The role of physical activity in the management of impaired glucose tolerance : a systematic review Although physical activity is widely reported to reduce the risk of type 2 diabetes in individuals with prediabetes , few studies have examined this issue independently of other lifestyle modifications . The aim of this review is to conduct a systematic review of controlled trials to determine the independent effect of exercise on glucose levels and risk of type 2 diabetes in people with prediabetes -LRB- IGT and/or IFG -RRB- . A detailed search of MEDLINE -LRB- 1966 -- 2006 -RRB- and EMBASE -LRB- 1980 -- 2006 -RRB- found 279 potentially relevant studies , eight of which met the inclusion criteria for this review . All eight studies were controlled trials in individuals with impaired glucose tolerance . Seven studies used a multi-component lifestyle intervention that included exercise , diet and weight loss goals and one used a structured exercise training intervention . Four studies used the incidence of diabetes over the course of the study as an outcome variable and four relied on 2-h plasma glucose as an outcome measure . In the four studies that measured the incidence of diabetes as an outcome , the risk of diabetes was reduced by approximately 50 % -LRB- range 42 -- 63 % -RRB- ; as these studies reported only small changes in physical activity levels , the reduced risk of diabetes is likely to be attributable to factors other than physical activity . In the remaining four studies , only one reported significant improvements in 2-h plasma glucose even though all but one reported small to moderate increases in maximal oxygen uptake . These results indicate that the contribution of physical activity independent of dietary or weight loss changes to the prevention of type 2 diabetes in people with prediabetes is equivocal . Introduction Given the growing prevalence of diabetes and the high economic cost of treating the condition and its comorbities , it is important to find effective ways of targeting those who are most at risk of developing the disease -LSB- 1 -RSB- . Prediabetes is the collective term for people with IGT and/or IFG -LSB- 2 -RSB- . Prediabetes is associated with an increased risk of development of type 2 diabetes -LSB- 3 -RSB- and cardiovascular disease -LSB- 4 -- 6 -RSB- . There is good evidence from cross-sectional and longitudinal studies for a link between levels of physical activity and the risk of type 2 diabetes -LSB- 7 -- 9 -RSB- . However , evidence from intervention studies in high-risk populations is limited , making it difficult to quantify the effectiveness of physical activity in reducing the risk of type 2 diabetes in individuals with prediabetes . Lifestyle intervention studies that have encouraged weight loss through a combination of dietary change and increased physical activity have reduced the risk of type 2 diabetes in individuals with IGT -LSB- 10 -- 14 -RSB- . However , because physical activity was not usually analysed independently of other variables , such as weight loss , it is difficult to determine the effectiveness of physical activity at protecting against the risk of diabetes in individuals with prediabetes . Therefore , the aim of this systematic review is to establish the effectiveness of physical activity independent of other variables at reducing the risk of diabetes or improving glucose parameters in people with prediabetes . Materials and methods Search strategy MEDLINE -LRB- 1966 to February week 4 , 2006 -RRB- and EMBASE -LRB- 1980 to week 8 , 2006 -RRB- were searched for articles examining the effect of an exercise or lifestyle intervention on individuals with prediabetes . The search was carried out using medical search headings -LRB- MeSH -RRB- and by searching titles and abstracts for relevant words . For example , studies including individuals with prediabetes were found by using the MeSH ` prediabetic state , ' ` insulin resistance , ' ` glucose intolerance ' and ` diabetes mellitus ' -LRB- subheading ` prevention and control ' -RRB- , and by searching titles and abstracts for ` prediabetes , ' ` impaired glucose tolerance , ' ` IGT , ' ` impaired