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Sub-Chronic Neuropathological and Biochemical Changes in Mouse Visual System after Repetitive Mild Traumatic Brain Injury.

Tzekov R, Dawson C, Orlando M, Mouzon B, Reed J, Evans J, Crynen G, Mullan M, Crawford F - PLoS ONE (2016)

Bottom Line: These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs.Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D.The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased.

View Article: PubMed Central - PubMed

Affiliation: The Roskamp Institute, Sarasota, FL, United States of America.

ABSTRACT
Repetitive mild traumatic brain injury (r-mTBI) results in neuropathological and biochemical consequences in the human visual system. Using a recently developed mouse model of r-mTBI, with control mice receiving repetitive anesthesia alone (r-sham) we assessed the effects on the retina and optic nerve using histology, immunohistochemistry, proteomic and lipidomic analyses at 3 weeks post injury. Retina tissue was used to determine retinal ganglion cell (RGC) number, while optic nerve tissue was examined for cellularity, myelin content, protein and lipid changes. Increased cellularity and areas of demyelination were clearly detectable in optic nerves in r-mTBI, but not in r-sham. These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs. Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D. Lipidomic analysis showed quantitative changes in a number of phospholipid species, including a significant increase in the total amount of lysophosphatidylcholine (LPC), including the molecular species 16:0, a known demyelinating agent. The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased. Results from the biochemical analysis correlate well with changes detected by histological and immunohistochemical methods and indicate the involvement of several important molecular pathways. This will allow future identification of therapeutic targets for improving the visual consequences of r-mTBI.

No MeSH data available.


Related in: MedlinePlus

Representative images of retina flatmounts and results from RGC counts.Representative images from the upper retina of a mouse after repetitive anesthesia alone (r-sham) (A) and r-mTBI (B). Retinal ganglion cells are labeled with anti-Brn-3a antibody. Scale bar = 50 μm. (C) Drawing depicting the retinal samples used to calculate the cell density; grey filled rectangles indicate the position of inner areas, while unfilled rectangles indicate the position of the outer retinal areas; ON—optic nerve. (D) Comparison between the average cells/field (cell densities) in the inner and outer areas.
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pone.0153608.g001: Representative images of retina flatmounts and results from RGC counts.Representative images from the upper retina of a mouse after repetitive anesthesia alone (r-sham) (A) and r-mTBI (B). Retinal ganglion cells are labeled with anti-Brn-3a antibody. Scale bar = 50 μm. (C) Drawing depicting the retinal samples used to calculate the cell density; grey filled rectangles indicate the position of inner areas, while unfilled rectangles indicate the position of the outer retinal areas; ON—optic nerve. (D) Comparison between the average cells/field (cell densities) in the inner and outer areas.

Mentions: Whole-mount retinal preparations from mice at 3 weeks after injury (n = 4, r-sham; n = 3, r-mTBI) were carried out as described [10] based on a slight modification of a published method [11]. Briefly, mice were deeply anesthetized, and the vertical orientation of the eye was marked by low-temperature cautery. Retinas were dissected and prepared as flattened whole mounts by making 4 radial cuts, postfixed for an additional hour, and then transferred into a 2 ml vial. Immunofluorescence staining with an antibody against brain-specific homeobox/POU domain protein 3A (Brn-3a) was performed similar to the methods previously described [12]. Briefly, retinas were permeated in 0.5% Triton X-100 by freezing for 15 minutes at -70°C, rinsed in fresh Triton X-100, and incubated overnight at 4°C with a primary goat anti-BRN3A (C-20) antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:100 in blocking buffer. Retinas were then washed 3 times in PBS and incubated for 2 hours at room temperature with the secondary antibody (Alexa Fluor 594-AffiniPure Rabbit Anti-Goat IgG; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted in blocking buffer. Finally, retinas were thoroughly washed in PBS and mounted vitreal side up on slides and covered with antifading solution. The slides were then imaged on Olympus BX63 fluorescent microscope (Olympus America, Center Valley, PA) with a motorized stage using x40 objective, Olympus DP72 camera and a Texas Red filter cube. Eighth images were acquired for each retina: four images acquired at a distance of 0.9 mm from the optic nerve head, one at each quadrant (inner images) and four were acquired at distance of 1.8 mm from the optic nerve head, one at each quadrant (outer images) (Fig 1). The acquired stacks of images were then exported and subsequently combined into one frame in ImageJ [13]. After background adjustment, the images were converted to 8-bit format and then adjusted for contrast, brightness, and threshold. Larger areas where the cells were perceived as fused were subdivided using a watershed procedure. The RGC nuclei were counted with an automated counting algorithm in ImageJ within the cell nucleus size range of 10 to 300 μm2.


