steady state metabolic measurement can be distinguished from each other. The metabolic maps from 13C-glucose imaging localized lactate production and overall glucose metabolism to different regions of some tumors. Such tumor heterogeneity would not be not detectable in FDG-PET., Competing Interests: SK, JB, DC, SM, TS, NO, HM, PL, JM, KS, KY, PC, JM, AL, TF, WL, MK No competing interests declared, GR, AC, JA is affiliated with GE HealthCare. The author has no other competing interests to declare.

Magnetic resonance imaging (MRI) diagnosis is better assisted by contrast agents that can augment the signal contrast in the imaging appearance. However, this technique is still limited by the inherently low sensitivity on the recorded signal changes in conventional T 1 or T 2 MRI in a qualitative manner. Here, we provide a new paradigm of MRI diagnosis using T 1 - T 2 dual-modal MRI contrast agents for contrast-enhanced postimaging computations on T 1 and T 2 relaxation changes. An albumin-binding molecule (i.e., truncated Evans blue) chelated with paramagnetic manganese ion was developed as a novel T 1 - T 2 dual-modal MRI contrast agent at high magnetic field (7 T). Furthermore, the postimaging computations on T 1 - T 2 dual-modal MRI led to greatly enhanced signal-to-noise ratios (SNR) and contrast-to-noise ratios (CNR) in both subcutaneous and orthotopic brain tumor models compared with traditional MRI methods. The T 1 - T 2 dual-modal MRI computations have great potential to eliminate suspicious artifacts and false-positive signals in mouse brain imaging. This study may open new avenues for contrast-enhanced MRI diagnosis and holds great promise for precision medicine.

Metabolic reprogramming is one of the defining features of cancer and abnormal metabolism is associated with many other pathologies. Molecular imaging techniques capable of detecting such changes have become essential for cancer diagnosis, treatment planning, and surveillance. In particular, 18 F-FDG (fluorodeoxyglucose) PET has emerged as an essential imaging modality for cancer because of its unique ability to detect a disturbed molecular pathway through measurements of glucose uptake. However, FDG-PET has limitations that restrict its usefulness in certain situations and the information gained is limited to glucose uptake only. 13 C magnetic resonance spectroscopy theoretically has certain advantages over FDG-PET, but its inherent low sensitivity has restricted its use mostly to single voxel measurements unless dissolution dynamic nuclear polarization (dDNP) is used to increase the signal, which brings additional complications for clinical use. We show here a new method of imaging glucose metabolism in vivo by MRI chemical shift imaging (CSI) experiments that relies on a simple, but robust and efficient, post-processing procedure by the higher dimensional analog of singular value decomposition, tensor decomposition. Using this procedure, we achieve an order of magnitude increase in signal to noise in both dDNP and non-hyperpolarized non-localized experiments without sacrificing accuracy. In CSI experiments an approximately 30-fold increase was observed, enough that the glucose to lactate conversion indicative of the Warburg effect can be imaged without hyper-polarization with a time resolution of 12s and an overall spatial resolution that compares favorably to 18 F-FDG PET.

Cancer is one of the leading causes of morbidity and mortality in the world, but more cancer therapies are needed to complement existing regimens due to problems of existing cancer therapies. Herein, we term ferroptosis therapy (FT) as a form of cancer therapy and hypothesize that the FT efficacy can be significantly improved via accelerating the Fenton reaction by simultaneously increasing the local concentrations of all reactants (Fe 2+ , Fe 3+ , and H 2 O 2 ) in cancer cells. Thus, Fenton-reaction-acceleratable magnetic nanoparticles, i.e., cisplatin (CDDP)-loaded Fe 3 O 4 /Gd 2 O 3 hybrid nanoparticles with conjugation of lactoferrin (LF) and RGD dimer (RGD2) (FeGd-HN@Pt@LF/RGD2), were exploited in this study for FT of orthotopic brain tumors. FeGd-HN@Pt@LF/RGD2 nanoparticles were able to cross the blood-brain barrier because of its small size (6.6 nm) and LF-receptor-mediated transcytosis. FeGd-HN@Pt@LF/RGD2 can be internalized into cancer cells by integrin α v β 3 -mediated endocytosis and then release Fe 2+ , Fe 3+ , and CDDP upon endosomal uptake and degradation. Fe 2+ and Fe 3+ can directly participate in the Fenton reaction, whereas the CDDP can indirectly produce H 2 O 2 to further accelerate the Fenton reaction. The acceleration of Fenton reaction generates reactive oxygen species to induce cancer cell death. FeGd-HN@Pt@LF/RGD2 successfully delivered reactants involved in the Fenton reaction to the tumor site and led to significant inhibition of tumor growth. Finally, the intrinsic magnetic resonance imaging (MRI) capability of the nanoparticles was used to assess and monitor tumor response to FT (self-MRI monitoring).

