Purinergic junctional transmission and propagation of calcium waves in cultured spinal cord microglial networks

In order to elucidate the mechanisms of purinergic transmission of calcium (Ca2 + ) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca2 + wave propagation. Microglia were confined to lanes 20–100 wide and Ca2 + waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 . The number of cells participating in propagation also decreased over this distance. Ca2 + waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 , indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca2 + wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y2 and P2Y12 at relatively low concentrations. Antibodies to P2Y12 showed these at very high density compared with P2Y2, indicating a role for P2Y12 receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.
Introduction
Microglia give a calcium (Ca2 + ) transient in mixed cultures of microglia and astrocytes, following mechanical stimulation of a single astrocyte [1, 2]. This transient in the microglia is dependent on the release of ATP by the astrocytes [1]. However, nothing is known about the mechanism of transmission of a Ca2 +  signal between microglial cells that allows for propagation of a Ca2 +  wave in populations of microglia [3]. In the absence of neurons, a Ca2 +  wave in astrocytes propagates for hundreds of microns from a point of mechanical stimulation (e.g., [4, 5]). It remains to be seen whether a Ca2 +  signal can be transmitted between microglia in such a way that there is propagation of a Ca2 +  wave.
The Ca2 +  waves in astrocytes, when initiated at a point in a culture, can jump cell-free gaps of different widths formed by scraping away cells [4, 6]. Such Ca2 +  waves propagate across these gaps with a delay that increases with gap width until this width reaches about 150 , when such propagation fails. More recent research has used micropatterned arrays of astrocytes in which lanes of cells about 110  wide alternate with cell-free lanes about 40  wide [4, 5]. If a Ca2 +  wave is initiated by mechanical stimulation in an astrocyte in just one lane it propagates both along the lane as well as transversely across the cell-free lanes into the adjacent astrocyte lanes with a delay of about 10 s. These observations support the idea that the transmission of Ca2 +  waves between astrocytes involves the release of a diffusible substance. The question of whether this wave is transmitted between microglia by a diffusible substance has not been addressed. It is known that microglia collecting in the vicinity of a stab wound express connexin CX43 and that these cells are coupled by such connexins in vitro under the control of certain cytokines [7]. However, given the relative low density of resting microglia in the brain compared to that of astrocytes [8–10], it seems unlikely that connexins are used by microglia in normal circumstances to transmit Ca2 +  waves. We have therefore used micropatterned arrays of microglia to ascertain if the Ca2 +  wave can propagate across cell-free regions, thus indicating that a diffusible substance is involved in transmission. As shown below, this turns out to be the case.
Microglia possess P2X7, P2Y1, P2Y2/4, P2Y6, P2Y12, P2Y13 and P2Y14 purinoceptors [11–14] with P2Y12 receptors and P2X7 receptors unique to microglia, at least in the hippocampus [15]. There is evidence that some microglia cells possess predominantly P2X receptors and others predominantly P2Y receptors [16]. P2X7 receptors on microglia are involved in apoptosis, transcription and microvesicle shedding [17]. Activation of P2X7 receptors can lead to the release of pro-inflammatory cytokines, such as TNFα [18, 19]. P2Y1 and P2Y2/4 receptors are involved in the release of the cytokine IL-10 that acts to markedly reduce the release of the pro-inflammatory cytokines [20]. Most (85%) resting microglia respond to ATP with a Ca2 +  transient [21] as a consequence of an action of ATP on P2X and P2Y receptors [11, 14]. The activation of P2X receptors leads to an influx of calcium ions whereas the activation of P2Y receptors releases calcium from internal stores [21–26]. Microglia can also release ATP using in part ATP-binding cassette (ABC) proteins, such as P-glycoproteins (mdr 1a and mdr 1b) and multi-drug resistant associated proteins (mrp1 and mrp4; [27]). Taken together, these observations on the action of ATP on purinergic receptors possessed by resting microglia suggest the hypothesis that the transmission of Ca2 +  waves between microglia is due to ATP, and this we have investigated.
The experimental work is supplemented by calculations using a theoretical model of extracellular communication in cellular networks, originally developed for astrocytes [28], and here modified for microglia.
Methods
The experimental methods for immunohistochemistry, mechanical stimulation of leading to Ca2 +  waves in cells, application of drugs, recording Ca2 +  waves and seeding cells into lanes were the same as that previously described [5]. The purification of microglia started when plated mixed glia culture from Sprague-Dawley rat pup spinal cord formed a confluent monolayer (usually between 1 and 2 weeks). The culture was shaken at 200 rpm for 1 h at 37°C using rotating shaker (IKA-Vibrax-VXR). During shaking, the astrocytes remained adhered to the poly-D-lysine coating whereas the microglia and oligodendrocytes detached from the astrocyte monolayer. Immediately after shaking, the medium containing the detached cells was transferred to a 15-ml centrifuge tube and centrifuged for 5 min at 500 rpm. The supernatant was discarded and the pellet was resuspended in 1 ml DMEM and triturated. Cell density was adjusted by adding fresh DMEM (typically 1-2 ml) after cell trituration and 300 μL of the cell suspension was pipetted onto each coverslip containing previously prepared microchannels and then incubated for 15 min. Incubating DMEM was replaced with fresh DMEM containing 50% of spinal cord astrocytic conditioned medium (ACM). After 15 min, microglia selectively adhere to the poly-D-lysine coating whereas other cell types that may be present, such as any remaining astrocytes and oligodendrocytes, take a longer period of time to adhere [29]. The purity of the microglial cultures was greater than 98% according to live staining of the cells with the microglial marker FITC-IB4 (Invitrogen). Microglia-plated microchannels were incubated in ACM supplemented DMEM for 48 h before use in experiments (Fig. 1). The medium was removed and replaced daily.
Fig. 1The distribution of microglia, immunostained with anti-CSF-1R, in parallel lanes of width 20  and separation 45 ; the calibration bar is 45
For Ca2 +  recording, the relative fluorescence amplitude (ΔF/F), was calculated using the formula
where F is the fluorescent intensity during the Ca2 +  transient,  is the intensity averaged over the interval immediately before the calcium transient and  is the average fluorescence intensity measured in several cell-free areas. Ca2 +  transients with a maximum ΔF/F value less than 0.3 (being 15% of the largest Ca2 +  transient observed in a lane of microglia) were discounted as being too close to the noise level to be reliable. All experiments were repeated at least three times and values are presented as mean ± s.d. Statistical significance was determined with the use of unpaired t-tests and ANOVA, and P < 0.05 was considered significant. All P2Y receptor antagonists were obtained from TOCRIS.
Mathematical model
A detailed description of a mathematical model of purinergic transmission in glial networks was given in [28] and subsequently applied to experimental results on such transmission between astrocytes [5]. This model has been adapted to the present case of microglial networks. The basic model is the same, so here we give only a brief summary of the main features, highlighting the changes that have been made.
Communication between the model microglia is mediated by ATP diffusing in the extracellular space. This ATP binds reversibly to metabotropic receptors (P2Y) on the surface of cells so that the ratio of bound to total receptors is given by
where [ATP] is the extracellular ATP concentration and KR is the dissociation constant for ATP binding. The usual meaning of KR is the concentration of ATP at which half the total receptors are bound; however, in the present context KR, as well as being a measure of the affinity of receptor types, also reflects additional variables such as spatial variations in receptor density, since ρ is a measure of the effective activity of ATP as a function of space and time. Thus KR is to be interpreted as an effective, rather than an actual, dissociation constant (see the section “Receptors” in [5]).
Each microglial cell is represented by a cube of side 8.3 , and these cubes are arranged in 2D arrays with their centres 25  apart. As explained in [28], this simplified geometry does not model the spatial complexity of a real cell, but is a lumped approximation. The Ca2 +  wave can be initiated either by increasing the IP3 concentration in a single cell, or by applying ATP extracellularly. In the present calculations, a fixed concentration of ATP (typically 20 μM) is applied for an extended time (typically 5 s) to the surface of one model microglial cell. The parameters used are those given in Table 1 of [28], except that the parameter governing the ATP release rate, VATP, has been reduced from 2×10 − 11 to 5×10 − 12μmol  − 2 s − 1 in order to obtain agreement with the experimental observations.
