Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation.

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re-establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full-length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9-week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell-based gene therapy provides a new approach to cardiac regeneration.

Polarimetry-based method to extract geometry-independent metrics of tissue anisotropy.

Recently, we have used polarimetry as a method for assessing the linear retardance of infarcted myocardium. While linear retardance reflects tissue anisotropy, experimental geometry has a confounding effect due to dependence of the linear retardance on the orientation of the sample with respect to the probing beam. Here, polarimetry imaging of an 8mm diameter birefringent polystyrene sphere of known anisotropy axis was used to test a dual-projection method by which the anisotropy axis and its true magnitude can be reconstructed, thus eliminating the confounding effect of anisotropy axis orientation. Feasibility is demonstrated in ex-vivo tissue imaging.

Depolarization of light in turbid media: a scattering event resolved Monte Carlo study.

Details of light depolarization in turbid media were investigated using polarization-sensitive Monte Carlo simulations. The surviving linear and circular polarization fractions of photons undergoing a particular number of scattering events were studied for different optical properties of the turbid media. It was found that the threshold number of photon scattering interactions that fully randomize the incident polarization (defined here as <1% surviving polarization fraction) is not a constant, but varies with the photon detection angle. Larger detection angles, close to backscattering direction, show lower full depolarization threshold number for a given set of sample's optical properties. The Monte Carlo simulations also confirm that depolarization is not only controlled by the number of scattering events and detection geometry, but is also strongly influenced by other factors such as anisotropy g, medium linear birefringence, and the polarization state of the incident light.

Proof-of-principle demonstration of a Mueller matrix decomposition method for polarized light tissue characterization in vivo.

We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.

Mueller matrix decomposition for extraction of individual polarization parameters from complex turbid media exhibiting multiple scattering, optical activity, and linear birefringence.

Linear birefringence and optical activity are two common optical polarization effects present in biological tissue, and determination of these properties has useful biomedical applications. However, measurement and unique interpretation of these parameters in tissue is hindered by strong multiple scattering effects and by the fact that these and other polarization effects are often present simultaneously. We have investigated the efficacy of a Mueller matrix decomposition methodology to extract the individual intrinsic polarimetry characteristics (linear retardance delta and optical rotation psi, in particular) from a multiply scattering medium exhibiting simultaneous linear birefringence and optical activity. In the experimental studies, a photoelastic modulation polarimeter was used to record Mueller matrices from polyacrylamide phantoms having strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres-induced scattering. Decomposition of the Mueller matrices recorded in the forward detection geometry from these phantoms with controlled polarization properties yielded reasonable estimates for delta and psi parameters. The confounding effects of scattering, the propagation path of multiple scattered photons, and detection geometry on the estimated values for delta and psi were further investigated using polarization-sensitive Monte Carlo simulations. The results show that in the forward detection geometry, the effects of scattering induced linear retardance and diattenuation are weak, and the decomposition of the Mueller matrix can retrieve the intrinsic values for delta and psi with reasonable accuracy. The ability of this approach to extract the individual intrinsic polarimetry characteristics should prove valuable in diagnostic photomedicine, for example, in quantifying the small optical rotations due to the presence of glucose in tissue and for monitoring changes in tissue birefringence as a signature of tissue abnormality.

Combined optical intensity and polarization methodology for analyte concentration determination in simulated optically clear and turbid biological media.

The use of a combined spectral intensity and polarization signals optically scattered by tissue to determine analyte concentration in optically clear and turbid biological media was explored in a simulation study. Blood plasma was chosen as the biological model and glucose as the analyte of interest. The absorption spectrum and optical rotatory dispersion were modeled using experimental data and the Drude's equation, respectively, between 500 and 2000 nm. A polarization-sensitive Monte Carlo light-propagation model was used to simulate scattering media. Unfold partial least squares and multiblock partial least squares were used as regression methods to combine the spectral intensity and polarization signals, and to predict glucose concentrations in both clear and scattering models. The results show that the combined approaches produce better predictive results in both clear and scattering media than conventional partial least squares analysis, which uses intensity or polarization spectra independently. This improvement was somewhat diminished with the addition of scattering to the model, since the polarization signals were reduced due to multiple scattering. These findings demonstrate promise for the combined approach in clear or moderately scattering biological media; however, the method's applicability to highly scattering tissues is yet to be determined. The methodology also requires experimental validation.

Monte Carlo study of pathlength distribution of polarized light in turbid media.

