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3D digital pathology for a chemical-functional analysis of glomeruli in health and pathology Hsiang-Hsin Chen, Tsung-Tse Lee, Ann Chen, Yeukuang Hwu, and Cyril Petibois Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04265 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018
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Analytical Chemistry
3D digital pathology for a chemical-functional analysis of glomeruli in health and pathology Hsiang-Hsin Chen1,3, Tsung-Tse Lee1, Ann Chen2, Yeukuang Hwu1, Cyril Petibois1,3(*) 1 = Academia Sinica, Institute of Physics, 128 Sec. 2, Academia Rd., Nankang, Taipei 11529, Taiwan 2 = Graduate Institute of Life Sciences, National Defense Medical Center, 161 Section 6, Minquan East Road, Neihu District, 114 Taipei City, Taiwan 3 = University of Bordeaux, Inserm U1029 LAMC, Allée Geoffroy Saint-Hillaire, Bat. B2, F33600 Pessac-Cedex, France * Corresponding author: Cyril Petibois,
[email protected] Abstract: Determining the filtration function and biochemical status of kidney at the single glomerulus level remains hardly accessible, even from biopsies. Here, we provide evidence that IR spectro-microscopy is a suitable method to account for the filtration capacity of individual glomeruli along with related physio-pathological condition. A ~4-µm voxel resolution 3D IR image reconstruction is built from consecutive tissue sections, thus providing a 3D IR spectrum matrix of an individual glomerulus. The filtration capacity of glomeruli was quantitatively determined after BaSO4 perfusion, and additional chemical data could be used to determined oxidative stress effects and fibrosis, thus combining functional and biochemical information from the same 3D IR spectrum matrix. This analytical approach was applied on mice with unilateral ureteral obstruction (UUO) inducing chronic kidney disease. Compared to the healthy condition, UUO induced a significant drop in glomeruli filtration capacity (-17±8% at day-4 and -48±14% at day-14) and volume (36±10% at day-4 and 67±13% at day-14), along a significant increase of oxidative stress (+61±19% at day-4 and +84±17% at day-14) and a change in the lipid-to-protein ratio (-8.2±3.6% at day-4 and -18.1±5.9% at day-14). Therefore, IR spectro-microscopy might be developed as a new 3D pathology resource for analyzing functional and biochemical parameters of glomeruli. KEY-WORDS: infrared microscopy, histology, quantitative analysis, 3D digital pathology, chemical imaging Introduction Chronic kidney disease (CKD) is a world health problem, which has rare symptoms detected before the appearance of severe pathological signs with the combined complication1. In the U.S., there is a higher prevalence of 10.6 % in the earlier stage (stage 1, 2 and 3) of CKD than the moderate stage (stage 4, 0.2 % prevalence) or late stage (stage 5, 0.1 % prevalence) by estimating glomerular filtration rate (GFR). More than 11 % of Americans aged over 65-year-old have CKD of at stage 3. ACS Paragon Plus Environment
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The progression of CKD eventually leads to the end-stage renal failure (classified as stage 5), which cause the permanent loss of kidney function, making the patients treated with the outcomes of taking the renal function replacement (either by dialysis or transplantation), which increases the loading on the financial issues in national policy. Many systematic disorders affect kidney function by attacking the glomeruli, the functional part of the renal unit called nephron, which is performing selective the ultrafiltration for body waste clearance from the blood. A variety of etiological factors are contributed to glomerular injury, such as genetic disorder, microenvironment stress, microorganism infection, uptake of harmful material as well as physiological decline of activity due to aging2. Progressive glomerular injury in CKD represents the consequences of which glomeruli are partially damaged, which generally are affected by tubular atrophy due to proliferated fibrosis in renal interstitium, and the response of compensatory hyperfiltration of glomeruli in remaining nephron due to the loss of sufficient nephrons. Histopathological patterns of glomerular change have two categories in morphological abnormalities with the variation in glomerular size and vasculature structure, and functional disorder with the impaired selectivity in filtrating materials3. Both types of features on glomerular disorder involves with the extent of severity in kidney damage. Hence, to diagnose and manage glomerular injury referring to impaired kidney function should incorporate the analysis in morphometric aberration and molecular dysfunction. The current method to evaluate the level of glomerular damage depends on either the level of glomerular filtration rate (GFR) or the appearance of proteinuria attributed to the impairment of glomerular barrier1. Nephron number and the filtration rate in each nephron determinate GFR. Both indicators are involved with body matrices. In some glomerular disease, the diagnosis requires the renal biopsies to estimates the expression and states of biomolecules correlated with the pathological change which cannot be indicated by blood or urine. However, lack of examination in morphometric information remains the insufficiency in the diagnosis of glomerular disease. The analytical weakness comes from the lack of multifactorial analysis, able to determine both the glomerular function and the biochemical features explaining its alteration. This would greatly enhance the outcome of biopsy analyses. To date, only a few imaging techniques have been able to characterize single glomeruli in 3D and at microscopic resolution, including high-resolution µ-CT4, µ-CT on corrosion casts5, and intravital imaging6, but these techniques are for research studies only, not for diagnosis. In vivo imaging cannot resolve the dimension of glomeruli (Ø < 100 µm) and their internal components (ex. blood vessels with Ø < 10 µm). Thus, 3D pathology involving labels for immunohistochemistry (IHC)7 have been proposed, which might comply with the anatomo-pathological expectations for clinical diagnostics. However, 3D pathology based on colorations (H&E) or IHC is still in its infancy and lacks providing consistent results with a diagnosis value8. This is mainly due to the limited number of data that IHC labels can provide and the lack of automation in 3D image reconstruction and interpretation. Recent developments of spectro-microscopy techniques have shown that 3D histological chemistry can provide many different results on a sample, taking benefit from spectral data translated into chemical and biological metadata9. Among the spectroscopy techniques ACS Paragon Plus Environment