Results
Quantitative characteristics of Ca2 +  wave propagation
The amplitude and velocity of propagating Ca2 +  waves in microglia at different positions along microglia lanes, from a point of mechanical initiation in a microglia, were determined. Figures 2A and B show the extent of propagation from the point of stimulation in two lanes, ~80  and ~40  wide, respectively. The Ca2 +  wave propagates with diminution in amplitude (Fig. 2C), at a velocity of about 5 s − 1, over a distance of at most 120  before becoming undetectable. This velocity is about one-quarter of that for Ca2 +  wave propagation in astrocyte lanes [5]. Quantitation of these observations for four different sets of microglia lanes in four cultures is shown in Fig. 3. Figure 3a shows that there is a continual decrease in amplitude of the Ca2 +  wave over 120  at which distance the amplitude falls below 15% to 20% of the initial amplitude and could no longer be reliably detected. The percentage of microglia cells across a lane that gives a Ca2 +  peak amplitude change that is greater than 15% of the peak amplitude at the site of initiation remains high for about the first 80  and then declines rapidly over the succeeding 40  (Fig. 3b). On the other hand, the peak Ca2 +  amplitude increases rapidly with an increase in the local density of cells that give an observable Ca2 +  transient response (Fig. 3c).
Fig. 2The Ca2 +  wave propagates along a lane of microglia with decreasing amplitude from the point of initiation. A and B show results for two different lanes of microglia, of widths 82±5  and 38±4 , respectively. In each case, (a) shows the site of Ca2 +  initiation by mechanical stimulation with a micropipette and (b) shows the microglia that responded with a Ca2 +  transient (indicated by open circles) after stimulation at the site indicated by the arrow; the calibration bar is 45 . C shows the timecourse of the Ca2 +  wave at different distances along a lane from the site of initiation, as indicated. The mechanical stimulus was applied for 1 s starting at time zeroFig. 3Quantitative characteristics of the Ca2 +  wave for a number of microglia lanes. a shows the average peak amplitude of Ca2 +  deliveries along the lanes before becoming undetectable at about 120  (values normalized to the peak Ca2 +  concentration at the stimulating electrode). b shows the number of microglia that give a Ca2 +  response at different positions along the length of a lane, expressed as a percentage of the total number of Ca2 +  indicator-labeled microglia at that position; this remains high for about 80  and then declines. c shows that the average amplitude of the peak Ca2 +  wave in equal-width segments of a microglia lane increases with the number of microglia that propagate Ca2 +  in the segment. Results in a, b and c are for three different lanes in three different cultures. In a and b the distance is from equal-width segments along a lane (for which the average peak Ca2 +  was calculated for all microglia in the segment) to the site of mechanical initiation of the Ca2 +  wave
The restricted local propagation of the Ca2 +  wave from the point of initiation is emphasized by experiments in which different sites of initiation along a single lane of microglia are determined. As Fig. 2 shows, in each case the Ca2 +  wave propagation is restricted to regions of about 100  diameter around the site of initiation.
Evidence for release and diffusion of ATP during Ca2 +  wave propagation
In order to test for the possibility that mechanically stimulated microglial cells release a diffusible substance, parallel lanes of microglia were constructed, separated by cell-free lanes (see Fig. 1). Tests were then made of the extent to which Ca2 +  waves could propagate across these cell-free lanes of widths 70  or more, independent of the width of the microglia lanes (Figs. 4A and B). No such propagation was observed in 20 experiments. On the other hand, if the cell-free lanes were less than ~60  wide there was always successful propagation of the Ca2 +  wave across the cell-free lanes (Fig. 4C). It appears then that a diffusible substance is released by the excited microglia and is able to initiate Ca2 +  transients in them.
Fig. 4Propagation of a Ca2 +  wave occurs between microglial lanes if these are not separated by distances greater than about 60 . A and B show parallel lanes of microglia in which the lane widths are 75±3  and 25±3 , respectively, separated by cell-free lanes of 165±3  and 70±4 , respectively; the open circles indicate the microglia that gave a Ca2 +  response following mechanical excitation of the microglial cell indicated by the arrow; there is no propagation of the Ca2 +  wave across these lanes. C shows parallel lanes of microglia in which lane widths are 50±6  and the cell-free lane 46±10 ; the open circles indicate that a Ca2 +  wave response was able to propagate across cell-free lanes as well as along the lanes. The calibration bar is 45  in A, B and C. The position of the micropipette in C(a) is not evident as it is out of focus
Since astrocytes use ATP as a chemical transmitter, and it is known that microglia initiate Ca2 +  transients in response to ATP, we determined if ATP was likely to be the diffusible substance released by microglia in order to promote Ca2 +  wave propagation. First, Ca2 +  wave propagation was blocked by the ATP-degrading enzyme apyrase (grade III, 60 units per mℓ; Fig. 5). Second, the effects of antagonists to the P2Y class of purinergic receptors on Ca2 +  wave propagation were tested. It is known that the pharmacological profile of P2Y receptor activation on spinal cord microglia and the expression of their mRNA clearly favours P2Y12 receptors, followed by P2Y6 and P2Y1 according to [12] and P2Y2, P2Y6, P2Y12 and P2Y14 according to [14]. Suramin (100 μM) completely blocked all propagation of the Ca2 +  wave along microglial lanes (compare Fig. 6B with 6A), indicating that P2Y6 and P2Y14 are not involved, and this was confirmed for P2Y6 using the specific P2Y6 antagonist MRS 2578 (30 μM). The specific P2Y1 antagonist MRS 2500 (100 μM) did not block the Ca2 +  wave. On the other hand, the P2Y12-specific antagonist 2-MeSAMP (300 μM) blocked Ca2 +  wave propagation. Any contribution of P2X receptors known to be present on microglial cells, such as P2X4 and P2X7 [11], to Ca2 +  wave propagation is likely to be minimal given the blocking effects of the P2Y12-specific antagonist 2-MeSAMP. We conclude that Ca2 +  wave propagation between microglial cells involves the release of ATP onto at least P2Y12 receptors. Visentin et al. [14] have also placed emphasis on the role of P2Y12 receptors in calcium signalling.
Fig. 5Propagation of Ca2 +  waves is blocked by the ATP-degrading enzyme apyrase. A and B show the extent of propagation of Ca2 + , from the point of mechanical stimulation of a microglial cell, to other microglial cells in a lane. In each case, the top panel (a) shows the position of the mechanically stimulating micropipette and the lower panel (b) shows the microglial cells that gave a Ca2 +  signal (open circles) in response to mechanical stimulation at the arrow. A is the control and in B apyrase (60 units/mℓ; grade III, Sigma) was present with only the stimulated cell now giving a Ca2 +  transient. The calibration bar is 45Fig. 6Transmission of the Ca2 +  wave between microglial cells is chemical due to the release of ATP. A and B show the extent of propagation of Ca2 +  from the point of mechanical stimulation of a microglial cell to other microglial cells in a lane. In each case, the top panel ((a)) shows the position of the mechanically stimulating micropipette and the lower panel ((b)) the microglial cell(s) that gave a Ca2 +  signal (open circles) in response to mechanical stimulation at the arrow. A is the control and in B suramin (100 μM) was present blocking P2Y receptors and only the stimulated cell responded with a Ca2 +  transient. The calibration bar is 40
Density of P2Y receptors on microglia
The theoretical requirement that KR take values from 25 to 45 μM may reflect different densities of P2Y receptors on the microglia, since in the present theory KR depends on this density as well as on the dissociation of ATP from the receptors (see Theory section above, and also the following section). Polyclonal antibody labelling of P2Y12 (Fig. 7a) receptors indicated that these are localized in clusters of average diameter 0.45 , as are P2Y receptors on astrocytes [5] and smooth-muscle cells [30]. The density of P2Y12 receptors, measured over nine microglial cells, varied about four-fold (Fig. 7b). We suggest that P2Y12 receptors mediate the Ca2 +  wave propagation. Since in the present model KR values need to vary over at least a two-fold range, it may be that this variability is partly due to differences in P2Y12 receptor density.
Fig. 7Density of P2Y receptors on microglia. a Distribution of anti-P2Y12 receptor immunofluorescence on single spinal-cord microglia in lanes; the shape of the cell is given by the borders of immunohisto chemical staining. b Histogram of the density of and anti-P2Y12 labelled receptors for different microglia. The error bars indicate ±SEM. At least ten areas on each cell were used to determine the P2Y receptor density
Modelling the quantitative characteristics of Ca2 +  wave propagation amongst microglia
The model of purinergic transmission of the Ca2 +  wave given in the Methods was used to give a quantitative description for comparison with the experimental results. A lane of microglia five cells wide and 528  long was considered in which the centre-to-centre distance between the microglia was 25 . The whole lane of microglia was placed on a 2D surface 528  by 528  (Fig. 8). Each row of five cells possessed KR values assigned by random permutations of the values 25, 30, 35, 40 and 45 μM. Note that these are effective KR values that take into account other properties besides dissociation of ATP from P2Y receptors. Activation of a microglial cell in the centre of the lane, by increasing the ATP concentration about the cell to 20 μM for 5 s, generated a Ca2 +  wave that propagated with diminution as shown in Fig. 8. The Ca2 +  wave varied in amplitude and velocity, both across the width of the lane as well as along its length (Fig. 8).
