3.3.2. Histological Staining for Calcium Deposits

Histology is a well-established standard lab technology and therefore, to date, is considered the gold standard. Staining for calcium like von Kossa and alizarin red have been established for decades [116,117]. To increase sensitivity, fluorescence dyes were established for the detection of calcium deposits in cells and tissue.

Classical histology often follows a standard workflow: tissue is dissected and either fixed, e.g., in paraformaldehyde or formalin, embedded, e.g., in paraffin, and sectioned; or alternatively, tissue is frozen after dissection and cryosectioned. Tissue sections of usually 4–6 µm thickness are then stained with the selected dye, imaged in an appropriate microscope and (semi-)quantification can then be conducted with dedicated software. Depending on the workflow, automatization is possible. The workflow and other characteristics of classical histology are summarized in Figure 1.

Characteristics of methods of classical microscopy employed for the detection of vascular calcification in mice. Key aspects for the critical appraisal of the applicability of classical histology are summarized with regard to the applicability of classical histology in vivo and ex vivo, the identifiable location and size of vascular calcification, the repeatability of analysis in longitudinal studies, the compatibility with additional methods, and the personal and technical expanse. The exemplary workflow presented is based on the publications of [116,117,118,121,122].

Due to the relatively inexpensive hardware requirements and the broad application of histology, many laboratories have equipment and histological procedures that are often included in routine lab education, thus making histology widely accessible. However, classical histology suffers from several drawbacks such as low specificity of dyes and high working expenditure for analysis of serial cuts of tissue or whole tissue sections. As the calcification foci could be spotty distributed, especially when only microcalcifications are present in the tissue, several tissue sections should be analyzed to avoid measuring bias. Extensive tissue processing can also result in post mortem modifications of morphology, including the drop out of hydroxyapatite crystals due to sectioning.

The method of von Kossa staining is widely used to detect the presence of abnormal calcium deposits in cell cultures and histological sections. It is based on a precipitation reaction where calcium ions, bound to phosphates, are replaced by silver ions from silver nitrate. Under a light source, silver phosphate undergoes a photochemical degradation and is thereby visualized as metallic silver deposits. If counterstained with nuclear fast red, the nuclei of the cells are colored in red and the cytoplasm in pink [118]. If counterstained with H&E staining, calcium appears in a deep-blue purple [117]. While commonly used for calcification assessment, von Kossa staining is somewhat limited by its reduced specificity for calcium crystals [117].

For decades, alizarin red S staining has been used to identify calcium deposits in cells and tissue sections. It reacts with calcium via its sulfonate and hydroxyl groups forming bright red deposits in aqueous solution requiring a pH of at least pH 4 [119]. This reaction is not strictly specific to calcium. Other cations like magnesium or manganese may interfere, but these elements normally do not occur in sufficient concentrations to interfere with the staining [116]. Nonetheless, it should be taken into account that this stain also detects calcium-binding proteins and proteoglycans without discriminating for the presence of hydroxyapatite [120].

Sim et al. developed a probe coupling fluorescein and the bisphosphonate alendronate. In comparison with different calcium salts, the probe preferentially binds to hydroxyapatite and shows higher specificity and signal than alizarin red staining, enabling visualization of micro-calcifications [121]. The application was possible both ex vivo and in vitro. Several other dyes targeting hydroxyapatite have been reported, including, for example, the long-established dye calcein [122,123], fluorescence-labeled osteocalcin [124] and an arylphosphonic acid-based dye [125]. The development of new fluorescent dyes may offer several advantages over von Kossa and alizarin red staining. A higher sensitivity and the opportunity for multiplexing are possible by likewise following a very similar protocol. Traditional workflows combined with fluorescent dyes still include tissue sectioning, thus prohibiting a further downstream analysis of tissues.

The NIRF calcium tracer is another dye for vascular calcification. This method is based on fluorescence optical imaging that uses excitation light from the near-infrared spectrum to stimulate fluorophores [126]. Based on the work of Zaheer et al. [127], Osteosense^® (PerkinElmer, Boston, MA, USA) is a NIRF calcium tracer consisting of a fluorophore and a bisphosphonate (pamidronate) that binds with great affinity to hydroxyapatite in mineral deposits. This dye is commercially available with fluorophores, exciting approximately at 680, 750 and 800 nm. In human samples and preclinical mouse models, this method has been demonstrated to show early microcalcifications [128]. As they bind to nanocrystals [129], very early lesions can be identified. Osteosense^® could discriminate the influence of a Bmp inhibitor on aortic mineralization in a d Ldlr^−/− mice model via ex vivo microscopy [130]. In addition, it is applied in several in vivo and ex vivo IVM studies [122,129].

For imaging of NIRF calcium tracers, special microscope equipment is required, depending on the dye employed, including, for example, suitable light sources providing light in the NIR range, cameras with a good response to NIR wavelengths and functioning objectives. Nevertheless, depending on the fluorophore coupled, imaging of Osteosense^® can also be possible with an appropriately equipped standard fluorescence microscope.