Background: Dengue fever is a mosquito-borne viral disease with a very high incidence in Southeast Asia. Most patients with dengue fever recover following a self-limiting febrile illness, while a small proportion may progress to develop severe disease with complications such as acute liver failure, acute kidney injury, and multiorgan failure. Secondary bacterial infections and thrombotic events are very rare., Case Presentation: A 38-year-old previously healthy Sri Lankan woman from Colombo, Sri Lanka, presented with dengue shock syndrome leading to acute liver failure and kidney injury. She was managed with intravenously administered fluid resuscitation with close monitoring of her hemodynamic status, and hemodialysis. Her renal and liver functions and platelet count improved gradually, but the fever persisted and there was a neutrophil leukocytosis. A clinical examination and investigations to identify a focus of secondary infection revealed staphylococcal infective endocarditis. She was started on intravenously administered vancomycin, but as the response was poor the antibiotic was changed to intravenously administered linezolid, to which the response was good. She also developed right proximal femoral deep vein thrombosis, and was commenced on subcutaneous enoxaparin and warfarin. Enoxaparin was stopped after her international normalized ratio reached the desirable range, and warfarin was continued for 3 months., Conclusions: Dengue virus is known to cause endothelial dysfunction that allows bacteria to invade tissues, defective functioning and reduction in the number of cells of the immune system, and alteration of cytokines leading to immune dysregulation, predisposing patients to develop secondary bacterial infections. Evidently, patients with dengue fever who have prolonged fever (more than 5 days) and acute kidney injury are at high risk for concurrent bacteremia. Dengue virus interferes with the components of the anti-clotting pathway, such as thrombomodulin-thrombin-protein C complex. It also activates endothelial cells and increases the expression of procoagulant factors. These factors may predispose patients with dengue viral infections to develop thrombotic complications. Therefore it is important to be aware of the possibility of serious secondary bacterial infections occurring following dengue viral infections, especially in patients with prolonged fever and acute kidney injury, and to keep in mind that thrombotic events may occur as complications of dengue viral infections.

