Supervisors: Prof Faisel Khan and Dr Mike MacDonald
The evaluation of oxidative stress and endothelial dysfunction in the microcirculation are novel modalities for the early preclinical assessment of Cardiovascular Disease (CVD) risk. Many studies have reported that oxidative stress and endothelial function in the peripheral skin microcirculation are a hallmark of the health status of the central coronary arteries. Skin microcirculation and the autofluorescence of cutaneous oxidative stress markers (e.g. nicotamide adenine dinucleotide, NAD(P)H), can be examined by the combination of non-invasive laser methods such as Laser Doppler Flowmetry (LDF), Reflectance Spectroscopy (RS), and Laser Fluorescence Spectroscopy (LFS) with vascular reactive tests. Therefore, the first task of this research was combining such minimally invasive technologies to develop protocols for the concurrent assessment of skin oxidative stress and microvascular function in both animal models and humans. The goal was achieved by using two single-point laser probes (LAKK-M, Spe Lazma, Russia) for the simultaneous measurement of NAD(P)H, microvascular blood flow and oxygen saturation during PORH (Post-Occlusive Reactive Hyperaemia) and iontophoresis reactive tasks, respectively by LFS, LDF and RS methods (Fig. 1-2).
Fig.1 Experimental setup for LFS, LDF and RS recordings. (a) Assessment of skin microvascular function and oxidative stress in anaesthetised mice during iontophoresis test. An iontophoresis chamber was attached on the mouse flank and filled with a solution of a vasoactive drug. LFS and LDF/RS laser probes were placed in two adjacent sites in the skin region inside the chamber to measure simultaneously NAD(P)H, blood flow and oxygen saturation. The vasoactive drug was delivered by applying a continuous anodal current of 100 µA. (b) Evaluation of microvascular function and oxidative stress in human subjects during PORH test. LDF/RS and LFS probes were placed in two adjacent sites at 1/3 length of the left volar forearm. A pressure cuff was placed in the upper left arm to occlude blood flow through the brachial artery by inflating the cuff at 200 mmHg.
Fig. 2 Example of simultaneous data recordings from the human forearm. (a) NAD(P)H 25 min time series reconstructed from 25 sequential discrete UV autofluorescence spectra (1 per min) collected by LFS method during PORH test. (b) 25 min LDF continuous tracing collected during PORH test.
Recently, scientists have demonstrated that the study of nonlinear dynamics of cardiovascular signals reveals ‘hidden’ information related to vascular function. For instance, the spectral analysis of LDF signal by Continuous Wavelet Transform (CWT) method revealed six distinct oscillatory components associated to multiple physiological functions. Monitoring these oscillatory components can provide information on the activity of key factors involved in the regulation of the microcirculation, i.e. endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and the sympathetic innervation. Moreover, scientists have drawn their attention also to the investigation of NAD(P)H nonlinear dynamics which may reveal specific oscillatory patterns of the cellular oxido-reductive reactions involved in ATP energy production, defining the potential switch of skin tissue from a normal to a pathologic metabolic phenotype that may affect micro-vessels reactivity. The examination of LDF and NAD(P)H nonlinear dynamics may be powerful either for the investigation of vasomotion (spontaneous rhythmic variations of micro-vessels diameter) or the prediction of CVD risk. Thus, the second task of this study was developing the techniques for the simultaneous in-vivo investigation of LDF and NAD(P)H nonlinear dynamics. The goal was achieved by implementing Matlab script codes for performing the CWT spectral analysis of LDF signal and NAD(P)H time series reconstructed from discrete autofluorescence measurements (Fig. 3). This was the first attempt to characterise metabolic NAD(P)H oscillations from mice and human in-vivo skin tissue.
