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.
1. BackgroundStroke, i.e. loss of brain function(s) due to disturbance in the blood supply to the brain, is the main cause of adult disability (e.g. paralysis) in the world, leaving more than half of the patients dependent on daily assistance. In Qatar, stroke is a major health problem with an estimated incidence of 238/100,000 per year for the population over 45 years old [1]. Stroke patients are often hospitalized and/or subjected to intensive rehabilitation programs for long periods of time, and their quality of life is severely affected socially and economically. Around 10% of the hospital beds in Qatar are occupied by stroke patients [1]. Thus, without major advances to improve prevention, treatment and rehabilitation of stroke, the social and economic costs of this disease will increase dramatically.There are pathological and physiological changes on the cellular and molecular levels associated with stroke. The objective of this work is to determine the molecular and structural changes occurring in the tissue of rat's brain. Vibrational spectroscopy, i.e. Fourier transform infrared (FTIR) imaging spectroscopy, was used as rapid and objective diagnostic platform to investigate the pathological and pathological changes in the rat's brain sections three weeks after stroke. FTIR spectroscopy was also used to differentiate between the biochemical makeup of the white and grey matters of a healthy control brain samples. Also, in the current study, scanning electron (SEM), energy dispersive X-ray spectroscopy (EDX), and atomic force microscopic (AFM) techniques were assessed to study the structural changes in the rat's brain tissues after experiencing an induced stroke.2. Experimental2.1. Sample preparationRats were anesthetized using 2–3% isoflurane. Experimental stroke was induced in rats by 90-min occlusion of the right middle cerebral artery with an intraluminal filament. Rats were euthanized with a lethal dose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. Rat's brains were extracted, embedded in paraffin and then serially sliced, using semi-automated rotary microtome, into 5 μm thickness sections for the FTIR imaging and AFM analysis and 35 μm thickness for the SEM and EDX analysis. The brain sections were mounted on MirrIR CFR, Low-e microscope slides for the FTIR imaging analysis, and on aluminum metal for the SEM analysis and EDX analysis. The paraffin was removed from the samples by using xylene and isopropanol.2.2. Instrumentation2.2.1. FTIR Imaging MeasurementsThe FTIR images were obtained using FTIR spectrometer (Agilent Technology) at a reflection mode within the range of 4000–700 cm–1. Spectral images were analyzed using Metlab software (The Mathworks Inc.). Origin 2015 software was used for graph drawing. Principal component analysis (PCA) was performed to study the spectral data variations between the FTIR spectra and images.2.2.2. Scanning Electron Microscopy (SEM)Rat's brain sections of 35 μm thickness were mounted on aluminum slides for SEM analysis. All the samples were viewed with a FEI Quanta 200, USA scanning electron microscope at 10 kV. SEM micrographs of the brain stroke and healthy rat's sections were compared. Elemental distribution in both healthy and induced stroke brain sections were investigated by using energy dispersive X-ray spectroscopy (EDX) equipped with SEM. The spectra provided a semi-quantitative view of the elemental composition of both weight and atomic percent.2.2.3. Atomic Force Microscopy (AFM)Bruker atomic force microscopy (AFM) was used for imaging and quantitatively determining the local elastic properties of healthy and induced stroke rat's brain sections. A controllable and constant force was applied at each data point and using the resulting force-distant curve for the formation the AFM images. Brain sections were scanned at 10 μm by 10 μm. About 100 force-distance curve were collected for each healthy and induced stroke brain sections and two random scan lines of force-distance curves was recorded.3. Results and DiscussionsThe FTIR spectroscopy results indicated that the white matter is richer in lipid content than the grey matter as shown in Figs. 1 and 2. The infrared spectrum images showed a decrease in the lipid content of the white matter associated with the induced stroke brain sections. FTIR bands assigned to the bio-chemical makeup such as proteins, lipids and ester varied in positions, line-shape, and intensity between control and induced stroke brain samples. The spectral images showed that there is a configuration changes is associated with the lipid bands in the rat's brain white matter that experienced stroke.The FTIR spectral images of the white matter in the induced stroke brain sections indicated that amide I and ester bands experienced a bio-chemical changes as shown in Fig. 3 and 4. Figure 5 shows the second derivative of the collected FTIR spectra from induced stroke brain sections. In Fig. 5, there are spectral differences that assigned to ester and protein regions. Figure 6a represents the loading spectra of the first three principal component analysis (PC1, PC2 and PC3). The variations principally were located in the regions of amide I band at (∼1695-1637 cm–1) and small variation in the amide II band