1.

INTRODUCTION

The sodium-related homeostasis processes are an important part of the vital functions of an organism. Sodium intake, retention and excretion are naturally regulated to balance between the salt and water content with a high accuracy of 12%. Any sodium concentration misbalances are known to cause serious diseases and malfunction of the body including a loss of myocardia function¹. Recent studies indicate that homeostasis of sodium is much more complex than previously thought; it includes not only kidneys but also skin and muscle tissues^2-4.

At the end of the 20^th century, a previously unknown type of “passive” sodium was suggested to exist following the fact that negatively charged glycosaminoglycans (GAGs) of the extracellular matrix may bind to positively charged cations of light metals. Thus, a part of sodium content becomes osmotically neutral enough to leave the active homeostasis process and to stop its influence on the values of arterial pressure (AP) and extracellular fluid volume³. However, the sodium deposits continue to affect the properties of adjacent tissues. Thus, an excessive concentration of sodium in GAG can initiate new biological processes involving the macrophages⁴.

In 2007-2012 the prolonged and accurate study of bodily sodium intake/excretion ratio⁵ discovered the amounts of excreted sodium not related directly to the sodium intake and arterial pressure values. This fact was explained by the existence of osmotically passive sodium deposits in skin and muscle tissues formed earlier that can deplete to contribute to the elevated level of sodium in urine. To visualize these sodium accumulations M. Hammon et al. used ²³Na Magnetic Resonance Imaging (MRI) of the lower legs⁶ and M Christa et al. observed an increased sodium MRI signal in myocardia⁷. However, low spatial resolution and weak intensity of sodium MRI signal did not allow studying the sodium concentration peaks at the cell scale.

Several years ago our group carried out a series of acoustical and X-ray florescence analysis (BRUKER S8 Tiger spectrometer) investigations of chemical and mechanical properties of the heart tissues of 18 deceased patients⁸. We found a correlation between elevated sodium concentration in heart muscles, patient’s age and arterial hypertension. Also there was evidence of sodium-related modification of muscle elasticity of myocardia and related diastolic function. However, these experiments also lacked high spatial resolution needed for studying the sodium localization at the cell level.

This paper presents the results of X-ray fluorescent and absorption microscopy experiments based on high brilliance X-ray source on detection and analysis of sodium depositions in the extracellular matrix of myocardial tissues of rats with the reference to their sodium intakes.

2.

THE SAMPLE PREPARATION

The samples of heat tissues were taken from different laboratory animals*. Two groups of 15 Wistar rats aged 15-16 weeks were grown, with the sodium consumption rate of these two groups being different from the third week. The low sodium diet limited the sodium intake by the value of 0.2 mEq per 200 g of body weight per day, while the high sodium diet ration contained 2.0 mEq or more per 200 g of body weight per day. In other words, two groups were distinguished by sodium intake rates as normal (control group) and extensive^{9, 10}. The water supply was always kept as ad libitum (25 ml of de-ionized water for 15 g of food) and the food ration was about 15 g per 200 g body weight per week for all the animals.

High sodium diet feeding was maintained in the corresponding group of animals during 8 weeks before the myocardial hypertrophy occurred¹¹. After 8 weeks the animals were intraperitoneally injected with methohexital at a dose of 100 mg/kg and sacrificed through decapitation.

The 3-μm thick slices of the paraffin-fixed myocardia were cut from the extracted heart tissues, then mounted onto slides and dried (T = 60 °C) in the air. Finally, paraffin was removed with ethanol and the slices were stained by hematoxylin and eosine in accordance with standard histological procedures. For X-ray studies, the slices were backed by thin (3-5 μm) polymer film.

It should be mentioned that due to the chemical fixation procedure the most dissolved sodium proves to be removed from interstitium and, to a lesser extent, from intracellular volumes restricted by cell membranes¹². However, the removal of osmotic sodium during the paraffin fixation has played a positive role in this particular case. It reduced the high background signal of osmotic sodium of the extracellular fluids to highlight the tracks of bound sodium deposits.

3.

X-RAY FLUORESCENCE MICROSCOPY

The experiments were conducted on TwinMic beamline at ELETTRA synchrotron (Italy). The X-ray fluorescence (XRF) microanalysis of the heart tissues was provided with the help of the X-ray microscope that was developed as a multi-mode instrument for high spatial resolution spectromicroscopy at low energy X-rays. In this particular experiments, the X-ray fluorescent microscopy was combined with scanning X-ray absorption microscopy (SXRM)^13-15, while the synchrotron ring operated with the electron energy of 2.4 GeV and the current of 160 mA.

For sodium mapping the TwinMic microscope was operated in scanning mode. A pinhole of 75 μm diameter was served as a secondary X-ray source, with the focal spot of 1.2 μm being produced by a zone plate. The micron-scale size of the probing X-ray spot was chosen as a compromise between the spatial resolution, duration of the scan run and dimensions of regions of interest (ROI).

