oa Combination novel modalities for enlightening global molecular elemental ultrastructure and morphological biochanges in subacute stroke in rat brain

Background and Purpose: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Novel and advanced diagnostic modalities are required to improve treatment in order to reduce the social and economic burden of this disease. Understanding the post-stroke bio-chemical changes is critical for patient treatment and survival. Unfortunately, conventional diagnostic procedures are used to investigate global changes in the stroke brain, rather than understanding the bio-chemical changes at the molecular and cellular level. Therefore, in the current study we used combined approaches to investigate the molecular, elemental, ultrastructure and morphological changes in stroke brain. Methods: Application of Fourier transform infrared (FT-IR) spectroscopy; laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS); transmission electron microscope (TEM), Raman micro-spectroscopy and atomic force microscopy (AFM) were used to examine stroke brain and were compared with the standard approach of immunohistochemistry. The areas investigated, one-week post sub-acute photo-thrombotic ischemic stroke in male rats, were primary stroke lesion gray matter (PS-GM), perilesional gray matter (PL-GM), lesioned white matter (L-WM), contra-lesioned white matter (CL-WM), contra-lesioned gray matter (CL-GM) corpus callosum (CC) and dorso-ventral cortices (DVC). Results: The infrared spectroscopy results revealed that total lipid and protein and the lipid CH2 content decreased significantly in the (PS-GM) while the total aggregated protein increased significantly relative to total protein, in particular we detected an increase in Aβ1-42. In contrast, both (PL-GM) and (L-WM) regions experienced an increase in carbonyl esters, olefinic = CH, and the CH3 groups of lipids. In contrast, both (PL-GM) and (L-WM) regions experienced an increase of carbonyl esters, olefinic = CH, and CH3 groups of lipids which is associated with decrease in CH2 lipid groups and lipid contents. Amongst the novel findings of the study was that the aggregated protein detected in the L-WM was Aβ1-42. FT-Raman micro-spectroscopy showed a reduction in lipid content and iron accumulation around the stroke lesion in the form of heme. Elemental analyses for divalent cations: Ca, Cu, Fe and Zn realized major changes in the different brain structures that may underscore functionality within these regions. In the elemental maps high Fe intensity around the stroke region correlated well with the presence of heme shown by Raman spectroscopy. Conclusions: The complementary methodologies revealed, at multiple-levels, the effects of stroke: edema, lipid peroxidation, as detected by increased olefinic = CH and carbonyl ester content, and enhanced protein aggregation. For the latter, an example was the identification of Aβ1-42 in the L-WM. These data point to the possibility that amyloid plaque formation is not exclusive to cortical neurons, but also affects the WM. One conclusion being that application of strategies developed for AD-treatment may be fruitful in treating stroke. Alternative therapies would include targeting edema, antagonizing lipid peroxidation for the protection of the blood brain barrier (BBB) post-stroke by the application of antioxidants. Alternatively application of free Fe sequestering molecules, as FT-Raman micro-spectroscopy detected the reduction in the lipid content and the iron accumulation around the stroke lesion.