Fig. 8Diagrammatic representation of the theoretical spatial and temporal changes in a Ca2 +  wave in a lane of microglia five cells wide following excitation of the central microglia, according to the mathematical model. The Ca2 +  wave is initiated by a 5-s pulse of ATP of concen tration 20 μM. The vertical bars give Ca2 +  in μM at times t = 7.5, 15, 22.5 and 30 s, as indicated. KR values range from 25 to 45 μM for different microglia across each lane
A quantitative analysis of Ca2 +  wave propagation in a lane, such as that shown in Fig. 8, gives the results summarised in Fig. 9. The peak amplitude of Ca2 +  in each microglial cell of the lane varied significantly both along the length and across the width of the lane (Fig. 9a). Normalizing the Ca2 +  to the largest amplitude observed at the site of initiation shows that many of the cells give a Ca2 +  amplitude that is less than 15% of the largest one (Fig. 9a). Using this as a cut-off for the ΔF/F value that would be observed experimentally (see Methods) gives a rate of decline of Ca2 +  with distance similar to that observed, from 100% to 20% over about 120  (compare Fig. 9a with Fig. 3a). The percentage of cells that gives a Ca2 +  amplitude greater than 15% of the largest amplitude at the site of initiation, for different rows of five cells along the length of the lane, remains high for the first 75  and then declines to about 40% of maximum at 120 . This is a similar pattern of Ca2 +  changes to that observed experimentally along a lane of microglia (compare Fig. 9b with Fig. 3b). The average amplitude of the peak Ca2 +  across rows of cells in a lane increases with the number of cells that are activated in a row (Fig. 9c). This is also observed experimentally (compare Fig. 9c with Fig. 3c).
Fig. 9Theoretical characteristics of a Ca2 +  wave in a microglia lane, according to the mathematical model as in Fig. 8, for quantitative comparison with the observed characteristics (see Fig. 3). a shows that the predicted peak amplitude of Ca2 +  for all cells in a lane, normalized to that at the site of stimulation, declines with distance along the length of the lane until it becomes undetectable at about 120  (compare to Fig. 3a); the horizontal line indicates the values of the Ca2 +  amplitude below which experimental detection is in the noise level (set at 15%). b shows the predicted number of microglia that give a Ca2 +  response greater than 15% of the maximum value at different positions along the length of a lane expressed as a percentage of the total number of cells at that position; this is maintained for about 70  and then declines rapidly (compare to Fig. 3b). c shows that the predicted amplitude of the average peak Ca2 +  in rows of a microglia lane increases with the number of microglia that propagate Ca2 +  in the row (compare to Fig. 3c)
These theoretical results highlight the fact that Ca2 +  wave propagation amongst microglial cells is very limited compared with that amongst astrocytes [5]. Experimentally, this was highlighted by mechanical stimulation of microglial cells at different well-separated sites along a single microglial lane, showing that Ca2 +  wave propagation was restricted to within about 90  of the stimulating micropipette (see Fig. 10). Such restricted propagation was also observed along the model lane following stimulation at well-separated sites (compare Fig. 11 with Fig. 10). The clustering of activated cells near the site of initiation of the Ca2 +  wave is, in the model, due to the large amount of ATP released in this region.
Fig. 10Stimulation of microglia at different sites in a lane leads to Ca2 +  wave propagation confined to the vicinity of the stimulating electrode. Shown in (a), (b) and (c) are three different sites, greater than 140  apart, of mechanical stimulation by a micropipette of a single cell in a single lane of microglia. In (d) is shown that the Ca2 +  waves initiated in each case ((a) to (c)) are confined to a region within 90  of the stimulating micropipette, with each set of symbols indicating the extent of Ca2 +  wave propagation. The calibration bar is 45 . The position of the micropipette in C(a) is not evident as it is out of focusFig. 11Theoretical predictions of the distribution of microglia in a lane five cells wide that gave a Ca2 +  response, with an amplitude greater than 15% of maximum, following excitation of a single microglial cell at three different sites along the length of the lane, indicated by filled circles (∙). The dots indicate the positions of microglia in the lane. The diamonds (⋄) indicate microglia that gave a response following stimulation of the microglia near the left-hand end of the lane (sixth cell from the left at − 250 ), the open circles (∘) responses following stimulation of the microglia near the right-hand end of the lane (sixth cell from the right at 250 ) and the plusses (+) microglia in the centre of the lane (at 0 ). (A longer lane (800 ) has been used for this calculation.) The KR values range from 25 to 45 μM for different microglia across each lane. Note that there is no overlap in the Ca2 +  wave domains of each stimulated microglial cell, the diamonds, crosses and circles designating discrete regions
The model of purinergic transmission of Ca2 +  waves was used to see if it could account for Ca2 +  propagation along and between lanes of microglia, such as those shown in Fig. 4. Figure 12 shows the propagation of Ca2 +  waves in three such parallel lanes of cells, separated by cell-free lanes 42  wide, following initiation of the Ca2 +  wave in the middle row of the middle lane by applying 20 μM of ATP for 5 s at a central cell. There is propagation of the Ca2 +  wave over about six cells of the middle lane before the side lanes are engaged, at about 7 s after application of the initiating stimulus (Fig. 12). Both side lanes first generate a Ca2 +  wave that is in cells in a row opposite or nearly opposite the row containing the initiating cell in the middle lane. By 13 s the crest of the Ca2 +  wave has reached the limits of Ca2 +  propagation at about 100  from the site of initiation, by which time it has travelled less than 75% of that distance along adjacent lanes (Fig. 12). Very few cells are engaged in Ca2 +  wave propagation in these adjacent lanes and propagation fails over distances of about 70  along the lane and 50  across it.
Fig. 12A diagrammatic representation of the theoretical spatial and temporal changes in Ca2 +  in three lanes of microglia each five cells wide, separated by cell-free lanes 42  wide, following excitation of a single microglial cell in the middle row of the middle lane. The Ca2 +  wave is initiated by a 5-s pulse of ATP of concentration 20 μM on the central microglial cell at time t = 0. The vertical bars give Ca2 +  in μM at times t = 7.5, 15, 22.5 and 30 s, as indicated. KR values range from 25 to 45 μM for different microglia across each lane. Note the limited propagation of the Ca2 +  wave in both the middle and side lanes
The question arises as to whether regeneration of ATP in each cell is necessary. Repeating calculations with regenerative release switched off, and thus only pure diffusion of ATP from its initial release site, gave travel distances along lanes reduced by about 20%. We investigated whether this reduced distance could be compensated for by increasing the initial release of ATP, but this then recruited cells in adjacent lanes in a way not observed experimentally. Thus the theoretical calculations indicate a role for intracellular regeneration of ATP, but at a much lower rate than in astrocytes.
Discussion
Although activation of P2X7 receptors by ATP leads to an influx of calcium ions in microglial cells [16, 31–33] it does not seem that these receptors mediate the Ca2 +  wave propagation in these cells as this is blocked by suramin which does not block P2X7 receptors. It seems likely that P2X7 receptors are activated at higher concentrations of ATP than is required for P2Y receptor activation [33], suggesting that the concentration of ATP reached at the receptors after its release from microglia is insufficient to excite P2X7 receptors. ATP acts on microglial cells to both release calcium from internal stores and to evoke an influx of calcium [34, 35]. P2Y receptors mediate Ca2 +  release from internal stores in microglial cells [14, 21, 32, 33, 36, 37], the extent of this release being under the modulatory control of P2X receptors [25, 32] and of toll-like receptors [38].
