Fourier-Transform Infrared Imaging Spectroscopy and Laser Ablation -ICPMS New Vistas for Biochemical Analyses of Ischemic Stroke in Rat Brain

Abstract

Objective: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Understanding the post-stroke biochemical and molecular changes are critical for patient survival and stroke management. The aim of this work was to investigate the photo-thrombotic ischemic stroke in male rats with particular focus on biochemical and elemental changes in the primary stroke lesion in the somatosensory cortex and surrounding areas, including the corpus callosum. Materials and Methods: FT-IR imaging spectroscopy and LA-ICPMS techniques examined stroke brain samples, which were compared with standard immunohistochemistry studies. Results: The FTIR results revealed that in the lesioned gray matter the relative distribution of lipid, lipid acyl and protein contents decreased significantly. Also at this locus, there was a significant increase in aggregated protein as detected by high-levels Aβ_1-42. Areas close to the stroke focus experienced decrease in the lipid and lipid acyl contents associated with an increase in lipid ester, olefin, and methyl bio-contents with a novel finding of Aβ_1-42 in the PL-GM and L-WM. Elemental analyses realized major changes in the different brain structures that may underscore functionality. Conclusion: In conclusion, FTIR bio-spectroscopy is a non-destructive, rapid, and a refined technique to characterize oxidative stress markers associated with lipid degradation and protein denaturation not characterized by routine approaches. This technique may expedite research into stroke and offer new approaches for neurodegenerative disorders. The results suggest that a good therapeutic strategy should include a mechanism that provides protective effect from brain swelling (edema) and neurotoxicity by scavenging the lipid peroxidation end products.

Keywords: FTIR imaging spectroscopy; LA-ICPMS; brain; ischemic; lipid peroxidation; neurodegeneration; photothrombotic; stroke model.

FIGURE 1

Photothrombotic lesion in rat somatosensory cortex results in cell death and astrigliosis. (A) Hematoxylin and Eosin (H&E) staining brain section of native control brain (magnification 5×). (B) H&E stained image 1-week post-stroke brain (magnification 5×). (C) Brain section scheme for the six regions of interest (ROI): primary stroke lesion gray matter (PS-GM), perilesional gray matter (PL-GM), lesioned white matter (L-WM) and contra-lesioned white matter (CL-WM), contra-lesioned gray matter (CL-GM), and corpus callosum white matter (CC). (D,E) Healthy control and lesioned brain sections labeled with GFAP and DAPI: primary lesion gray matter (PS), perilesional gray matter (PL) indicated activated astrocytes (GFAP, red stain) around the ischemic region (peri-infarct region) and degenerated neurons with shrunken nuclei (DAPI, blue stain). Infiltration of the astrocytes around the primary lesion PS-GM region with GFAP (red). The DAPI labels (blue) degenerated cells. (F,G) Healthy control and affected stroke rat brains sections were labeled with MAP2 and DAPI. (H,I) Healthy control and affected stroke rat brains sections labeled with Tau and DAPI to assist in comparisons. Scale bar = 100 μm.

FIGURE 2

Immunohistochemistry staining identifying altered APP and Aβ_1-42 in infarcted brain sections. (A,B) Contra-lesioned and lesioned hemispheres labeled with APP and DAPI: primary lesion gray matter (PS) shows amyloid precursor protein (APP) and contra-lesioned gray matter (CL-GM) with normal homogenous distribution of APP (red). (C,D) Contra-lesioned and affected stroke hemispheres were labeled with APP (red), DAPI (Blue) and Aβ_1-42 (green). (E–H) Native control and 1-week affected stroke rat brains sections labeled with myelin basic protein (MBP, red), DAPI (blue), and Aβ_1-42 (green). (F,G) Primary lesion gray matter (PS-GM) contains degenerated neurons (DAPI, blue) and disorganization of the myelin sheath (MBP, red) of the axonal neurons in the lesioned white matter (L-WM) scale bar = 100 μm for (A–H).

