CellBoost: A pipeline for machine assisted annotation in Neuroanatomy.

One of the important yet labor intensive tasks in neuroanatomy is the identification of select populations of cells. Current high-throughput techniques enable marking cells with histochemical fluorescent molecules as well as through the genetic expression of fluorescent proteins. Modern scanning microscopes allow high resolution multi-channel imaging of the mechanically or optically sectioned brain with thousands of marked cells per square millimeter. Manual identification of all marked cells is prohibitively time consuming. At the same time, simple segmentation algorithms suffer from high error rates and sensitivity to variation in fluorescent intensity and spatial distribution. We present a methodology that combines human judgement and machine learning that serves to significantly reduce the labor of the anatomist while improving the consistency of the annotation. As a demonstration, we analyzed murine brains with marked premotor neurons in the brainstem. We compared the error rate of our method to the disagreement rate among human anatomists. This comparison shows that our method can reduce the time to annotate by as much as ten-fold without significantly increasing the rate of errors. We show that our method achieves significant reduction in labor while achieving an accuracy that is similar to the level of agreement between different anatomists.

A lone spike in blood glucose can enhance the thrombo-inflammatory response in cortical venules.

How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.

Constructing an adult orofacial premotor atlas in Allen mouse CCF.

Premotor circuits in the brainstem project to pools of orofacial motoneurons to execute essential motor action such as licking, chewing, breathing, and in rodent, whisking. Previous transsynaptic tracing studies only mapped orofacial premotor circuits in neonatal mice, but the adult circuits remain unknown as a consequence of technical difficulties. Here, we developed a three-step monosynaptic transsynaptic tracing strategy to identify premotor neurons controlling vibrissa, tongue protrusion, and jaw-closing muscles in the adult mouse. We registered these different groups of premotor neurons onto the Allen mouse brain common coordinate framework (CCF) and consequently generated a combined 3D orofacial premotor atlas, revealing unique spatial organizations of distinct premotor circuits. We further uncovered premotor neurons that simultaneously innervate multiple motor nuclei and, consequently, are likely to coordinate different muscles involved in the same orofacial motor actions. Our method for tracing adult premotor circuits and registering to Allen CCF is generally applicable and should facilitate the investigations of motor controls of diverse behaviors.

Brain microvasculature has a common topology with local differences in geometry that match metabolic load.

The microvasculature underlies the supply networks that support neuronal activity within heterogeneous brain regions. What are common versus heterogeneous aspects of the connectivity, density, and orientation of capillary networks? To address this, we imaged, reconstructed, and analyzed the microvasculature connectome in whole adult mice brains with sub-micrometer resolution. Graph analysis revealed common network topology across the brain that leads to a shared structural robustness against the rarefaction of vessels. Geometrical analysis, based on anatomically accurate reconstructions, uncovered a scaling law that links length density, i.e., the length of vessel per volume, with tissue-to-vessel distances. We then derive a formula that connects regional differences in metabolism to differences in length density and, further, predicts a common value of maximum tissue oxygen tension across the brain. Last, the orientation of capillaries is weakly anisotropic with the exception of a few strongly anisotropic regions; this variation can impact the interpretation of fMRI data.

Source apportionment of PM2.5 at two Seattle chemical speciation sites.

Positive Matrix Factorization analysis of PM2.5 chemical speciation data collected from 2015-2017 at Washington State Department of Ecology's urban NCore (Beacon Hill) and near-road (10th and Weller) sites found similar PM2.5 sources at both sites. Identified factors were associated with gasoline exhaust, diesel exhaust, aged and fresh sea salt, crustal, nitrate-rich, sulfur-rich, unidentified urban, zinc-rich, residual fuel oil, and wood smoke. Factors associated with vehicle emissions were the highest contributing sources at both sites. Gasoline exhaust emissions comprised 26% and 21% of identified sources at Beacon Hill and 10th and Weller, respectively. Diesel exhaust emissions comprised 29% of identified sources at 10th and Weller but only 3% of identified sources at Beacon Hill. Correlation of the diesel exhaust factor with measured concentrations of black carbon and nitrogen oxides at 10th and Weller suggests a method to predict PM2.5 from diesel exhaust without a full chemical speciation analysis. While most PM2.5 sources exhibit minimal change over time, primary PM2.5 from gasoline emissions is increasing on average 0.18 µg m-3 per year in Seattle. IMPLICATIONS: This study utilizes Positive Matrix Factorization to determine PM2.5 sources from chemical speciation measurements at two urban Seattle sites from 2015-2017. The paper reports PM2.5 source trends, and extends previous source apportionment analyses in Seattle to the present day. The study also quantifies diesel PM2.5 at a near-road site, and describes a predictive model that allows estimation of the contribution of diesel PM2.5 to the total measured PM2.5 at near-road sites across the country without a full chemical speciation analysis.

An active texture-based digital atlas enables automated mapping of structures and markers across brains.

