Transretinal degeneration in ageing human retina: a multiphoton microscopy analysis.

AIM: Retinal cell remodelling has been reported as a consistent feature of ageing. However, the degree to which this results in transretinal degeneration is unclear. To address this, the authors used multiphoton microscopy to quantify retinal degeneration in post-mortem human eyes of two age groups. METHODS: Retinas from six young subjects (18-33 years old) and six older subjects (74-90 years old) were prepared as wholemount preparations. All retinas were stained with 4,6-diamidino-2-phenylindole and imaged by multiphoton confocal microscopy to quantify neuron densities in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL) and outer nuclear layer (ONL). Neurons were counted using automated cell identification algorithms. All retinas were imaged hydrated to minimise tissue artefacts. RESULTS: In both groups, 56% of the area within the central 4 mm eccentricity and 27% of the area with eccentricity between 4 mm and 7 mm were imaged. Compared with young subjects, the peak RGCL neuron loss in the aged subjects (25.5%) was at 1 mm eccentricity. INL and ONL neuron densities significantly decreased at 1-2 mm eccentricity (8.7%) and 0.5-4 mm eccentricity (15.6%) respectively (P <0.05). The reduction in neuron density in the INL corresponded, spatially, to the region with the greatest neuron loss in the RGCL and ONL. CONCLUSIONS: This is the first study to correlate neurodegeneration in different populations of cells in the ageing retinas. These data confirm that the greatest neuronal loss occurs in the RGCL and ONL in human ageing retinas, whereas the INL is relatively preserved.

Topography of neuron loss in the retinal ganglion cell layer in human glaucoma.

AIM: To determine if retinal ganglion cell (RGC) loss influences the loss of surrounding RGCs to generate clustered patterns of cell death in human glaucoma. It is hypothesised that retinal ganglion cell loss accelerates the loss of surrounding cells to generate, at a local, cellular scale, clustered patterns of retinal of RGC death. The absence of these interactions would result in a diffuse pattern RGC loss. METHOD: Six glaucomatous retinas (67-83 years old) and six age-matched control retinas (61-89 years old) were prepared as wholemounts and stained by 4',6-diamidino-2-phenylindole (DAPI) solution (3 microg/ml in PBS). An area corresponding to central 14 degrees of the visual field was imaged. The nearest-neighbour distribution was determined for cells in both normal and glaucomatous RGCL. RESULTS: Clustered RGC loss in human glaucoma was observed on a background of diffuse loss. The mean nearest-neighbour distance (NND) of the glaucomatous retinas was significantly higher than with controls (p<0.001). The distribution of NND in glaucomatous retinas was skewed to the higher values with a higher positive kurtosis relative to controls. The quantitative analysis of the pattern of cell loss is supported by the visual inspection of the patterns of cell loss. DISCUSSION: The nearest-neighbour analysis is consistent with the presence of two patterns of cell loss in the RGCL in glaucoma. While the diffuse of cell loss can account for an overall reduction in the RGC population, an additional non-random pattern is consistent with the hypothesis that RGC loss has a local influence on the viability of surrounding cells.

The effects of hormone replacement therapy on cortical bone in postmenopausal women. A histomorphometric study.

Investigations of the actions of estrogen on the skeleton have mainly focused on cancellous bone and there are no reported histomorphometric studies of the effects of oestrogen on cortical bone in humans. The aim of this study was to investigate the effects of both conventional hormone replacement therapy (HRT) and high-dose oestradiol on cortical bone in postmenopausal women. Transiliac biopsies were obtained from nine postmenopausal women aged 54-71 yr before and after 2 yr (mean, 23.5 months) of conventional HRT and in seven postmenopausal women aged 52-67 yr after long-term, high-dose oestradiol implant therapy (at least 14 yr). Indices of bone turnover, remodeling, and cortical structure were assessed by image analysis. Cortical width was highest in the women treated with high-dose oestrogen therapy (2.29 +/- 0.78 mm; mean +/- SD) and lowest in untreated women (1.36 +/- 0.60 mm; P=0.014). The proportion of canals with an eroded surface was significantly lower in the high-dose oestrogen group than in women before or after conventional HRT (3.03 +/- 3.7% vs. 11.1 +/- 7.1% and 10.5 +/- 8.6%; P=0.017 and 0.05, respectively). Bone formation rate (microm2/microm/day) in untreated women was significantly higher than in the high-dose oestrogen group (0.121 +/- 0.072 vs. 0.066 +/- 0.045, respectively; P=0.05), values in women treated with conventional HRT being intermediate. Our results provide the first histomorphometric evidence in postmenopausal women of dose-dependent oestrogen-induced suppression of bone turnover in iliac crest cortical bone. There was also a trend toward higher wall width with increasing dose of oestrogen, consistent with the previously reported anabolic effect in cancellous bone.

