Silver diagnosis in neuropathology: principles, practice and revised interpretation

Silver-staining methods are helpful for histological identification of pathological deposits. In spite of some ambiguities regarding their mechanism and interpretation, they are widely used for histopathological diagnosis. In this review, four major silver-staining methods, modified Bielschowsky, Bodian, Gallyas (GAL) and Campbell–Switzer (CS) methods, are outlined with respect to their principles, basic protocols and interpretations, thereby providing neuropathologists, technicians and neuroscientists with a common basis for comparing findings and identifying the issues that still need to be clarified. Some consider “argyrophilia” to be a homogeneous phenomenon irrespective of the lesion and the method. Thus, they seek to explain the differences among the methods by pointing to their different sensitivities in detecting lesions (quantitative difference). Comparative studies, however, have demonstrated that argyrophilia is heterogeneous and dependent not only on the method but also on the lesion (qualitative difference). Each staining method has its own lesion-dependent specificity and, within this specificity, its own sensitivity. This “method- and lesion-dependent” nature of argyrophilia enables operational sorting of disease-specific lesions based on their silver-staining profiles, which may potentially represent some disease-specific aspects. Furthermore, comparisons between immunohistochemical and biochemical data have revealed an empirical correlation between GAL+/CS-deposits and 4-repeat (4R) tau (corticobasal degeneration, progressive supranuclear palsy and argyrophilic grains) and its complementary reversal between GAL-/CS+deposits and 3-repeat (3R) tau (Pick bodies). Deposits containing both 3R and 4R tau (neurofibrillary tangles of Alzheimer type) are GAL+/CS+. Although no molecular explanations, other than these empiric correlations, are currently available, these distinctive features, especially when combined with immunohistochemistry, are useful because silver-staining methods and immunoreactions are complementary to each other.
Introduction
The year 2006 was the centenary anniversary of the first clinical description of a presenile patient with progressive cognitive decline, later defined as Alzheimer’s disease (AD) [7]. It was the same year (1906) when Santiago Ramón y Cajal and Camillo Golgi were awarded the Nobel prize for their contributions on neuroanatomy achieved by using silver-staining methods, especially Golgi-silver technique. In the field of neuropathology as well, silver staining was the principal method of choice at that time, which visualized senile plaques (SPs), neurofibrillary tangles (NFTs) and Pick bodies [2]. More than 80 years have passed before biochemical compositions of these pathological structures were unveiled. During this period, technical trials to identify and localize these molecules on histological sections showed the effectiveness of immunohistochemistry and revolutionized not only research but also routine diagnosis in the field of neuropathology. Probably because of the apparent success in the molecular explanation of diseases, recent attention to silver staining has not been very enthusiastic. Although most silver-staining methods are still left with some ambiguities, neuropathologists have profited by their efficacies. Silver-staining methods have been steadily improved and one of the recent advances is undoubtedly Gallyas method [41]. In practice, it allows stable, reproducible and sensitive visualization with low background. However, it is unfortunate that their principles, technical details and interpretations, which are different according to each method, are sometimes not sufficiently shared among neuropathologists and technicians. It frequently happens that some time-honored protocols are left without possible explanations and their interpretation is often restricted to “argyrophilic or not”. This naive interpretation is based on the assumption that “argyrophilia” is a homogeneous phenomenon irrespective of the staining method and the lesion. Recent studies have demonstrated, on the contrary, that “argyrophila” is heterogeneous and dependent on the staining method and the lesion [85, 119–122]. Here, established silver-staining methods in the field of neuropathology are reconsidered with the intension of improving their use and interpretation.
The first part of this review summarizes basic principles and outlines protocols about four principal silver-staining methods for formalin-fixed, paraffin-embedded samples from human autopsy brains mainly with degenerative diseases, currently in use in our laboratory and their modifications. Although other silver-staining methods have also been invented to visualize normal neuronal structures, astrocytes, microglia and experimental tissue samples, it is not possible to encompass the whole range of these techniques mainly because of the limitation of my own experience. The second part provides an overview of similarities and differences between silver-staining methods and immunohistochemistry, so that a comprehensive interpretation, including their pitfalls results. Nevertheless, this review is not intended to establish a relative grading of these methods by identifying the possible superiority of a method (for example, immunohistochemistry) over another (for example, a silver-staining method). Instead, it is designed to show how unique each staining method can be, by providing a distinct point of view, each independent of the other. If we are prepared to take these possible differences of staining methods into account, such an approach will provide a less biased, multi-faceted and fuller stereoscopic picture than can be achieved with immunohistochemistry alone.
General principle of silver staining
The coloration in conventional histology is based on the selective affinity of a dye to some tissue elements relative to surrounding elements and their permeability. This differential affinity to specific tissue elements is finally represented by the distinctive original color of the dye as seen with its solution. This is essentially a single step procedure, called dyeing, and does not involve chemical transformation of the dye. Subsequent washing step is also selective in dissolving out the attached dye, which is called differentiation [5]. Various combinations (for example, hematoxylin and eosin, the Klüver-Barrera method, etc.) are currently in use for neuropathological examinations. The mechanism of silver staining is, however, completely different. Because the solutions containing coloring agents used for silver-staining methods are usually colorless, simple application of these solutions to tissue sections, by itself, does not allow visualization through light microscope even if the reagent has some affinity to the tissue elements. Subsequent chemical transformation is necessary for visualization. In silver solutions, most of the silver is present as ion or complex salt. The silver ions should be first attached to the target elements as silver ions or silver salts (step 1). This attachment, however, does not allow visualization by itself. The attached silver ions (Ag+) have to be reduced (=accept electrons) to yield, in situ, metallic silver particles (Ag) now visualized only if their amount and size are sufficient for microscopic detection (step 2). Therefore, not only the differential affinity of the silver ion to some tissue elements (step 1) but also its chemical transformation for visualization (step 2) is essential. Although the attachment of silver ions to tissue elements is based on the “in situ” formation of silver salt, these silver ions, either in the solution or as salts, are reducible. They undergo reduction to form metallic silver. Gallyas demonstrated that there is a time lag between the rapid attachment of silver ions onto the section (step 1, within 15 min) and delayed formation of metallic silver particles (step 2) there [42]. Because these two steps are independently affected by pH, temperature, the concentration of silver ion and coexisting molecules [42], these steps are independent even if both take place in a single manipulation. This dual function of silver, that enables “site-directed chemical transformation”, is fundamental to various silver-staining methods.
Because some tissue elements (glutathione, creatine, adrenaline etc.) exhibit an intrinsic reducing capacity, which may be strong enough to reduce silver ions into metallic silver particles in situ, these two steps (selective attachment and visualization after reduction of silver ions) may take place in a single manipulation. This is called silver reaction or “argentaffin” reaction [35, 102]. When the reducing capacity of the tissue elements is absent or not sufficient, it is necessary to provide reducing agents externally after silver ions have been attached to the tissue elements. It is also possible to facilitate or modify these steps by some reagents [40]. Anyway, this two-step procedure is called postblackening (Nachschwärzen) or “argentophilic” reaction [35, 102] and most of the current silver-staining methods for neuropathology consist, in essence, of at least these two steps. If sufficient amount of silver ions have been attached to the tissue elements but not have been reduced spontaneously after step 1, it is possible to visualize them simply by applying an appropriate reducing agent externally. This is called chemical development, during which the amount of deposited silver in the tissue section remains unchanged. However, it is not easy to increase the amount of silver deposits during step 1. For example, increasing the concentration of silver reagents or prolongation of incubation time is frequently hampered by nonspecific staining. This trade off between sensitivity and specificity, principally encountered in all kinds of staining, is especially troublesome with silver-staining methods because reactions in the silver solutions and those in reducing agents are both capricious and difficult to standardize. This is one of the reasons why more sensitive, stable and reproducible silver solutions and staining methods have been a matter of considerable endeavor for a long period, as will be discussed later.
Particle size and possible determinants of selective staining
It has been reported that the apparent color of silver-stained elements is correlated with the size of final silver particles. In the test tube, the smallest range in their diameter (10–20 nm) is correlated with yellow color. The largest range (>100 nm) is associated with black color. Intermediate ranges are correlated with red (>30 nm) or blue-purple (50–60 nm) [102, 103], as reported by Liesegang [70]. The emission spectra of fluorogenic semiconductor nanocrystals, recently introduced for fluorolabeling, are dependent not only on their materials but also on their particle size [20, 22]. Although the size range of semiconductor nanocrystals (2–6 nm) is much smaller than that of silver particles, it is interesting to note that these differences in their color spectra are dependent on the particle size in both silver particles and the nanocrystals. Others reported, however, that black color is correlated with a larger amount of deposits [35]. Although the deposition of silver particles is primed by an affinity of the silver ion to some tissue elements, is this affinity the only major determinant of the selective staining? According to Seki [102], some colloidal substance in the tissue may hinder formation and deposition of metallic silver particles (Schutzkolloid). In addition, packing density of tissue ultrastructures (for example, fiber density) may also be responsible, because an excessive looseness of the structure may not allow secure settlement of silver particles. It is also possible that its extreme tightness does not permit appropriate growth of silver particles. He then concluded that a small quantity of this intervening colloid (eine Armut an Schutzkolloid) and an appropriate density of the structure (eine passende Strukturdichte) of the target structure are also important for the selectivity of silver staining. This idea that some spatial constraints around silver particles, in addition to the primary affinity of the silver ion, are essential in determining the selectivity of silver staining may be related to the current concept of disease-specific “conformational status” of pathological deposits and their constituent molecules.