ATP released following stimulation of astrocytes can generate Ca2 +  transients in nearby microglial cells [2]. Repeated stimulation of the astrocytes releases sufficient ATP to activate P2X7 receptors on the microglial cells, greatly increasing membrane permeability in the microglial cells [1] and leading to the release of inflammatory cytokines, such as IL-2, from the cells [39]. The present work suggests that in these experiments the ATP released in moderate amounts from singly stimulated astrocytes most likely acts on P2Y12 receptors on microglial cells. We could block the ATP-dependent Ca2 +  propagation in these cells with suramin, which blocks P2Y1, P2Y2 and P2Y12 receptors, but could not be blocked by P2Y1 receptor antagonists. Given that the predominant receptor on microglial spinal cord cells shows the pharmacological profile of P2Y12 [12] (see also [14]), we conclude that this receptor, which we found in relatively high density, is most likely to be mediating the effect of ATP. Thus there is no evidence that P2X7 receptors are engaged in Ca2 +  propagation in microglia. It is interesting to note in this regard that microglial cells rapidly re-orientate their processes towards a site of ATP release in vivo as a consequence of the action of the released ATP on P2Y receptors, an action that is blocked by apyrase [40] and that nucleotides acting on P2Y12 receptors of microglia exert a chemotactic effect [41].
We have used soft lithography techniques of micro-fabrication to allow controlled and discrete patterning of microglia so that quantitative investigations can be carried out [42]. Such an approach avoids the difficulties inherent in the random seeding of microglial cells on a homogeneous substrate for carrying out quantitative measurements of the properties of propagating calcium waves [43]. The technique allows lanes of microglial cells with controlled widths of from 15  to over 150 , separated by cell-free regions with the same range of widths [44]. It is unlikely that this fabrication method affects the seeded microglial cells, since when applied to astrocytes the rates of propagation of the Ca2 +  waves remain about the same as determined in random seeded cultures [4, 5].
The amplitude of the Ca2 +  wave, and the percentage of microglial cells excited to give a Ca2 +  response, were correlated along the length of a microglial lane, both decreasing from near the site of initiation over a distance of about 120  before the wave ceased to be detectable. Decreases in amplitude of Ca2 +  waves from a site of mechanical initiation have often been observed in randomly seeded astrocytes over a homogeneous substrate, but in this case over several hundred microns [45–47]. The purinergic gliotransmission model suggests that the decline in amplitude of the Ca2 +  wave is due to the relatively high level of ATP released from the stimulated microglial cell, which then dominates the concentration profile of ATP within approximately 100 . The decline in amplitude of the wave approximately follows this concentration gradient of ATP. The decline in the percentage of microglial cells excited to give a Ca2 +  response within this 100  range is then attributed to a failure of microglial cells possessing a relatively high KR to generate a Ca2 +  response as the concentration of ATP declines over the same range. The more than two-fold range in the KR values used in our model is comparable to the range of P2Y12 receptor densities found in microglial cells using immunohistochemistry.
Our work shows that there can be propagation of Ca2 +  waves between parallel lanes of microglial cells when these are about 30  wide and separated by cell-free lanes about 40  wide, but this does not occur until many microglial cells in the initiating lane undergo a Ca2 +  response. It seems likely that a certain minimum amount of ATP must be released from the initiating lane, involving a certain minimum number of microglial cells undergoing a Ca2 +  response, before nearby microglial lanes are excited. Since ATP can diffuse across cell-free lanes as wide as 150  from astrocyte-seeded lanes [5], it seems likely that the ATP released from microglial cells is much less than that from astrocytes since the P2Y12 receptors in the model for microglia possessed lower KR values (25 − 45 μM) than in the model for astrocytes (25 − 125 μM; [5]).
The purinergic transmission model can account for the observed extent of the Ca2 +  wave provided pure diffusion of ATP from the stimulation site is supplemented by regenerative release of ATP from the microglia. Fluctuations in the density of excited microglia along a lane leads to local fluctuations in the amplitude of the Ca2 +  wave due to changes in the local ATP concentration, consequential on the changes in the local number of microglial cells excited. This is modelled by assigning a range of KR values to the P2Y receptors on the microglia, and this also reflects the density of receptors on individual microglial cells.
Stimulation of three different microglial cells some hundreds of microns apart gave a Ca2 +  response in microglia at highest density closest to the site of stimulation. Our model quantitatively explains these observations as arising from the large amount of ATP released by the stimulated microglial cell diffusing to activate microglia within about 100 .

transmission
propagation
calcium waves
microglia
purines


Canal wall reconstruction and mastoid obliteration with composite multi-fractured osteoperiosteal flap

We used inferior pedicled composite multi-fractured osteoperiosteal flap (CMOF), our original and new surgical approach, to obliterate the mastoid cavity and reconstruct the external auditory canal (EAC) to prevent the open cavity problems. CMOF was used to obliterate the mastoid cavity and reconstruct the EAC in 24 patients (13 women, 11 men; age span 12–51 years) who underwent radical mastoidectomy to treat the chronic otitis media between 1998 and 2004. Small meatoplasty was done in all 24 patients to relive their aesthetical concerns. Temporal bone CT scanning was done to observe the neo-osteogenesis in the mastoidectomy cavity and the CMOF, and the EAC volume was measured postoperatively. All our patients were followed-up for 2 years. The epithelization of the new EAC in our patients was complete at the end of the second month. Cholesteatoma, granulation, and recurrence of osteitis did not occur in any of the patients. We saw the new bone formation filling the mastoid cavity in the postoperative temporal bone CT scanning images. The mean volume of the new EAC on the 24th month was 1.83 ± 0.56 cm3. We had an almost natural EAC, which owed its existence to the neo-osteogenesis that grows behind the CMOF, which we use to obliterate the mastoid cavity and to reconstruct the EAC.
Introduction
Open cavity mastoidectomy techniques applied for chronic otitis media cause some cavity problems. These patients need life-long otologic care for the debris that accumulates due to the insufficient self-cleaning mechanism of mastoidectomy cavities. Sixteen percent of ears without cavities needs cleaning, whereas this ratio climbs to 42% for ears with cavities [11]. These patients complain of dizziness in cold weather and during swimming. Other problems are non-aesthetic meatoplasty and difficulties in the placement of hearing-aid devices [2, 7].
Various obliteration and external auditory canal (EAC) reconstruction techniques have been recommended to eliminate open cavity problems. Mosher was the first to use soft tissue flap in 1911 and has described the postaural subcutaneous flap technique for cavity obliteration [10]. In the following years soft tissue flaps, free bone, cartilage grafts, autograft and synthetic filling substances have been used to reduce mastoid cavities [1]. Mastoid cavity obliteration by neo-osteogenesis under the periosteal flap has been first shown by Kahramanyol in 1992 [8]. This result has been my start-point and I have used inferior pedicled CMOF, which is our modification for EAC reconstruction and to reduce the volume of radical mastoidectomy cavity. In addition, small meatoplasty was done to lessen aesthetic concerns.
Materials and methods
Twenty-four patients (13 women and 11 men ranging in age from 12 to 51 years) were operated between 1998 and 2004. Various stages of cholesteatoma were detected during preoperative otoscopy and intraoperative observations in all those patients who had otorrhea and hearing loss for years. Four patients had complications such as facial paralysis, labyrinthine fistula and intracranial abscess. Ossicle chain injury of various severities was present in all these participants.
All patients were operated under general anaesthesia. Prior to operation, each patient had otoscopic examination, pure tone and speech audiometry and temporal bone CT scans. Approval for radical mastoidectomy indication was granted after simple mastoidectomy and atticotomy.
Operative technique
The incision line that was 1.5–2 cm posterior to the postauricular sulcus was kept as a margin as the subcutaneous tissues were dissected into fascia. Dissection was advanced to the skin of the posterior meatal wall along the fascia. The skin of the posterior meatal wall was horizontally transected between 6 and 12 O’ clock. Then, the periosteum was incised till the bone was reached about 1–2 mm anteriorly to the postauricular incision. The superior part of the incision runs parallel to the inferior border of the temporalis muscle. From the upper margin of the incision that extends to the bone, the periosteum was elevated to the inferior pedicle with a 7-mm chisel. During this procedure bone lamellae with utmost 1-mm thickness were raised as a part of a fractured bone panel attached to the periosteum. By this way the mastoid apex was reached inferiorly and EAC skin posteriorly. Periosteum was dissected from the EAC posterior wall skin. Thus, CMOF with an inferior base was formed. The bone lamellae under the periosteum were fractured into fragments as much as possible (Fig. 1). The flap was given the functionality of a flexible metal watch belt to place it properly into the mastoid cavity and let it reconstruct the EAC. The flap was retracted inferiorly to be able to work on the mastoid cortex.
Fig. 1Preparation of the composite multi-fractured osteoperiosteal flap; CMOF composite multi-fractured osteoperiosteal flap, MT musculus temporalis, BL bone lamellae, EAC external auditory canal, MC mastoid cortex, C chisel
The following rules were attended during radical mastoidectomy. (1) All bony buttresses were excised and lowered as much as possible. This is called saucerization or skeletonization. (2) The mastoid apex was excised. (3) All related anatomic structures and loci were visualized. (4) All diseased tissues were removed. (5) White cortical bony tissues were reached in all directions. Moreover, large defects at the dural plate and or the sigmoid sinus were reconstructed with modelled autologous bone chips obtained from the operative field.