Mechanism of bone loss after liver transplantation: A histomorphometric analysis.

Organ transplantation is associated with increased bone loss and high fracture risk, but the pathophysiological mechanisms responsible have not been established. We have performed a histomorphometric analysis of bone remodeling before and 3 months after liver transplantation in 21 patients (14 male, 7 female) aged 38-68 years with chronic liver disease. Eight-micrometer undecalcified sections of trans-iliac biopsies were assessed using image analysis. Preoperatively, bone turnover was low with a tendency toward reduced wall width and erosion depth. The bone formation rate increased from 0.021 +/- 0.016 (mean +/- SD) to 0.067 +/- 0.055 microm2/microm/day after transplantation (p < 0.0002) and activation frequency from 0.24 +/- 0.21/year-1 to 0.81 +/- 0. 67/year-1 (p < 0.0001). No significant change was observed in wall width, but there was a trend toward an increase in indices of resorption cavity size. There was a small increase in osteoid seam width postoperatively (p< 0.02) and decrease in mineralization lag time (p < 0.001). No significant changes in indices of cancellous bone structure were observed in the postoperative biopsies. These results demonstrate a highly significant and quantitatively large increase in bone turnover in the first 3 months after liver transplantation. Although no significant disruption of cancellous bone structure was demonstrated during the time course of the study, the observed changes in bone remodeling predispose to trabecular penetration and may thus result in long-term adverse effects on bone strength.

Regional differences in cortical porosity in the fractured femoral neck.

Although bone mass is a contributory risk factor for intracapsular hip fracture, its distribution and porosity within the femoral neck is also important for bone strength. In femoral neck biopsies from 13 women with intracapsular hip fracture (mean +/- SEM: 75.4+/-2.1 years, OP) and 19 cadaveric samples (9 men and 10 women [control], aged 79.4+/-1.7 years), a segmental analysis was used to quantify circumferential variations in the characteristics of cortical bone haversian systems. In female control femoral necks, there was an increasing porosity gradient between the inferior (I) (7.7+/-0.6%) and superior regions (S) (16.05+/-1.8%,p < 0.005). In walking, these regions undergo compression and tension, respectively. In men, a similar trend was observed, but the differences were not significant (I: 11.1+/-1.2%; S: 14.1+/-1.7%; p = 0.133). This porosity gradient was not maintained in the fracture group (I: 10+/-1%; S: 12.65+/-1.2%). In contrast, porosity in the fracture group was greatest in the anterior cortex, being 41% higher in that quadrant than in controls (p = 0.06). The areal density of haversian canals ranged from 16.7 to 21.3 canals/mm2 with no significant differences between fractures and controls. In the control women, mean canal diameter was highest in the superior region (60+/-2.8 microm), and these canals were significantly larger than those in the inferior region (49.4+/-1.4 microm, p < 0.05). This difference was less marked in the fracture cases (I: 53.21+/-2.5 microm; S: 59.1+/-2.8 microm; p = 0.0878). Although the mean canal diameter in the anterior quadrant of the fracture cases was higher than in the control women this did not reach significance (OP/F: 59.5+/-3 microm; control/F: 52.7+/-2.6 microm; p = 0.106). However, the proportion of "giant" canals with diameters >385 microm (defined as the top 0.5% in the controls) was doubled in the anterior region of the fracture cases (OP/F: 1.28%; control/F: 0.69%; p < 0.005). Porosity is related to the square of the canal radius; therefore, such canals make a substantial contribution to cortical porosity. Previous work has shown that the elastic modulus of bone decreases approximately as the square root of porosity. Therefore, the increased porosity and the higher prevalence of "giant" canals have a markedly negative influence on the ability of the cortical shell to withstand stresses associated with a fall. The mechanisms responsible for the localized generation of "giant" haversian canals, and ultimately the "trabecularization" of the cortex, require further investigation.