These explanations are, however, far from complete for the majority of silver-staining methods. Furthermore, because these experiments have been performed mainly in animal tissues, in which sampling and handling of tissue materials are optimal, it remains to be clarified whether similar explanations are applicable to human samples obtained for neuropathological examinations, often containing distinct pathological lesions and suffering from suboptimal handling such as postmortem delay, suboptimal fixation and embedding in paraffin wax. Even though various methods for celloidin- or paraffin-embedded autopsy samples have been invented. Table 1 shows fundamental steps and contents of the solutions for the silver-staining methods currently in use in our laboratory.
Table 1Protocols for different silver-staining methodsBielschowsky method (original) for frozen sections [8]Modified Bielschowsky method for paraffin sections [131]Bodian method [9,10]Gallyas method [41]Campbell–Switzer method [21]PretreatmentPyridine (24–48 h)––0.25% potassium permanganate (RT, 15 min), then2% oxalic acid (RT, 2 min) [114], then0.4% lanthanum nitrate/2% sodium acetate (RT, 60 min) [47]–WashingDW––DW–Silver solution I3% silver nitrate(RT, 24 h)20% silver nitrate(RT, 20 min)1% silver proteinate + metallic copperf (37°C, 24 h)Alkaline silver iodate (RT, 4 min) 0.035% silver nitrate 10% potassium iodide 4% sodium hydroxidePyridine silver (RT, 40 min) 0.52% silver nitrate 8.7% (volume) pyridine 0.39% potassium carbonateWashingDWaDWDW0.5% acetic acid 3 min × 3 times0.5% acetic acid 3 min × 3 timesSilver solution II4–5%b ammoniacal silver20%c ammoniacal silver(RT, 15 min in the dark)–Physical developerg (RT, 5–10 min) 0.1% silver nitrate 0.5% tungsto-silicic acid 0.1% ammonium nitrate 2.5% sodium carbonate 0.03–0.2% formalinPhysical developergWashingDWDW + ammoniad–Chemical developer20% formalin in tap waterDevelopere in 20% ammoniacal silver 1% hydroquinone/5% sodium sulfiteDW distilled water, RT room temperaturea20% formalin in tap water is optional [50]b5 ml of 20% silver nitrate + five drops of 40% sodium hydroxide, followed by titration with ammonia to dissolve the precipitates. DW (20 ml) is then added [8]cAmmonium hydroxide was added to 20% silver nitrate drop by drop until the precipitates turn clear. Additional two drops of ammonium hydroxide is recommended [131]dAdd three drops of ammonium hydroxide to DW [131].eAdd three drops of the developer (formalin 20 ml, DW 100 ml, concentrated nitric acid one drop, citric acid 0.5 g) to the 20% ammoniacal silver used as silver solution II [131].f4–6 g of metallic copper in 100 ml of the silver proteinate solution is optimal [9, 10]gThe physical developers reported for Gallyas and Campbell–Switzer methods are essentially identical. In our laboratory, equal amount of stock solutions A (0.5 g sodium carbonate-anhydrous, 100 ml DW) and B (0.2 g silver nitrate, 1 g tungusto-silicic acid, 0.2 g ammonium nitrate, formalin 0.73 ml, 100 ml DW) are mixed immediately before use
Bielschowsky methods
Improvements in silver-staining methods have often been derived from chemistry. The ammoniacal silver solution had been used for production of mirror, based on the nature of the complex “silver diamine [Ag(NH3)2]+ “ in the solution that is liable to precipitate, after being reduced, at the interface between the glass surface and the solution. This is called the silver mirror reaction and it was Fajersztajn who initially applied this phenomenon to tissue staining [34]. Later, Max Bielschowsky [8] improved this method by incubating formalin-fixed frozen sections in the silver nitrate prior to the ammoniacal silver solution (Table 1). Although the exact mechanism still remains to be clarified [100], it is plausible that this prior treatment with the silver nitrate provides active foci for subsequent silver deposition in the ammoniacal silver solution [50]. Anyway, it is reasonable to suppose that this stepwise impregnation (silver nitrate-ammoniacal silver) improved the staining probably by increasing the amount and the size of silver precipitates [66]. Early modifications of Bielschowsky method were summarized by Beech and Davenport [6]. Garven and Gairns [50] provided a comprehensive review on the possible mechanism of the so-called Bielschowsky-Gros method and its modifications. The presence of a huge number of modifications, however, indicates that this method is highly unstable and still awaits improvements. A modification for paraffin-embedded samples of normal neural tissues from various animal species was first proposed by Sevier and Munger [105]. Practically, it is probably Yamamoto–Hirano’s modification [131], which is currently accepted as the standard Bielschowsky method for neuropathological examination of paraffin-embedded autopsy samples (Table 1). This Yamamoto–Hirano’s modification is different from other conventional modifications of Bielschowsky method because the usual silver nitrate–ammoniacal silver sequence is followed by another ammoniacal silver solution containing developers.
Bodian method
Bodian established a new method, applicable to paraffin-embedded sections, for staining nerve fibers and nerve endings [9, 10]. This method is rather stable and reproducible and is currently in use for neuropathology. He used 1% silver proteinate (which usually contains 8% of silver) solution and placed metallic copper in it. Because the staining does not work well in the absence of metallic copper, he speculated that metallic copper might regulate the deposition of metallic silver by decreasing the concentration of silver ion [9, 10]. However, the mixture of silver nitrate and powdered egg albumin was reportedly successful even in the absence of metallic copper [92]. Nevertheless, the measured concentration of silver ion gradually decreases after addition of metallic copper, as demonstrated by two independent groups [64, 93]. Concomitant and gradual increase in the concentration of copper ion [64, 93] is accompanied by a decrease in pH [93]. These reciprocal changes between concentrations of silver and copper ions are mainly explained by a primary difference in ionization energy between copper and silver. In other words, copper is more liable to be ionized than silver. Because their concentrations are not influenced by the presence of sections in the solution, the amount of silver ion trapped in the section is practically negligible. While the addition of metallic copper up to 5 g/100 ml progressively decreases the concentration of silver ion in dose-dependent fashion (from 1,000 to 250 μg/ml), further increase in the amount of metallic copper up to 15 g/100 ml does not lead to further increase in the copper ion nor further decrease in the silver ion in the solution [64]. Indeed, this amount of copper, 5 g/100 ml, is in perfect agreement of the amount, 4–6 g/100 ml, originally recommended by Bodian [9, 10] and the amount of metallic copper more or less than this range does not give optimal results. Interestingly, not only the amount of metallic copper but also the shape of metallic copper (foil, plate, chip or granules) influences the concentration of copper and silver ions and the results of staining. The metallic copper as “foil” is correlated with the highest concentration of copper ion and the most intense staining [64, 93]. Moreover, these reactions in the silver proteinate solution occur slowly and the equilibrium is reached after 12–16 h. Figure 1 illustrates putative chronological events during incubation in the silver proteinate solution and development. In the early phase of silver impregnation, the high concentration of silver ion facilitates its attachment to the section (step 1, Fig. 1). This initial attachment is apparently not selective, because early interruption of incubation yields an overall nonselective nerve staining [93]. Gradual deposition of silver ion as metallic silver on the surface of metallic copper (step 2, Fig. 1) leads to a gradual decrease in the concentration of silver ion and a concomitant increase in the concentration of copper ion in the solution. Continued incubation in the solution with a decreasing concentration of silver ion facilitates liberation of the silver ion, once trapped in the section, back into the solution (step 3, Fig. 1). The selective staining of Bodian method is explained if this rate of liberation of silver ion back to the solution (step 3, Fig. 1) is different according to the tissue elements. This chronological change may be necessary to yield the selective staining and explains why an equivalent concentration of copper ion provided as copper sulfate instead of metallic copper does not yield similar results [64]. Some copper ions are also deposited onto the section (step 4, Fig. 1). Residual silver proteinate as colloidal fraction may release silver ions gradually throughout the incubation (step 5, Fig. 1). For practice, (1) 5 g of metallic copper as foil in 100 ml of 1% silver proteinate solution and (2) incubation for 16–24 h are recommended as optimal for Bodian method [64].