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Analytical Chemistry
available for histological analyses, infrared (IR) microscopy seems the most promising since it is providing a global chemical information of the sample (from molecular bonds IR absorptions) and it is quantitative10, thus allowing the 3D reconstruction of chemical data on a single intensity scale of contrasts (translatable into molecular concentrations)11. IR spectro-microscopy is also able to combine a chemical analysis of the tissue sample and highlight the presence of contrast agents in the blood system, such as gold and barium-sulfate (BaSO4) nanoparticles12, thus promising a functional plus chemical analysis of the sample in 3D. Numerous analytical algorithms such as spectral clustering and classification, principle component analysis (PCA), and curve fitting on IR spectra can be applied for extracting relevant information from sample contents. In this study, we want to demonstrate that FTIR microscopy might be considered as a CKD diagnosis method by analyzing kidney biopsies, thus achieving to provide an information of glomeruli condition in terms of perfusion level and chemical alterations. To that end, we used a mouse model of CKD with unilateral ureteral obstruction (UUO) to alter the function and micro-environment of glomeruli. Our aim was to show that IR spectro-microscopy can reveal multifactorial alterations of glomeruli at the single specie level. We provide functional and biochemical (oxidative stress, fibrosis, metabolism) results significantly correlated to the worsening of the disease.
Experimental section Animal experiment and tissue sample preparation All animal experiments were performed in male B6/C57 male mice strain of 8 weeks purchased from National Laboratory Animal Center, Taipei, Taiwan. All the procedures involving animals were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (AS IACUC), Approval Number: Protocol #RMiPHYHY2010039. Complete UUO surgery was operated in the right kidney of each mouse after being anesthetized with the mixture of isoflurane and oxygen. 3 groups of mice (n = 6) were used for the study, sacrificed at days 4, 9 and 14 after the operation. The non-operated kidneys (left ones) were used as controls. A solution of BaSO4 nanoparticles (Ø 500 nm; 20 mMol.L-1) was perfused into both kidneys of mice, after being perfused with PBS. After sacrifice, the kidneys excised were immediately frozen in liquid nitrogen. Serial sections of kidney frozen tissue blocks were obtained by cryomicrotomy (−20 °C, 3050-TM, Leica-Microsystems, France) for a 5 µm thickness and then deposited on IR-transparent CaF2 substrates (25x75 mm, 2-mm thick, CrystalTechno, Moscow, Russia). Series of 20 to 30 sections were performed from the surface of kidney samples to cover the first layers of glomeruli. Visible images of tissue sections were acquired before to perform IR imaging. IR data acquisition and data processing Location of glomeruli on histological sections were revealed by visible images of tissue. For every glomerulus location, a single FPA IR image data acquisition (341x341 µm) was performed. Spectra ACS Paragon Plus Environment
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acquisition covered the 900-4000 cm-1 spectral interval in transmission on a Hyperion-3000 IR microscope, equipped with a 128x128 FPA detector and using a 15X magnification objective (Bruker, France). A final 2.66x2.66 µm pixel resolution was obtained on IR images. The background was recorded before each sample image acquisition on the blank area of the CaF2 window. The microscope and spectrometer were constantly purged with N2 and the room where was installed the microscope was air-conditioned at 20°C and 55% humidity. For all IR image acquisitions, 5000 scans were co-added at a 4 cm-1 spectral resolution (975 data points per IR spectra). All spectral processing and classification was performed using a mathematical software module (Kinetics) provided by E.Goormaghtigh’s laboratory (SFMB, Université Libre de Bruxelles, Brussels, Belgium) modified by our team members and working on the MATLAB2012 software (Mathworks, France). The data processing was operated on the entire spectra (not truncated) of image data sets. The subtraction of baseline correction in each spectrum used 6 reference points at 925, 1800, 2100, 2500, 2700 and 3700 cm-1. Standard spectral data treatments were applied on 2D and 3D IR spectrum matrices, such as defining the lipid-to-protein ratio using the 3000-2800 cm-1 and 1700-1600 cm-1 spectral intervals (details provided in the results section and figure legends). Spectra clustering Clustering classification provides a direct identification of discriminant spectral parameters via the reduction of complexity in spectra13. The advantage is that the data treatment can be supervised or unsupervised, depending on a possible a priori selection of spectral data or intervals. We first used the spectral interval covering the most intense IR absorptions of BaSO4 (1160-1220 