Photon pathlength distributions as a function of the number of scattering events in cylindrical turbid samples are studied using a polarization-sensitive Monte Carlo model with linearly polarized light input. Sample scattering causes extensive depolarization, yielding a photon field comprised of polarized and depolarized sub-populations. It is found that the pathlength of polarization-preserving photons is distributed within a defined spatial range with strong angular dependence. This pathlength, averaged over the range, is 2-3X smaller than the one averaged over the widely-spread range of all (polarized + depolarized) collected photons. It is also demonstrated that changes in optical properties of the media affect the pathlength distributions.

Polarized light propagation in multiply scattering media exhibiting both linear birefringence and optical activity: Monte Carlo model and experimental methodology.

A Monte Carlo model for polarized light propagation in birefringent, optically active, multiply scattering media is developed in an effort to accurately represent the propagation of polarized light in biological tissue. The model employs the Jones N-matrix formalism to combine both linear birefringence and optical activity into a single effect that can be applied to photons as they propagate between scattering events. Polyacrylamide phantoms with strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres as scattering particles are used for experimental validation. Measurements are made using a Stokes polarimeter that detects scattered light in different geometries, and compared to the results of Monte Carlo simulations run with similar parameters. The results show close agreement between the experimental measurements and Monte Carlo calculations for phantoms exhibiting turbidity and birefringence, as well as for phantoms exhibiting turbidity, birefringence, and optical activity. Other scattering-independent polarization properties can be incorporated into the developed Jones N-matrix formalism, enabling quantification of the polarization effects via an accurate polarization-sensitive Monte Carlo model.

Angular measurements of light scattered by turbid chiral media using linear Stokes polarimeter.

The effects of turbid chiral media on light polarization are studied in different directions around the scattering samples using a refined linear Stokes polarimeter, which simplifies the signal analysis, and allows for the detailed investigations of scattered light. Because no moving parts are involved in a measurement at a specific detection direction, the determination accuracy of polarization states is increased. The results show that light depolarization increases with both turbidity and detection angle for low and moderately turbid samples; however, the angular dependence decreases with increasing turbidity. When the turbidity is increased to approximately 100 cm(-1), the depolarization becomes higher in the forward than in the backward direction. Polarization sensitive Monte Carlo simulations are used to verify some experimental observations. The results also demonstrate that surviving linear polarization fraction and overall intensity are more sensitive to the increase of glucose concentration in backward than in the forward direction in highly turbid media, indicating that backward geometry may be preferable for potential glucose detection in a biomedical context. Comparison measurements with optically inactive glycerol suggest that the refractive index matching effect, and not the chiral nature of the solute, dominates the observed optical rotation engendered by glucose in highly turbid media.

Quantitative correlation between light depolarization and transport albedo of various porcine tissues.

We present a quantitative study of depolarization in biological tissues and correlate it with measured optical properties (reduced scattering and absorption coefficients). Polarized light imaging was used to examine optically thick samples of both isotropic (liver, kidney cortex, and brain) and anisotropic (cardiac muscle, loin muscle, and tendon) pig tissues in transmission and reflection geometries. Depolarization (total, linear, and circular), as derived from polar decomposition of the measured tissue Mueller matrix, was shown to be related to the measured optical properties. We observed that depolarization increases with the transport albedo for isotropic and anisotropic tissues, independent of measurement geometry. For anisotropic tissues, depolarization was higher compared to isotropic tissues of similar transport albedo, indicating birefringence-caused depolarization effects. For tissues with large transport albedos (greater than ~0.97), backscattering geometry was preferred over transmission due to its greater retention of light polarization; this was not the case for tissues with lower transport albedo. Preferential preservation of linearly polarized light over circularly polarized light was seen in all tissue types and all measurement geometries, implying the dominance of Rayleigh-like scattering. The tabulated polarization properties of different tissue types and their links to bulk optical properties should prove useful in future polarimetric tissue characterization and imaging studies.

Do different turbid media with matched bulk optical properties also exhibit similar polarization properties?

We here investigate polarimetric behavior of thick samples of porcine liver, Intralipid, and microsphere-based tissue phantoms whose absorption and scattering properties are matched. Using polarized light we measured reflection mode Mueller matrices and derived linear/circular/total depolarization rates, based on polar decomposition. According to our results, phantoms exhibit greater depolarization rates in the backscattering geometry than the liver sample. The enhanced tissue polarization preservation differs from previous reports of polarimetric transmission studies, with the likely cause of this difference being the angular dependence of the single-scattering phase function. Also, Intralipid approximated polarimetric liver behavior well, whereas the polystyrene phantoms did not.