Fig. 1Mechanism of the Bodian method. Step 1: Attachment of silver ions to the section driven by high concentration of silver ions; single asterisk: a fraction of silver ions are already reduced to metallic silver even before chemical development. Step 2: Deposition of silver ions as metallic silver leads to a decrease in the concentration of silver ion and reciprocal liberation of copper ions. Step 3: Delayed liberation of silver ions from the section back to the solution with decreased concentration of silver ion after step 2. Step 4: Copper ions are now available in the solution after step 2 to be attached on the section. Step 5: Silver proteinate gradually releases silver ions during steps 1–4. Step 6: Not only silver but also copper ions/salts on the section are reduced to metallic particles. Step 7: These metallic particles are replaced with metallic gold; double asterisk: retrieval of copper ions in the gold chloride solution indicates that metallic copper has been deposited on the section (steps 4 and 6). Symbols: circle: silver; square: copper; pentagon: gold; blank symbols: reducible (ion/salts); filled symbols: metallic
After being incubated in the silver proteinate solution, sections are treated with 1% hydroquinone with 5% sodium sulfite for reduction (step 6, Fig. 1) [9, 10]. It has been reported that formaldehyde, suitable for reducing the silver diamine of Bielschowsky method, is not appropriate by itself for reducing the silver ions in Bodian method. In contrast, hydroquinone suitable to reduce the silver ions in Bodian method is not appropriate for reducing the silver diamine of Bielschowsky method [35]. However, some recommended formaldehyde in combination with hydroquinone for the reducing step of Bodian method [9, 10]. Because it is usually after the incubation in the developer when the sections appear contrasted for visualization, it seems reasonable to suppose that deposited silver ions are reduced to form metallic silver only after exposure to hydroquinone. If this is the case, treatment of the silver impregnated sections with sodium sulfite, which dissolves out silver salt but not metallic silver, will diminish the staining. On the contrary, Peters [93] demonstrated the presence of sodium sulfite-resistant metallic silver and suggested that at least some fraction of silver ions have already been reduced to metallic silver during incubation in the silver proteinate solution (asterisk, step 4, Fig. 1). They may serve as nuclei that attract surrounding reducible silver ions/salts to form larger metallic silver particles during subsequent development. Indeed, Gallyas demonstrated that radiolabeled silver ions in the silver proteinate solution are deposited to the section in the form of metallic silver even before the development. This is based on the finding that the radiolabeled silver on the section is resistant to the treatment with 1% silver nitrate/10% acetic acid that washes out reducible silver ions but not metallic silver particles. Because the localization of this radiolabeled silver, now deposited as metallic silver, is in good agreement with the final image of the adjacent section stained by Bodian method, it is likely that these metallic silver deposits play a determinant role in defining final image obtained with Bodian method [44]. At any rate, it is possible to intensify the staining through subsequent gold toning by incubating in 1% gold chloride with glacial acetic acid (three drops for 100 ml). Although the mechanism of Bodian method, especially that of copper ion, is not yet completely clarified, not only silver but also copper ions are retrieved in the gold chloride solution for toning (double asterisk, step 7, Fig. 1) [64]. This indicates that the ionized copper in the silver proteinate solution has been deposited on the tissue and that the deposited copper, in addition to silver, is replaced with gold [64]. This dual replacement may explain why this gold toning is necessary and particularly effective for Bodian method. After gold toning, it is now possible and necessary to treat the sections with 5% sodium thiosulfate to dissolve out silver ions that remain unreduced in the tissue. Otherwise, these remaining ions may be reduced after exposure to light to form undesirable precipitates. Because Bodian method was originally invented to stain normal axons and neurofibrils, these normal structures are always superimposed on pathological structures such as SPs and NFTs. These intervening normal structures are one of the major hindrances of Bodian method especially when examining silver-stainable deposits overlaid on normal structures.
Gallyas method
Another way of improvement had been achieved based on the chemical principle of photography, “physical development”. As opposed to the chemical development, additional silver ions are provided with reducing agents to form silver particles around the reactive foci, which have been already activated after exposure to light in dose-dependent manner. This greatly increases the amount of metallic silver or oxidized silver that are now visible as photographic images. It was Liesegang who first introduced this idea to histological staining in an attempt to establish a more consistent and reproducible method of silver staining [70, 88]. The protocols with physical development were later customized successfully for neuropathology by Gallyas [40]. Because physical developer solutions contain, in general, silver ions and reducing agents that together react rapidly, protecting colloid (tungsto-silicic acid for Gallyas method) is included in order to control by retarding this reaction [43].
As with other silver-staining methods, the attachment of silver ion to specific targets determines final localization of silver particles. According to Gallyas, this specific attachment is mediated by some determinant substance, which binds to the target tissue elements or the tissue elements themselves, followed by their chemical modifications. These foci are enabled to attract silver ions to form silver salts. Otherwise, the determinant substance renders these foci capable of reducing silver ions to form submicroscopic metallic silver particles bound to the target elements. The chemical nature of the original determinant substance may determine which components (for example, fibrous astrocytes, microglia or oligodendrocytes) in the section are finally silver-stained [37–39]. For visualization of NFTs, sections are incubated with silver iodide in alkaline condition that exhibits affinity to NFTs [41] (Table 1). Although Gallyas customized protocols according to various target structures other than NFTs [37–39], this silver iodide protocol for NFTs, currently in routine use for neuropathological diagnosis, is defined in this review as “Gallyas method” for simplicity and convenience. If this silver solution is prepared with radiolabeled silver, it is possible to quantify the silver ions/particles after each step of the staining protocol. Gallyas demonstrated that most of the silver attached to the section after incubation in this silver iodide solution is reducible silver salt but not metallic silver, because it is washed out after incubation in nonlabeled 1% silver nitrate/10% acetic acid (Table 1 of Ref. [44]), a treatment that expels reducible silver ions/salts from the sections. Incubation in sodium thiosulfate or sodium cyanide, alternate ways to expel these reducible silvers, diminishes the final image, again compatible with the assumption that the attached silver after incubation in the silver iodide solution is silver ions/salts but not metallic silver [41].
Is a separate step necessary to transform these attached silver ions/salts into metallic silver particles so that they may subsequently work as nuclei for subsequent growth of silver particles in the physical developer? Because the reducing agent in the physical developer is ready to reduce these attached silver ions/salts into metallic silver nuclei, an isolated step for reduction is not necessary before the treatment in the physical developer. However, a brief exposure of the silver-impregnated sections to formalin prior to the physical development leads to an instantaneous completion of the physical development [41]. However, it is possible that silver ions contained in the physical developer potentially attaches to the tissue and are physically developed to form separate metallic silver particles. Because this may happen independently of the silver from the initial alkaline silver iodide solution [45], a prolonged incubation in the physical developer may be hampered by this unexpected staining. Therefore, pretreatments have been invented in order to minimize this background. A pretreatment initially proposed by Gallyas was 5% periodic acid for 30 min. He tried various pretreatments [46] and finally proposed an alternative pretreatment with 0.4% lanthanum nitrate/2% sodium acetate in H2O2 for 1 h, which is more effective in eliminating the background [47]. An additional pretreatment with sodium permanganate followed by oxalic acid [114] (initially intended for melanin bleaching [71]) prior to this lanthanum nitrate/sodium acetate treatment of Gallyas [47] was found highly effective in eliminating the background and is currently in use as the standard in our laboratory. The background staining is practically negligible because the expected target staining is usually obtained before long and this untoward background reaction is generally slow. Similar oxidative pretreatments are not beneficial for other silver-staining methods [68]. The subsequent gold toning is essentially similar to that for Bodian method.
Compared with Bodian and Bielschowsky methods, Gallyas method is characterized by its stability, reproducibility and ability to visualize abundant deposits [15]. Moreover, normal structures are not stained in practice. This allows easy and reproducible examination especially when looking for pathological deposits such as NFTs or glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) [90, 119]. However, amyloid deposits are hardly visualized and it is usually neuritic components of SPs that are labeled by Gallyas method.
Campbell–Switzer method
The initial impregnation solution of Campbell–Switzer method contains pyridine (Table 1). Pyridine was initially used for tissue fixation. Bielschowsky [8] used pyridine for postfixation of formaldehyde-fixed frozen sections and found an enhanced selectivity. In order to avoid instability and risk of explosion of the ammoniacal silver solution, Hicks [57] added pyridine (4.4 vol%) to the silver nitrate solution (0.55%) buffered with potassium carbonate at alkaline range. Thereafter, Campbell et al. [21] combined this silver-pyridine (2.5 vol%)-carbonate solution with the physical developer invented by Gallyas and found that this staining is not only highly sensitive in detecting SPs and NFTs in AD brains but also as stable and reproducible as Gallyas method. The solution was later modified by increasing the concentration of pyridine up to 8.7% [13] and was found to be equally very powerful in detecting α-synuclein-related pathologies [101]. Gallyas demonstrated that the presence of pyridine markedly decreases the amount of silver, either metallic or reducible, attached to the section [42]. This is compatible with the observation that pyridine enhances the selectivity of Bielschowsky method [8] and that a higher concentration of pyridine is associated with reduced background [21]. Because the standard pretreatment (potassium permanganate/oxalic acid followed by lanthanium nitrate) for Gallyas method does not affect the results obtained with Campbell–Switzer method and there are essentially no differences between the physical developers used for Gallyas and Campbell–Switzer methods, the only significant difference between them is the silver solutions [86]. The reason for these differences between Gallyas and Campbell–Switzer methods remains unexplained.