Fig. 3 CWT spectral analysis of LDF and NAD(P)H time series measured from the human forearm during PORH test. (a) Time-frequency domain scalogram (left) and corresponding time-averaged CWT spectrum (right) from LDF signal. (b) CWT scalogram and corresponding time-averaged spectrum from NAD(P)H time series. The scalogram indicates the distribution of the spectral energy of the signal in the time-frequency domain by using a gradient coloured map. The time-averaged chart allows discriminating the spectral energy peaks associated to the activity of various biological oscillators located at different frequency intervals. We detected the typical LDF oscillations described in literature: (I) cardiac, (II) respiratory, (III) myogenic, (IV) neurogenic, (V) endothelial NO-dependent, (VI) endothelial NO-independent (EDHF). Moreover, three in-vivo skin metabolic low frequency oscillators contributing to NAD(P)H fluctuations were identified for the first time, which may reflect the health status of the cutaneous tissue: Metabolic Oscillator-1 (MO-1), MO-2, and MO-3.
The final task of the study was the application of the implemented protocols and CWT analysis for the investigation of microvascular (LDF) and metabolic (NAD(P)H) nonlinear dynamics in a mouse model characterized by increased oxidative stress (Nrf2-/-) and in a cohort of human smokers.
The analysis of data from mice revealed correlations between metabolic and microvascular oscillations, which might support the theory of cell metabolic oscillators as drivers of vasomotion. Moreover, the results evidenced an important role of the endothelial modulation in vasomotion, and helped to better clarify the physiological origin of the wavelet oscillation in the 5 x 10-3 Hz frequency interval, suggesting an association with the Endothelial-Derived Hyperpolarizing Factor (EDHF) pathway. Finally, mice results revealed also opposite trends of vascular and metabolic dynamic biomarkers between control and Nrf2-/- animals, indicating that metabolic and vascular oscillators may find application to distinguish healthy and impaired vascular conditions.
The analysis of data from human individuals revealed that healthy smokers compared to non-smokers are characterised by hyper-stimulation of the micro-vessels reactivity, probably due to the effect of substances contained in cigarettes smoke (e.g. nitric oxide and nicotine). This may lead to an increase of endothelial, neurogenic and myogenic activities causing an initial damage to micro-vessels, leading to the cutaneous endothelial dysfunction observed in chronic elder smokers. These findings suggest that the increase of the oscillatory activity of LDF spectral components in healthy smokers represent a preclinical biomarker associated with the onset of microvascular dysfunction. The metabolic dynamic biomarkers did not show discriminatory differences between smokers and non-smokers, indicating that healthy smokers maintain an overall normal energy metabolism.
Journal and Conference Papers and Posters
[January 2018] In-vivo assessment of microvascular functional dynamics by combination of cmOCT and wavelet transform. Oral presentation at Photonics West 2018, San Francisco, USA, 29th January 2018. The paper will be published in Proc. SPIE 10493, Dynamics and Fluctuations in Biomedical PHotonics XV, 10493-24.
[April 2017] In-vivo monitoring of mice skin metabolic fluctuations by fluorescence spectroscopy. Paper presented at the Fluoro Fest Workshop, Glasgow, Scotland, 24th-26th April 2017.
[November 2016] Tissue optics measurements of novel CVD risk factors. Poster presented at University of Dundee School of Medicine Research Symposium, Dundee, Scotland, 10th November 2016.
[October 2016] Simultaneous assessment of skin microcirculation and auto-fluorescence to predict cardiovascular dysfunction. Poster presented at IUA SFMV 2016 Congress, Lyon, France, 6th October 2016.
[September 2016] Tissue optics measurements of novel CVD risk factors. Poster presented at the Bi-annual Biophotonics and Imaging Summer School, Galway, Ireland, 8th September 2016.
[September 2016] Combined Optical Flowmetry, Oximetry and Fluorescence measurement of novel markers for vascular dysfunction. Poster presented at PHOTON16, Leeds, UK, 5th September 2016.
[August 2016] Combined Optical Flowmetry, Oximetry and Fluorescence measurement of novel markers for vascular dysfunction. Poster presented at the Biophotonic approaches: From molecules to living systems conference, Dundee, Scotland, 23rd August 2016.
[April 2016] Non-invasive approach to investigate novel surrogate markers of Cardiovascular Disease (CVD) risk. Poster presented at 66th British Microcirculation Society Annual Meeting, Newcastle, England, 7th April 2016.
[February 2016] Non-invasive laser-based scans in mice to investigate novel surrogate markers of Cardiovascular Disease risk. Poster presented at University of Dundee School of Medicine Research Symposium, Crieff, Scotland, 25th February 2016.