Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository.

Selecting and implementing a tissue-clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue-clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that many articles do not provide sufficient detail to replicate or reproduce experimental results. To help address this issue, we have developed a novel, freely available repository of tissue-clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue-clearing protocols did not perform well (negative results). The goal of T-CLEARE is to provide a forum for the community to share evaluations and modifications of tissue-clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.

Standard metadata for 3D microscopy.

Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments' images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for three-dimensional (3D) microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting 3D Microscopy Metadata Standards (3D-MMS) includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration.

Effects of spot parameters in pencil beam scanning treatment planning.

BACKGROUND: Spot size σ (in air at isocenter), interspot spacing d, and spot charge q influence dose delivery efficiency and plan quality in Intensity Modulated Proton Therapy (IMPT) treatment planning. The choice and range of parameters varies among different manufacturers. The goal of this work is to demonstrate the influence of the spot parameters on dose quality and delivery in IMPT treatment plans, to show their interdependence, and to make practitioners aware of the spot parameter values for a certain facility. Our study could help as a guideline to make the trade-off between treatment quality and time in existing PBS centers and in future systems. METHODS: We created plans for seven patients and a phantom, with different tumor sites and volumes, and compared the effect of small-, medium-, and large-spot widths (σ = 2.5, 5, and 10 mm) and interspot distances (1σ, 1.5σ, and 1.75σ) on dose, spot charge, and treatment time. Moreover, we quantified how postplanning charge threshold cuts affect plan quality and the total number of spots to deliver, for different spot widths and interspot distances. We show the effect of a minimum charge (or MU) cutoff value for a given proton delivery system. RESULTS: Spot size had a strong influence on dose: larger spots resulted in more protons delivered outside the target region. We observed dose differences of 2-13 Gy (RBE) between 2.5 mm and 10 mm spots, where the amount of extra dose was due to dose penumbra around the target region. Interspot distance had little influence on dose quality for our patient group. Both parameters strongly influence spot charge in the plans and thus the possible impact of postplanning charge threshold cuts. If such charge thresholds are not included in the treatment planning system (TPS), it is important that the practitioner validates that a given combination of lower charge threshold, interspot spacing, and spot size does not result in a plan degradation. Low average spot charge occurs for small spots, small interspot distances, many beam directions, and low fractional dose values. CONCLUSIONS: The choice of spot parameters values is a trade-off between accelerator and beam line design, plan quality, and treatment efficiency. We recommend the use of small spot sizes for better organ-at-risk sparing and lateral interspot distances of 1.5σ to avoid long treatment times. We note that plan quality is influenced by the charge cutoff. Our results show that the charge cutoff can be sufficiently large (i.e., 106 protons) to accommodate limitations on beam delivery systems. It is, therefore, not necessary per se to include the charge cutoff in the treatment planning optimization such that Pareto navigation (e.g., as practiced at our institution) is not excluded and optimal plans can be obtained without, perhaps, a bias from the charge cutoff. We recommend that the impact of a minimum charge cut impact is carefully verified for the spot sizes and spot distances applied or that it is accommodated in the TPS.

Impact of spot charge inaccuracies in IMPT treatments.

BACKGROUND: Spot charge is one parameter of pencil-beam scanning dose delivery system whose accuracy is typically high but whose required value has not been investigated. In this work we quantify the dose impact of spot charge inaccuracies on the dose distribution in patients. Knowing the effect of charge errors is relevant for conventional proton machines, as well as for new generation proton machines, where ensuring accurate charge may be challenging. METHODS: Through perturbation of spot charge in treatment plans for seven patients and a phantom, we evaluated the dose impact of absolute (up to 5× 106 protons) and relative (up to 30%) charge errors. We investigated the dependence on beam width by studying scenarios with small, medium and large beam sizes. Treatment plan statistics included the Γ passing rate, dose-volume-histograms and dose differences. RESULTS: The allowable absolute charge error for small spot plans was about 2× 106 protons. Larger limits would be allowed if larger spots were used. For relative errors, the maximum allowable error size for small, medium and large spots was about 13%, 8% and 6% for small, medium and large spots, respectively. CONCLUSIONS: Dose distributions turned out to be surprisingly robust against random spot charge perturbation. Our study suggests that ensuring spot charge errors as small as 1-2% as is commonly aimed at in conventional proton therapy machines, is clinically not strictly needed.

Dose uncertainties in IMPT for oropharyngeal cancer in the presence of anatomical, range, and setup errors.

PURPOSE: Setup, range, and anatomical uncertainties influence the dose delivered with intensity modulated proton therapy (IMPT), but clinical quantification of these errors for oropharyngeal cancer is lacking. We quantified these factors and investigated treatment fidelity, that is, robustness, as influenced by adaptive planning and by applying more beam directions. METHODS AND MATERIALS: We used an in-house treatment planning system with multicriteria optimization of pencil beam energies, directions, and weights to create treatment plans for 3-, 5-, and 7-beam directions for 10 oropharyngeal cancer patients. The dose prescription was a simultaneously integrated boost scheme, prescribing 66 Gy to primary tumor and positive neck levels (clinical target volume-66 Gy; CTV-66 Gy) and 54 Gy to elective neck levels (CTV-54 Gy). Doses were recalculated in 3700 simulations of setup, range, and anatomical uncertainties. Repeat computed tomography (CT) scans were used to evaluate an adaptive planning strategy using nonrigid registration for dose accumulation. RESULTS: For the recalculated 3-beam plans including all treatment uncertainty sources, only 69% (CTV-66 Gy) and 88% (CTV-54 Gy) of the simulations had a dose received by 98% of the target volume (D98%) >95% of the prescription dose. Doses to organs at risk (OARs) showed considerable spread around planned values. Causes for major deviations were mixed. Adaptive planning based on repeat imaging positively affected dose delivery accuracy: in the presence of the other errors, percentages of treatments with D98% >95% increased to 96% (CTV-66 Gy) and 100% (CTV-54 Gy). Plans with more beam directions were not more robust. CONCLUSIONS: For oropharyngeal cancer patients, treatment uncertainties can result in significant differences between planned and delivered IMPT doses. Given the mixed causes for major deviations, we advise repeat diagnostic CT scans during treatment, recalculation of the dose, and if required, adaptive planning to improve adequate IMPT dose delivery.