Structure of the femoral neck in hip fracture: cortical bone loss in the inferoanterior to superoposterior axis.

Although bone mass is a contributory risk factor for hip fracture, its distribution about the femoral neck is also important. Femoral neck biopsies were obtained from 13 females with intracapsular hip fracture (fracture: mean age 74.3 +/- 2.3 years [SEM]) and 19 cadaveric samples (control: 9 males and 10 females 79.4 +/- 1.7 years) and the areas of cortical and cancellous bone were quantitated in octants. In the control group, although males had larger bones than females, the proportions of cortical and cancellous bone were not different (p > 0.05) between the genders. The total amount of bone, as a proportion of bone + marrow, was significantly reduced in the fractures compared with the female controls (%Tt.Ar: fracture 27.83 +/- 1.18, female control 33.62 +/- 1.47; p = 0.0054). Reductions in cortical bone area occurred in all regions but particularly in the inferior, inferoanterior, and anterior octants (p < 0.05). There were no differences between cases and controls in the regional amount of cancellous bone (all regions, p > 0.178). Marked reductions in mean cortical bone width between the fracture and female control group occurred in the anterior, inferoanterior (31%), and superoposterior (25%) regions. Representing cortical widths as simple Fourier functions of the angle about the center of area (R2adj = 0.79) showed in the cases that there was preservation of the cortical bone in the inferior region, with the proportional loss of cortical bone being greatest in the inferoanterior and superoposterior regions. It is concluded that loss of cortical, rather than cancellous, bone predominates in cases of femoral neck fracture. This loss occurs primarily along the inferoanterior to superoposterior axis. As this axis bears the greatest strain during a fall, it is hypothesized that specific thinning of the cortex in these regions leads to an exaggerated propensity to fracture in those so affected, above that resulting from an equivalent general decrease in bone mass.

Cortical remodeling following suppression of endogenous estrogen with analogs of gonadotrophin releasing hormone.

The effects of estrogen suppression on osteonal remodeling in young women was investigated using transiliac biopsies (eight paired biopsies + four single pre; three single post biopsies) taken before and after treatment for endometriosis (6 months) with analogs of gonadotrophin releasing hormone (GnRH). Estrogen withdrawal increased the proportion of Haversian canals with an eroded surface (106%, p = 0.047), a double label (238%, p = 0.004), osteoid (71%, p = 0.002), and alkaline phosphatase (ALP) 116%, p = 0.043) but not those showing tartrate-resistant acid phosphatase (TRAP) activity (p = 0.25) or a single label (p = 0.30). Estrogen withdrawal increased TRAP activity in individual osteoclasts in canals with diameters greater than 50 microns (p = 0.0089) and also the number of osteons with diameters over 250 microns (p = 0.049). ALP activity in individual osteoblasts was increased but not significantly following treatment (p = 0.051). Wall thickness was significantly correlated with osteon diameter (p < 0.001). In a separate group of patients (four pairs + one post biopsy) on concurrent treatment with tibolone, there was no significant increase in the osteon density, cortical porosity, median canal diameter, or the markers of bone formation and resorption. Enzyme activities and numbers of active canals were also not increased with the concurrent treatment, but there was still an increase in the osteon diameter. As previously shown for cancellous bone, estrogen withdrawal increased cortical bone turnover. We have now shown that resorption depth within Haversian systems was also increased with treatment. The enhanced TRAP activity in individual osteoclasts supports the concept that osteoclasts are more active following estrogen withdrawal in agreement with theoretical arguments advanced previously. Understanding the cellular and biochemical mechanisms responsible for increased depth of osteoclast resorption when estrogen is withdrawn may allow the development of new strategies for preventing postmenopausal bone loss.

Cortical and cancellous bone in the human femoral neck: evaluation of an interactive image analysis system.

