Elucidating the neuropathologic mechanisms of SARS-CoV-2 infection

Acknowledgements We want to express our gratitude to the Union Medical University Clinic, Dominican Republic, for their support and collaboration in the development of this research project. We also want to express our gratitude to the Mexican families who have donated the brain of their loved ones affected with Alzheimer's disease and made our research possible. This work is dedicated to the memory of Professor Dr. José Raúl Mena López†.Peer reviewedPublisher PD

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Acute Neuroinflammatory Response in the Substantia Nigra Pars Compacta of Rats after a Local Injection of Lipopolysaccharide

Models of Parkinson’s disease with neurotoxins have shown that microglial activation does not evoke a typical inflammatory response in the substantia nigra, questioning whether neuroinflammation leads to neurodegeneration. To address this issue, the archetypal inflammatory stimulus, lipopolysaccharide (LPS), was injected into the rat substantia nigra. LPS induced fever, sickness behavior, and microglial activation (OX42 immunoreactivity), followed by astrocyte activation and leukocyte infiltration (GFAP and CD45 immunoreactivities). During the acute phase of neuroinflammation, pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-4, and IL-10) responded differentially at mRNA and protein level. Increased NO production and lipid peroxidation occurred at 168 h after LPS injection. At this time, evidence of neurodegeneration could be seen, entailing decreased tyrosine hydroxylase (TH) immunoreactivity, irregular body contour, and prolongation discontinuity of TH+ cells, as well as apparent phagocytosis of TH+ cells by OX42+ cells. Altogether, these results show that LPS evokes a typical inflammatory response in the substantia nigra that is followed by dopaminergic neurodegeneration

Neurturin overexpression in dopaminergic neurons induces presynaptic and postsynaptic structural changes in rats with chronic 6-hydroxydopamine lesion.

The structural effect of neurturin (NRTN) on the nigrostriatal dopaminergic system in animals remains unknown, although NRTN has been shown to be effective in Parkinson's disease animal models. Herein, we aimed to demonstrate that NRTN overexpression in dopaminergic neurons stimulates both neurite outgrowths in the nigrostriatal pathway and striatal dendritic spines in aging rats with chronic 6-hydroxydopamine (6-OHDA) lesion. At week 12 after lesion, pTracer-mNRTN-His or pGreenLantern-1 plasmids were intranigrally transfected using the NTS-polyplex nanoparticles system. We showed that the transgenic expression in dopaminergic neurons remained until the end of the study (12 weeks). Only animals expressing NRTN-His showed recovery of tyrosine hydroxylase (TH)+ cells (28 ± 2%), their neurites (32 ± 2%) and the neuron-specific cytoskeletal marker β-III-tubulin in the substantia nigra; striatal TH(+) fibers were also recovered (52 ± 3%), when compared to the healthy condition. Neurotensin receptor type 1 levels were also significantly recovered in the substantia nigra and striatum. Dopamine recovery was 70 ± 4% in the striatum and complete in the substantia nigra. The number of dendritic spines of striatal medium spiny neurons was also significantly increased, but the recovery was not complete. Drug-activated circling behavior decreased by 73 ± 2% (methamphetamine) and 89 ± 1% (apomorphine). Similar decrease was observed in the spontaneous motor behavior. Our results demonstrate that NRTN causes presynaptic and postsynaptic restoration of the nigrostriatal dopaminergic system after a 6-OHDA-induced chronic lesion. However, those improvements did not reach the healthy condition, suggesting that NRTN exerts lesser neurotrophic effects than other neurotrophic approaches

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

26 octobre 19201920/10/26 (A21,N7255)

NRTN gene transfection increase spine density and total number of intersections in MSN.

<p><b>A</b>. Sholl diagram for the morphological study of dendrites and dendritic spines. <b>B</b>. Representative micrograph of a medium spiny neuron (MSN) of a healthy striatum. The arrow shows the 50 μm segment (primary dendrite) where dendritic spines were analyzed. Upper calibration bar = 25 μm. The small panels show different types of spines (thin, mushroom, stubby and wide, from left to right) indicated by the arrow. Calibration bar = 5 μm. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion and the Sholl analysis was performed at the end of the study (12 weeks after transfection or 24 weeks after lesion). UT = untransfected rats with lesion. <b>C.</b> Sholl analysis of total number of intersections along dendritic trees in MSNs at all distances in 200-μm radius from the soma. <b>D.</b> Plot of mean spine density analyzed per 50-μm primary dendrite from cell body (proximal segment, upper arrow in <b>B</b>) from six neurons per rat. All values represent the mean ± SEM (<i>n</i> = 6 independent rats in each experimental condition). Statistical analysis (<b>C</b> and <b>D</b>) was performed using one-way ANOVA and Tukey <i>post-hoc</i> test.</p