Enhancement of silver staining by methenamine
Methenamine has been used for histological demonstration of glycogen and mucin [52]. Periodic acid-methenamine silver stain has been used for staining of the basement membrane of the glomeruli [61]. Dekura et al. [30] first described the use of the methenamine-silver solution for neurofibrils without prior periodic acid. Further modification of this methenamine-silver method was found highly sensitive in detecting SPs, which is equivalent to immunohistochemistry for amyloid β-protein (Aβ) [1, 53]. Interestingly, formic acid pretreatment, which enhances immunoreactivity for Aβ of SPs, completely diminishes argyrophilia of SPs by the methenamine-silver method or congophilia, suggesting that the methenamine-silver method detects specific structures, such as β-pleated sheets of amyloid, which are destroyed by formic acid [130]. It also stains perivascular amyloid but fails to stain Kuru plaque of Gerstmann–Sträussler–Scheinker disease [130] and renal deposits of amyloid [53]. This is in agreement with the early speculation by Seki [102] that factors other than direct affinity of silver to some tissue elements, such as the quantity of intervening colloid or packing density of tissue components are also responsible for final results of the silver staining. The paucity of normal neurofibrils with this method is helpful in delineating SPs on electron microscopy [76]. It is then possible to detect even sparse aggregations of amorphous, often ramified structures with fine granular silver deposits, suggesting that this highly sensitive method is capable of detecting the earliest stage of Aβ deposition [58]. Although the mechanism of lesion-specific enhancement remains unknown, introduction of 5% of methenamine in the silver proteinate solution of Bodian method is similarly effective in enhancing the selective affinity to SPs and NFTs [65]. It is interesting that this methenamine-silver method exhibits more affinity to Pick bodies when they are extracellular [89], while intracellular counterparts are more readily stained by conventional Bodian method even with electron microscopy [91].
Pretreatment with microwave
A microwave treatment of sections in silver solution was first reported by Brinn [19]. This treatment causes an increase in molecular movements across the tissue. This presumably facilitates quicker silver impregnation. At the same time, shortened incubation time for impregnation and activated molecular movements may minimize precipitate formation. Microwave activation of silver impregnation for SPs and NFTs was first introduced in order to apply King’s method [63] (originally designed for frozen sections) to paraffin-embedded sections [74]. Microwave activation is effective with Bielschowsky [23] and the methenamine silver [36] methods in shortening the incubation time for silver impregnation and eliminating the background. Although the elevated temperature may partly explain the improvements of these silver-staining methods, it has also been reported that a lowered temperature at 5°C for initial impregnation in 20% silver nitrate for Yamamoto–Hirano’s modification of Bielschowsky method gives more reproducible results in visualizing a larger number of diffuse plaques [73].
Molecular species detected by silver staining
It is interesting to identify isolated molecular species detected by silver-staining methods, but these attempts have been so far limited. Gambetti et al. used Bodian method and anti-neurofilament antibodies in parallel to stain an extract from rat spinal cord electrophoresed on the SDS-polyacrylamide gel. The typical triplet of 200, 145 and 68 kDa identified on the Bodian-stained gel is in agreement with the neurofilament triplet identified on the counterpart stained with anti-neurofilament antibodies [48]. They confirmed similar parallelism in other vertebrate and nonvertebrate species as well [94]. Additional less intense bands at a lower molecular weight range may correspond to tubulin or degradation products of neurofilament proteins [4]. Because these findings have been obtained with normal nerve tissue, further analyses based on brains containing pathological lesions are mandatory to identify components responsible for their argyrophilia on the pathological lesions. Iqbal et al. [60] used Gallyas method to stain the SDS-gel after electrophoresis of the paired helical filament (PHF) fraction extracted from AD brain. The electrophoretic mobilities of the Gallyas-positive bands correspond to those of PHF tau (intense), normal tau (less intense) and high molecular weight MAPs (weak). The absence of Gallyas-positive bands in the range of neurofilament proteins is in sharp contrast with the Bodian-positive triplet of neurofilament [48]. This difference may explain why Gallyas method is practically free from the staining of normal structures such as neuronal soma and neurites. This discrepancy suggests that molecular species detected by different silver-staining methods can be completely different, even though both of the methods label apparently identical structures such as NFTs on histological sections. Because several silver-staining methods have been modified to stain SDS gels [62, 78, 107], it may be possible to identify molecular species, either normal or specific to some pathological structures, which have a selective affinity to some silver-staining methods. Because extracted proteins electrophoresed on gel are necessarily modified or significantly degenerated, this approach may be fruitful only if the disease-specific nature, if detectable with silver-staining methods, is readily represented by a single isolated molecule even when it is modified for electrophoresis.
Quantitative comparison of silver-staining methods and immunohistochemistry
Even after the introduction of immunohistochemistry, modification of classic silver-staining methods or invention of new silver-staining methods have been attempted [11, 25, 26, 98]. Two major deposits targeted by these silver-staining methods are undoubtedly NFTs and SPs of AD [31, 32, 79, 82]. Different silver-staining methods [24, 27, 31, 54, 56, 67, 99, 124, 125, 127] have been compared quantitatively for their relative ability to detect NFTs or SPs, as represented in Fig. 2 [56]. In respect to detection of NFTs, these comparative studies are in agreement that Gallyas method is most powerful, followed by one of the three methods: modified Bielschowsky, Bodian or Cross methods. The methenamine-silver method is rated as least powerful for NFTs. There are two studies, each claiming a superior or equivalent number of NFTs detected with Reusche method [99] or with the nickel peroxidase method [27] compared with Gallyas method. This rating of putative sensitivity, however, is not shared when SPs are examined. SPs are well stained by modified Bielschowsky [125, 131], the methenamine-silver [1], periodic acid methenamine silver [128] or thioflavin-S [124] to similar extent. Bodian method is less powerful in detecting SPs , especially diffuse deposits [54, 56, 67, 127, 128]. Gallyas, Cross [56, 67] and Garvey [27] methods are least powerful in detecting SPs.
Fig. 2Density of NFTs (a: upper panel) and that of SPs (b: lower panel) as a function of intellectual status evaluated by the Blessed Test Score. Both are dependent on the staining methods. Reproduced with permission [56]
It is needless to say that a more sensitive detection is preferable in order to identify specific deposits in more detail with ease. However, if one deals with clinicopathological correlation of a disease such as AD, where symptoms and deposits are more or less quantifiable under the background of normal aging, it is possible to identify a threshold of the deposits with any of the methods that may correlate with a threshold of the cognitive decline [118]. For this purpose, stability and reproducibility of the method are preferable [31–33, 54, 79–82]. Reliable agreement between the conventional Braak staging of neurofibrillary pathology based on Gallyas method [17] and the revised protocol based on AT8 immunohistochemistry [18] is a good example. On the other hand, if one is looking for specific pathological deposits, such as Pick bodies, GCIs or astrocytic plaques not observed in normal aging [118], specificity is the primary concern for the staining method. Some of these comparative studies included immunohistochemistry for tau [31, 56, 99], Aβ [27, 54, 56, 99, 124, 127], ubiquitin [56] or neurofilament [24] for comparison with silver-staining methods. Some studies with highly sensitive Aβ immunohistochemistry demonstrated that it visualizes a larger number of deposits than with conventional silver-staining methods [1, 56]. This suggests that amorphous Aβ deposition, not identified by conventional silver-staining methods, represents the earliest stage of Aβ deposition [59, 95, 129]. On the other hand, it is noteworthy that none of the series found any quantitative superiority of immunohistochemistry in identifying NFTs over the best-possible silver-staining method. For example, it is rather frequent that pathological structures in the form of NFT do not exhibit tau-like immunoreactivity any more, while most of them are stained by some silver-staining methods or fluorochromes [116]. This is explained if some tau epitopes in NFTs are truncated out during their evolution from pretangle to extracellular NFTs [12, 116]. Furthermore, immunohistochemical detection is highly dependent on the antibody and experimental procedures. These technical uncertainties make it difficult to place immunohistochemical detection as a standard for neuropathological diagnosis, especially when the quantity of deposits is of primary importance as with the histological diagnostic criteria for AD [32, 79]. It is needless to say that the molecular specificity of immunohistochemistry is a potentially reliable and logical way in order to sort different diseases under the diagnostic flags of molecules. This highly specific detection by pinpointing a single target molecule is, however, “a very restricted, monochromic view” as pointed out by Switzer [108]”. Identification of the disease-specific molecular species and their reliable probes applicable to histological sections are desirable. Even if this kind of probes are available, immunohistochemical procedures are also difficult to standardize and much more costly than most of silver-staining methods [67]. Immunohistochemistry is highly costly especially when staining large histological samples, for example, hemispheric sections of human brain. Moreover, penetration of antibodies is sometimes not sufficient when staining thick histological sections. Silver-staining methods are more readily applicable even to large and thick histological sections [13]. It is of note as well that archival materials preserved for a long period, especially in formaldehyde, sometimes fail to exhibit immunoreactivity. It is frequently possible to visualize the lesions with silver-staining methods even in these archival materials [13, 123].