Importance: Poorly controlled hypertension is a leading global public health problem requiring new treatment strategies., Objective: To assess whether a low-dose triple combination antihypertensive medication would achieve better blood pressure (BP) control vs usual care., Design, Setting, and Participants: Randomized, open-label trial of a low-dose triple BP therapy vs usual care for adults with hypertension (systolic BP >140 mm Hg and/or diastolic BP >90 mm Hg; or in patients with diabetes or chronic kidney disease: >130 mm Hg and/or >80 mm Hg) requiring initiation (untreated patients) or escalation (patients receiving monotherapy) of antihypertensive therapy. Patients were enrolled from 11 urban hospital clinics in Sri Lanka from February 2016 to May 2017; follow-up ended in October 2017., Interventions: A once-daily fixed-dose triple combination pill (20 mg of telmisartan, 2.5 mg of amlodipine, and 12.5 mg of chlorthalidone) therapy (n = 349) or usual care (n = 351)., Main Outcomes and Measures: The primary outcome was the proportion achieving target systolic/diastolic BP (<140/90 mm Hg or <130/80 mm Hg in patients with diabetes or chronic kidney disease) at 6 months. Secondary outcomes included mean systolic/diastolic BP difference during follow-up and withdrawal of BP medications due to an adverse event., Results: Among 700 randomized patients (mean age, 56 years; 58% women; 29% had diabetes; mean baseline systolic/diastolic BP, 154/90 mm Hg), 675 (96%) completed the trial. The triple combination pill increased the proportion achieving target BP vs usual care at 6 months (70% vs 55%, respectively; risk difference, 12.7% [95% CI, 3.2% to 22.0%]; P < .001). Mean systolic/diastolic BP at 6 months was 125/76 mm Hg for the triple combination pill vs 134/81 mm Hg for usual care (adjusted difference in postrandomization BP over the entire follow-up: systolic BP, -9.8 [95% CI, -7.9 to -11.6] mm Hg; diastolic BP, -5.0 [95% CI, -3.9 to -6.1] mm Hg; P < .001 for both comparisons). Overall, 419 adverse events were reported in 255 patients (38.1% for triple combination pill vs 34.8% for usual care) with the most common being musculoskeletal pain (6.0% and 8.0%, respectively) and dizziness, presyncope, or syncope (5.2% and 2.8%). There were no significant between-group differences in the proportion of patient withdrawal from BP-lowering therapy due to adverse events (6.6% for triple combination pill vs 6.8% for usual care)., Conclusions and Relevance: Among patients with mild to moderate hypertension, treatment with a pill containing low doses of 3 antihypertensive drugs led to an increased proportion of patients achieving their target BP goal vs usual care. Use of such medication as initial therapy or to replace monotherapy may be an effective way to improve BP control., Trial Registration: anzctr.org.au Identifier: ACTRN12612001120864; slctr.lk Identifier: SLCTR/2015/020.

To examine the relationship between local oxygen partial pressure and energy metabolism in the tumor, electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) with hyperpolarized [1- 13 C] pyruvate were performed. SCCVII and HT29 solid tumors implanted in the mouse leg were imaged by EPRI using OX063, a paramagnetic probe and 13 C-MRI using hyperpolarized [1- 13 C] pyruvate. Local partial oxygen pressure and pyruvate metabolism in the two tumor implants were examined. The effect of a single dose of 5-Gy irradiation on the pO 2 and metabolism was also investigated by sequential imaging of EPRI and 13 C-MRI in HT29 tumors. A phantom study using tubes filled with different concentration of [1- 13 C] pyruvate, [1- 13 C] lactate, and OX063 at different levels of oxygen confirmed the validity of this sequential imaging of EPRI and hyperpolarized 13 C-MRI. In vivo studies revealed SCCVII tumor had a significantly larger hypoxic fraction (pO 2 < 8 mmHg) compared to HT29 tumor. The flux of pyruvate-to-lactate conversion was also higher in SCCVII than HT29. The lactate-to-pyruvate ratio in hypoxic regions (pO 2 < 8 mmHg) 24 hours after 5-Gy irradiation was significantly higher than those without irradiation (0.76 vs. 0.36) in HT29 tumor. The in vitro study showed an increase in extracellular acidification rate after irradiation. In conclusion, co-imaging of pO 2 and pyruvate-to-lactate conversion kinetics successfully showed the local metabolic changes especially in hypoxic area induced by radiation therapy., Competing Interests: CONFLICTS OF INTEREST The authors have no potential conflicts of interest to disclose.