Cleansing of the tympanic cavity was emphasized. Patients with bone conduction thresholds better than 35 dB and an open eustachian ostium and a healthy peri-eustachian mucosa were treated with tympanic reconstruction. The prepared CMOF was placed over the facial ridge and attached to the anterior attic wall in all cases. The flap could be elongated to the attic with the help of small incisions. The bone lamellae beneath the periosteum are supportive material to our flap and thus became a skeleton for the expected EAC. Absorbable gelatine sponge was laid over the mastoid bone behind the CMOF. The EAC skin was incised longitudinally at the 6 O’ clock direction and the resultant skin flap having its nourishing root at the zygoma was laid posteriorly and inferiorly over the CMOF. We cared to keep the skin margins over the fascial flap. That is how the new EAC was composed. Then EAC was filled with absorbable gelatine sponge (Fig. 2). Siebenmann technique was used to form a small meatoplasty. A ‘‘Y” incision was used with its concavity looking to the posterior to elevate a flap of choncal skin and a sufficient part of the choncal cartilage was excised. The triangular skin flaps were just laid down such that they could touch the CMOF. The EAC in expectation was filled with absorbable gelatine sponge and extraphore with antibiotic ointment. The subcutaneous and cutaneous postauricular incisions were closed (Fig. 3).
Fig. 2Transposition of the composite multi-fractured osteoperiosteal flap (above), and fixation of it (below); SF skin flap, MC mastoid cavity, CMOF composite multi-fractured osteoperiosteal flap, SS sigmoid sinus, BL bone lamellae, EAC external auditory canal, S spongeFig. 3Shematic represantation of surgical procedure. Pr Periosteum, BL bone lamella, E extraphore, S sponge, FG fascia graft, SF skin flap, SS sigmoid sinus
Postoperative antibiotics and anti-inflammatory medicines were continued for 1 week. Skin sutures were removed on the 7th postoperative day. The cavity dressings were removed on the 14th day. Antibiotic and steroid drops were used at the end of the second postoperative month. In all patients epithelization of the cavity completed at the end of the 2nd month.
Temporal bone CT scanning was done to observe the neo-osteogenesis in the mastoidectomy cavity and the CMOF on the 1st, 12th and 24th months. The EAC volume was measured on the second, 6th, 12th and 24th months. The volume measurements were done with saline, warmed up to the body temperature that was filled into the EAC up to the level of the posterior line of the meatoplasty. The head position was horizontal during the measurement. Data of the study were analysed by the statistical package for social sciences (SPSS) for Windows 10.0.
Results
This study was done between the years 1998 and 2004 in 24 patients. All patients were followed-up for at least 2 years. EACs were smooth and healthy. In all patients epithelization was complete after the 2nd month of the operation. None had recurrent osteitis, cholesteatoma or granulation. Otoscopic examination could reveal the entire cavity in all these cases and debris and cerumen cleaning was feasible.
The mean volume of the new EAC on the 2nd, 6th, 12th and 24th months were 0.93 ± 0.32, 1.43 ± 0.48, 1.66 ± 0.53, 1.83 ± 0.56 cm3, respectively. The canal volume measurements of the patients that we were able to follow after 24 months revealed no significant difference. The evaluations of the postoperative temporal bone CT scanning revealed no retraction or displacement around, but demonstrated new bone formation obliterating the mastoid cavity behind the CMOF that was used to build the posterior wall of the EAC (Fig. 4). The CMOF never displaced toward the EAC or the sigmoid sinus, and it always remained where it was placed during the operation.
Fig. 4Postoperative temporal bone CT images 1 month later (a), and 2 years later (b)
None of our patients returned with vertigo induced by cold air or swimming. No aesthetical disapproval occurred. Introducing the hearing aid placements into the EAC was easy and almost natural. The therapeutical results in four patients with preoperative complications were successful. In one patient conchal perichondritis emerged soon after the operation and was treated with medical therapy.
Discussion
Various treatments have been proposed for mastoid cavity obliteration and reconstruction [1, 10]. Extended meatoplasty has been used for auto-cleansing and sufficient aeration [4, 8, 13]. Some authors offered a small meatoplasty to avoid the inevitable aesthetic concerns stemming from a wider meatoplasty [6]. However, small meatoplasty has restricted the cavity drainage and aeration by increasing the cavity depth posteriorly. In our technique a small meatoplasty became a functional continium of the almost a normal EAC anatomy that was obtained by the placement of the CMOF to the posterior edge of the meatoplasty. This newly formed EAC has provided sufficient drainage and aeration, and a preferable aesthetic appearance (Fig. 5).
Fig. 5The external ear meatus in the second postoperative year
The bilateral perfusion obtained by the postauricular artery supplying the inferior pedicled CMOF and the branches of internal maxillary artery supplying the superior pedicled EAC flap have accelerated cavity healing and epithelization. The absorbable gelatine sponges placed into the mastoidectomy cavity and new EAC have helped the immobilization of the CMOF by supporting it placed at the facial ridge. Moreover, they have contributed to the formation of a homogenous haematoma under the CMOF. Many authors have detected that haematoma at the fracture site has aided in fracture healing [5, 12]. Numerous callus areas emerging in interlamellar fracture lines under the CMOF have supplied both skeletal support and obliteration of the mastoidectomy cavity by neo-osteogenesis.
Histological investigations relating fracture-healing process have shown that intramembranous bone formation begins under the periosteum in a few days after fracture formation and bridging between fracture fragments begins in 4 weeks [3]. We observed calcification foci indicating osteoblastic activity in histological investigations of a cavity wall biopsy taken from a patient in 6 months after surgery (Fig. 6). New bone formation filling the mastoidectomy cavity under the CMOF was detected in the postoperative temporal bone CT images taken in the 1st, 12th and 24th months.
Fig. 6Calcification foci indicating osteblastic activity are seen in histologic investigations 6 months after surgery (HE ×400)
In a study where Palva flap was used for mastoid obliteration, the ear canal was found enlarged at both the early and the late postoperative check-up compared to the preoperative values [11]. The results of the studies, reported by Kahramanyol et al. [8] in which mastoid cavities were obliterated with fascioperiosteal flap, have shown that the mastoid cavity volume was decreased by 39% due to neo-osteogenesis under fascioperiosteal flap, and that the obliteration has stayed in long term has shown radiologically [9]. In our study it has shown a nearly twofold increase in EAC volume and no significant difference after 24th months. Thus the difference in EAC volumes between the 2nd month and the 2nd year of the postoperative period was assessed as a result of shrinking of the tissues, composing the EAC, especially the periosteum.
Conclusion
Some large meatoplasties performed for open cavity problems are not preferable due to non-aesthetic appearance. On the other hand, small meatoplasties that are preferred to overcome this concern cause aeration and drainage problems by increasing the depth of mastoidectomy cavity posteriorly. Therefore, in this study we both performed small meatoplasties to avoid the aesthetic concerns and at the same time filled the mastoid cavity up to the posterior edge of the meatoplasty to obtain efficient aeration and drainage.

canal wall reconstruction
mastoid obliteration
osteoperiosteal flap
radical mastoidectomy
neo-osteogenesis


Polymorphisms in the Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5) Gene Are Associated with Peak Bone Mass in Non-sedentary Men: Results from the Odense Androgen Study

Purpose To investigate the impact of the Ala1330Val (rs3736228, exon 18) and Val667Met (rs4988321, exon 9) polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene on peak bone mass in young men.
Family and twin studies have demonstrated that genetic factors account for 50–80% of the inter-individual variation in bone mineral density (BMD) in both women and men [1–9]. Polymorphisms in a number of genes affect the bone phenotype regarding BMD, bone size, or fracture risk [10]. Only a small part of the overall variation in BMD, however, has been explained by the polymorphisms identified so far.
The transmembrane proteins low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 are essential in Wnt signaling. They are expressed in many tissues including osteoblasts and have been implicated in the adaptive response of bone to mechanical load [11]. Located on chromosome 11q12–14, activating mutations in the LRP5 gene are responsible for rare conditions with a “high bone mass phenotype” [12, 13] as well as autosomal dominant osteopetrosis type 1 [14], and transgenic mice carrying the LRP5 G171V mutation have increased bone mass and bone biomechanical properties [15]. Conversely, inactivating mutations are responsible for the osteoporosis pseudoglioma syndrome [16] and this syndrome is recapitulated by LRP5 inactivation in mice [17].