Qualitative representation by silver-staining methods
As summarized in the previous section, differences in the quantity of argyrophilic AD lesions are dependent on the silver-staining method. As has been considered frequently, it may be “the sensitivity” of a staining method that governs the quantity of the argyrophilic lesions. This is probably in agreement with our general impression that “modified Bielschowsky method is more powerful than Bodian method in detecting AD lesions.” [131] This interpretation is based on the assumption that so-called “argyrophilia” is a homogeneous phenomenon regardless of the staining method and the nature of lesion, which is not the case. For example, Gallyas method is one of the most “sensitive” methods in detecting NFTs and neuropil threads in AD, while it is far less “sensitive” in detecting SPs. This empirical but evident discrepancy is not readily explained by a mere difference in the “sensitivity” of the methods and rather indicates that argyrophilic properties are dependent not only on the staining methods but also on the target lesions. Careful neuropathologists have been aware of these “method- and lesion-dependent natures” of various argyrophilic deposits in neurodegenerative diseases [16, 85, 96]. Table 2 provides a summary of the heterogeneity of argyrophilia according to the staining method and the lesion. Gallyas method is one of the most “sensitive” silver-staining method that clearly labels NFTs of AD, as well as neuronal and glial lesions of corticobasal degeneration/progressive supranuclear palsy (CBD/PSP) [113, 120]. This “sensitive” method, however, fails to stain Pick bodies, another distinct tau-positive deposit [16, 85, 96, 117, 118]. On the other hand, Campbell–Switzer method labels Pick bodies but not CBD/PSP-related lesions [120, 122]. This reversed and complementary profile with Gallyas and Campbell–Switzer methods is hardly explained by a superior sensitivity of Gallyas method over Campbell–Switzer method or vise versa. These discrepancies between Gallyas and Campbell–Switzer methods are readily demonstrable if mirror section pairs are initially fluorolabeled and one of the section pair is subsequently stained with Gallyas and the counterpart with Campbell–Switzer method, as shown in Fig. 3. It is now known that molecular species of tau are different according to the disease-specific lesions. Namely, Pick bodies are usually composed of three-repeat (3R) tau, while CBD/PSP-related tau pathologies are positive for four-repeat (4R) tau [28]. Because Pick bodies are usually positive with Campbell–Switzer method but negative with Gallyas method (Fig. 3a–d), one of the possible interpretations is that 3R tau deposits, such as Pick bodies, exhibit argyrophilia with Campbell–Switzer but not with Gallyas method. In contrast, the lesions of 4R tauopathy (CBD/PSP and argyrophilic grain disease [110, 111]) are related to the argyrophilia with Gallyas but not with Campbell–Switzer method (Fig. 3i–l) [120]. Indeed, NFTs of AD, Down syndrome, and diffuse neurofibrillary tangles with calcification, all containing both 3R and 4R isoforms, similarly exhibit argyrophilia with either method (Fig. 3e–h) [120–122]. It is interesting that Gallyas-positive Pick-like bodies were found to be positive for 4R tau but sparsely positive for 3R tau [132], which corroborates the empirical correlation between 4R tau and Gallyas method. Moreover, in one of the largest studies on sporadic and familial frontotemporal degeneration with tau-positive deposits, “Pick bodies” defined as “inclusions stained by Bielschowsky but not by Gallyas” were found in 17 cases. All exhibited the immunoreactivity with a 3R-tau specific antibody as well as 3R tau predominance in tau biochemistry except for a single case with E342V mutation in the tau gene [29]. This particular case is characterized by diffuse cytoplasmic tau staining in neurons and PHF-like electron microscopic features [72], both of which are not typical of Pick bodies. In another sporadic case, Bodian-positive inclusions positive for 4R but not 3R tau have electron microscopic features with twisted ribbon distinct from those of typical Pick bodies [83]. These apparent heterogeneities necessitate a revision of the classical definition of Pick bodies “round argyrophilic, tau-positive inclusions in neuronal cytoplasm”, because argyrophilic features and tau-species are both heterogeneous in this classical category. Among argryophilic cytoplasmic inclusions in neuron, it seems that those positive for 3R-tau [84] and those negative with Gallyas method [29] are correlated to form a cluster to be defined as Pick bodies. Argyrophilia with the Campbell–Switzer method [122] is helpful for more precise and straightforward definition of Pick body. In contrast with Gallyas and Campbell–Switzer methods, argryrophilia with Bielschowsky and Bodian methods are less dependent on the target lesions (Table 2). It is noteworthy that tau-positive cortical lesions of PSP are silver-stained with Bodian method [55], while those of CBD frequently fail to exhibit argyrophilia with Bodian method [69, 109, 115] especially in the cerebral cortices. Although possible biochemical distinctions of tau between CBD and PSP have been described [3, 104], it remains to be clarified how these biochemical differences are related to these morphological differences between CBD and PSP. Another possible limitation is related to the observations that the expression of tau-isoforms is not homogeneous throughout the brain [51, 126]. Because most of the data on tau-positive deposits summarized in Table 2 and Fig. 3 have been obtained on cerebral cortices, it remains to be determined whether these findings are similarly applicable to extracortical areas such as the brainstem. Yoshida [132] demonstrated that 3R tau immunoreactivity is represented in the brainstem even in so-called 4R tauopathies such as CBD/PSP. Because neurofibrillary pathologies related to AD or aging are sometimes found in the brainstem nuclei and usually positive for both 3R and 4R isoforms, it remains to be clarified whether the presence of 3R tau in brainstem nuclei in CBD/PSP is ascribed to tauopathies independent of normal or accelerated aging.
Table 2Heterogeneity of argyrophilia according to disease and methodStaining methodsCampbell–SwitzerBodianBielschowskyGallyasPick bodies [16, 85, 96, 117, 118]++++++−AD-NFTs++++++++Down-NFTs++++++++DNTC-NFTs++++++++PSP/CBD-neurons−+a+b++PSP/CBD-glia−±c+b++Argyrophilic grains [14, 77, 112]-++++AD-SPs+++++++AD-diffuse deposits++-±-Lewy bodies [75, 119]+++++-GCIs [87, 90]++++++AD Alzheimer’s disease, NFTs neurofibrillary tangles, DNTC diffuse NFTs with calcification, PSP progressive supranuclear palsy, CBD corticobasal degeneration, SP senile plaques, GCIs glial cytoplasmic inclusions, ++: easily recognizable, +: positive, ±: questionably positive, −: negativeaPositive in PSP cases [55] but less evident in CBD cases [67, 109, 115]bNot consistent[97], positive in some cases, not appropriate for histological examinationcThe argyrophilia (Bodian) is less evident in glia than in neurons, especially in CBD casesFig. 3Silver-staining profiles of various deposits in neurodegenerative diseases compared with immunofluoresence images. (a–d Pick bodies[122]; e–h neurofibrillary tangles; i–l argyrophilic grains [121]; m–p glial cytoplasmic inclusions; q–t Lewy bodies [119]). Mirror section pairs were initially multi-fluorolabeled for PHF-tau (AT8, green for b, c, f, g, j, k) or for α-synuclein (green for n, o, r, s), with thiazin red (red, fluorochrome that labels fibrillary structure such as neurofibrillary tangles for b, c, f, g, j, k, n, o, r, s) and for ubiquitin (blue for f, g, j, k, n, o, r, s). After recording the fluorescent images from the same area of the mirror section pairs, one of the section pair was stained with Campbell–Switzer (CS: a, e, i, m, q) and the other with Gallyas (GAL: d, h, l, p, t) method. Fluorescent images and silver staining profiles of the same area from the mirror section pair were compared. Pick bodies (a–d) in the pyramidal layer of hippocampus are stained with CS (a) but not with GAL (d) [122]. Neurofibrillary tangles (NFTs, e–h) from the frontal cortex of diffuse neurofibrillary tangles with calcification are stained with both CS (e) and GAL (h) [121]. This staining profile is shared with NFTs of AD [122]. Argyrophilic grains (i–l, in the square) and tau-positive neurons are present (j, k) in the parahippocampal gyrus. The tau-positive neuron (double asterisk) and neuropil thread (arrow) stained with both CS and GAL are similar to those observed in DNTC (e–h), representing neurofibrillary pathology. Grains and pretangle neuron (single asterisk) are stained with GAL (l) but not with CS (i) in contrast. This staining profile is shared with cortical lesion of corticobasal degeneration/progressive supranuclear palsy [120]. Glial cytoplasmic inclusions (m–p) in the putamen, positive for α-synuclein (n, o), are stained with both CS (m) and GAL (p) [119]. Lewy bodies (arrowhead, q–t) and Lewy neurites (arrow, q–t) in the dorsal motor nucleus of vagus, positive for α-synuclein (r, s), are stained with CS (q) but not with GAL (t) [119]. The same blood vessel is indicated with asterisk (q–t). Bar  50 μm (a–d, m–p, q–t), bar  30 μm (e–l). Reproduced with permission. a–d [122]; e–l [121]; m–t [119]
Interestingly, argyrophilic differentiation with Gallyas and Campbell–Switzer methods is not limited to tau-positive deposits. Both of the two staining methods label GCIs of MSA (Fig. 3m–p) [119]. Although GCIs are positive for some restricted tau epitopes [106], their major constituent is now considered to be α-synuclein. Indeed, Campbell–Switzer method clearly labels Lewy bodies and their neurites, another representative deposits of α-synuclein [13]. It is interesting that Gallyas method fails to label Lewy bodies and Lewy neurites (Fig. 3q–t). Therefore, these two silver-staining methods, when performed in parallel, provide staining profiles that may allow qualitative distinction between GCIs and Lewy bodies [119]. Because no biochemical markers that enable distinction between Lewy bodies and GCIs are not readily available, these differential profiles in silver staining provides an additional qualitative distinction possibly linked to underlying molecular differences.