Near-infrared photoimmunotherapy (NIR PIT) employs the photoabsorbing dye IR700 conjugated to antibodies specific for cell surface epidermal growth factor receptor (EGFR). NIR PIT has shown highly selective cytotoxicity in vitro and in vivo. Cell necrosis is thought to be the main mode of cytotoxicity based mainly on in vitro studies. To better understand the acute effects of NIR PIT, molecular imaging studies were performed to assess its cellular and vascular effects. In addition to in vitro studies for cytotoxicity of NIR PIT, the in vivo tumoricidal effects and hemodynamic changes induced by NIR PIT were evaluated by 13 C MRI using hyperpolarized [1,4- 13 C 2 ] fumarate, R 2 * mapping from T 2 *-weighted MRI, and photoacoustic imaging. In vitro studies confirmed that NIR PIT resulted in rapid cell death via membrane damage, with evidence for rapid cell expansion followed by membrane rupture. Following NIR PIT, metabolic MRI using hyperpolarized fumarate showed the production of malate in EGFR-expressing A431 tumor xenografts, providing direct evidence for photosensitized tumor necrosis induced by NIR PIT. R 2 * mapping studies showed temporal changes in oxygenation, with an accompanying increase of deoxyhemoglobin at the start of light exposure followed by a sustained decrease after cessation of light exposure. This result suggests a rapid decrease of blood flow in EGFR-expressing A431 tumor xenografts, which is supported by the results of the photoacoustic imaging experiments. Our findings suggest NIR PIT mediates necrosis and hemodynamic changes in tumors by photosensitized oxidation pathways and that these imaging modalities, once translated, may be useful in monitoring clinical treatment response., (Published by Elsevier Inc.)

Rationale: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30% to 40% of strokes. Inflammation has been suggested as a critical etiologic factor. However, there is lack of experimental evidence., Objective: In this study, we investigated inflammation-associated stroke using a mouse model that developed spontaneous stroke because of myeloid deficiency of TGF-β (transforming growth factor-β) signaling., Methods and Results: We report that mice with deletion of Tgfbr2 in myeloid cells ( Tgfbr2 Myeko ) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild-type mice via Tgfbr2 Myeko bone marrow transplant and can be rescued in Tgfbr2 Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation and cerebral endotheliopathy, characterized by elevated NF-κB (nuclear factor-κB) activation and TNF (tumor necrosis factor) production by myeloid cells. A high-fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence., Conclusions: Our studies show that TGF-β signaling in myeloid cells is required for maintenance of vascular health and provide insight into inflammation-mediated cerebrovascular disease and stroke., (© 2017 American Heart Association, Inc.)

The recently emerged exceedingly small magnetic iron oxide nanoparticles (ES-MIONs) (<5 nm) are promising T 1 -weighted contrast agents for magnetic resonance imaging (MRI) due to their good biocompatibility compared with Gd-chelates. However, the best particle size of ES-MIONs for T 1 imaging is still unknown because the synthesis of ES-MIONs with precise size control to clarify the relationship between the r 1 (or r 2 /r 1 ) and the particle size remains a challenge. In this study, we synthesized ES-MIONs with seven different sizes below 5 nm and found that 3.6 nm is the best particle size for ES-MIONs to be utilized as T 1 -weighted MR contrast agent. To enhance tumor targetability of theranostic nanoparticles and reduce the nonspecific uptake of nanoparticles by normal healthy cells, we constructed a drug delivery system based on the 3.6 nm ES-MIONs for T 1 -weighted tumor imaging and chemotherapy. The laser scanning confocal microscopy (LSCM) and flow cytometry analysis results demonstrate that our strategy of precise targeting via exposure or hiding of the targeting ligand RGD 2 on demand is feasible. The MR imaging and chemotherapy results on the cancer cells and tumor-bearing mice reinforce that our DOX@ES-MION3@RGD 2 @mPEG3 nanoparticles are promising for high-resolution T 1 -weighted MR imaging and precise chemotherapy of tumors.