fasting glucose ' and ` IFG . ' Studies that included an exercise intervention were found by using the MeSH ` lifestyle , ' ` sports , ' ` exercise therapy ' and ` physical fitness , ' and by searching titles and abstracts for ` exercise , ' ` physical activity , ' ` physical fitness , ' ` resistance training , ' ` strength training , ' ` circuit training , ' ` endurance training ' and ` aerobic training . ' In addition , the reference lists of relevant published original articles and reviews were hand-searched . One reviewer -LRB- T. Yates -RRB- performed the electronic and hand-searches and reviewed the results . Studies that clearly did not meet the inclusion criteria were rejected during the initial review . Where uncertainty existed , the full text of the article was obtained and reviewed . Two reviewers -LRB- T. Yates and K. Khunti -RRB- independently assessed all potentially relevant studies and performed data extraction . Disagreement was resolved by discussion and , where necessary , third party adjudication . Subjects Participants were adults -LRB- age ≥ 18 years -RRB- diagnosed with prediabetes . Prediabetes was defined as IGT and/or IFG using one of the sets of criteria previously recommended by the WHO -LSB- 15 , 16 -RSB- or the American Diabetes Association -LRB- ADA -RRB- -LSB- 17 , 18 -RSB- . Studies that defined IGT or IFG using other criteria were included if the mean value of the participants ' plasma glucose fell within the range of IGT or IFG as defined by the WHO or ADA criteria -LRB- 2-h plasma glucose ≥ 7.8 mmol/l and < 11.1 mmol/l , and fasting glucose < 7.8 mmol/l for IGT ; 2-h plasma glucose < 7.8 mmol/l , and fasting glucose ≥ 5.6 mmol/l and < 7.0 mmol/l for IFG -RRB- . Interventions Interventions that included an exercise programme were included . ` Exercise programme ' was taken to mean any intervention that actively promoted and supported physical activity or a structured exercise training regimen . Studies that only provided individuals with brief written or verbal physical activity advice were excluded . Studies investigating the effect of a single or acute episode of exercise were also excluded . Outcome measures Only studies with an outcome measure of physical activity and a relevant clinical measure were included . A relevant clinical measure was defined as progression to diabetes or a suitable measure of plasma glucose -LRB- 2-h plasma glucose for IGT , or fasting glucose for IFG -RRB- . Type of study Randomised and non-randomised controlled trials were included . Analysis As the heterogeneity of the type of exercise interventions and outcome measures did not lend itself to quantitative methods of analysis , a systematic narrative review was undertaken . Baseline and follow-up exercise , body mass and glucose parameters were reported using mean ± SEM , or median -LRB- interquartile range -RRB- . Results reporting the SD or the 95 % CI were converted to SEM using the formulas -LRB- where t is the t distribution value for a 95 % CI -RRB- , respectively . Where the SEM , SD or 95 % CI for the change from baseline to follow-up was not reported , only the mean value is reported because of the potential error involved in calculating SEM for this figure . When available , the relative risk of diabetes in the intervention group compared with the control group was also reported . Results The search produced 307 hits , from which 279 potential studies were identified , of these , eight trials met the criteria for inclusion -LRB- see Fig. 1 -RRB- . Study details for the eight included studies are shown in Table 1 ; the main outcomes are presented in Table 2 . Fig. 1Flow diagram of the literature search . Duplicates : where several studies reported on the same trial and cohort , only one published study for each trial was included for the purposes of this review ; the included studies were those that reported on the cohort as a whole , had relevant follow-up measures and included the most recently published data . Where a relevant study was identified in more than one publication , the study was included only onceTable 1Characteristics of included studiesAuthors , yearStudy