At present, these empirical distinctions are useful for diagnostic differentiation but await further explanations preferably at molecular or ultrastructural level. These distinctive features of silver-staining profiles may be explained if (1) molecular species specific to each subcategory (for example, 3R vs. 4R tau or LBs vs. GCIs) has different affinity to each silver-staining method; (2) some disease-specific ultrastructures of pathological deposit are related to each staining profile; (3) other independent molecules in these deposits play some roles in characterizing the pathological deposits so that distinctive silver-staining profiles are engendered; or (4) some disease-specific conformational changes are related to a silver-staining profile specific to a disease. Because Campbell–Switzer and Gallyas methods are useful in discriminating not only tau-positive deposits but also α-synuclein-positive deposits even with their molecular and ultrastructural diversities, it is plausible that factors such as (3) or (4) shared by tau-positive deposits and synuclein-positive deposits may play roles, at least partly, in these discriminating abilities with these silver-staining methods.
Conclusions and perspective
Even with my limited experience on these silver-staining methods, it is apparent that the argyrophilic profiles are dependent not only on the silver-staining method but also on the target lesion. This indicates that “argyrophilia” is highly heterogeneous. However, this heterogeneity is not capricious but rather representative of some disease-specific aspects. The apparent empirical correlations between the argyrophilic profiles and the disease-specific pathological structures are useful in sorting these neurodegenerative disorders. Combined silver-staining profiles, for example with Gallyas and Campbell–Switzer methods, are more convenient than modified Bielschowsky and Bodian method for sorting different disease-specific lesions. Although these staining profiles are discussed in this review as if disease-specific, more detailed comparison at cellular level demonstrated that they can be different from cell to cell even in the same section (intercellular differences) [117]. Furthermore, staining profiles are heterogeneous even in a single cell (intracellular differences) [49]. These silver-staining and immunohistochemical profiles are much more informative especially when combined together than when used in isolation because such combination is not a mere summation of the results obtained with different methods. It will rather provide a more stereoscopic and multi-faceted view, which may allow more precise morphological delineation of disease-specific deposits. In my view, comments such as “Silver-staining methods are unreliable, nonreproducible, nonscientific and out-dated.” are now out-dated. Awareness of their utility and reliability of silver-staining methods, different from immunohistochemistry, may facilitate diagnosis and accelerate research, which hopefully clarifies molecular basis of each silver-staining method and improves our understanding of diseases.

gallyas
campbell–switzer
argyrophilia
senile plaques
methenamine
tauopathies
synucleinopathies


Progressive vertebral deformities despite unchanged bone mineral density in patients with sarcoidosis: a 4-year follow-up study

Summary To evaluate the incidence of new and/or progressive vertebral deformities and changes in bone mineral density, we re-examined 66 patients with sarcoidosis after a follow-up period of four years. In 17 subjects (26%) new and/or progressive vertebral deformities were found, though BMD did not change significantly.
Introduction
Sarcoidosis is a T-cell driven chronic inflammatory disease. Although chronic inflammation has been associated with decreased bone mineral density as a result of the effects of cytokines on bone metabolism [1–4], we and others could not demonstrate changes in BMD in subjects with this condition, even if treated with glucocorticoids (GCs). In a cross-sectional study of 124 subjects with sarcoidosis, BMD values similar to an age- and sex-matched reference population were found [5]. Comparable observations were made in three small studies in untreated patients [6–8]. These studies also found a normal BMD relative to age and sex-matched controls, except for a small group of postmenopausal women in which BMD was moderately decreased at the spine in longstanding sarcoidosis only [7].
Although in our cross-sectional study normal BMD values were observed, increased levels of the bone resorption marker serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) and the bone formation marker serum procollagen type I amino-terminal propeptide (PINP) suggestive of increased bone turnover were found [5]. ICTP levels correlated with markers of disease activity such as soluble IL-2 receptor (sIL2R) and angiotensin converting enzyme (ACE). In addition, vertebral deformities suggestive of fracture were demonstrated in 20% of the subjects studied in this series. This may imply that the fracture risk in sarcoidosis is increased due to an increased bone turnover with consequent changes in microarchitecture and decrease of bone strength which is not reflected by changes in BMD [9, 10].
If so, this may result in progressive vertebral deformities during the course of the disease. For this reason we re-examined individuals with sarcoidosis four years after the initial measurements to determine the incidence of new and/or progressive vertebral deformities and their relation with changes in BMD.
Subjects and methods
Subjects
Sixty-six of the 124 subjects with sarcoidosis that were studied in 2002 [5] agreed to participate in the follow-up study performed in 2006. None of the 56 subjects who declined or were unable to participate had impaired mobility or a history of vertebral fractures. The mean age of this group was 45 years and did not differ with respect to gender or glucocorticoid (GC) use from the group of subjects that were re-examined in 2006.
Demographic, clinical and treatment data of the subjects studied in 2002 and 2006 are summarized in Table 1. The group consisted of 22 pre-menopausal women, 11 post-menopausal women, and 33 men; median age of the total group (all Caucasian) was 43 years (20–66 y). The clinical records of all patients were reviewed. In 2002 patients were evaluated according to a standard protocol that included questionnaires, measurement of height and weight, lung function, measurement of BMD, a single energy densitometry of the spine, and laboratory evaluation [5]. In 2006 the same protocol was repeated. Informed consent was obtained from all participants and the study was approved by the medical ethics committee of our institution.
Table 1Baseline and follow-up demographic, clinical, and treatment variables (n = 66)VariableBaseline (n=66)Follow-up (n=66)P*Demographic variablesFemale sex33 (50%)Postmenopausal11 (17%)14 (21%)nsAge, years43 (20–66)Body mass index, kg/m226.9 ± 5.727.2 ± 5.3nsSmoking7 (11%)Daily dietary calcium intake, mg740 (110–2360)758 (150–1340)nsClinical variablesDisease duration, years3 (1–22)7 (5–26)Chest X-ray stage (0-I-II-III-IV)24/11/12/16/327/5/12/15/7nsFEV1,% of predicted87 ± 2891 ± 26nsDLCO,% of predicted87 ± 1692 ± 18nsPhysical activity8.6 ± 3.78.1 ± 3.7nsLaboratory values (in serum)Calcium, mmol/l (2.1–2.6)2.4 ± 0.12.4 ± 0.08ns1,25(OH)2D3, nmol/l (0.040–0.200)0.14 ± 0.03ACE, U/l (9–25)22.5 ± 9.815.3 ± 7.90.001sIL-2R, kU/l (241–846)654 (188–4315)Hs-CRP, mg/l (<10)3.2 (0.2–191)2.0 (1–16)<0.05Z-score ICTP0.7 ± 1.4Z-score PINP−0.1 ± 0.9Treatment variablesGC use never31 (47)26 (39)nsGC use previous14 (21)25 (38)<0.01GC use current21 (32)15 (23)nsLifetime GC dose, mg9240 (200–48750)11187 (200–56700)<0.001Daily dose, mg12.4 ± 6.210.5 ± 3.3<0.05Started on bisphosphonates after baseline measurement6 (9)Clinical risk factors for osteoporosisFracture2 (2/24 = 8%)5 (5/28 = 18%)nsLow body weight (< 60 kg)8 (12)7 (11)nsSevere immobilization00nsLow physical activity index ≤ 518 (27)18 (27)nsMother with hip fracture6 (9)7 (11)nsData are given as mean ± SD, median (range) or number (%); *= p value between baseline and follow-up measurement. Reference parameters in bracketsAbbreviations: GC, glucocorticoid; FEV1, forced expiratory volume in one second; DLCO, diffusion capacity for carbon monoxide; 1,25(OH)2D3, 1,25 dihydroxyvitamin D; ACE, angiotensin converting enzyme; sIL-2R, soluble interleukin-2 receptor; Hs-CRP, high-sensitivity C-reactive protein; ICTP, carboxy-terminal cross-linked telopeptide of type I collagen; PINP, procollagen type I amino-terminal propeptide
Pulmonary evaluation
Lung function measurements, including forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), were measured with a pneumotachograph. The diffusion capacity for carbon monoxide (DLCO) was measured using the single-breath method (both Masterlab, Jaeger, Würzburg, Germany). Values were expressed as a percentage of those predicted [11].