Brain atlases enable the mapping of labeled cells and projections from different brains onto a standard coordinate system. We address two issues in the construction and use of atlases. First, expert neuroanatomists ascertain the fine-scale pattern of brain tissue, the 'texture' formed by cellular organization, to define cytoarchitectural borders. We automate the processes of localizing landmark structures and alignment of brains to a reference atlas using machine learning and training data derived from expert annotations. Second, we construct an atlas that is active; that is, augmented with each use. We show that the alignment of new brains to a reference atlas can continuously refine the coordinate system and associated variance. We apply this approach to the adult murine brainstem and achieve a precise alignment of projections in cytoarchitecturally ill-defined regions across brains from different animals.

Nerve-targeted probes for fluorescence-guided intraoperative imaging.

A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury.

Circuits in the rodent brainstem that control whisking in concert with other orofacial motor actions.

The world view of rodents is largely determined by sensation on two length scales. One is within the animal's peri-personal space; sensorimotor control on this scale involves active movements of the nose, tongue, head, and vibrissa, along with sniffing to determine olfactory clues. The second scale involves the detection of more distant space through vision and audition; these detection processes also impact repositioning of the head, eyes, and ears. Here we focus on orofacial motor actions, primarily vibrissa-based touch but including nose twitching, head bobbing, and licking, that control sensation at short, peri-personal distances. The orofacial nuclei for control of the motor plants, as well as primary and secondary sensory nuclei associated with these motor actions, lie within the hindbrain. The current data support three themes: First, the position of the sensors is determined by the summation of two drive signals, i.e., a fast rhythmic component and an evolving orienting component. Second, the rhythmic component is coordinated across all orofacial motor actions and is phase-locked to sniffing as the animal explores. Reverse engineering reveals that the preBötzinger inspiratory complex provides the reset to the relevant premotor oscillators. Third, direct feedback from somatosensory trigeminal nuclei can rapidly alter motion of the sensors. This feedback is disynaptic and can be tuned by high-level inputs. A holistic model for the coordination of orofacial motor actions into behaviors will encompass feedback pathways through the midbrain and forebrain, as well as hindbrain areas.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

OBJECTIVE: Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. METHODS: K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. RESULTS: In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. CONCLUSION: RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression.

Primary and Secondary Sources of Gas-Phase Organic Acids from Diesel Exhaust.

Organic acids have primary and secondary sources in the atmosphere, impact ecosystem health, and are useful metrics for identifying gaps in organic oxidation chemistry through model-measurement comparisons. We photooxidized (OH oxidation) primary emissions from diesel and biodiesel fuel types under two engine loads in an oxidative flow reactor. formic, butyric, and propanoic acids, but not methacrylic acid, have primary and secondary sources. Emission factors for these gas-phase acids varied from 0.3-8.4 mg kg-1 fuel. Secondary chemistry enhanced these emissions by 1.1 (load) to 4.4 (idle) × after two OH-equivalent days. The relative enhancement in secondary organic acids in idle versus loaded conditions was due to increased precursor emissions, not faster reaction rates. Increased hydrocarbon emissions in idle conditions due to less complete combustion (associated with less oxidized gas-phase molecules) correlated to higher primary organic acid emissions. The lack of correlation between organic aerosol and organic acid concentrations downstream of the flow reactor indicates that the secondary products formed on different oxidation time scales and that despite being photochemical products, organic acids are poor tracers for secondary organic aerosol formation from diesel exhaust. Ignoring secondary chemistry from diesel exhaust would lead to underestimates of both organic aerosol and gas-phase organic acids.

18F-positron-emitting/fluorescent labeled erythrocytes allow imaging of internal hemorrhage in a murine intracranial hemorrhage model.

An agent for visualizing cells by positron emission tomography is described and used to label red blood cells. The labeled red blood cells are injected systemically so that intracranial hemorrhage can be visualized by positron emission tomography (PET). Red blood cells are labeled with 0.3 µg of a positron-emitting, fluorescent multimodal imaging probe, and used to non-invasively image cryolesion induced intracranial hemorrhage in a murine model (BALB/c, 2.36 × 108 cells, 100 µCi, <4 mm hemorrhage). Intracranial hemorrhage is confirmed by histology, fluorescence, bright-field, and PET ex vivo imaging. The low required activity, minimal mass, and high resolution of this technique make this strategy an attractive alternative for imaging intracranial hemorrhage. PET is one solution to a spectrum of issues that complicate single photon emission computed tomography (SPECT). For this reason, this application serves as a PET alternative to [99mTc]-agents, and SPECT technology that is used in 2 million annual medical procedures. PET contrast is also superior to gadolinium and iodide contrast angiography for its lack of clinical contraindications.

Linking Load, Fuel, and Emission Controls to Photochemical Production of Secondary Organic Aerosol from a Diesel Engine.

