Journal: Neural Regeneration Research

Article Title: A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts

doi: 10.4103/NRR.NRR-D-24-00422

Figure Lengend Snippet: Characterization of cellular proportions and morphologies in control (uninjured) cultures at 8 days in vitro . Representative fluorescence micrographs showing: (A) β-Tubulin (Tuj-1) + neurons with a high density of axonal growth. (B) Glial fibrillary acidic protein (GFAP) + astrocytes with fibrous morphologies. (C) Ramified ionized calcium binding adaptor molecule 1 (Iba1) + microglia. (D) Neuronal glial antigen 2 (NG2) + oligodendrocyte precursor cells expressing multipolar morphologies. (E) Processed myelin basic protein (MBP) + oligodendrocytes. (F) Double immunostaining of NG2 and MBP revealed distinct OPC and oligodendrocyte populations. (G) Triple immunostaining with GFAP, Tuj-1, and Iba1 demonstrated a bed layer of fibrous astrocytes with neuronal processes throughout and microglia residing at the top. (H) Proportion of cells immunopositive for each marker ( n = 5). n = number of cultures, each from a different litter of mice pups. Data are presented as mean ± SEM. DAPI: 4′,6-Diamidino-2-phenylindole.

Article Snippet: Primary antibodies were rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Cat# Z0334, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-β-tubulin (Tuj-1) (1:500, Cat# 801202, BioLegend, San Diego, CA, USA), goat anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Cat# ab5076, Abcam, Cambridge, UK), mouse anti-neuronal glial antigen2 (NG2) (1:500, Cat# ab275024, Abcam), rat anti-myelin basic protein (MBP) (1:200) (Cat# 160223, BioRad, Hercules, CA, USA).

Techniques: Control, In Vitro, Fluorescence, Binding Assay, Expressing, Double Immunostaining, Triple Immunostaining, Marker

Journal: Neural Regeneration Research

Article Title: A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts

doi: 10.4103/NRR.NRR-D-24-00422

Figure Lengend Snippet: Reproducible induction of transecting lesions. Representative micrographs demonstrating the defined injury margins with minimal intralesional debris immediately post-injury (day 0) showing β-tubulin (Tuj-1) (A), glial fibrillary acidic protein (GFAP) (B), DAPI positive cells (C) and lesion in phase contrast (D). White arrows indicate lesion margins. (E) Graph shows consistent reproducible lesion widths were generated ( n = 25 lesion measurements per biological repeat (multiple coverslips)). Data are presented as mean ± SEM. DAPI: 4′,6-Diamidino-2-phenylindole.

Article Snippet: Primary antibodies were rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Cat# Z0334, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-β-tubulin (Tuj-1) (1:500, Cat# 801202, BioLegend, San Diego, CA, USA), goat anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Cat# ab5076, Abcam, Cambridge, UK), mouse anti-neuronal glial antigen2 (NG2) (1:500, Cat# ab275024, Abcam), rat anti-myelin basic protein (MBP) (1:200) (Cat# 160223, BioRad, Hercules, CA, USA).

Techniques: Generated

Journal: Neural Regeneration Research

Article Title: A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts

doi: 10.4103/NRR.NRR-D-24-00422

Figure Lengend Snippet: Characterization of astrogliosis at the lesion site. (A, B) Representative micrographs of perilesional astrocytes at 1 and 3 DPL, respectively. These exhibit reactive astrocytic profiles with ruffles extending out from the injury border and/or enhanced GFAP. White dashed lines indicate lesion margins. (C, D) Representative control astrocytes. (E) Graph shows more reactive morphologies at the lesion edge. (F, G) Graphs show enhanced GFAP reactivity at the lesion margins 1 (F) and 3 DPL (G), respectively. Data are presented as mean ± SEM. n = 3. n = number of cultures, each from a different litter of mouse pups. * P < 0.05, *** P < 0.001 (unpaired t -test for E; one-way analysis of variance with Tukey’s post hoc test for F and G). DAPI: 4′,6-Diamidino-2-phenylindole; DPL: days post-lesion; GFAP: glial fibrillary acidic protein.

Article Snippet: Primary antibodies were rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Cat# Z0334, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-β-tubulin (Tuj-1) (1:500, Cat# 801202, BioLegend, San Diego, CA, USA), goat anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Cat# ab5076, Abcam, Cambridge, UK), mouse anti-neuronal glial antigen2 (NG2) (1:500, Cat# ab275024, Abcam), rat anti-myelin basic protein (MBP) (1:200) (Cat# 160223, BioRad, Hercules, CA, USA).

