Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes—laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-µm histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEImax), which includes collagen and elastin, increases with animal weight (R2=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R2=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEImax at R2=0.60).
Background: Arterial bifurcations are commonly the sites of developing atherosclerotic plaque that lead to arterial occlusions and plaque rupture (myocardial infarctions and strokes). Laser induced fluorescence (LIF) spectroscopy provides an effective nondestructive method supplying spectral information on extracellular matrix (ECM) protein composition, specifically collagen and elastin.
Purpose: To investigate regional differences in the ECM proteins -- collagen I, III and elastin in unstable plaque by analyzing data from laser-induced fluorescence spectroscopy of human carotid endarterectomy specimens.
Methods: Gels of ECM protein extracts (elastin, collagen types I & III) were measured as reference spectra and internal thoracic artery segments (extra tissue from bypass surgery) were used as tissue controls. Arterial segments and the endarterectomy specimens (n=21) were cut into 5mm cross-sectional rings. Ten fluorescence spectra per sampling area were then recorded at 5 sites per ring with argon laser excitation (357nm) with a penetration depth of 200 μm. Spectra were normalized to maximum intensity and analyzed using multiple regression analysis. Tissue rings were fixed in formalin (within 3 hours of surgery), sectioned and stained with H&E or Movat's Pentachrome for histological analysis. Spectroscopy data were correlated with immunohistology (staining for elastin, collagen types I, III and IV).
Results: Quantitative fluorescence for the thoracic arteries revealed a dominant elastin component on the luminal side -- confirmed with immunohistology and known artery structure. Carotid endarterectomy specimens by comparison had a significant decrease in elastin signature and increased collagen type I and III. Arterial spectra were markedly different between the thoracic and carotid specimens. There was also a significant elevation (p<0.05) of collagen type I distal to the bifurcation compared to proximal tissue in the carotid specimens.
Conclusion: Fluorescence spectroscopy is an effective method for evaluating ECM (collagen and elastin) associated with vascular remodeling despite the considerable variability in the plaque structure. Consistent regional differences were detected in the carotid specimens.
Atherosclerosis is the underlying vascular pathology that initiates arterial thromboembolic occlusions (myocardial infarctions, strokes and peripheral vessel blockage). Two imaging modalities, Optical Coherence Tomography (OCT) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), were investigated for detection and compositional analysis of unstable plaque associated with plaque erosion and sudden occlusion. OCT produces high resolution images
whereas mass spectrometry images provide information on the spatial distribution of chemical elements. Diseased carotid arteries taken from patients with high-risk lesions were imaged with OCT and ToF-SIMS to give molecular and metabolic information, and matched with histopathology. OCT results show clear indications of vascular remodeling by the presence of fatty acid deposits, fibrous tissue and calcifications. ToF-SIMS further characterized changes based on
secondary ion topography analysis where a high 23Na/39K ratio was indicative of arterial tissue degradation and the amount of 40Ca corresponded with late stage atherosclerosis. This pilot experiment has demonstrated that in vitro OCT imaging and ToF-SIMS of diseased carotid arteries have scientific merit for targeting clinically relevant morphology and metabolic changes to compare stable and unstable plaque. These optical techniques provide complimentary metabolic and molecular information on unstable plaque, specifically cell break-down with altered ion ratios of 23Na, 39K and 40Ca.