FIGURE 3

Whole brain section-FTIR imaging of biochemical changes within contralateral and ipsilateral hemispheres following photothrombotic focal ischemic insult to the somatosensory cortex. (A) H&E stained brain section captured by Digital Scanning Microscope Bright field (20×). (B). Representative unstained FTIR light microscopic image of the healthy control rat brain (10×). (C). Representative FTIR chemical image of the unstained brain section showing main biochemical components: lipid, phospholipid, protein, carbohydrates and nucleic acids. (D,E) FTIR image that represent the total lipid and total protein distribution in the healthy control rat brain. (F,G) FTIR image that represent the total lipid and total protein distribution in the stroke affected rat brain. Scale bars = 100 μm. (H) Representative FTIR averaged spectra in the range of 4000–400 cm^-1 acquired from the cortical region of the PS-GM, L-WM, CL-GM, and CL-WM. (I) Representative FTIR averaged spectra in the range of 4000–400 cm^-1 acquired from the cortical region of the PS-GM, PL-GM, CL-GM and native control GM. (J) Representative average second-derivative spectra of the amide I band in the spectral range of 1700–1600 cm^-1 acquired from the primary lesion gray matter (PS-GM), perilesional gray matter (PL-GM), and contra-lesioned gray matter (CL-GM) regions post ischemic. The spectra show α- helical secondary protein structure at 1655 cm^-1 and β-sheets protein conformation at 1630 cm^-1. (K,L) Representative curve fitting of the amide I band in the spectral range of 1700-1600 cm^-1 of the CL-GM and PS-GM, respectively, to quantify the aggregated protein relatively. (M) Histogram of the aggregated protein comparing different regions of interest: native control, PS-GM, PL-GM, L-WM with CL-GM. The FTIR images were colored-coded: red color corresponds to the highest content and blue color corresponds to the lowest content as shown on the color bars in the figures. Statistical significance was determined from six animals with a paired t-test and the 95% confidence interval. Bars represent mean ± SD. ^∗p > 0.05, ^∗∗p > 0.01 relative to CL-GM. ^σp > 0.05, ^σσp > 0.01 relative to PL-GM.

FIGURE 4

Functional group (macromolecular/sub-cellular/biochemical) images obtained of the native control ischemic stroke brain sections. (A–I). Representative FTIR images of lipid/protein, lipid acyl group (CH₂), olefin = CH, methyl (CH₃) and lipid ester (C = O) distribution in the native healthy control brain tissue sections, respectively. (B–J) The same function groups (above) obtained from the ischemic stroke brain sections. (K–P) Histogram of specific bio-molecules content such as total protein, total lipid, lipid acyl group (CH₂), olefin = CH, methyl (CH₃), and lipid ester (C = O), respectively. The regions of interest were native control rat brain GM and ischemic brain PS-GM, PL-GM, L-WM, and CL-GM. The FTIR images were color coded, red color corresponds to the highest content and blue color corresponds to the lowest content as shown on the color bars in the figures. Statistical significance was determined from six animals. Statistical significance was determined from six animals with a paired t-test and the 95% confidence interval. Bars represent mean ± SD. ^∗p > 0.05, ^∗∗p > 0.01 relative to CL-GM. ^σp > 0.05, ^σσp > 0.01 relative to PL-GM.

FIGURE 5

Principle component analysis (PCA) analyses of averaged FTIR spectroscopic studies. (A) Representative 3D score plot for PCA analysis based on the average FTIR spectral data in the region of 4000–700 cm^-1 (with the range of 2500–2000 cm^-1 removed) collected from the time point of native healthy control and stroke affected rat brain. (B). The score plot of the PCA shows that there are three main PCs that separate the time point native control healthy and the affected stroke rat brains and are accounted for PC1 (93%), PC2 (4%), and PC3 (1%). (C) Represents the PCs plot that shows that there are the three main PCs that separate PS-GM, PL-GM, and CL-GM spectra. (D) The score plot of the PCA and shows that there are three main PCs that separate the PS-GM, PL-GM, and CL-GM spectra are counted for PC1 (95%), PC2 (1.5%), and PC3 (1%). (E) Represents the hierarchical dendrogram that shows a clear separation PS-GM, PL-GM, and CL-GM regions in the ipsilateral side of 1-week post ischemic stroke. The hierarchical dendrogram shows that PS-GM is located in one group while the PL-GM and CL-GM regions are located in the second group.

FIGURE 6

Qualitative elemental maps for whole rat brain sections. (A–G) Represent images of the elemental distributions of C, P, S, Cu, Fe, Zn, and Ca, respectively, in the rat brain section 1-week post-ischemic stroke measured by LA-ICP-QMS. (H–N) Elemental concentration of C, P, S, Cu, Fe, Zn, and Ca, respectively, from four regions of interest such as PS-GM, PL-GM, L-GM, and CL-GM. These LA images were color-labeled according to the calculated concentration values, where red corresponds to the highest concentration and white corresponds to the lowest concentration as shown on the color bars in the figures. Statistical significance was determined from six animals with a paired t-test and the 95% confidence interval. Bars represent mean ± SD. ^∗p > 0.05, ^∗∗p > 0.01 relative to CL-GM. ^σp > 0.05, ^σσp > 0.01 relative to PL-GM.