Techniques: Control

Journal: Neural Regeneration Research

Article Title: A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts

doi: 10.4103/NRR.NRR-D-24-00422

Figure Lengend Snippet: Activated astrocytes, neurons, or OPCs within or proximal to the lesion site do not show obvious uptake of PEG/CMX MPs. (A–C) Representative micrographs showing the detection of CMX (red) or (D–E) PEG (red) nanoparticles with astrocytes, neurons, or OPCs respectively ((GFAP, Tuj-1, or NG2 + (green)). White dashed lines indicate lesion margins. Yellow arrows indicate cellular regions in close proximity to MP aggregates with no clear signs of internalization (i.e. MP aggregate falls outside the boundary of the cell body/process). White asterisks indicate MP aggregates near nuclei of GFAP, Tuj-1 or NG2 negative cells (these are likely to be microglial nuclei). (A–A2) Low magnification (A) and high magnification (A1 and A2) of astrocytes with CMX in the lesion area. There is no clear internalization of the MPs; MPs sit in clusters close to negatively stained nuclei, or particles appear to be attached to the surface of the astrocytes. Note, for astrocytic internalization we would expect to see clear association of MPs within the astrocyte cell body or clustered around the nuclei. (B–B2) Neurons with close association of CMX aggregates yet no clear internalization as aggregates appear larger than neurites. (C–C2) OPCs show no internalization of CMX, yellow arrow indicates a CMX aggregate in close proximity to cells, however the aggregate staining falls outside the OPC cell body and is more likely to be attached to the cell surface. (D–D2) Astrocytic response to PEG particles with no clear internalization and PEG aggregates clustered around GFAP negative nuclei. (E–E2) Neuronal interaction with PEG where aggregates associated with processes are large and likely to be membrane-bound. There is no clear internalization of MPs observed. (F–F2) OPCs have no interaction with PEG particles. DAPI: 4′,6-Diamidino-2-phenylindole; DPL: days post-lesion; GFAP: glial fibrillary acidic protein; MPs: magnetic particles; NG2: neuronal glial antigen 2; OPC: oligodendrocyte precursor cell; PEG: poly-ethylene-glycol; Tuj-1: β-tubulin.

Article Snippet: Primary antibodies were rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Cat# Z0334, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-β-tubulin (Tuj-1) (1:500, Cat# 801202, BioLegend, San Diego, CA, USA), goat anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Cat# ab5076, Abcam, Cambridge, UK), mouse anti-neuronal glial antigen2 (NG2) (1:500, Cat# ab275024, Abcam), rat anti-myelin basic protein (MBP) (1:200) (Cat# 160223, BioRad, Hercules, CA, USA).

Techniques: Staining, Membrane

Journal: Neural Regeneration Research

Article Title: A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts

doi: 10.4103/NRR.NRR-D-24-00422

Figure Lengend Snippet: Histological and electrophysiological characterization of cultures using extracellular MEA recordings. (A–E) Representative immunofluorescence staining of cultures on the MEA: DAPI nuclear stain, GFAP + astrocytes, Iba-1 + microglia (arrows indicate ramified microglia), Tuj-1 + neurons, and merge (scale bars: 50 µm). Images show a widespread neuronal and astrocytic network on the electrodes (black squares) with microglia distributed throughout. (F) Representative array-wide 60-second raster plots depicting the development of spiking events and synchronized bursting events over 35 DIV. (G) ASDR shows a sigmoidal-type increase in the number of spikes throughout the array over 28 DIV. (H) The average synchronized burst rate (bursts/min) increases over time with complex characteristics per culture. (I) The average spike amplitude of the 4 independent cultures. (J) Demonstrates the average ASDR of large amplitude spikes (spikes/s), showing an increasing frequency of large amplitude spikes over time in culture. All cultures show signs of electrophysiological maturation over time (7–28 DIV) as their spiking/bursting activity and spike amplitudes increase. n1–4 represent separate biological repeats conducted on four separate MEA devices. Data are presented as mean ± SEM. ASDR: Array-wide spike detection rate; DAPI: 4′,6-diamidino-2-phenylindole; DIV: days in vitro ; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1; MEA: multielectrode array; Tuj-1: β-tubulin.

Article Snippet: Primary antibodies were rabbit anti-glial fibrillary acidic protein (GFAP) (1:500, Cat# Z0334, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-β-tubulin (Tuj-1) (1:500, Cat# 801202, BioLegend, San Diego, CA, USA), goat anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Cat# ab5076, Abcam, Cambridge, UK), mouse anti-neuronal glial antigen2 (NG2) (1:500, Cat# ab275024, Abcam), rat anti-myelin basic protein (MBP) (1:200) (Cat# 160223, BioRad, Hercules, CA, USA).

Techniques: Immunofluorescence, Staining, Activity Assay, In Vitro, Binding Assay