Intranigral Administration of β-Sitosterol-β-D-Glucoside Elicits Neurotoxic A1 Astrocyte Reactivity and Chronic Neuroinflammation in the Rat Substantia Nigra

Chronic consumption of β-sitosterol-β-D-glucoside (BSSG), a neurotoxin contained in cycad seeds, leads to Parkinson’s disease in humans and rodents. Here, we explored whether a single intranigral administration of BSSG triggers neuroinflammation and neurotoxic A1 reactive astrocytes besides dopaminergic neurodegeneration. We injected 6 μg BSSG/1 μL DMSO or vehicle into the left substantia nigra and immunostained with antibodies against tyrosine hydroxylase (TH) together with markers of microglia (OX42), astrocytes (GFAP, S100β, C3), and leukocytes (CD45). We also measured nitric oxide (NO), lipid peroxidation (LPX), and proinflammatory cytokines (TNF-α, IL-1β, IL-6). The Evans blue assay was used to explore the blood-brain barrier (BBB) permeability. We found that BSSG activates NO production on days 15 and 30 and LPX on day 120. Throughout the study, high levels of TNF-α were present in BSSG-treated animals, whereas IL-1β was induced until day 60 and IL-6 until day 30. Immunoreactivity of activated microglia (899.0±80.20%) and reactive astrocytes (651.50±11.28%) progressively increased until day 30 and then decreased to remain 251.2±48.8% (microglia) and 91.02±39.8 (astrocytes) higher over controls on day 120. C3(+) cells were also GFAP and S100β immunoreactive, showing they were neurotoxic A1 reactive astrocytes. BBB remained permeable until day 15 when immune cell infiltration was maximum. TH immunoreactivity progressively declined, reaching 83.6±1.8% reduction on day 120. Our data show that BSSG acute administration causes chronic neuroinflammation mediated by activated microglia, neurotoxic A1 reactive astrocytes, and infiltrated immune cells. The severe neuroinflammation might trigger Parkinson’s disease in BSSG intoxication

NRTN gene transfection increase spine density and total number of intersections in MSN.

<p><b>A</b>. Sholl diagram for the morphological study of dendrites and dendritic spines. <b>B</b>. Representative micrograph of a medium spiny neuron (MSN) of a healthy striatum. The arrow shows the 50 μm segment (primary dendrite) where dendritic spines were analyzed. Upper calibration bar = 25 μm. The small panels show different types of spines (thin, mushroom, stubby and wide, from left to right) indicated by the arrow. Calibration bar = 5 μm. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion and the Sholl analysis was performed at the end of the study (12 weeks after transfection or 24 weeks after lesion). UT = untransfected rats with lesion. <b>C.</b> Sholl analysis of total number of intersections along dendritic trees in MSNs at all distances in 200-μm radius from the soma. <b>D.</b> Plot of mean spine density analyzed per 50-μm primary dendrite from cell body (proximal segment, upper arrow in <b>B</b>) from six neurons per rat. All values represent the mean ± SEM (<i>n</i> = 6 independent rats in each experimental condition). Statistical analysis (<b>C</b> and <b>D</b>) was performed using one-way ANOVA and Tukey <i>post-hoc</i> test.</p

Proportional density of the different types of spines in medium spiny neurons from the rats of the four groups studied.

<p>Proportional density of the different types of spines in medium spiny neurons from the rats of the four groups studied.</p

Neurotrophic effect of NRTN gene transfection on TH(+) cells and neurites of the substantia nigra with chronic 6-OHDA lesion.

<p><b>A.</b> Representative micrographs of TH immunohistochemistry. Headings = Anterior-posterior coordinates of Paxinos and Watson Rat Atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188239#pone.0188239.ref054" target="_blank">54</a>]. 24WLUT = Untransfected rats with 24 weeks of 6-OHDA lesion. 12WL4WT = 12 weeks after lesion and 4 weeks after transfection, 12WL12WT = 12 weeks after lesion and 12 weeks after transfection. The scale bar of 300 μm is common for slices at different levels and of 50 μm for details. <b>B.</b> TH(+) neuron counting. <b>C.</b> Densitometry of TH(+) relative area. 4WT and 12 WT = 4 and 12 weeks after transfection. 24WL = untransfected rats with 24 weeks of lesion. 12WL = 12 weeks after lesion. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion. All values are the mean ± SEM (<i>n</i> = 3 independent rats for each experimental condition). One-way ANOVA and Newman-Keuls post-test. * <i>P</i> < 0.001, ** <i>P</i> < 0.0001. ns = no statistical significance, <i>P</i> > 0.05.</p