OBJECTIVE To study peritumoral brain edema (PTBE), it is necessary to create a model that accurately simulates vasogenic brain edema (VBE) without introducing a complicated tumor environment. PTBE associated with brain tumors is predominantly a result of vascular endothelial growth factor (VEGF) secreted by brain tumors, and VEGF infusion alone can lead to histological blood-brain barrier (BBB) breakdown in the absence of tumor. VBE is intimately linked to BBB breakdown. The authors sought to establish a model for VBE with chronic infusion of VEGF that can be validated by serial in-vivo MRI and histological findings. METHODS Male Fischer rats (n = 182) underwent stereotactic striatal implantation of MRI-safe brain cannulas for chronic infusion of VEGF (2-20 µg/ml). Following a preinfusion phase (4-6 days), the rats were exposed to VEGF or control rat serum albumin (1.5 µl/hr) for as long as 144 hours. Serial MRI was performed during infusion on a high-field (9.4-T) machine at 12-24, 24-36, 48-72, and 120-144 hours. Rat brains were then collected and histological analysis was performed. RESULTS Control animals and animals infused with 2 µg/ml of VEGF experienced no neurological deficits, seizure activity, or abnormal behavior. Animals treated with VEGF demonstrated a significantly larger volume (42.90 ± 3.842 mm 3 ) of T2 hyper-attenuation at 144 hours when compared with the volume (8.585 ± 1.664 mm 3 ) in control animals (mean difference 34.31 ± 4.187 mm 3 , p < 0.0001, 95% CI 25.74-42.89 mm 3 ). Postcontrast T1 enhancement in the juxtacanalicular region indicating BBB breakdown was observed in rats undergoing infusion with VEGF. At the later time periods (120-144 hrs) the volume of T1 enhancement (34.97 ± 8.99 mm 3 ) was significantly less compared with the region of edema (p < 0.0001). Histologically, no evidence of necrosis or inflammation was observed with VEGF or control infusion. Immunohistochemical analysis demonstrated astrocyte activation, vascular remodeling, and increased claudin-5 expression in juxtacanalicular regions. Aquaporin-4 expression was increased in both control and VEGF animals in the juxtacanalicular regions. CONCLUSIONS The results of this study show that chronic brain infusion of VEGF creates a reliable model of VBE. This model lacks necrosis and inflammation that are characteristic of previous models of VBE. The model allows for a precise investigation into the mechanism of VBE formation. The authors also anticipate that this model will allow for investigation into the mechanism of glucocorticoid action in abrogating VBE, and to test novel therapeutic strategies targeting PTBE.

TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Reported procedures on the synthesis of gold nanoshells with smooth surfaces have merely demonstrated efficient control of shell thickness and particle size, yet no branch and nanoporous features on the nanoshell have been implemented to date. Herein, we demonstrate the ability to control the roughness and nanoscale porosity of gold nanoshells by using redox-active polymer poly(vinylphenol)-b-(styrene) nanoparticles as reducing agent and template. The porosity and size of the branches on this branched nanoporous gold nanoshell (BAuNSP) material can be facilely adjusted by control of the reaction speed or the reaction time between the redox-active polymer nanoparticles and gold ions (Au 3+ ). Due to the strong reduction ability of the redox-active polymer, the yield of BAuNSP was virtually 100%. By taking advantage of the sharp branches and nanoporous features, BAuNSP exhibited greatly enhanced physico-optical properties, including photothermal effect, surface-enhanced Raman scattering (SERS), and photoacoustic (PA) signals. The photothermal conversion efficiency can reach as high as 75.5%, which is greater than most gold nanocrystals. Furthermore, the nanoporous nature of the shells allows for effective drug loading and controlled drug release. The thermoresponsive polymer coated on the BAuNSP surface serves as a gate keeper, governing the drug release behavior through photothermal heating. Positron emission tomography imaging demonstrated a high passive tumor accumulation of 64 Cu-labeled BAuNSP. The strong SERS signal generated by the SERS-active BAuNSP in vivo, accompanied by enhanced PA signals in the tumor region, provide significant tumor information, including size, morphology, position, and boundaries between tumor and healthy tissues. In vivo tumor therapy experiments demonstrated a highly synergistic chemo-photothermal therapy effect of drug-loaded BAuNSPs, guided by three modes of optical imaging.