Polarization birefringence measurements for characterizing the myocardium, including healthy, infarcted, and stem-cell-regenerated tissues.

Myocardial infarction leads to structural remodeling of the myocardium, in particular to the loss of cardiomyocytes due to necrosis and an increase in collagen with scar formation. Stem cell regenerative treatments have been shown to alter this remodeling process, resulting in improved cardiac function. As healthy myocardial tissue is highly fibrous and anisotropic, it exhibits optical linear birefringence due to the different refractive indices parallel and perpendicular to the fibers. Accordingly, changes in myocardial structure associated with infarction and treatment-induced remodeling will alter the anisotropy exhibited by the tissue. Polarization-based linear birefringence is measured on the myocardium of adult rat hearts after myocardial infarction and compared with hearts that had received mesenchymal stem cell treatment. Both point measurement and imaging data show a decrease in birefringence in the region of infarction, with a partial rebound back toward the healthy values following regenerative treatment with stem cells. These results demonstrate the ability of optical polarimetry to characterize the micro-organizational state of the myocardium via its measured anisotropy, and the potential of this approach for monitoring regenerative treatments of myocardial infarction.

Mueller matrix decomposition for polarized light assessment of biological tissues.

The Mueller matrix represents the transfer function of an optical system in its interactions with polarized light and its elements relate to specific biologically or clinically relevant properties. However, when many optical polarization effects occur simultaneously, the resulting matrix elements represent several "lumped" effects, thus hindering their unique interpretation. Currently, no methods exist to extract these individual properties in turbid media. Here, we present a novel application of a Mueller matrix decomposition methodology that achieves this objective. The methodology is validated theoretically via a novel polarized-light propagation model, and experimentally in tissue simulating phantoms. The potential of the approach is explored for two specific biomedical applications: monitoring of changes in myocardial tissues following regenerative stem cell therapy, through birefringence-induced retardation of the light's linear and circular polarizations, and non-invasive blood glucose measurements through chirality-induced rotation of the light's linear polarization. Results demonstrate potential for both applications.

Interstitial Doppler optical coherence tomography as a local tumor necrosis predictor in photodynamic therapy of prostatic carcinoma: an in vivo study.

We have tested the feasibility of real-time localized blood flow measurements, obtained with interstitial (IS) Doppler optical coherence tomography (DOCT), to predict photodynamic therapy (PDT)-induced tumor necrosis deep within solid Dunning rat prostate tumors. IS-DOCT was used to quantify the PDT-induced microvascular shutdown rate in s.c. Dunning prostate tumors (n=28). Photofrin (12.5 mg/kg) was administered 20 to 24 hours before tumor irradiation, with 635 nm surface irradiance of 8 to 133 mWcm(-2) for 25 minutes. High frequency ultrasound and calipers were used to measure the thickness of the skin covering the tumor and the location of the echogenic IS probe within it. A two-layer Monte Carlo model was used to calculate subsurface fluence rates within the IS-DOCT region of interest (ROI). Treatment efficacy was estimated by percent tumor necrosis within the ROI, as quantified by H&E staining, and correlated to the measured microvascular shutdown rate during PDT treatment. IS-DOCT measured significant PDT-induced vascular shutdown within the ROI in all tumors. A strong relationship (R2=0.723) exists between the percent tumor necrosis at 24 hours posttreatment and the vascular shutdown rate: slower shutdown corresponded to higher treatment efficacy, i.e., more necrosis. Controls (needle+light, no drug, n=3) showed minimal microvascular changes or necrosis (4%+/-1%). This study has correlated a biological end point with a direct and localized measurement of PDT-induced microvascular changes, suggesting a potential clinical role of on-line, real-time microvascular monitoring for optimizing treatment efficacy in individual patients.

Stokes polarimetry in multiply scattering chiral media: effects of experimental geometry.

The spatial distribution of optical rotation alpha and surviving linear polarization fraction beta(L) of light scattered from cylindrical turbid chiral (glucose-containing) and achiral samples is studied using a linear Stokes polarimeter. alpha and beta(L) are measured in and off the incident plane as the detection angle changes from the forward to the backward direction. The experimental results exhibit a complex dependence on the detection geometry: alpha is more sensitive to glucose presence off the incident plane, whereas beta(L) exhibits larger effects in-plane, as validated by polarization sensitive Monte Carlo simulations. A rigorous methodology is presented for optimizing the experimental geometry in the polarimetric examinations of complex random systems.