An interactive image analysis package was developed to examine whole cross-sections from the femoral neck. The package quantifies cortical width (Ct.Wi), cortical porosity (Ct.Po), and proportions of cortical, cancellous bone as a percentage of bone plus marrow area. Segmental analysis was used to quantify circumferential variations in bone distribution within the femoral cross-section. To evaluate reproducibility of data four independent operators analyzed previously prepared femoral neck sections from a 2000 BC population. Differences in total and circumferential distributions of cortical and cancellous bone with respect to gender and age of samples were demonstrated. Reproducibility was assessed using coefficients of variation (CV). Analysis of sections using a variable magnification, giving largest possible image size, rather than a set magnification reduced variation between operators for all measurements. Use of a calculated threshold significantly decreased variation between operators for the proportions of cortical and cancellous bone (p < or = 0.026). Dividing the image into 8 rather than 16 segments also improved reproducibility. There was little agreement between operators in the determination of cortical porosity. The mean CV for the other quantitative indices such as cortical width and proportions of cortical and cancellous bone ranged from 4.87% to 13.52%. The genders showed similar patterns in circumferential distribution of bone. Cortical width was significantly greater in the inferior region compared to the other areas, whereas percent cortical bone was lowest at the superior region. The center of mass (COM) for the younger age group was located anteriorly, whereas in the older samples the COM was located posteriorly of the center of area (p = 0.041). Basic data relating to cortical and cancellous bone of acceptable reproducibility in comparison with current standards in iliac histomorphometry can now be provided at modest cost in operator time and resources.

Effects of hormone replacement therapy on cancellous bone microstructure in postmenopausal women.

Menopausal bone loss is associated with disruption of cancellous bone architecture which has adverse mechanical effects and is believed to be irreversible. The aim of this study was to examine the effects of long-term hormone replacement therapy on cancellous bone structure in women with postmenopausal osteoporosis. Iliac crest biopsies from 22 women with osteopenia or osteoporosis were obtained before and after hormone replacement therapy (mean duration 23.5 months). Cancellous bone architecture was assessed by strut analysis, trabecular bone pattern factor, and marrow star volume. Post-treatment biopsies showed no significant changes in any of the structural indices assessed. Our results suggest that hormone replacement therapy preserves existing cancellous bone structure but provide no evidence that this treatment is able to reverse structural disruption in women with postmenopausal osteopenia or osteoporosis.

Assessment of cancellous bone structure: comparison of strut analysis, trabecular bone pattern factor, and marrow space star volume.

Cancellous bone architecture is an important determinant of bone strength. Recently, several approaches to the assessment of bone structure in histological sections have been described; however, no comparative studies of these different methods have been reported. We have developed computerized methods for the simultaneous assessment of several indices of bone structure, including trabecular bone pattern factor, marrow space star volume, node-to-terminus ratio, trabecular number, and trabecular separation. The relationships between these variables has been examined in iliac crest cancellous bone obtained from 41 healthy subjects, 17 male and 24 female, aged 20-80 years (mean 47.9 years). All structural indices assessed showed significant correlations with cancellous bone area (p < 0.0001). Values for trabecular bone pattern factor and marrow space volume were highly correlated (r = 0.789; p < 0.0001). A comparison of indices obtained by strut analysis with trabecular bone pattern factor and marrow space star volume also revealed significant relationships, especially for the terminus-to-terminus strut length (r = 0.704 and r = 0.634, respectively; p < 0.0001) and node to terminus ratio (r = -0.947 and r = -0.788, respectively; p < 0.0001). The node-to-terminus ratio and trabecular bone pattern factor showed significant relationships with age which were independent of sex, cancellous bone area and trabecular width (p < 0.01 and p < 0.005, respectively). Our results demonstrate strong correlations between the different two-dimensional indices of bone structure in cancellous bone from healthy subjects. Trabecular penetration is likely to be an important determinant of all these variables, which may therefore reflect connectivity; however, direct comparison of these methods with three-dimensional techniques is required to establish their true relationship to bone structure.

The effects of gonadotrophin-releasing hormone agonists on iliac crest cancellous bone structure in women with endometriosis.

The administration of gonadotrophin-releasing hormone (GnRH) analogs to premenopausal women causes hypoestrogenism and bone loss, but the effects on cancellous microstructure have not been determined. In this study we have assessed bone structure in transiliac biopsies obtained from women before and after treatment for endometriosis with GnRH analogs. Twenty-one premenopausal women were studied, paired biopsies being obtained in 13; five women received both GnRH analogs and Org OD 14 (Tibolone, Livial). Comparison of pre- and post-treatment biopsies in women treated only with GnRH analogs showed a reduction in indices related to connectivity (node-to-terminus ratio, node-to-loop strut length, p < 0.02) and increase in inversely related indices (terminus-to-terminus and node-to-terminus strut length, p < 0.03). No significant changes were seen in any of the structural indices in women receiving both GnRH and Org OD 14 therapy. Activation frequency and bone formation rate at tissue level increased in women treated with GnRH agonists alone, although this change was not statistically significant. Our results suggest that bone loss induced by GnRH analogs may be associated with adverse effects on cancellous microstructure which are unlikely to be reversed following cessation of therapy. Concurrent treatment with Org OD 14 appears to prevent these changes.