Multimodal imaging strategies integrating manifold images have improved our ability to diagnose, to guide therapy, and to predict outcomes. Magnetic resonance imaging (MRI) is among the most widely used imaging technique in the clinic and can enable multiparameter anatomical demonstration of diagnosis. Due to the inherent black-and-white production of MR images, however, MRI detection is largely hampered by the occurrence of false-positive diagnoses. In this Perspective, we introduce the paradigm of manipulating the multiparameter MRI, T 1 -T 2 dual-modal MRI, along with enhancement by specific contrast agents. We hope this discussion will promote emerging research interest in T 1 -T 2 dual-modal MRI and provoke the rational design of contrast agents for sophisticated MRI applications.

Hereditary spastic paraplegias (HSPs; SPG1-76 plus others) are length-dependent disorders affecting long corticospinal axons, and the most common autosomal dominant forms are caused by mutations in genes that encode the spastin (SPG4), atlastin-1 (SPG3A) and REEP1 (SPG31) proteins. These proteins bind one another and shape the tubular endoplasmic reticulum (ER) network throughout cells. They also are involved in lipid droplet formation, enlargement, or both in cells, though mechanisms remain unclear. Here we have identified evidence of partial lipoatrophy in Reep1 null mice in addition to prominent spastic paraparesis. Furthermore, Reep1-/- embryonic fibroblasts and neurons in the cerebral cortex both show lipid droplet abnormalities. The apparent partial lipodystrophy in Reep1 null mice, although less severe, is reminiscent of the lipoatrophy phenotype observed in the most common form of autosomal recessive lipodystrophy, Berardinelli-Seip congenital lipodystrophy. Berardinelli-Seip lipodystrophy is caused by autosomal recessive mutations in the BSCL2 gene that encodes an ER protein, seipin, that is also mutated in the autosomal dominant HSP SPG17 (Silver syndrome). Furthermore, REEP1 co-immunoprecipitates with seipin in cells. This strengthens the link between alterations in ER morphogenesis and lipid abnormalities, with important pathogenic implications for the most common forms of HSP., (Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.)

Background: In this feasibility study, a mechanically loaded in-vitro tissue culture model of joint morphogenesis using the isolated lower extremity of the 8 day old chick embryo was developed to assess the effects of mechanical loading on joint morphogenesis., Methods: The developed in-vitro system allows controlled flexion and extension of the chick embryonic knee with a range of motion of 20 degrees from a resting position of 90-100 degrees of flexion. Joint morphogenesis at 2, 3, 4 and 7 days of culture was assessed by histology and micro MRI in 4 specimen types: undisturbed in-ovo control embryos, in-ovo paralyzed embryos, in-vitro unloaded limb cultures, and in-vitro loaded limb cultures. Relative glycosaminoglycan (GAG) concentration across the joint was assessed with an MRI technique referred to as dGEMRIC (delayed gadolinium enhanced MRI of cartilage) where T1 is proportional to glycosaminoglycan concentration., Results: Average T1 over the entire tissue image for the normal control (IC) knee was 480 msec; for the 4 day loaded specimen average T1 was 354 msec; and for the 7 day loaded specimens T1 was 393 msec. The 4 day unloaded specimen had an average T1 of 279 msec while the 7 day unloaded specimen had an average T1 of 224 msec. The higher T1 values in loaded than unloaded specimens suggest that more glycosaminoglycan is produced in the loaded culture than in the unloaded preparation., Conclusion: Isolated limb tissue cultures under flexion-extension load can be viable and exhibit more progression of joint differentiation and glycosaminoglycan production than similarly cultured but unloaded specimens. However, when compared with controls consisting of intact undisturbed embryos in-ovo, the isolated loaded limbs in culture do not demonstrate equivalent amounts of absolute growth or joint differentiation.

Disseminated fungal infections caused by Candida species are associated with homing of the pathogen to specific organs in human and murine hosts. Kidneys are a primary target organ of Candida albicans, and invasion into the kidney medulla can lead to loss of renal function and death. Therefore, development of noninvasive methods to assess kidney infections could aid in the management of disseminated candidemia. We describe a magnetic resonance imaging method utilizing iron oxide-based contrast agents to noninvasively assess recruitment of phagocytes and kidney inflammation. C. albicans also colonizes the brain and can cause meningoencephalitis. We describe additional imaging methods to assess loss of the blood-brain barrier function that initiates brain infections.

Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies., (Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGF-β signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Olfactomedin 1 (Olfm1) is a secreted glycoprotein that is preferentially expressed in neuronal tissues. Here we show that deletion of exons 4 and 5 from the Olfm1 gene, which encodes a 52 amino acid long region in the N-terminal part of the protein, increased neonatal death and reduced body weight of surviving homozygous mice. Magnetic resonance imaging analyses revealed reduced brain volume and attenuated size of white matter tracts such as the anterior commissure, corpus callosum, and optic nerve. Adult Olfm1 mutant mice demonstrated abnormal behavior in several tests including reduced marble digging, elevated plus maze test, nesting activity and latency on balance beam tests as compared with their wild-type littermates. The olfactory system was both structurally and functionally disturbed by the mutation in the Olfm1 gene as shown by functional magnetic resonance imaging analysis and a smell test. Deficiencies of the olfactory system may contribute to the neonatal death and loss of body weight of Olfm1 mutant. Shotgun proteomics revealed 59 candidate proteins that co-precipitated with wild-type or mutant Olfm1 proteins in postnatal day 1 brain. Olfm1-binding targets included GluR2, Cav2.1, teneurin-4 and Kidins220. Modified interaction of Olfm1 with binding targets led to an increase in intracellular Ca(2+) concentration and activation of ERK1/2, MEK1 and CaMKII in the hippocampus and olfactory bulb of Olfm1 mutant mice compared with their wild-type littermates. Excessive activation of the CaMKII and Ras-ERK pathways in the Olfm1 mutant olfactory bulb and hippocampus by elevated intracellular calcium may contribute to the abnormal behavior and olfactory activity of Olfm1 mutant mice., (© 2013.)

Disseminated candidiasis primarily targets the kidneys and brain in mice and humans. Damage to these critical organs leads to the high mortality associated with such infections, and invasion across the blood-brain barrier can result in fungal meningoencephalitis. Candida albicans can penetrate a brain endothelial cell barrier in vitro through transcellular migration, but this mechanism has not been confirmed in vivo. MRI using the extracellular vascular contrast agent gadolinium diethylenetriaminepentaacetic acid demonstrated that integrity of the blood-brain barrier is lost during C. albicans invasion. Intravital two-photon laser scanning microscopy was used to provide the first real-time demonstration of C. albicans colonizing the living brain, where both yeast and filamentous forms of the pathogen were found. Furthermore, we adapted a previously described method utilizing MRI to monitor inflammatory cell recruitment into infected tissues in mice. Macrophages and other phagocytes were visualized in kidney and brain by the administration of ultrasmall iron oxide particles. In addition to obtaining new insights into the passage of C. albicans across the brain microvasculature, these imaging methods provide useful tools to study further the pathogenesis of C. albicans infections, to define the roles of Candida virulence genes in kidney versus brain infection and to assess new therapeutic measures for drug development., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Background and Purpose: Enhanced angiogenesis facilitates neurovascular remodeling processes and promotes brain functional recovery after stroke. Previous studies from our laboratory demonstrated that valproate (VPA), a histone deacetylase inhibitor, protects against experimental brain ischemia. The present study investigated whether VPA could enhance angiogenesis and promote long-term functional recovery after ischemic stroke., Methods: Male rats underwent middle cerebral artery occlusion for 60 minutes followed by reperfusion for up to 14 days. Assessed parameters were: locomotor function through the Rotarod test; infarct volume through T2-weighted MRI; microvessel density through immunohistochemistry; relative cerebral blood flow through perfusion-weighted imaging; protein levels of proangiogenic factors through Western blotting; and matrix metalloproteinase-2/9 activities through gelatin zymography., Results: Postischemic VPA treatment robustly improved the Rotarod performance of middle cerebral artery occlusion rats on Days 7 and 14 after ischemia and significantly reduced brain infarction on Day 14. Concurrently, VPA markedly enhanced microvessel density, facilitated endothelial cell proliferation, and increased relative cerebral blood flow in the ipsilateral cortex. The transcription factor hypoxia-inducible factor-1α and its downstream proangiogenic factors, vascular endothelial growth factor and matrix metalloproteinase-2/9, were upregulated after middle cerebral artery occlusion and significantly potentiated by VPA in the ipsilateral cortex. Acetylation of histone-H3 and H4 was robustly increased by chronic VPA treatment. The beneficial effects of VPA on Rotarod performance and microvessel density were abolished by hypoxia-inducible factor-1α inhibition., Conclusions: Chronic VPA treatment enhances angiogenesis and promotes functional recovery after brain ischemia. These effects may involve histone deacetylase inhibition and upregulation of hypoxia-inducible factor-1α and its downstream proangiogenic factors vascular endothelial growth factor and matrix metalloproteinase-2/9.

