Fig 1.
Chemical Structure and Metabolic Pathways Involved in Ganglioside Synthesis and Catabolism.
A group of transferase enzymes add either a sugar unit (ex: along A-series pathway) or a sialic acid residue (ex: A to B series pathways) to synthesize different ganglioside species. A different set of enzymes breaks down these components to increase levels of simpler species thus a homeostatic level of each ganglioside species is maintained in a healthy organism.
Fig 2.
Following surgery, rats are euthanized via anesthetic overdose and decapitated. Fresh brains are isolated and sectioned at 10μm on a cryostat onto MALDI plates. Sections sprayed with CMBT matrix using an airbrush are processed on a MALDI mass spectrometer. Regions of interest (ROIs) are chosen based on adjacently sectioned and thionin stained brain sections. Stroke regions are identified and mass spectra are averaged from this infarct as well as from the corresponding non-injured striatum on the contralateral hemisphere. Mass spectra are generated from each ROI, and the areas under the curve for the species of interest is calculated. Quantified data are represented as the ratio of the area under the curve between the ipsilateral (stroke) and contralateral ROIs.
Fig 3.
Increased GM3 Expression Within Infarcted Region of Combined Aβ/ET-1 Group Persists at 21 d.
(A) Representative MALDI IMS images of ganglioside GM3 d18:1 in stroke (ET-1 alone) and combined Aβ/ET-1 animals 3 and 21 d following surgery. Arrows indicate region of stroke induced infarct. Light regions in images represent areas of high expression while dark regions represent low levels of expression as indicated by intensity bar. (B) MALDI IMS quantification of ROIs from the striatum of control, sham surgery, Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 3 d and Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 21 d. Data are expressed as the ratio of ipsilateral to contralateral ROIs. * indicates statistical significance over control and sham surgical groups, one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 4 for each group). There was an increase in expression observed in both the ET-1 alone and combined Aβ/ET-1 groups from 3 to 21 d, however this was only statistically significant in the ET-1 alone group as the expression at 3 d was at control levels. (C,D) IHC detection and quantification of GM3 within the infarct region. Panel (D) is a higher magnification image of panel (C). * indicates statistical significance between groups, one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 6 for each group).
Fig 4.
Effects of GM3 Accumulation on Neurodegeneration Following Stroke.
(A) Photomicrographs of fluorojade-B immunofluorescence staining within the infarcted striatum of ET-1 and combined Aβ/ET-1 coronal rat brain sections. (B and C) Quantification of cell counts of FJB and NeuN cell counts. At 21 d following surgery, there was more FJB positive cells and less NeuN positive cells in the combined Aβ/ET-1 group compared to the ET-1 group according to one-way ANOVA and Tukey’s post-hoc, p<0.05 (n = 6 for each group). (D) Photomicrographs of immunofluorescence dual labelling showing co-localization between NeuN and GM3 (shown individually and overlayed) and FJB and GM3 (shown individually and overlayed) in the infarct region of a 21 d combined Aβ/ET-1 animal.
Fig 5.
Elevated GM2 Expression at 3 d Remains Increased only in Combined Aβ/ET-1 Group.
(A and C) Representative MALDI IMS images of GM2 d18:1 (A) and d20:1 (C) in stroke (ET-1 alone) and combined Aβ/ET-1 animals 3 and 21 d following surgery. Arrows indicate region of stroke induced infarct. (B and D) MALDI IMS quantification of ROI’s from the striatum of control, sham surgery, Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 3 d and Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 21 d. Data are expressed as the ratio of ipsilateral to contralateral ROIs. Light regions in images represent areas of high expression while dark regions represent low levels of expression as indicated by intensity bar. There was no statistical increase in GM3 expression between 3 and 21 d in any group, however, GM2 expression in the ET-1 group decreased significantly from 3 to 21 d (back to control levels). * indicates statistical significance over control and sham surgical groups, one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 4 for each group).
Fig 6.
Elevated GM1 Expression in Combined Aβ/ET-1 Group.
(A and C) Representative MALDI IMS images of GM1 d18:1 (A) and d20:1 (C) in stroke (ET-1 alone) and combined Aβ/ET-1 animals 3 and 21 d following surgery. Arrows indicate region of stroke induced infarct. (B and D) MALDI IMS quantification of ROI’s from the striatum of control, sham surgery, Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 3 d and Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 21 d. Data are expressed as the ratio of ipsilateral to contralateral ROIs. Light regions in images represent areas of high expression while dark regions represent low levels of expression as indicated by intensity bar. There were no statistically significant changes in GM1 expression between 3 and 21 d in any of the groups. * indicates statistical significance over control and sham surgical groups in panel (B) and statistical significance over control, sham and ET-1 alone surgical groups in panel (D), one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 4 for each group).
Fig 7.
Increased GD1a [Na+] Expression at 3 d in Combined Aβ/ET-1 Group.
(A and C) Representative MALDI IMS images of GD1a [Na+] d18:1 (A) and d20:1 (C) in stroke (ET-1 alone) and combined Aβ/ET-1 animals 3 and 21 d following surgery. Arrows indicate regions of stroke induced infarct. (B and D) MALDI IMS quantification of ROI’s from the striatum of control, sham surgery, Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 3 d and Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 21 d. Data are expressed as the ratio of ipsilateral to contralateral ROIs. Light regions in images represent areas of high expression while dark regions represent low levels of expression as indicated by intensity bar. GD1a [Na+] expression significantly decreased from 3 to 21 d in the d18:1 species. There were no other significant changes in expression between 3 and 21 d in any of the groups. * indicates statistical significance over control and sham surgical groups one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 4 for each group).
Fig 8.
Decreased Expression of GD1a [K+] at 3 d in ET-1 Alone Group.
(A and C) Representative MALDI IMS images of GD1a [K+] d18:1 (A) and d20:1 (C) in stroke (ET-1 alone) and combined Aβ/ET-1 animals 3 and 21 d following surgery. Arrows indicate regions of stroke induced infarct. (B and D) MALDI IMS quantification of ROI’s from the striatum of control, sham surgery, Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 3 d and Aβ alone, ET-1 alone, and combined Aβ/ET-1 animals at 21 d. Data are expressed as the ratio of ipsilateral to contralateral ROIs. Light regions in images represent areas of high expression while dark regions represent low levels of expression as indicated by intensity bar. There were no statistically significant changes in expression between 3 and 21 d in any of the surgical groups. * indicates statistical significance over control groups, one-way ANOVA, Tukey’s post-hoc, p<0.05 (n = 4 for each group).