at (1543 cm–1). Figure 6b represents the loading spectra of the PC4, PC5 and PC6. The variations principally were located in the protein region, mainly amide I band at (∼1695-1637 cm–1) and ester band at about 1730 cm–1. The use of FTIR imaging and chemometric analyses such as principal component analysis (PCA) of spectral data allows to investigate and differentiate spectral images pattern collected from control and stroke rat's brain samples.The scanning electron microscope results showed that lesion region in the induced stroke brain sections are enriched by the selected elements such as Fe and Ca as shown in Fig. 7 (a & b). Scanning electron microscope (SEM) micrographs indicated that there is structure change in the induced stroke brain section. The structure of stroked brain sample in the nanometer scale appeared to be significantly rough compared to the control brain sample (Fig. 8 a & b).Atomic force microscope (AFM) images showed that the stroke brain section is swollen compared to healthy brain sections. The AFM images of the induced stroke brain sections appeared more stretched when compared to the control brain section image as shown in Fig. 9 (a & b). AFM results also showed that the force-distance curves in Fig. 10, recorded using control (healthy) brain sections (blue) and induced stroke brain sections (red). The force-distance showed that the AFM cantilelver deflection of the healthy brain samples is higher than the induced stroke brain section. This indicate that the healthy brain section are softer and elastic than the induced stroke brain sections.4. ConclusionFTIR imaging spectroscopy, scanning electron and atomic force microscopy techniques were able to analyze and differentiate between the healthy and induced stroke rat's brain sections on the molecular, structural and global levels making them valuable tools to investigate, diagnose and study the structural plasticity of the stroke induced brain. FTIR imaging spectroscopy in combination with multivariate analysis such as principal component analysis (PCA) is a non-destructive technique that proves to be rapid, accurate and straightforward to be performed. It constitutes a powerful approach to be used as a medical diagnosis tool to investigate the pathological changes associated with stroke in the brain tissues.KeywordsFourier Transform Infrared (FTIR) imaging spectroscopy, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Brain tissue, Stroke, Chemometric AnalysisAcknowledgmentsThis article was made possible by a NPRP award [5 - 381 - 3 - 10] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors. We would like to acknowledge the Center for Advanced Materials at Qatar University for performing the AFM analysis, and Central Laboratories Unit at Qatar University for performing the SEM and EDX analysis.Reference[1] Hamad, A., Sokrab, T.E., Momeni, S., Mesraoua, B., and Lingren, A., Stroke in Qatar: a one-year, hospital-based study. J Stroke Cerebrovasc Dis, 2001. 10(5): p. 236–41.
Background: Coronary artery disease remains one of the leading causes of morbidity and mortality in developed countries and is projected to be the leading cause of death in the developing world by 2010. Affecting nearly 12 million people in the USA, it accounts for 50% of all cardiovascular deaths and is the leading cause of congestive heart failure as well as premature permanent disability in workers. About 1.1 million myocardial infarctions and >400,000 new cases of congestive heart failure each year, cardiovascular disease severely impacts men and women as well as various ethnic groups. Objectives: The aim of this study is to evaluate the results of transmyocardial laser revascularization (TMLR) in patients with coronary artery disease, in whom percutaneous transluminal coronary angiography (PTCA) or coronary artery bypass grafting (CABG) cannot be done. Method: Fifty patients were included in the study: 1) Canadian cardiovascular angina class (CCS) III or IV patients (only 5 patients with CCS class II were included and those patients were the early trial in year 1997 where the recommendations for TMLR was not yet established); 2) Ejection fraction of 30%; 3) Those with evidence of reversible ischemia (based on stress thallium perfusion scanning); 4) Candidates of neither CABG or PTCA (according to the results of coronary angiography). The laser system used was a heart laser system (PLC medical systems). Results: There was a significant improvement in the severity of angina as assessed by Canadian Cardiovascular Society grading after the procedure. Improvement was noticed after 1 month and the improvement was most significant after 3 months and then slightly declined after 6 months but still significant in comparison to the pre-procedural values (P <0.05). Also, there was a significant improvement in the grade of dyspnoea as assessed by the New York Heart Association functional dyspnoea class and the most significant improvement was after 3 months and the degree of improvement decreased but still significantly better than the pre-procedural values (P <0.05). Conclusion: TMLR offers consistent amelioration of severe angina in patients having no conventional therapeutic alternative. Surgeons should recognize that the procedure is intended only for the purpose of reducing angina symptoms.