Disruption of the blood-brain barrier (BBB) is a serious complication frequently encountered in neurodegenerative disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating childhood neurodegenerative lysosomal storage disorder caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. It remains unclear whether BBB is disrupted in INCL and if so, what might be the molecular mechanism(s) of this complication. We previously reported that the Ppt1-knockout (Ppt1-KO) mice that mimic INCL manifest high levels of oxidative stress and neuroinflammation. Recently, it has been reported that CD4(+) T-helper 17 (T(H)17) lymphocytes may mediate BBB disruption and neuroinflammation, although the precise molecular mechanism(s) remain unclear. We sought to determine: (i) whether the BBB is disrupted in Ppt1-KO mice, (ii) if so, do T(H)17-lymphocytes underlie this complication, and (iii) how might T(H)17 lymphocytes breach the BBB. Here, we report that the BBB is disrupted in Ppt1-KO mice and that T(H)17 lymphocytes producing IL-17A mediate disruption of the BBB by stimulating production of matrix metalloproteinases (MMPs), which degrade the tight junction proteins essential for maintaining BBB integrity. Importantly, dietary supplementation of resveratrol (RSV), a naturally occurring antioxidant/anti-inflammatory polyphenol, markedly reduced the levels of T(H)17 cells, IL-17A and MMPs, and elevated the levels of tight junction proteins, which improved the BBB integrity in Ppt1-KO mice. Intriguingly, we found that RSV suppressed the differentiation of CD4(+) T lymphocytes to IL-17A-positive T(H)17 cells. Our findings uncover a mechanism by which T(H)17 lymphocytes mediate BBB disruption and suggest that small molecules such as RSV that suppress T(H)17 differentiation are therapeutic targets for neurodegenerative disorders such as INCL.

The intracellular enzyme urea amidolyase (Dur1,2p) enables C. albicans to utilize urea as a sole nitrogen source. Because deletion of the DUR1,2 gene reduces survival of C. albicans co-cultured with a murine macrophage cell line, we investigated the role of Dur1,2p in pathogenesis using a mouse model of disseminated candidiasis. A dur1,2Δ/dur1,2Δ strain was significantly less virulent than the wild-type strain, showing significantly higher survival rate, better renal function, and decreased and less sustained fungal colonization in kidney and brain. Complementation of the mutant restored virulence. DUR1,2 deletion resulted in a milder host inflammatory reaction. Immunohistochemistry, flow cytometry, and magnetic resonance imaging showed decreased phagocytic infiltration into infected kidneys. Systemic cytokine levels of wild-type mice infected with the dur1,2 mutant showed a more balanced systemic pro-inflammatory cytokine response. Host gene expression and protein analysis in infected kidneys revealed parallel changes in the local immune response. Significant differences were observed in the kidney IL-1 inflammatory pathway, IL-15 signaling, MAP kinase signaling, and the alternative complement pathway. We conclude that Dur1,2p is important for kidney colonization during disseminated candidiasis and contributes to an unbalanced host inflammatory response and subsequent renal failure. Therefore, this Candida-specific enzyme may represent a useful drug target to protect the host from kidney damage associated with disseminated candidiasis.