Diesel engines are important sources of fine particle pollution in urban environments, but their contribution to the atmospheric formation of secondary organic aerosol (SOA) is not well constrained. We investigated direct emissions of primary organic aerosol (POA) and photochemical production of SOA from a diesel engine using an oxidation flow reactor (OFR). In less than a day of simulated atmospheric aging, SOA production exceeded POA emissions by an order of magnitude or more. Efficient combustion at higher engine loads coupled to the removal of SOA precursors and particle emissions by aftertreatment systems reduced POA emission factors by an order of magnitude and SOA production factors by factors of 2-10. The only exception was that the retrofitted aftertreatment did not reduce SOA production at idle loads where exhaust temperatures were low enough to limit removal of SOA precursors in the oxidation catalyst. Use of biodiesel resulted in nearly identical POA and SOA compared to diesel. The effective SOA yield of diesel exhaust was similar to that of unburned diesel fuel. While OFRs can help study the multiday evolution, at low particle concentrations OFRs may not allow for complete gas/particle partitioning and bias the potential of precursors to form SOA.

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization.

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Anthropogenic Sulfur Perturbations on Biogenic Oxidation: SO2 Additions Impact Gas-Phase OH Oxidation Products of α- and ß-Pinene.

In order to probe how anthropogenic pollutants can impact the atmospheric oxidation of biogenic emissions, we investigated how sulfur dioxide (SO2) perturbations impact the oxidation of two monoterpenes, α-and ß-pinene. We used chemical ionization mass spectrometry to examine changes in both individual molecules and gas-phase bulk properties of oxidation products as a function of SO2 addition. SO2 perturbations impacted the oxidation systems of α-and ß-pinene, leading to an ensemble of products with a lesser degree of oxygenation than unperturbed systems. These changes may be due to shifts in the OH:HO2 ratio from SO2 oxidation and/or to SO3 reacting directly with organic molecules. Van Krevelen diagrams suggest a shift from gas-phase functionalization by alcohol/peroxide groups to functionalization by carboxylic acid or carbonyl groups, consistent with a decreased OH:HO2 ratio. Increasing relative humidity dampens the impact of the perturbation. This decrease in oxygenation may impact secondary organic aerosol formation in regions dominated by biogenic emissions with nearby SO2 sources. We observed sulfur-containing organic compounds following SO2 perturbations of monoterpene oxidation; whether these are the result of photochemistry or an instrumental artifact from ion-molecule clustering remains uncertain. However, our results demonstrate that the two monoterpene isomers produce unique suites of oxidation products.

Identification of High-Risk Plaques by MRI and Fluorescence Imaging in a Rabbit Model of Atherothrombosis.

INTRODUCTION: The detection of atherosclerotic plaques at risk for disruption will be greatly enhanced by molecular probes that target vessel wall biomarkers. Here, we test if fluorescently-labeled Activatable Cell Penetrating Peptides (ACPPs) could differentiate stable plaques from vulnerable plaques that disrupt, forming a luminal thrombus. Additionally, we test the efficacy of a combined ACPP and MRI technique for identifying plaques at high risk of rupture. METHODS AND RESULTS: In an atherothrombotic rabbit model, disrupted plaques were identified with in vivo MRI and co-registered in the same rabbit aorta with the in vivo uptake of ACPPs, cleaved by matrix metalloproteinases (MMPs) or thrombin. ACPP uptake, mapped ex vivo in whole aortas, was higher in disrupted compared to non-disrupted plaques. Specifically, disrupted plaques demonstrated a 4.5~5.0 fold increase in fluorescence enhancement, while non-disrupted plaques showed only a 2.2~2.5 fold signal increase. Receiver operating characteristic (ROC) analysis indicates that both ACPPs (MMP and thrombin) show high specificity (84.2% and 83.2%) and sensitivity (80.0% and 85.7%) in detecting disrupted plaques. The detection power of ACPPs was improved when combined with the MRI derived measure, outward remodeling ratio. CONCLUSIONS: Our targeted fluorescence ACPP probes distinguished disrupted plaques from stable plaques with high sensitivity and specificity. The combination of anatomic, MRI-derived predictors for disruption and ACPP uptake can further improve the power for identification of high-risk plaques and suggests future development of ACPPs with molecular MRI as a readout.

Fluorescently labeled peptide increases identification of degenerated facial nerve branches during surgery and improves functional outcome.

Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL), complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41) can improve intraoperative visualization of chronically (up to 9 months) denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair.

Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture.

We review the organizational principles of the cortical vasculature and the underlying patterns of blood flow under normal conditions and in response to occlusion of single vessels. The cortex is sourced by a two-dimensional network of pial arterioles that feeds a three-dimensional network of subsurface microvessels in close proximity to neurons and glia. Blood flow within the surface and subsurface networks is largely insensitive to occlusion of a single vessel within either network. However, the penetrating arterioles that connect the pial network to the subsurface network are bottlenecks to flow; occlusion of even a single penetrating arteriole results in the death of a 500 µm diameter cylinder of cortical tissue despite the potential for collateral flow through microvessels. This pattern of flow is consistent with that calculated from a full reconstruction of the angioarchitecture. Conceptually, collateral flow is insufficient to compensate for the occlusion of a penetrating arteriole because penetrating venules act as shunts of blood that flows through collaterals. Future directions that stem from the analysis of the angioarchitecture concern cellular-level issues, in particular the regulation of blood flow within the subsurface microvascular network, and system-level issues, in particular the role of penetrating arteriole occlusions in human cognitive impairment.

Early detection of thrombin activity in neuroinflammatory disease.

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.