Sub-Chronic Neuropathological and Biochemical Changes in Mouse Visual System after Repetitive Mild Traumatic Brain Injury.

Tzekov R, Dawson C, Orlando M, Mouzon B, Reed J, Evans J, Crynen G, Mullan M, Crawford F - PLoS ONE (2016)

Representative images of retina flatmounts and results from RGC counts.Representative images from the upper retina of a mouse after repetitive anesthesia alone (r-sham) (A) and r-mTBI (B). Retinal ganglion cells are labeled with anti-Brn-3a antibody. Scale bar = 50 μm. (C) Drawing depicting the retinal samples used to calculate the cell density; grey filled rectangles indicate the position of inner areas, while unfilled rectangles indicate the position of the outer retinal areas; ON—optic nerve. (D) Comparison between the average cells/field (cell densities) in the inner and outer areas.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835061&req=5

pone.0153608.g001: Representative images of retina flatmounts and results from RGC counts.Representative images from the upper retina of a mouse after repetitive anesthesia alone (r-sham) (A) and r-mTBI (B). Retinal ganglion cells are labeled with anti-Brn-3a antibody. Scale bar = 50 μm. (C) Drawing depicting the retinal samples used to calculate the cell density; grey filled rectangles indicate the position of inner areas, while unfilled rectangles indicate the position of the outer retinal areas; ON—optic nerve. (D) Comparison between the average cells/field (cell densities) in the inner and outer areas.
Mentions: Whole-mount retinal preparations from mice at 3 weeks after injury (n = 4, r-sham; n = 3, r-mTBI) were carried out as described [10] based on a slight modification of a published method [11]. Briefly, mice were deeply anesthetized, and the vertical orientation of the eye was marked by low-temperature cautery. Retinas were dissected and prepared as flattened whole mounts by making 4 radial cuts, postfixed for an additional hour, and then transferred into a 2 ml vial. Immunofluorescence staining with an antibody against brain-specific homeobox/POU domain protein 3A (Brn-3a) was performed similar to the methods previously described [12]. Briefly, retinas were permeated in 0.5% Triton X-100 by freezing for 15 minutes at -70°C, rinsed in fresh Triton X-100, and incubated overnight at 4°C with a primary goat anti-BRN3A (C-20) antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:100 in blocking buffer. Retinas were then washed 3 times in PBS and incubated for 2 hours at room temperature with the secondary antibody (Alexa Fluor 594-AffiniPure Rabbit Anti-Goat IgG; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted in blocking buffer. Finally, retinas were thoroughly washed in PBS and mounted vitreal side up on slides and covered with antifading solution. The slides were then imaged on Olympus BX63 fluorescent microscope (Olympus America, Center Valley, PA) with a motorized stage using x40 objective, Olympus DP72 camera and a Texas Red filter cube. Eighth images were acquired for each retina: four images acquired at a distance of 0.9 mm from the optic nerve head, one at each quadrant (inner images) and four were acquired at distance of 1.8 mm from the optic nerve head, one at each quadrant (outer images) (Fig 1). The acquired stacks of images were then exported and subsequently combined into one frame in ImageJ [13]. After background adjustment, the images were converted to 8-bit format and then adjusted for contrast, brightness, and threshold. Larger areas where the cells were perceived as fused were subdivided using a watershed procedure. The RGC nuclei were counted with an automated counting algorithm in ImageJ within the cell nucleus size range of 10 to 300 μm2.

Bottom Line: These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs.Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D.The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased.

View Article: PubMed Central - PubMed

Affiliation: The Roskamp Institute, Sarasota, FL, United States of America.

ABSTRACT
Repetitive mild traumatic brain injury (r-mTBI) results in neuropathological and biochemical consequences in the human visual system. Using a recently developed mouse model of r-mTBI, with control mice receiving repetitive anesthesia alone (r-sham) we assessed the effects on the retina and optic nerve using histology, immunohistochemistry, proteomic and lipidomic analyses at 3 weeks post injury. Retina tissue was used to determine retinal ganglion cell (RGC) number, while optic nerve tissue was examined for cellularity, myelin content, protein and lipid changes. Increased cellularity and areas of demyelination were clearly detectable in optic nerves in r-mTBI, but not in r-sham. These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs. Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D. Lipidomic analysis showed quantitative changes in a number of phospholipid species, including a significant increase in the total amount of lysophosphatidylcholine (LPC), including the molecular species 16:0, a known demyelinating agent. The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased. Results from the biochemical analysis correlate well with changes detected by histological and immunohistochemical methods and indicate the involvement of several important molecular pathways. This will allow future identification of therapeutic targets for improving the visual consequences of r-mTBI.

No MeSH data available.


Related in: MedlinePlus