Background: Unwanted hair is one of the most common medical problems affecting women of reproductive age inducing a lot of psychological stress and threatening their femininity and self-esteem. Old methods of removing unwanted hair include shaving, waxing, chemical epilation, and electrolysis, all of which have temporary results. However laser-assisted hair removal is the most efficient method of long-term hair removal currently available. It is desirable to develop a reduced-cost photodynamic therapy (PDT) system whose properties should include high efficiency and low side-effects. Objectives: The aim is to develop an adequate PDT system including helium-neon (He-Ne) laser and its effect on the photodynamic activity of methylene blue (Figs. 1 and 2) in biological experiments. Method: Mice skin tissues were used in this study and divided into the following six groups: controls (Fig.3), free methylene blue (MB) incubation, liposomal MB incubation, laser without MB, free MB for 3 and 4 hrs and laser, liposome MB for 3 hrs and laser. MB was applied to wax epilated areas. The areas were irradiated with CW He-Ne laser system that emits orange-red light with wavelength 632.8 nm and 10 mW at energy density of 5 J/cm2 for 10 minutes. The UV-visible spectrum was collected by Cary spectrophotometer. Results: MB is selectively absorbed by actively growing hair follicles due to its cationic property (Fig.4). MB untreated sections showed that hair follicles and sebaceous glands are intact and there is no change due to the laser exposure (Fig.5). Free MB sections incubated for 3 hrs showed that He:Ne laser induced destruction in hair follicles, leaving an intact epidermis (Fig. 6). Treated section with free MB for 4 hrs showed degeneration and necrosis in hair follicles, leaving an intact epidermis (Fig. 7). Liposomal MB sections incubated for 3 hrs showed He:Ne laser induced destruction in hair follicles with intradermal leukocytic infiltration (Fig. 8). Conclusion: Low power He:Ne laser and MB offers a successful PDT system in selectively damaging hair follicles, leaving an intact epidermis. The current PDT system provides better outcome than hair destruction through laser heat transfer procedures and laser-mediated hair removal, due to complete destruction of hair follicles.
Background: Unwanted hair is one of the most common medical problems affecting women of reproductive age inducing a lot of psychological stress and threatening their femininity and self-esteem. Old methods of removing unwanted hair include shaving, waxing, chemical depilation, and electrolysis, all of which have temporary results. However laser-assisted hair removal is the most efficient method of long-term hair removal currently available. It is desirable to develop a reduced cost photodynamic therapy (PDT) system whose properties should include high efficiency and low side-effects. Method: Mice skin tissues were used in this study and divided into six groups such as controls, free methylene blue (MB) incubation, liposome methylene blue (MB) incubation, laser without methylene blue (MB), free methylene blue (MB) for 3 and 4 hrs and laser, liposome methylene blue (MB) for 3 hrs and laser. Methylene blue (MB)was applied to wax epilated areas. The areas were irradiated with CW He-Ne laser system that emits orange-red light with wavelength 632.8 nm and 10 mW at energy density of 5 J/ cm2 for 10 minutes. The UV-visible absorption spectrum was collected by Cary spectrophotometer. Results: Methylene blue (MB) is selectively absorbed by actively growing hair follicles due to its cationic property. Methylene blue (MB)untreated sections showed that hair follicle and sebaceous gland are intact and there is no change due to the laser exposure. Free methylene blue (MB) sections incubated for 3 hrs showed that He:Ne laser induced destruction in hair follicles, leaving an intact epidermis. Treated section with free methylene blue (MB) for 4 hrs showed degeneration and necrosis in hair follicle, leaving an intact epidermis. Liposomal methylene blue (MB) sections incubated for 3 hrs showed He:Ne laser induced destruction in hair follicles with intradermal leucocytic infiltration. Conclusions: Low power CW He:Ne laser and methylene blue (MB) offered a successful PDT system in selectively damaging hair follicles, leaving an intact epidermis. The current PDT system provides better outcome than hair destruction through laser heat transfer procedures and laser-mediated hair removal, due to complete destruction of hair follicles
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