In addition to these mutations, a number of polymorphisms have been described in the LRP5 gene (http://www.ncbi.nlm.nih.gov/SNP). One of these, the Ala1330Val (rs3736228, exon 18) polymorphism, has been associated with attenuated bone gain in prepubertal boys [18], decreased BMD in Japanese [19], American [20], Australian [21], and Dutch [22] women as well as in elderly Dutch men [22]. It has also been associated with an increased risk of fragility fractures in postmenopausal Australian women [21] and in elderly Dutch men [22]. No association, however, was found between this polymorphism and bone loss in elderly men and women [22]. Similarly, the Val667Met (rs4988321, exon 9) polymorphism was associated with attenuated bone gain in prepubertal boys but not in girls participating in a 1-year longitudinal Swiss study [18]. In the same study, this polymorphism was also associated with decreased lumbar spine bone mineral content and projected bone area and there was a trend toward lower BMD in the spine [18]. These associations were mainly driven by men [18] and in haplotype analysis including Ala1330Val, most of the effect was conferred by the Val667Met polymorphism. Moreover, in a case-control study by Ferrari et al. [23] on idiopathic osteoporosis in males, both LRP5 polymorphisms significantly increased the odds ratio for osteoporosis. Additionally, Val667Met was associated with low stature in adults of both sexes [18]. Thus, current evidence suggests that polymorphisms in the LRP5 gene may be involved in the control of peak bone mass and may increase the risk of osteoporosis in males [16,18, 23].
In the present study, we investigated the association between the Ala1330Val and Val667Met polymorphisms and peak bone mass in men in a population-based study.
Subjects and Methods
The Odense Androgen Study (OAS) is a population-based, prospective, observational study on the interrelationship between endocrine status, body composition, muscle function, and bone metabolism in young men. Details on the design and inclusion of participants are reported elsewhere [24]. In brief, 3,000 men aged 20–30 years were randomly selected from the civil registration database in Fuenen County, Denmark, and invited by mail to participate in the study. These subjects also received a questionnaire regarding smoking, medication, chronic diseases, drug abuse, etc. A total of 2,042 questionnaires were returned, and from these 859 men expressed interest in participation in the clinical part of the study. Nineteen withdrew before an appointment for inclusion was made. Ten men did not meet with the inclusion criteria (Caucasian and age between 20–29 years), and another 3 men gave their notice after the inclusion had stopped. Aside from these latecomers none of the responders were excluded. Of the remaining 827 eligible subjects, 43 did not attend for their appointment and did not wish to reschedule, but informed consent was obtained from 784 men, with dropout from 1 subject. Thus 783 Caucasian men gave written informed consent and were included in the OAS. These participants had similar age, body mass index (BMI), lifestyle, socio-economic status, and educational level as the background population, as evaluated by questionnaires and demographic data (manuscript in preparation). The protocol stipulated that subjects with massive substance or alcohol abuse, malignant disease or severe chronic disease be excluded; however, none of the responders fulfilled any of these criteria. The study was approved by the local Ethics Committee of Fuenen and Vejle Counties (file number 20010198), conducted according to the Helsinki Declaration, and declared in ClinicalTrials.gov: NCT00150163, registered September 6, 2005.
Genotyping
DNA was isolated from whole blood samples using QIAamp DNA Blood Midi kits (Qiagen, Valencia, CA, USA).
The Ala1330Val polymorphism was analyzed using Taqman primers and probes that were designed using Primer Express software (Applied Biosystems):Sense: 5′-GACGGCGAGGCAGACTGT-3′Antisense: 5′-GCAGGGCCAGGGTCTTG-3′C probe: 5′-FAM-TCAAAGTCCGCCTCGT-NFQ-3′T probe: 5′-VIC-CACAGTCCACCTCGTC-NFQ-3′The real-time analysis was performed using the ABI PRISM 7700 Sequence Detection System. To validate the method, approximately 10% of the samples were repeated and in total 17 samples—at least 5 of each genotype—were sequenced.
The Val667Met polymorphism was analyzed using fluorescence polarization as described previously [25], using AcycloPrime-FP SNP Detection kits (Perkin Elmer Life Sciences, Boston, MA, USA). PCR fragments containing the polymorphisms were generated using standard conditions with the following PCR primers:Sense: 5-gccTTcTTggTcTTcAccAg-3′Antisense: 5′-cTTTgAggcAggAAcAgAgg-3′Enzymatic clean-up and subsequent AcycloPrime-FP reaction with SNP primer (sense 5′-ccTcgAgAccAATAAcAAcgAc-3′) were performed according to the instructions of the kit manufacturer. Fluorescence polarization was read in a Wallac Victor multilabel plate reader (Perkin Elmer Life Sciences, Boston, MA, USA). Blank samples and samples with known genotype were included as negative and positive controls, respectively. A total of 10% of all samples were sequenced and genotyping confirmed in all cases.
Anthropometrics and Lifestyle
Body weight, body height, sitting height, and arm span were measured. Data regarding exercise habits were collected using a self-administered questionnaire. Non-sedentary lifestyle was defined as participation in any form of regular exercise.
Dual-Energy X-ray Absorptiometry (DXA)
BMD of the lumbar spine, hip, and whole-body was measured using a Hologic 4500 DXA-scanner (Waltham, MA, USA). The coefficient of variation (CV) for BMD is 1.5%, 1.5%, and 0.7% in the lumbar spine, total hip, and whole body, respectively. Lean body mass (LBM) and fat mass was obtained from the whole-body scans.
Biochemistry
Serum samples were drawn after an overnight fast between 08.00 and 10.00 hours and stored for later analyses at −80°C. Serum concentrations of osteocalcin were measured by a luminoimmunoassay (BRAHMS Diagnostica, Berlin, Germany) with an intra-assay and inter-assay CV of 5% and 8%, respectively.
Serum levels of type 1 collagen C-terminal telopeptide (1CTP) were measured using radioimmunoassay (RIA) (Orion Diagnostica, Espoo, Finland) with an intra-assay and inter-assay CV of 4% and 6%, respectively. Bone-specific alkaline phosphatase (bone-AP) concentration in serum was determined enzymatically after monoclonal antibody capture (Metra Biosystems, Mountain View, CA, USA). The intra-assay CV was 2% and the inter-assay CV 12% for this analysis. Serum levels of 25-OH-vitamin D were measured using a competitive radioimmunoassay (DiaSorin, Stillwater, MN, USA) with an intra-assay CV of 6% and an inter-assay CV of 15%. Serum IGF-I was determined in acid–ethanol extracts as described previously [26]. Intra- and inter-assay variation averaged less than 5% and less than 10%, respectively.
Statistics
Data are reported as the mean ± SD, median (range), or median [95% CI], as appropriate. BMD values are shown in absolute values. Regarding the allele dose-analyses, BMD values were transformed to Z-scores using the SD observed in our population. Hardy-Weinberg equilibrium was checked using chi-square analysis. Linkage disequilibrium (D’) between polymorphisms was evaluated by using GOLD software [27]. Haplotype analysis was undertaken using FAMHAP software for constructing haplotypes [28]. Comparisons between groups were performed using one-way ANOVA, unpaired t-test or similar nonparametric tests. Associations between parameters were analyzed using Pearson’s regression analysis and multiple linear regression analysis. Dichotomies were tested using the chi-square test. A p value of less than 0.05 was considered significant. Statistical analyses were performed using SPSS 11.5 and SPSS 12.0.
Results
Genotype Frequencies
For the Ala1330Val polymorphism, the CC genotype was found in 589 (75.6%), the CT genotype in 170 (21.8%), and the TT genotype in 20 (2.6%), respectively (Table 1) allele frequencies of 0.87 and 0.13 for the C- and T-alleles, respectively.
