Measurement-oriented deep-learning workflow for improved segmentation of myelin and axons in high-resolution images of human cerebral white matter.

BACKGROUND: Standard segmentation of high-contrast electron micrographs (EM) identifies myelin accurately but does not translate easily into measurements of individual axons and their myelin, even in cross-sections of parallel fibers. We describe automated segmentation and measurement of each myelinated axon and its sheath in EMs of arbitrarily oriented human white matter from autopsies. NEW METHODS: Preliminary segmentation of myelin, axons and background by machine learning, using selected filters, precedes automated correction of systematic errors. Final segmentation is done by a deep neural network (DNN). Automated measurement of each putative fiber rejects measures encountering pre-defined artifacts and excludes fibers failing to satisfy pre-defined conditions. RESULTS: Improved segmentation of three sets of 30 annotated images each (two sets from human prefrontal white matter and one from human optic nerve) is achieved with a DNN trained only with a subset of the first set from prefrontal white matter. Total number of myelinated axons identified by the DNN differed from expert segmentation by 0.2%, 2.9%, and -5.1%, respectively. G-ratios differed by 2.96%, 0.74% and 2.83%. Intraclass correlation coefficients between DNN and annotated segmentation were mostly >0.9, indicating nearly interchangeable performance. COMPARISON WITH EXISTING METHOD(S): Measurement-oriented studies of arbitrarily oriented fibers from central white matter are rare. Published methods are typically applied to cross-sections of fascicles and measure aggregated areas of myelin sheaths and axons, allowing estimation only of average g-ratio. CONCLUSIONS: Automated segmentation and measurement of axons and myelin is complex. We report a feasible approach that has so far proven comparable to manual segmentation.

Resilience Is Associated With Larger Dentate Gyrus, While Suicide Decedents With Major Depressive Disorder Have Fewer Granule Neurons.

BACKGROUND: Early life adversity (ELA) increases major depressive disorder (MDD) and suicide risk and potentially affects dentate gyrus (DG) plasticity. We reported smaller DG and fewer granular neurons (GNs) in MDD. ELA effects on DG plasticity in suicide decedents with MDD (MDDSui) and resilient subjects (ELA history without MDD or suicide) are unknown. METHODS: We quantified neural progenitor cells (NPCs), GNs, glia, and DG volume in whole hippocampus postmortem in four groups of drug-free, neuropathology-free subjects (N = 52 total): psychological autopsy-defined MDDSui and control subjects with and without ELA (before 15 years of age). RESULTS: ELA was associated with larger DG (p < .0001) and trending fewer NPCs (p = .0190) only in control subjects in whole DG, showing no effect on NPCs and DG volume in MDDSui. ELA exposure was associated with more GNs (p = .0003) and a trend for more glia (p = .0160) in whole DG in MDDSui and control subjects. MDDSui without ELA had fewer anterior and mid DG GNs (p < .0001), fewer anterior DG NPCs (p < .0001), and smaller whole DG volume (p = .0005) compared with control subjects without ELA. In MDDSui, lower Global Assessment Scale score correlated with fewer GNs and smaller DG. CONCLUSIONS: Resilience to ELA involves a larger DG, perhaps related to more neurogenesis depleting NPCs, and because mature GNs and glia numbers do not differ in the resilient group, perhaps there are effects on process extension and synaptic load that can be examined in future studies. In MDDSui without ELA, smaller DG volume, with fewer GNs and NPCs, suggests less neurogenesis and/or more apoptosis and dendrite changes.

Blood Vessels and Perivascular Phagocytes of Prefrontal White and Gray Matter in Suicide.

Inflammatory processes may contribute to psychiatric disorders and suicide. Earlier, we reported greater densities of perivascular phagocytes in dorsal prefrontal white matter (DPFWM) in suicide than in non-suicide deaths. To distinguish between greater vascularity and greater coverage of vessels by perivascular phagocytes, and to determine whether the excess of perivascular phagocytes is derived from microglia or from non-parenchymal immune cells, we made stereological estimates of vascular surface area density (AVTOTAL) by staining for glucose transporter Glut-1, and the fraction of vascular surface area (AF) immunoreactive (IR) for CD163 (CD163 AF) in dorsal and ventral prefrontal white and gray matter. Manner of death or psychiatric diagnosis showed no association with CD163 AF in any region. Suicide was associated with a lower AVTOTAL compared with non-suicides in DPFWM (p = 0.018) but not with AVTOTAL in the 3 other regions of interest. Thus, the earlier observation of increased density of perivascular phagocytes in DPFWM after suicide cannot be attributed to infiltration by peripheral monocytes or to increased vascularity. Greater AVTOTAL ventrally than dorsally (p = 0.002) was unique to suicide and white matter.

Reliable and durable Golgi staining of brain tissue from human autopsies and experimental animals.

BACKGROUND: Golgi stains are notoriously capricious, particularly when applied to human brain. The well-known difficulties, which include complete failure of impregnation, patchy staining, unstable staining, and extensive crystalline deposits in superficial sections, have discouraged many from attempting to use these techniques. A reliable method that produces uniform impregnation in tissue from human autopsies and experimental animals is needed. NEW METHOD: The method described, "NeoGolgi", modifies previous Golgi-Cox protocols (Glaser and Van der Loos, 1981). Changes include: much longer time (>10 weeks) in Golgi solution, agitation on a slowly rocking platform, more gradual infiltration with Parlodion, more thorough removal of excess staining solution during embedding, and shorter exposure to ammonia after infiltration. RESULTS: The procedure has successfully stained over 220 consecutive frontal or hippocampal blocks from more than 175 consecutive human autopsy cases. Dendritic spines are easily recognized, and background is clear, allowing examination of very thick (200 µm) sections. Stained neurons are evenly distributed within cortical regions. The stain is stable for at least eight years. Most importantly, all stained neurons are apparently well-impregnated, eliminating ambiguity between pathology and poor impregnation that is inherent to other methods. COMPARISON WITH EXISTING METHODS: Most methods of Golgi staining are poorly predictable. They often fail completely, staining is patchy, and abnormal morphology is often indistinguishable from poor impregnation. "NeoGolgi" overcomes these problems. CONCLUSION: Starting with unfixed tissue, it is possible to obtain Golgi staining of predictably high quality in brains from human autopsies and experimental animals.

Air gun injury with deadly aftermath--case report.

In Republic of Macedonia the use of air guns is quite widespread. They are used mainly for target practice. They are regulated by the Law of Arms, where they are defined as pneumatic weapons. There is no legal limit on type or quantity of ammunition that one may possess. Our Institute performs at least 90% of the forensic autopsies in Macedonia. In this report we describe the only fatality by pneumatic weapon to come to our attention over the past 10 years. A 6-year-old girl was accidentally wounded by her brother when he and his father were trying a new air gun, a 4.5mm single shot, break barrel, spring piston air rifle manufactured in China under the brand "Westlake". She died within minutes. Autopsy showed cardiac tamponade due to penetration of the aorta. A 0.5g metal projectile, 4.5mm in diameter, with a pointed, conical shape, was recovered from the pericardial sac.