The incident X-ray photon energy was chosen to be 1.472 keV to ensure the best excitation and detection of the K_α lines of sodium atoms (E = 1.041 keV). The X-ray flux through the pinhole was about 3·10⁹ photons/s that enabled to produce a proper XRF signal on seven silicon drift detectors with an exposure time of 5 s. The measured XRF K-line signals of chemical elements were processed by using open source PyMCA software¹⁶ to compose the chemical element mapping.

The absorption contrast SXRM images were recorded with fast readout CCD camera (Andor Technology) to provide SXRM images of cardiomyocytes and interstitial space as supplementary information to X-ray fluorescence maps.

The TwinMic configuration and experimental conditions enabled a successful visualization of the myocardial cellular structure and chemical element mapping of sodium, magnesium, oxygen, and other elements with precise overlapping of obtained morphological and chemical data. The XRF/SXRM image sizes of ROIs varied from 50х50 to 72x72 pixels, i.e. from 60 х 60 μm² to 86.4 × 86.4 μm². All 19 ROIs selected on 7 samples (3 ROIs per sample on all the samples except two) of myocardial tissues have been studied with using both XRF and SXRM microscopy modes (see Table 1).

Table 1.

Sodium XRF maps (top row) and absorption contrast X-ray images (bottom row) of different regions of interest (ROI) of myocardia slices taken from rats of two groups: samples K5 and K7 are heart tissues of normal (control) diet group, samples B1-B4, B7 are heart tissues of high sodium diet group. The ROI dimensions are defined as (60…86.4) x (60…86.4) μm².

Thus, Table 1 shows a set of X-ray absorption images and corresponding sodium maps acquired with the help of synchrotron based STXM and XRF microscopy for heart tissue slices of high and low sodium diet animals: 5 samples were extracted from 5 rats of high sodium diet group and 2 samples were taken from 2 rats of the control group. The obtained STXM and XRF microscopy images enabled a clear visual segmentation of cardiomyocytes and intercellular spaces in the heart muscle tissues. In all samples the XRF analysis revealed a higher concentration of the sodium content inside the cells than in the interstitium space. This inverted ratio between intracellular and extracellular sodium concentrations can be explained by a strong wiping out of light metals during the chemical fixation procedure (see Section 2).

The main challenge in the statistical processing was to perform an accurate data sampling inside and outside boundaries of cardiomyocytes in the context of thin slice 3D-to-2D transformation of muscle fibers and possible interstitium perforation. This problem has been solved by using the SXRM images for careful manual sampling of six 3x3 pixels (3.6х3.6 μm²) zones inside every ROI to present 3 values of intracellular and 3 values of extracellular sodium content. The sampling generated the dataset of local sodium K-line emission values that could be used for the calculation of averaged intracellular and extracellular values in the ROI. The detailed description of our statistical processing of the data is beyond the scope of this paper and will be published later¹⁷.

Preliminary estimation of the relative sodium concentration results in a statistically significant difference of the sodium content in interstitium outside cardiomyocytes: 718.0 ± 402.4 for high sodium diet in comparison of 496.6 ± 283.5 for low sodium diet, i.e it demonstrates an elevated level in the heart tissues of high sodium diet animals. Note that we did not measure the real values of sodium concentration in the tissues because our interest was focused at study relative change of sodium content affected by the sodium diet type. We have to mention also poor statistics of the experiments due to the small number of the sampling points and samples that made us use complicated methods for statistical analysis, such as Kruskal-Wallis and Mann-Whitney tests.

To sum-up, we were able to present an experimental demonstration of elevated sodium content inside interstitium of myocardia of high sodium diet animals as compared to animals of the control group with low sodium diet. The obtained elevation of the sodium concentration cannot be explained in the frames of the normal balance of osmotic sodium in interstitium fluids and is considered to be the results of sodium depositions.

4.

CONCLUSIONS

To our knowledge, this paper presents the world’s first high resolution X-ray imaging of myocardial cells by means of transmission and fluorescence X-ray microscopy techniques. The results show a statistically relevant increase of sodium content in interstitium of myocardia that can be explained by the new concept of sodium depositing and confirm the original assumption on the existence of osmotically inactive sodium allegedly bound with negatively charged glycosaminoglycans inside the interstitium network.

We consider this work as a beginning of new research of previously unknown structures and their effects on heart function. The next step will be a new series of similar X-ray experiments to improve the statistical properties of the sampling and a combined study of sodium and GAG structures by using XRF and FTIR-spectromicroscopy methods to validate glycosaminoglycan-sodium relation.

The authors thank Dr. N.L.Popov (LPI RAS) for his help and useful comments regarding the study and manuscript. This work was supported by CalipsoPlus EU network travel grant (20185066). The FISR Project “Tecnopolo di nanotecnologia e fotonica per la medicina di precisione” (funded by MIUR/CNR, CUP B83B17000010001) and the TECNOMED project (funded by Regione Puglia, CUP B84I18000540002) are acknowledged for financial support as well. Part of the research reported in this publication was also supported in the frames of HORIZON 2020 Program.