Table 1Covariates, BMD, and lifestyle (sedentary/non-sedentary) by LRP5 genotypes (Ala1330Val and Val667Met polymorphisms)PolymorphismsAla1330ValVal667MetCCCTTTp values (ANOVA)GGGAAAp values (ANOVA)n58917020–699764–Age (years)25.5 ± 2.825.3 ± 2.825.9 ± 2.7NS25.5 ± 2.825.3 ± 2.627.0 ± 1.8NSBody height (cm)181.7 ± 6.8181.5 ± 6.1182.8 ± 7.7NS181.7 ± 6.8181.3 ± 5.6190.7 ± 6.30.02Arm span (cm)183.4 ± 8.2 183.1 ± 7.0185.4 ± 8.9NS183.3 ± 8.0182.6 ± 6.9193.5 ± 6.80.04Body weight (kg)81.9 ± 11.881.1 ± 12.485.9 ± 16.7NS81.7 ± 12.182.2 ± 12.990.4 ± 12.2NSBody mass index (kg/m2)24.8 ± 3.324.6 ± 3.725.6 ± 4.1NS24.8 ± 3.325.1 ± 4.324.8 ± 1.8NSTotal fat mass (kg)14.9 ± 6.214.9 ± 7.317.7 ± 8.5NS15.3 ± 6.215.1 ± 6.717.9 ± 7.9NSLean body mass (kg)64.0 ± 7.363.3 ± 6.865.3 ± 9.0NS64.7 ± 6.9 64.4 ± 6.070.4 ± 7.0NSUnits alcohol/week (n)11.4 ± 10.210.7 ± 8.612.3 ± 8.7NS11.0 ± 9.410.8 ± 8.16.5 ± 7.3NSCigarettes/day (n)0 [0–45]0 [0–35]0 [0–25]NS0 [0–35]0 [0–30]0 [0–1]NSSerum 25-OH-D3 (nmol/l)65.1 ± 27.964.2 ± 26.967.9 ± 28.6NS65.2 ± 27.563.0 ± 29.160.0 ± 25.4NSSerum IGF-I (μg/l)199.9 ± 52.8202.4 ± 50.3191.0 ± 36.1NS27.6 ± 7.525.5 ± 6.627.3 ± 7.8NSSerum osteocalcin (mmol/l)3.0 ± 1.13.1 ± 1.33.0 ± 1.0NS3.1 ± 1.22.8 ± 1.02.3 ± 0.5NSSerum 1CTP (μg/l)5.0 ± 1.44.9 ± 1,55.1 ± 1.3NS5.0 ± 1.44.9 ± 1.44.9 ± 0.9NSBone-AP (U/l)26. 6 ± 8.128.4 ± 10.026.7 ± 8.3NS26.7 ± 8.225.8 ± 7.033.4 ± 16.4NSBMDspine (g/cm2)1.08 ± 0.121.07 ± 0.171.05 ± 0.13NS1.08 ± 0.121.07 ± 0.221.07 ± 0.15NSBMDhip (g/cm2)0.95 ± 0.140.94 ± 0.140.94 ± 0.15NS0.95 ± 0.140.96 ± 0.150.84 ± 0.070.03BMDWB (g/cm2)1.22 ± 0.10*1.21 ± 0.091.21 ± 0.11NS1.22 ± 0.101.21 ± 0.111.20 ± 0.08NSSedentary/non-sedentary (n)150/44037/1334/16NS171/52818/581/3NSNo significant differences were found between the groups (ANOVA). This was true also if the CT and TT genotypes were pooled (t-test). Genotypes were in Hardy-Weinberg equilibrium (Ala1330Val: χ2 = 3.2, p = 0.07; Val667Met: χ2 = 1.48, p = 0.22)*p = 0.05 comparing the CC genotype with CT + TT pooled; NS, not significant
Similarly, the GG, GA, AA genotypes of the Val667Met polymorphism were found in 699 (89.7%), 76 (9.8%), and 4 (0.5%), respectively. This corresponds to allele frequencies of 0.95 and 0.05 for the G- and A-alleles, respectively.
Both allele distributions were compatible with Hardy-Weinberg equilibrium (χ2 = 3.2, p = 0.07 and χ2 = 1.48, p = 0.22). The two polymorphisms were not in complete linkage disequilibrium (D’ = 0.955, R² = 0.33).
Covariates
Data on covariates in relation to genotypes are shown in Table 1. No significant differences regarding covariates (age, body height, body weight, BMI, sitting height (not shown), total fat mass, lean body mass, alcohol consumption, smoking, serum 25-OH-D3, or serum IGF-I) were seen between the genotypes of the two polymorphisms. Regarding the Val667Met genotypes, however, subjects with the AA genotype had significantly increased body height (190.7 ± 6.6 vs. 181.7 ± 6.8 and 181.3 ± 5.6 cm, p = 0.02) and arm span (193.5 ± 6.8 vs. 183.3 ± 8.0 and 182.6 ± 6.9 cm, p = 0.04).
BMD in Relation to Age
In the spine, no relationship between BMD and age could be detected. Maximum BMD in the total hip was observed around the age of 22 years. In subjects above this age BMD was significantly lower (ANOVA, p < 0.05). In subjects aged 30 years, BMD was 5.5% lower that the observed maximum.
Genotypes in Relation to BMD
Regarding the Ala1330Val polymorphism, no significant differences between BMD in the spine, hip, or whole body were found between subjects with the CC, CT, and TT genotypes. When the CT and TT genotypes were pooled, whole-body BMD was significantly lower in the CT + TT compared with the CC genotype (1.21 ± 0.10 vs. 1.22 ± 0.10, p = 0.05).
Regarding the Val 667Met polymorphism, a significant difference in hip BMD was observed, since subjects with the AA genotype had lower BMD (0.84 ± 0.07 vs. 0.95 ± 0.14 and 0.96 ± 0.15, p = 0.03).
Table 1 also shows the distribution of genotypes in relation to sedentary/non-sedentary lifestyle, but no significant difference in lifestyle was found between any of the genotypes.
Table 2 shows the gene-dose effect on BMD expressed as the change in Z-score of each copy of the T- or A-allele, respectively. The analysis was performed both with and without adjustment for BMI, smoking (0/1), any continuous medication (0/1), and serum 25-OH-D3 in the overall study population (multiple regression analysis). Regarding the Ala1330Val polymorphism, whole-body BMD tended to decrease with each copy of the T-allele (p = 0.07) in the overall study population (n = 779). No significant association between number of T-alleles and BMD at the other skeletal sites was found. Regarding the Val667Met polymorphism, no significant gene-dose effect could be demonstrated in the overall population.
Table 2Multiple regression analysis determining the change in BMD Z-score for each copy of the T- or A-allelePolymorphismAla1330ValVal667MetZ-scoreUnadjustedAdjustedaUnadjustedAdjustedaTotal population (n = 779)BMDspine−0.11 (−0.29; 0.06)NS−0.10 (−0.27; 0.06)NS−0.044 (−0.28; 0.19)NS−0.054 (−0.27; 0.16)NSBMDhip−0.03 (−0.20; 0.11)NS−0.02 (−0.17; 0.11)NS0.018 (−0.20; 0.23)NS0.002 (−0.22; 0.21)NSBMDWB−0.13 (−0.27; 0.11)p = 0.07−0.12 (−0.25; 0.01)p = 0.07−0.085 (−0.30; 0.13)NS−0.11 (−0.33; 0.10)NSNon-sedentary (n = 589)BMDspine−0.21 (−0.40; −0.03)p = 0.02−0.20 (−0.37; −0.02)p = 0.01−0.26 (−0.51; −0.01)p = 0.04−0.23 (−0.46; −0.002)p = 0.048BMDhip−0.08 (−0.26; 0.09)NS−0.06 (−0.22; 0.10)NS−0.08 (−0.33; 017)NS−0.07 (−0.32; 0.19)NSBMDWB−0.17 (−0.33; −0.01)p = 0.04−0.15 (−0.30; 0.01)p = 0.07−0.17 (−0.41; 0.08)NS−0.18 (−0.44; 0.07)NSData are shown as mean effect and 95% confidence interval. Z-scores were derived from the study population itselfaAdjusted for BMI (ln-transformed), lean body mass (ln-transformed), smoking (0/1), any continuous medication (0/1), and serum 25-OH-D3
When analysis was restricted to non-sedentary men (n = 589), however, a significant correlation between the number of T-alleles at the Ala1330Val polymorphism and BMD in the spine was found in both the unadjusted (−0.21 [95% CI: −0.40; −0.03], p = 0.02) (Fig. 1) and adjusted analysis (−0.20 [95% CI: −0.37; −0.02], p = 0.01) (Table 2). Similarly, each copy of the A-allele in the Val667Met polymorphism decreased the lumbar spine BMD in unadjusted (−0.26 [95% CI: −0.51; −0.01], p = 0.04) and adjusted (−0.23 [95% CI: −0.46; −0.002], p = 0.048) analysis.