Structural mechanisms of trabecular bone loss in primary osteoporosis: specific disease mechanism or early ageing?

Osteoporosis is characterised by reduced bone mass and disruption of cancellous bone architecture; however, it is unknown whether these changes arise from a specific disease process or represent one extreme of physiological bone loss. We have quantitatively assessed cancellous structure in 35 patients with primary osteoporosis and 41 normal subjects. Cancellous microstructure was assessed by computerised strut analysis and by calculation of trabecular width, separation and number. Node to terminus ratio, node to node and node to loop strut length were significantly decreased in patients with osteoporosis when compared to normal subjects (P < 0.001), whereas terminus count and terminus to terminus strut length were significantly increased (P < 0.001). When two subgroups were matched for age these differences remained highly significant (P < 0.005). However, when two subgroups were matched for cancellous area, no significant differences were observed in any of the structural indices except terminus count (P < 0.05). Mean trabecular width and number were significantly lower and trabecular separation significantly higher in the patients with osteoporosis before and after age-matching but their differences disappeared after matching for cancellous area. Multiple regression analysis confirmed highly significant correlations between cancellous bone area and structural indices after adjustment for age, sex and disease status (P < 0.001). Our data demonstrate that for a given cancellous area, structural changes in primary osteoporosis are similar to those observed during age-related bone loss in normal subjects. These findings support the hypothesis that primary osteoporosis is the result of greater biological ageing rather than a specific disease process and are consistent with evidence from other sources that low bone mass is associated with increased mortality from diseases unrelated to osteoporosis.

Bone turnover in non-steroid treated rheumatoid arthritis.

OBJECTIVE: To examine whether changes in cancellous bone turnover and resorption cavity depth contribute to bone loss in patients with non-steroid treated rheumatoid arthritis. METHODS: Iliac crest biopsies were obtained from 37 patients with non-steroid treated rheumatoid arthritis, 13 male and 24 female, aged 37-71 years. Bone turnover and resorption cavity characteristics were quantitatively assessed using semiautomated computerised techniques. RESULTS: When compared with age- and sex-matched control values, there was a significant reduction in bone formation rate at tissue level and activation frequency (P < 0.001) in the patient group. The eroded perimeter, mean and maximum eroded depth and cavity area were also significantly reduced (P < 0.01, < 0.005, < 0.01 and < 0.005 respectively). CONCLUSION: These results demonstrate low bone turnover in non-steroid treated rheumatoid arthritis and indicate that the reduced bone mass in these patients is due mainly to a negative remodelling balance.

Effect of long-term tamoxifen therapy on cancellous bone remodeling and structure in women with breast cancer.

The effects of long-term tamoxifen therapy on bone remodeling were studied in 41 women with breast cancer, 22 treated with tamoxifen for a minimum of 15 months (mean 33) and 19 untreated. Transiliac crest bone biopsies were obtained and a comprehensive histomorphometric analysis performed using a semiautomatic image analysis system. There were no statistically significant differences between the two groups in bone area, osteoid perimeter and area, or osteoid width. Mineral appositional rate, adjusted appositional rate, and mineralization lag time were also similar in the two groups; however, tissue-based bone formation rate was significantly lower in the tamoxifen-treated women (p = 0.05) and the remodeling period significantly longer (p < 0.05). Mean and maximum resorption cavity depth and cavity area were significantly reduced in the tamoxifen-treated patients compared to the untreated patients (p < 0.01, p < 0.01, and p < 0.03, respectively). Calculated and directly measured indices of cancellous bone structure were similar in the two groups, although the data indicated a trend toward greater connectedness in the tamoxifen-treated group. These data indicate that tamoxifen does not exert an antiestrogenic effect on bone remodeling in the human and are consistent with a weak estrogenic effect.

