Investigation of Biochemical Alterations in Ischemic Stroke Using Fourier Transform Infrared Imaging Spectroscopy-A Preliminary Study

Abstract

Objective: Brain damage, long-term disability and death are the dreadful consequences of ischemic stroke. It causes imbalance in the biochemical constituents that distorts the brain dynamics. Understanding the sub-cellular alterations associated with the stroke will contribute to deeper molecular understanding of brain plasticity and recovery. Current routine approaches examining lipid and protein biochemical changes post stoke can be difficult. Fourier Transform Infrared (FTIR) imaging spectroscopy can play a vital role in detecting these molecular alterations on a sub-cellular level due to its high spatial resolution, accuracy and sensitivity. This study investigates the biochemical and molecular changes in peri-infract zone (PIZ) (contiguous area not completely damaged by stroke) and ipsi-lesional white matter (WM) (right below the stroke and PIZ regions) nine weeks post photothrombotic ischemic stroke in rats.

Materials and methods: FTIR imaging spectroscopy and transmission electron microscopy (TEM) techniques were applied to investigate brain tissue samples while hematoxylin and eosin (H&E) stained images of adjacent sections were prepared for comparison and examination the morphological changes post stroke.

Results: TEM results revealed shearing of myelin sheaths and loss of cell membrane, structure and integrity after ischemic stroke. FTIR results showed that ipsi-lesional PIZ and WM experienced reduction in total protein and total lipid content compared to contra-lesional hemisphere. The lipid/protein ratio reduced in PIZ and adjacent WM indicated lipid peroxidation, which results in lipid chain fragmentation and an increase in olefinic content. Protein structural change is observed in PIZ due to the shift from random coli and α-helical structures to β-sheet conformation.

Conclusion: FTIR imaging bio-spectroscopy provide novel biochemical information at sub-cellular levels that be difficult to be obtained by routine approaches. The results suggest that successful therapeutic strategy that is based on administration of anti-oxidant therapy, which could reduce and prevent neurotoxicity by scavenging the lipid peroxidation products. This approach will mitigate tissue damage in chronic ischemic period. FTIR imaging bio-spectroscopy can be used as a powerful tool and offer new approach in stroke and neurodegenerative diseases research.

Keywords: Fourier transform infrared (FTIR) imaging spectroscopy; photothrombotic stroke; transmission electron microscope (TEM), brain ischemia.

Figure 1

The hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) images. (A) Contra-lesional and Ipsi-lesional hemispheres of nine weeks stroke brain tissues showing the regions of interest—peri-infract zone (PIZ) and white matter (WM). (B_a) Representative TEM images of contra-lesional cortex (unaffected neurons), whereas (B_b) representing ipsi-lesional PIZ (affected side) and (B_c–d) representing ipsi-lesional WM showing the ultrastructural changes—axonal shearing, formation of vacuoles and swelling marked with black arrows on the images. Scale bar = 100 µm for H&E and 1 µm for TEM.

Figure 2

Fourier Transform Infrared (FTIR) biochemical images of Total Lipid (C-H region) and Total Protein (Amide I + II) on (A) Contra-lesional and (B) Ipsi-lesional hemisphere of nine weeks post stroke brain tissues. The images show significant changes in distribution of lipid and protein at peri-infract zone (PIZ) and white matter (WM). The stars in the bright field images shows where the spectra where taken. The color bars showing the intensity of the biochemical components—red as maximum and blue as minimum. Scale bar = 100 µm.

Figure 3

Fourier Transform Infrared (FTIR) images of different lipid components—Olefin=CH/Lipid, Lipid ester/Lipid, CH₂/Lipid, CH₃/Lipid and Lipid/Protein at (A) Contra-lesional and (B) Ipsi-lesional of nine weeks post stroke brain tissue. The color bars showing the intensity of the biochemical components—red as maximum and blue as minimum. Scale bar = 100 µm.

Figure 4

Comparison of Fourier Transform Infrared (FTIR) spectra from (A) White matter—WM and (B) Peri-infract zone—PIZ at contra-lesional (black) and ipsi-lesional (red) hemisphere of nine weeks post stroke showing the changes of peaks in Protein and Lipid regions separately. (C) Representative curve fitting spectra from– contra-lesional gray matter (GM) (left) and ipsi-lesional PIZ (right) quantifying changes in relative amount of aggregated protein from α-helix and β-sheet variations. (D) Representative second derivative spectra from PIZ showing α-helix and β-sheet variations. The peak assignments (name + wavenumbers) are specified by blue arrow.