In conclusion, histological analysis with the classical dyes alizarin red and von Kossa, imageable in the transmitted light microscope, remains the gold standard with fluorescence dyes offering a valuable alternative and with dye-dependent higher specificity towards hydroxyapatite, which comes with increased technical requirements and higher experimental costs. The calcification area can be localized (intimal/medial) and, depending on the sensitivity of the method, a discrimination between micro- and macrocalcifiation is also possible. Sectioning, staining and imaging of substantial tissue sections is required for quantification and location of calcification, thus resulting in tissue destruction. The requirement of animal sacrifice for histological analysis impedes longitudinal analysis. However, as shown by an elegant experimental design by Hutcheson et al., with an intravenous application of alizarin red and calcein at different time points prior to animal sacrifice and post mortem histological analysis [122], an implementation of a longitudinal approach employing classical histology seems possible.

IVM imaging is carried out in a living species and allows imaging in a physiological in vivo setting. IVM is predominantly used with the application of fluorescent dyes. The application route of the dye depends on the experimental model, for example via intravenous injection. As an alternative to dyes, the application of genetically altered animal models with the expression of a fluorescence reporter is also possible.

The selected procedures for IVM are summarized in Figure 2. Before starting IVM, the effectiveness of a sound anesthesia and a firm restrainment must be tested to prevent tissue movement. Depending on the time frame of IVM and on the tissue imaged, a prolonged surgery time have to be conducted, which can include, but is not limited to, ventilation, thermal control, infusions, and maintenance or intensification of anesthesia. The desired tissue is isolated, accessed by surgery and imaged. The microscopic options include two-photon microscopy (TPM, also known as laser scanning microscopy or multiphoton microscopy) and confocal microscopy. In TPM, the fluorophore is excited by two or more photons of lower energy levels that have to be absorbed by the fluorophore simultaneously. This offers several advantages, as the lower energy levels of photons permit a deeper tissue penetration and results in less out-of-plane light, thus reducing background and improving the signal-to-noise ratio [131]. TPM also causes less phototoxicity and photobleaching than confocal microscopy, where a single photon at a specific wavelength excites the fluorophore [131].

Characteristics of protocols of intravital microscopy (IVM) employed for the detection of vascular calcification in mice. Key aspects for the critical appraisal of the applicability of IVM are summarized with regard to the applicability of IVM in vivo and ex vivo, the location and size of vascular calcification identifiable by IVM, the repeatability of the read out in longitudinal studies, the compatibility with additional methods, and the personal and technical expanse. The exemplary workflow presented is based on the publications of [81,127,129,130,132,133].

Up to now, most of the experimental procedures using IVM work with an ApoE^−/− mouse model and analyze plaque progression with or without a treatment procedure. For studying skeletal development and atherosclerosis, a bone tracer was conjugated using the bisphosphonate pamidronate to the NIR fluorophore IRDye78 [127]. Apart from commercially available Osteosense^®, an application of other dyes discussed earlier in this article seems possible, e.g., for a fluorescein-coupled alendronate dye recently published by Sim et al., where the authors predicted the potential for a future in vivo application [121].

A study by Aikawa et al. combined IVM with µCT to study inflammation and calcification [129]. IVM was conducted repeatedly in mice aged 20 and 30 weeks on an atherogenic diet. They applied bisphosphonate-derived dyes to detect calcification (Osteosense^®), iron oxide fluorescent nanoparticles detecting macrophage accumulations and an imaging agent for cathepsin K activated upon enzymatic cleavage. That proof-of-principle study clearly highlighted the potential for applying repeated read-out techniques in a longitudinal study in mice [129].

Following the principle of those established protocols [129], several other experimental procedures were conducted. In a further study on valve calcification by the same group, IVM was followed by euthanasia, subsequent ex vivo imaging and further histological analysis [132]. To assess the effect of cathepsin S inhibition, mice received coinjection of a protease activatable dye (ProSense^®) and a bone tracer (Osteosense^®), followed by fluorescence microscopy both in vivo and ex vivo after dissection of the aorta [133]. With Osteosense^®, the authors found increased vascular calcification in ApoE^−/− mice with CKD compared to ApoE^−/− mice with normal kidney function [133]. A longitudinal approach was followed in another protocol. The first IVM was conducted at the beginning of the diet switch, followed by an additional IVM 10 weeks later, which depicted a significant increase in calcification over the prolonged continuation of atherogenic diet [122].

Apart from cardiovascular calcifications, Köppert et al. published an interesting protocol on the clearance of CPP [81]. They created fluorescent CPP particles by labeling bovine fetuin-A with the fluorescent dyes Alexa 488 or Alexa 546, which were then used to prepare primary and secondary CPP. Mice were soundly anesthetized, the liver was extraventralized and the mouse was mounted on a thermostatic microscope with the liver downward. Mice were injected with Hoechst 33258, tetramethylrhodamine ethylester, primary and secondary CPP, and intravital TPM was continuously recorded. The authors found liver sinusoidal endothelial cells to clear primary CPP, whereas liver Kupffer cells preferentially cleared secondary CPP [81].

In summary, IVM requires extensive and in part customized technical equipment as well as extensive researcher training. However, IVM, especially TPM, offers an excellent resolution for studying calcification and underlying pathophysiological processes in vivo, providing researchers with images in the biological setting. IVM can enable longitudinal study design [122]. After image acquisition, post-operational care by suturing of the wounds and post-operative analgesia is necessary. Euthanasia of the animal at the end of IVM and tissue dissection allows further post mortem analysis. Under the limitation of good tissue preservation, further analysis of the tissue in downstream analytical processes is possible, considering that the applied fluorophore does not interact with the downstream analysis procedure.