Chest radiographs were graded according to the radiographic staging of DeRemee (0 to III), adding stage IV, the end stage of lung fibrosis [12, 13]. All interpretations were made by a radiologist who was blinded to the patient’s history.
Laboratory assays
Serum 1,25-dihydroxyvitamin D concentration was determined by radioimmuno-assay using a commercially available kit [(IDS Ltd, Boldon, England, interassay coefficient of variation (IE-CV) 18%, intra-assay CV (IA-CV) 15%)]. High-sensitivity C-reactive protein (hs-CRP) was measured by particle-enhanced immunonephelometry on the BN Prospec (Dade Behring). The detection limit is 0.175 mg/L and the measuring range is 0.175–1100 mg/L. Soluble IL-2 receptor (sIL-2R) was determined on the IMMULITE automated analyzer, by means of a two-site chemiluminescent enzyme immunometric assay with a measuring range of 50–7500 kU/L (Diagnostic Product Corporation, Los Angeles, CA, cat no LKIP1). Serum angiotensin converting enzyme (ACE) was measured using a colorimetric method. The precision of the ACE assay was < 5.6% and the reference interval for ACE was 9–25 U/L.
As a marker for bone formation, serum procollagen type I amino-terminal propeptide (PINP) was measured. As a marker for bone resorption, serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) was assessed. Both PINP (IE-CV 3.2%, IA-CV 2.5%, lowest detectable concentration 0.4 μg/l) and ICTP (IE-CV 3.5%, IA-CV 2.3%, lowest detectable concentration <0.1 μg/l) were measured using commercial RIA kits (Orion Diagnostica Oy, Espoo, Finland). To adjust for age and gender Z-scores for these bone markers were obtained using a Dutch reference group (300 women, 150 men), checked for normal BMD of the lumbar spine and femur and normal 25-hydroxyvitamin-D levels [14, 15].
Bone mineral density and vertebral morphometry
Bone mineral density (BMD) was measured by dual X-ray absorptiometry (DXA, Hologic QDR 4500). In 2002 only the BMD of the hip was measured. In 2006 the BMD of both the hip and of the lumbar spine were determined. As reference group for the hip the NHANES III database (sex- and age-matched) was used. A standard protocol as described previously was used for measurement of BMD. To adjust for age and gender, Z-scores were used. To examine changes in Z-scores between baseline and follow-up measurements a Δ Z-score was calculated reflecting the difference between the Z-score at follow-up and the Z-score at baseline.
Furthermore, after bone density measurement a lateral single energy densitometry of the thoracic and lumbar spine for vertebral fracture assessment (VFA) was performed (also called Morphometric X-ray absorptiometry (MXA)) [16]. The scans obtained were analyzed twice by one trained operator (intra-observer correlation: 0.85), using the semi-quantitative method of Genant [17]. In addition we measured every vertebra quantitatively. The observer was blinded to the T-score values and to the values of the first set of measurements. After visual examination six points were placed on each vertebral body from T4 to L4. From these points three vertebral heights were measured anterior (Ha), mid (Hm) and posterior (Hp). On the basis of the average score of these morphometric measurements, ratios were calculated and a prevalent vertebral deformity was defined as a reduction of height of 20% or more (Ha/Hp; Hm/Hp and Hp/Hp below). Severity of deformities was assessed using the scoring system of Genant [17]. A score of ‘0’ was assigned to normal, non-fractured vertebra; ‘1’ for a mild deformity (20–25% reduction in anterior, middle or posterior vertebral height); ‘2’ for a moderate deformity (25–40% reduction) and ‘3’ for a severe deformity (>40% reduction). A new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade ≥ 1) and a progressive deformity if the grade increased [17].
Questionnaires
Calcium intake of all patients was scored in 2002 as well as 2006 on the basis of a detailed dietary list. Known clinical risk factors for osteoporosis (weight below 60 kg, mother with hip fracture, history of fractures after age 50, menopausal status and severe immobilization) as well as daily activities and exercise were assessed by a validated questionnaire [18], in which sports, daily and work activities are scored with a minimum of zero and a maximum of eighteen. GC therapy was evaluated by means of a patient questionnaire and verified using all the records of the patient’s pharmacist. It was scored as never, previous or current use and if subjects were currently using GCs, the daily dose was noted.
Statistics
Student t-tests, chi-square tests, and one-way ANOVAs were used, depending on the variables and subgroups tested. Depending on the analysis, change scores or actual scores were used. Patients with new and/or progressive vertebral deformity were clustered for the multivariate and the receiver-operating characteristics (ROC) analysis. Multivariate logistic regression analyses was performed to assess the strength of association between the incidence of new and/or progressive vertebral deformities and gender, age, weight, clinical risk factors, GC use, lifetime GC dose, daily GC dose, disease activity, bone markers, calcium intake, physical activity and BMD measurements. The variables that were entered in the multivariable analysis were those variables that appeared related (p < 0.10) to this outcome measure in univariate analyses. Odds ratio (OR) and its 95% confidence intervals (CI) were calculated by using SPSS version 12.0. ROC analysis was used to assess the ability of various levels of the T-score femoral neck to predict the incidence of a new and/or progressive vertebral deformity. The ROC curve indicates the probability of a true-positive result as a function of the probability of a false-positive result for all possible threshold values [19]. A p value < 0.05 was considered statistically significant.
Results
Bone mineral density and bone turnover parameters
The results of BMD measurements are shown in Table 2. The BMD of the total group remained unchanged after a median follow-up of 45 months (range 35–49 months). When stratifying patients according to GC use, no decrease in each of the subgroups was found. Patients that never used GCs showed a Δ Z-score of the femoral neck (FN) of 0.03 ± 0.36 and a Δ Z-score of the trochanter of −0.08 ± 0.37. In patients with previous use of GCs these Δ Z-scores were 0.10 ± 0.36 and 0.22 ± 0.43, respectively. Even the group currently on GCs revealed no decrease of Z-score (Δ Z-score FN: 0.06 ± 0.30 and Δ Z-score trochanter: 0.00 ± 0.18) and also the subgroup of postmenopausal women (n = 11) did not show significant bone loss (Δ Z-score FN: 0.06 ± 0.45 and Δ Z-score trochanter: −0.05 ± 0.52). In the total group, bone turnover parameters at baseline showed an increased Z-score of ICTP compared to norm scores (0.7, 95% confidence interval (CI):0.4–1.1; p < 0.001). on the other hand, the marker of bone formation (Z-score PINP) did not differ from the reference population.
Table 2BMD variables at baseline and follow-up for the total group (n = 66, median follow-up duration 45 months (range 35–49 months)VariableBaseline measurementFollow-up measurementP*BMD, mean ± SD gm/cm2Femoral neck0.84 ± 0.120.83 ± 0.12nsTrochanter0.74 ± 0.130.74 ± 0.12nsTotal hip0.97 ± 0.130.98 ± 0.14nsLumbar spine1.04 ± 0.14Z-score, mean ± SDFemoral neck0.17 ± 1.00.23 ± 1.1nsTrochanter0.27 ± 1.10.32 ± 1.1nsTotal hip0.18 ± 1.00.32 ± 1.00.001Lumbar spine0.03 ± 1.4T-score, mean ± SDFemoral neck−0.42 ± 1.0−0.46 ± 1.0nsTrochanter−0.02 ± 1.1−0.03 ± 1.0nsTotal hip−0.14 ± 1.0−0.10 ± 1.0nsLumbar spine−0.50 ± 1.3Abbreviations: BMD, bone mineral densityP* = p value between baseline and follow-up measurement
Clinical fractures and vertebral deformities
Three new non-vertebral fractures occurred during the follow-up period. These included a hip fracture (twice in the same patient), an ankle fracture and a fracture of the thumb. All these fractures were related to trauma and occurred in subjects older than 50 years.
Morphometric data are summarized in Table 3. In 2002 vertebral deformities (ratio of <0.80) were found in 19 vertebrae of 13 subjects. Seventeen of these were wedge and two biconcave deformities. No crush deformities were seen. The majority of these deformities were found in the low thoracic region. At follow-up a new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade ≥ 1) and a progressive deformity if the grade increased [17]. With this method, 36 vertebral deformities were found in 21 subjects. In one subject a vertebral deformity (ratio 0.78 of T11) found in 2002 was not found at follow-up (ratio 0.81). So, in total nine new subjects revealed one or more vertebral deformities, which means an increase of vertebral deformities from 20 to 32% of the subjects studied (p < 0.05). From the 21 subjects with a vertebral deformity in 2006, 17 subjects (26% of total group) were diagnosed with one or more new or progressive vertebral deformities and in four subjects the deformity was unchanged compared to baseline. Data on number and severity of the deformities can be found in Table 3. Six patients were started on a bisphosphonates after baseline measurement and from these six patients, two had a new or progressive vertebral deformity at follow-up.