Reduced bone formation in patients with osteoporosis associated with inflammatory bowel disease.

The pathophysiology of bone loss associated with inflammatory bowel disease has not been clearly defined. In this study we have performed a detailed histomorphometric analysis of iliac crest bone obtained from 19 patients with inflammatory bowel disease in whom a diagnosis of osteoporosis had been made. Eleven subjects were receiving prednisolone at the time of their biopsy. Comparison with control values demonstrated a highly significant reduction in trabecular bone area in the patient group (p < 0.001). Wall width, adjusted appositional rate and bone formation rate were all significantly reduced in the patient group (p < 0.001) and the formation period was significantly increased (p < 0.001). Resorption cavities were slightly smaller in the patient group, differences in maximum cavity depth and cavity length achieving statistical significance (p < 0.005 and p < 0.05 respectively). The mineral appositional rate was significantly reduced in the patients with inflammatory bowel disease (p < 0.001) and the mineralization lag time significantly increased (p < 0.001); however, osteoid area, perimeter and seam width were not significantly different from controls. These results demonstrate that osteoporosis associated with inflammatory bowel disease is characterized by reduced bone formation at the cellular and tissue level; the proportionately greater change in wall width than in resorption cavity depth is consistent with a negative remodelling balance. Although none of the patients had osteomalacia as defined by the criteria of increased osteoid seam width and mineralization lag time, the higher mineralization lag time in the patient group indicates a mild mineralization defect.

Bone remodeling in hip fracture.

Hip fracture incidence has shown strong upward secular trends in many societies with wide differences in age adjusted incidence between nations. Falls and reduced physical activity have emerged as the strongest risk factors in epidemiological studies, while clinical investigations have pointed to secondary hyperparathyroidism as an important candidate cause of the loss of femoral cortical bone in old age. Until recently there have been few studies performed directly on the region of interest in the proximal femur. Non-invasive methodology using 85Sr has now been developed by our group for measuring bone formation and (with concurrent serial DXA densitometry) resorption in the femoral neck. Bone turnover averaged about 8% annually in controls. A group of younger cases of femoral fracture showed similar indices of total and regional bone formation to a control group; but their resorption was higher. To further investigate this, a femoral neck bone biopsy technique has been developed which can be applied to fracture cases treated by arthroplasty. Preliminary studies have established that the anatomical asymmetry of the neck in cross-section is considerable and imposes restraints on the interpretation of smaller or incomplete femur biopsies. Prospects are quite good that, in the absence of tetracycline pre-labeling, mineralization can be studied by assessment of alkaline phosphatase-positive surfaces in cryostat sections. Moreover, such sections will permit study of other anatomically localized metabolic activities as well as antigen expression and osteocyte viability. Candidate mechanisms for the regional decline in bone quality as well as bone mass in subjects suffering hip fracture can now be investigated more effectively.

Epithelial--stromal interactions in tumors. A morphologic study of fibroepithelial tumors of the breast.

BACKGROUND AND METHODS: The known spatial interaction between normal breast epithelium and its surrounding stroma prompted an investigation of the spatial relationship between stromal mitoses and the epithelial component of fibroepithelial tumors of the breast. The authors applied a novel computerized morphometric technique to routinely processed histologic sections of 23 fibroepithelial tumors (13 fibroadenomas and 10 phyllodes tumors). The proportional area of epithelium in successive concentric annuli surrounding stromal mitoses was measured, and its distribution was compared with that around suitable control points. RESULTS: The authors found that stromal mitotic activity in these tumors was significantly more likely to occur close to rather than remote from the epithelial component, with a significant excess of epithelium around mitoses compared with control points within a range of 79 microns. Essentially similar findings were obtained when randomly identified fibroblast nuclei were used as control points, thus obviating variations in stromal cell density with distance from epithelium as an explanation for the findings. CONCLUSIONS: These findings support the hypothesis that stromal growth in fibroepithelial tumors depends, to a variable extent, on the epithelial component. An interaction in the opposite direction (i.e., the stroma providing the growth support to the epithelium) also may occur, but this was not investigated. It is suggested that there is an interdependence of growth between the epithelial and stromal components in these tumors that explains their complex morphology and that stromal dependence on epithelium is lost with increasing malignancy of the stromal elements.