cm-1, Supplementary figure-S1) to highlight its distribution in glomeruli’ vascular network. IR spectra for each tissue section were extracted for form a 128×128×875 datapoints matrix per FPA. k-means clustering (J.B. McQueen algorithm14) is a nonhierarchical clustering method that uses an iterative algorithm to update randomly selected initial cluster centers between spectra, and to obtain the class membership for each spectrum, with maximal variance between clusters15. It has the advantage over other classification methods that it considers spectra as variables rather than datapoints in spectra. Curve fitting Curve-fitting of FTIR spectra was performed using a sub-routine of Matlab 2012/a. Curve fitting algorithm provides detailed information of chemical components attributed to individual bands in IR spectra. The spectral parameters including wavelength of bands, amplitude (peak height), bandwidth and band shape possess unique features representing a discriminant vectors of chemical variations in tissue. The performance of curve-fitting in spectra of IR image data is operated point to point by auto-algorithm in a MatLab software subroutine (using the fmicon minimization algorithm). The second derivative spectrum was used to determine the number and the position (maximal IR intensity wavenumber) of the bands constituting the spectral interval of fatty acyl chains (3050-2800 cm-1) as previously described16. In brief, with this method, the bands constituting a given spectral interval are revealed by the successive minima of the second derivative spectrum. All bands were positioned at maximal intensities revealed by second derivative spectrum and shape ACS Paragon Plus Environment
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Analytical Chemistry
parameters were set free to any combination of 80% Gaussian / 20% Lorentzian functions. Band width at half height was set fixed for each band while maximal intensity was set free for curve fitting calculations. Spectral curve-fitting quality was assessed by a RMSE value set at 0.1% of the total spectral interval area. 3D IR image reconstruction The visible images of tissue sections were first obtained to determine the area of interest using anatomical details that could be easily recognized between sections (typically on a 1 mm² area, equivalent to a 3x3 FPA acquisition sequence). The proper realignment of tissue sections for a 3D stacking of 2D visible and IR images was performed using several anatomical features in the tissue area. The x,y orientation of the tissue sections was recorded for IR image acquisition, thus making visible and IR images superimposable (Figure 1). The 3D IR image was further reduced to the 3D block of IR spectra corresponding to the double of a glomerulus volume (typically a cube of 70x70x70 spectra or x,y,z ≈180-190 µm). The Amira® (Thermo Fisher Scientific - FEI) graphics software was used for 3D reconstruction of images. Statistics All results were expressed as mean ± SE. Comparison between healthy and pathological tissues (UUO models at 4, 9, and 14 days post-surgery) were performed with multivariate analysis of variance (MANOVA; SPSS 15.0, SPSS Inc., France) applied on the results obtained from the spectral curve fitting data. All data obtained from fatty acyl chains absorption bands were used for the comparison between healthy and pathological conditions. Multiple comparisons were performed using Dunnett’s T3 Post Hoc analysis for the determination of significant difference between these groups for each absorption band. Dunnett’s test analysis commonly used when ANOVA has rejected the hypothesis of the equality of the means distributions. It compares group means and is specially designed for the situations where all group means are to be checked against one reference group. Its purpose is to identify the groups whose means are significantly different from the means of this reference group. P values were fixed at 0.05 and 0.01 to consider the significant level of difference between series of data.
Results 3D image reconstructions Visible and IR images obtained on tissue sections were aligned according to recognizable anatomical features (border of kidney, distribution of glomeruli, blood vessels, etc.). A high number of scans was required to obtain sufficient S/N level and make quantitative analyses between IR images reliable. The minimum number of scans making S/N consistent between images was 2500 and a number of 5000 was chosen to ensure that we obtained high-quality IR images. The 3D stacks of 2D images were further reduced to single glomeruli volumes and registered as separate 3D ACS Paragon Plus Environment
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spectrum matrices. For each 3D spectrum matrix, the corresponding 2D IR images were also registered for applying spectral data treatments. A total of 40 3D images of glomeruli were obtained, with 10 per condition (healthy and 3 time-points in CKD development). Example of 3D IR images of glomeruli are given as supplementary material (videos). CKD-induced changes in biochemical contents A first evaluation of pathology-induced changes on chemical contents of glomeruli was performed by calculating the proteins-to-lipids ratio (L/P), using the spectral intervals of fatty acyl chains (3050-2800 cm-1) and amide I (1700-1600 cm-1), fixing the scale for all IR images (as indicated on figure 1). The L/P ratio is a standard measurement used in FTIR spectroscopy for assessing a general patho-physiological condition change, which can results from a global imbalance between major molecular contents induced by fibrosis17, inflammation18, oxidative stress11, metabolic aberrations19, etc. The distribution of L/P values on IR images of glomeruli shows that a significant loss of lipid contents could be observed along pathology development, with an average value dropping from 0.34 ± 0.14 for the normal condition to 0.25 ± 0.09 (P