Table 3Number and grade of deformitiesBaselineFollow-upNo. of subjects with deformity13 (20%)21 (32%)*No. of deformities Mild1728 Moderate28 Severe00Total1936*P < 0.05 between number of subjects with deformity at baseline and follow-up
Comparing the groups with and without new or progressive vertebral deformities at follow-up, no differences in Δ Z-scores of BMD of the trochanter or femoral neck (FN) were found (Δ Z-score trochanter −0.02 ± 0.41 and 0.08 ± 0.38 respectively and Δ Z-score FN 0.01 ± 0.32 and 0.08 ± 0.35). In addition no differences in baseline Z-scores of ICTP and PINP were seen between these groups. Multivariable logistic regression analysis, including factors that correlated in the univariate analysis, revealed that a T-score of the femoral neck at baseline (OR per 1 SD T-score reduction = 2.5 (CI: 1.0–5.9), p = 0.04), and a mother with a hip fracture (OR = 14.1 (CI:1.4–142,6), p = 0.02) were determinants of a new and/or progressive morphometric vertebral deformity at follow-up measurement. Factors such as age, gender, calcium in take, GC use, daily GC dose, lifetime GC dose, disease activity, bone markers, radiographic stage and disease duration at baseline did not predict new and/or progressive vertebral deformities.
The threshold level of the T-score FN that maximized the combined specificity and sensitivity on the ROC curve (Fig. 1) was < −0.45 for predicting a new and/or progressive deformity (sensitivity 88%, specificity 51%).
Fig. 1ROC curve using Femoral neck T-score to identify patients with new and/or progressive vertebral deformity. Area under the curve (AUC): 0.72. Arrow: The threshold level of T-score FN that maximized combined specificity and sensitivity was < −0.45 (sensitivity 88%, specificity 51%)
Discussion
In this cohort of subjects with sarcoidosis, a high prevalence of morphometric vertebral deformities suggestive of fracture was found, as well as a substantial increase in vertebral deformities during a follow-up period of four years. In 2002 20% of subjects were diagnosed with vertebral deformities according to the criteria of Genant [17], which increased to 32% of all subjects in 2006. In parallel, the total number of deformities in these subjects almost doubled. However, BMD of the trochanter and femoral neck did not change over time and BMD of the lumbar spine at follow-up measurement did not differ from the reference population. These data are suggestive of an increased risk of progressive vertebral deformities in individuals with sarcoidosis despite preservation of BMD.
Although data on prevalent or incident fractures in younger healthy populations are lacking, data from other studies suggest that the incidence and prevalence of vertebral deformities in this population are indeed high. Prevalence rates of 30% asymptomatic vertebral fractures are demonstrated in elderly post-menopausal women on chronic GC therapy using the same techniques [20]. In a previous study in 60 subjects (mean age 49 ± 13 years) with differentiated thyroid carcinoma we found vertebral deformities in 7% of patients [21]. Data from the European Vertebral Osteoporosis Study (EVOS), a very large cross-sectional population based study on European subjects aged 50 to 79 years, showed a prevalence of vertebral deformities of 12% (range 6–21%) in males and females [22]. In the Rotterdam study, in which 3469 men and women aged 55 years and older were studied, the prevalence of vertebral deformity suggestive of fracture was 6.9% in men and 7.5% in women [23]. The epidemiology of vertebral fractures in women aged 50–54 years turned out to vary in different countries from 4.7% – 11.5% [24]. All these studies indicate that the fracture risk in subjects with sarcoidosis is substantial, regardless the differences in populations studied and differences in methodology.
A new vertebral deformity was found in 15 subjects (23%). To identify incident deformities several approaches can be followed. Measurement of changes in vertebral heights of the same vertebral body from a baseline to a later radiograph in which a decrease in height of 15 or 20% or 4 mm is suggestive of fracture [25, 26], changes in indices of vertebral area [27] or changes in the number or presence of prevalent deformities [17, 28]. Black and coworkers evaluated these different approaches and concluded that none of these were consistently better than any other method [29]. As we aimed to assess the change of numbers of subjects with one or more vertebral deformities over time we used the last method [17], in which changes in number of prevalent deformities are scored. A comparable approach was followed in the European Prospective Osteoporosis Study (EPOS) [30], which revealed an incidence of new deformities of 3.4% after a similar follow-up period. As the mean age of subjects included in this study was substantially higher than that of our cohort, these data cannot be used as a reference, although it is likely that in younger age groups even lower incident deformities would be observed. The high prevalence of vertebral deformities at baseline, the significant increase of more than 50% of subjects after follow-up with one or more deformity and the increase of severity of prevalent deformities all imply that sarcoidosis is a relevant risk factor for vertebral deformity.
What is the underlying mechanism of this predisposition to vertebral deformities in view of the lack of effects on BMD in sarcoidosis? The load bearing capacity of bone, also referred to as ‘whole bone strength’, depends on the amount of bone, the spatial distribution of the bone mass, and the intrinsic properties of the materials that comprise the bone. Thus, properties at the cellular, matrix, micro- and macro-architectural levels may all impact the mechanical properties of bone [31, 32]. Apparently, in sarcoidosis mechanisms are involved that influence bone strength without having a significant impact on bone mass. As we found in the total group an increased marker of bone resorption (Z-score ICTP) at baseline, one of the possible mechanisms could be increased bone remodelling with a negative effect on bone micro-architecture that is not reflected by a change in BMD. It is well known that chronic inflammatory diseases influence bone physiology by the production of cytokines stimulating bone turnover [1, 2, 33]. Increased bone remodelling is associated with an increased bone fragility and thus fracture risk [34, 35] and in postmenopausal women the level of bone turnover turned out to be an as strong and independent predictor of fractures as BMD [36, 37]. These data may support the hypothesis that the chronic inflammatory state in sarcoidosis results in increased bone remodelling with a negative effect on bone strength and thus an increased fracture risk.
No changes in BMD in the group currently treated with GCs were found. This is unexpected as GCs are known to effect BMD via several mechanisms with consequent decrease of BMD. It may well be that this is due to intermittent GC use, as most of our patients were on intermittent glucocorticoids. Other studies have demonstrated that intermittent GC use has no major effects on BMD [38]. A recent large retrospective cohort study on clinical fracture risk among patients from the UK General Practice Research Database showed that intermittent use of high dose of oral GCs was associated with only a small increase in the risk of osteoporotic fractures [39].
Despite the on average normal BMD, we found T-score of the femoral neck and a family history of hip fractures to be predictors of a new and/or progressive vertebral deformity. This suggests that the combination of a lower BMD in combination with the increased bone turnover in sarcoidosis predisposes to progressive vertebral deformity. If so, this would mean that in these high risk individuals preventive treatment should be considered to reduce fracture risk. Controlled trials are needed, however, to substantiate this suggestion.
One of the limitations of our study is the lack of an age and sex matched control population. Unfortunately data on vertebral deformities in younger populations are at present not available. The aim of this study was, however, not to compare sarcoidosis patients with healthy subjects, but to follow a cohort of these patients and to compare follow-up with baseline measurements. Another limitation is the use of morphometric X-ray absorptiometry (MXA) instead of spine radiographs. MXA is less reliable for the detection of deformities at the upper thoracic spine, where deformities are less frequent as compared to the lumbar and mid-thoracic spine. A recent study comparing MXA with lateral spine X-ray found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine, where vertebral fractures are most common [40]. The advantage of MXA is the low dose of radiation and the convenience of the technique for patients. The present quality of the images, with ongoing refinement of this technology, is considered sufficient to be used for the diagnosis of vertebral deformity consistent with fracture [41]. Furthermore there is a lack of a “gold standard” for VFA. We followed the method of Genant [17], which is based on a reduction of the ratios of anterior, middle or posterior heights and all measurements were performed twice to improve accuracy. This is the simplest and most practical method [42] and an association with future fracture risk is documented [43, 44]. The above mentioned EVOS study, however, applied the methodology described by McCloskey and Eastell and co-workers in which measurements are corrected for normal variations in vertebral shape [25]. Relative to the method of Genant, the method of Eastell [25] or McCloskey [28] may have resulted in lower prevalences of vertebral deformities. This does not, however, explain the differences in prevalence of vertebral deformities reported elsewhere and in this paper. The restrictions of the methodology are also the limited ability to provide a differential diagnosis for the detected deformities, a lower sensitivity for milder fractures and the inability to evaluate the uppermost thoracic levels. Other disorders that may cause changes in vertebral shape involve congenital abnormalities and conditions as severe osteoarthritis [45] and Scheuermann’s disease. We have, however, no indications that these relatively rare conditions may have interfered with our observations.
In conclusion, we have shown that in subjects with sarcoidosis the number of vertebral deformities, diagnosed with morphometric assessment, increases during the course of this disease despite preservation of BMD. Although this is an uncontrolled study, it appears that subjects with sarcoidosis have an increased fracture risk, even if BMD is normal. High risk individuals can be identified by a low-normal BMD and by a family history of hip fractures. Probably these individuals will benefit from therapies that increase bone strength. A T-score FN below −0.45 may be used to identify these individuals with a high sensitivity and an acceptable specificity. Studies evaluating the effects of such therapies in individuals with sarcoidosis are however clearly needed.

vertebral deformities
bone mineral density
sarcoidosis