Fig. 1BMD of the lumbar spine, total hip, and whole body in relation to the genotype regarding the Ala1330Val (upper panel) and the Val667Met (lower panel) polymorphisms in the LRP5 gene. Participants with a non-sedentary (left) and sedentary lifestyle (right) are displayed. Data are shown as the mean ± SE. (*)p < 0.10, *p < 0.05
Genotypes in Relation to Bone Markers
No significant differences were observed between genotypes of the Ala1330Val or Val667Met polymorphisms regarding bone markers (Table 1).
Haplotype Analysis
The effect of haplotypes of the Ala1330Val and Val667Met polymorphisms on BMD adjusted for BMI, lean body mass, smoking, drugs, and vitamin D are shown in Table 3. No significant association between BMD and haplotypes was found. This was also the case when sedentary and non-sedentary men were analyzed separately (not shown).
Table 3The effect of haplotypes of Ala1330Val and Val667Met polymorphisms on BMD adjusted for BMI, lean body mass, smoking, drugs, and vitamin DHaplotypesAla1330ValCTVal667MetGAGApn1331312981BMDspine (g/cm2)0.008 ± 0.96−0.42 ± 0.65−0.09 ± 0.90−0.04 ± 1.60NSBMDhip (g/cm2)−0.002 ± 1.010.97 ± 1.48−0.0005 ± 0.95−0.04 ± 1.0NSBMDWB (g/cm2)0.02 ± 1.010.70 ± 0.59−0.10 ± 0.87−0.14 ± 1.04NSData are shown as the mean ± SD. No significant associations between BMD and haplotypes were found when sedentary and non-sedentary men were analyzed separately (not shown)
Discussion
We found a significant association between the Ala1330Val and the Val667Met polymorphisms in the LRP5 gene and BMD in young men, especially when physical activity was taken into account as confounder. This finding supports a number of recent papers reporting significant associations between polymorphisms in this gene and BMD [18, 19, 21, 22]. Some of these studies involved or were restricted to men [18, 22, 29, 30]. Moreover, Ferrari et al. [18] suggested that the polymorphisms in the LRP5 gene are associated with bone gain in prepubertal boys. Only one study [25] failed to demonstrate an association between variations in the LRP5 gene and BMD, but most likely this is due to their restricted sample size (n = 219).
Major disruption of the LRP5 gene (homozygous nonsense or frame-shift mutations) resulting in loss-of-function has been demonstrated to be responsible for the osteoporosis pseudoglioma syndrome [16]. Conversely, a number of dominantly inherited gain-of-function mutations result in high bone mass phenotypes [12–14] and autosomal dominant osteopetrosis type 1 [14]. Minor changes in this gene, i.e., frequently occurring polymorphisms, may also be of importance in the regulation of bone mass as suggested by the association studies mentioned above. Further support comes from mapping a quantitative trait locus for BMD to chromosome 11q12–13 in some [31, 32] but not all studies [7, 33, 34]. In a case-control study comprising 78 men with idiopathic osteoporosis and 86 controls, Ferrari et al. [23] found that Ala1330Val and Val667Met conferred an odds ratio for osteoporosis of 2.98 (95% CI: 1.03–8.81) and 3.27 (95% CI: 1.43–7.44), respectively. Also, a single prospective study [21] comprising 1,301 elderly Australian women demonstrated that the c.3357A > G (rs556442) polymorphism was associated with decreased BMD and increased incidence of fractures. This polymorphism located in exon 15 is synonymous but was found to be in linkage disequilibrium with Ala1330Val [21]. Similarly, Mizuguchi et al. [19] found a linkage equilibrium block comprising exons 7 to 18. In our study, no significant effect of Ala1330Val or Val667Met on BMD was found apart from the ANOVA analysis which suggested that the AA genotype at Val667Met was associated with low BMD of the hip and increased body height. The number of participants with this genotype, however, was low (n = 4) and we found no significant gene-dose effect to back this finding. In non-sedentary subjects, however, we found a significant association between Ala1330Val genotype and the Val667Met and Z-score BMD in the spine both in unadjusted analysis and when adjusted for BMI, smoking, continuous medication, and serum levels of 25-OH-D3. Further, we observed an association between Ala1330Val and whole-body BMD in the unadjusted analysis. This interaction between lifestyle (sedentary/non-sedentary), LRP5 genotype, and BMD has not previously been reported. Such an association is fully in line with the proposed role of LRP5 as a player in adapting bone to weight-bearing. LRP5 acts as a co-receptor for Wnt proteins and is expressed in the osteoblast and a number of other cell types. The Ala1330Val (exon 18) polymorphism results in a substitution of alanine with the chemically similar valine. No functional studies have been performed on this polymorphism; however, the polymorphism is located within the second LDL-repeat of LRP5 [22] and this region is involved in the ligand binding of the LDL-receptor [35]. Also, preliminary results have shown that drugs that inhibit Dkk binding to LRP5 increase bone formation in vitro [36] and LRP5 seems to be involved in the adaptive response of bone to mechanical load and may be partly responsible for the “mechanostat” [11]. It is presently unclear why association between the Ala1330Val and Val667Met genotypes and BMD was found only in the spine and not in the hip in the non-sedentary group. Differences between the skeletal sites regarding loading pattern (compression vs. bending) and bone architecture (trabecular vs. cortical) may be important.
In our study, the numbers of T-alleles and A-alleles of the Ala1330Val and Val667Met polymorphisms, respectively, were associated with a decrease in BMD of the lumbar spine of 0.2 SD in the non-sedentary participants (i.e., approximately 2.5%). Previous studies have assessed this impact to be from 1% [20] to 15% [18] in women and men and women, respectively. While a number of genes have been associated with bone mass in women (e.g., PTH [37], alpha-1 chain of type 1 collagen [10], vitamin D receptor [38], transforming growth factor beta-1 [39], interleukin-6 [40], and osteoprotegerin [41]), only a few genes have been shown to be associated with peak bone mass in men. We have previously reported that the MTHFR gene is associated with peak bone mass in the same series of men as reported here [42]. Each of these polymorphisms including the LRP5 gene only accounts for a fraction of the variability in BMD; however, the 2.5% impact suggested by our study is not trivial.
We found no association between either projected bone area at the spine or hip, or body weight and any of the polymorphisms; however, our data demonstrated a significant impact of the AA genotype of the Val667Met polymorphism and body height as previously reported by Ferrari et al. [18] and Koay et al. [29]. In our study, the number of subjects with the AA genotype was, however, very low (n = 4). Thus, this finding clearly needs confirmation in other study designs.
The allele frequencies found in our study corresponded closely to those reported in Dutch, Australian, Japanese, and Korean populations [19, 21, 22, 30].
Our study has some limitations. We only tested two polymorphisms in the LRP5 gene. We cannot, therefore, rule out the possibility that the associations found in our study could be due to other polymorphisms in the LRP5 gene or even in neighboring genes in linkage with the polymorphisms tested. Indeed, Bollerslev et al. [21] found that several of the polymorphisms in the LRP5 gene were in linkage disequilibrium with each other in Australian women. In our study, haplotype analyses were negative. This might be due to the limited number of participants. Testing large arrays of polymorphisms or haplotypes in relation to many outcome variables (i.e., bone mass at several skeletal sites), however, carry an increased risk of detecting spurious associations. Also, our study had a limited size. Given the observed genotype frequency and BMD values, our study had the power to detect differences in BMDof the spine between CC and CT + TT of 0.037 g/cm2 (alpha = 0.05 and beta = 0.80). Similarly, our study had the power to detect differences of 0.070 g/cm2 between GG and GA + AA in the overall analysis.
Our study also has several strengths. First, it was population-based. In contrast to most similar studies [18, 20] that recruited participants by advertisements, we recruited our participants by direct mailing on the basis of the National Personal Registry that includes all Danish citizens. This enabled us to account meticulously for the recruitment of our participants. Thus, our sample was not skewed with respect to key socio-economic parameters [24]. Second, our population had a relatively homogeneous genetic background both due to a small influx to our population and since participants with a first- or second-generation immigrant background were excluded on the basis on data from our national registry. Third, we selected young men aged 20–30 years since these were expected to have reached their peak bone mass. This was confirmed by our data demonstrating an infinitesimal decrease in BMD by age. Moreover, very few were afflicted with chronic diseases or had long-term exposure of environmental factors that may affect bone mass.
We conclude that the Ala1330Val (exon 18) and Val667Met (exon 9) polymorphisms of the LRP5 gene are significantly associated with lumbar spine peak bone mass in physically active men, potentially accounting for 2.5% of BMD in the spine. This gene-environment interaction provides support for LRP5 as a mediator of load-induced bone formation and suggests that this gene is involved in the pathogenesis of osteoporosis in men.