location/nameStudy designIntervention durationNumber of subjects -LRB- men/women -RRB- Inclusion criteriaType of interventionType of dietary interventionType of exercise interventionMethod of physical activity measurementLindström et al. 2003 -LSB- 21 -RSB- Finland/Finnish Diabetes Prevention StudyRCT3 years522 -LRB- 172/350 -RRB- IGT -LRB- WHO criteria , 1985 -RRB- , age 40 -- 64 years , BMI ≥ 25 kg/m2Exercise and dietWeight reduction through a healthy dietParticipants individually encouraged to increase their overall level of physical activity . Circuit-type exercise sessions were also offered.Self-report -- Kuopio 12 month leisure time physical activity questionnaireKnowler et al. 2002 -LSB- 12 -RSB- USA/Diabetes Prevention Research GroupRCTAverage follow-up 2.8 -LRB- range 1.8 -- 4.6 -RRB- years2 ,161 -LRB- 680/1481 -RRB- aIGT -LRB- ADA criteria , 1997 -RRB- , age ≥ 25 years , BMI ≥ 24 kg/m2 -LRB- ≥ 22 kg/m2 if Asian -RRB- , fasting plasma glucose ≥ 5.3 mmol/lExercise and dietWeight reduction through a healthy , low-energy , low-fat dietParticipants individually encouraged to accumulate at least 150 min/week of moderate intensity exercise.Self-report -- Modified Activity Questionnaire and activity logPan et al. 1997 -LSB- 10 -RSB- China/The Da Qing IGT and Diabetes Study RCT6 years530 -LRB- 283/247 -RRB- IGT -LRB- WHO criteria , 1985 -RRB- , age ≥ 25 years1 . Exercise 2 . Exercise and dietWeight maintenance for those with a BMI < 25 kg/m2 through a healthy energy-balanced diet ; weight reduction for those with a BMI ≥ 25 kg/m2 through reduced energy intakeParticipants were encouraged to increase their physical activity to 1 unit per dayb . Those who were aged < 50 years and were able to were encouraged to accumulate 2 units per day.Self-report -- type not reportedEriksson and Lindgärde , 1991 -LSB- 24 -RSB- Sweden/The 6-year Malmö feasibility studyNon-randomised controlled trial5 years260 -LRB- 260/0 -RRB- IGT -LRB- 2-h post-challenge glucose values 7 -- 11 mmol/l , and fasting plasma glucose < 7.8 mmol/l -RRB- Exercise and dietHealthy dietary advice givenParticipants were encouraged to increase their physical activity levels . Participants given the option of training in organised groups for a 6-month period in the first year.VO2max -- heart rate response to submaximal workload using bicycle ergometerOldroyd et al. 2006 -LSB- 19 -RSB- EnglandRCT2 years69 -LRB- 39/30 -RRB- IGT -LRB- WHO criteria , 1985 -RRB- Exercise and dietWeight reduction through a healthy , low-energy , low-fat dietParticipants were encouraged to undertake 20 -- 30 min of aerobic activity 2 -- 3 days/week . In addition , all participants were given a discount at local gyms.Cardiovascular fitness -- shuttle testSelf-report -- type not reportedMensink et al. 2003 -LSB- 22 -RSB- Netherlands/Study on lifestyle-intervention and impaired glucose tolerance MaastrichtRCT2 years114 -LRB- 64/50 -RRB- IGT -LRB- 2-h post-challenge glucose values 7.8 -- 12.5 mmol/l , and fasting plasma glucose < 7.8 mmol/l -RRB- , age > 40 years , BMI ≥ 25.0 kg/m2Exercise and dietWeight reduction through a healthy , low-energy , low-fat dietParticipants were encouraged through goal setting to undertake 30 min of moderate intensity exercise per day and were given use of free exercise classes.VO2max -- incremental exhaustive test using bicycle ergometerLindahl et al. 1999 -LSB- 23 -RSB- SwedenRCT1 year186 -LRB- 69/117 -RRB- IGT -LRB- WHO criteria , 1985 -RRB- , age 30 -- 60 years , BMI ≥ 27 kg/m2Exercise and dietWeight reduction through a healthy , low-energy , low-fat diet.Participants were encouraged to increase their physical activity . Supervised exercise sessions were available in the first monthVO2max -- heart rate response to submaximal and maximal workload using bicycle ergometerCarr et al. 2005 -LSB- 20 -RSB- USARCT2 years62 -LRB- 29/33 -RRB- IGT -LRB- WHO criteria , 1998 -RRB- Structured exercise and dietParticipants were encouraged to follow the energy-balanced American Heart Foundation Step 2 dietWalking/jogging at > 70 % of heart rate reserve for 1 h on 3 days/weekVO2max -- modified branching treadmill protocolVO2max , maximal oxygen consumptionaData for lifestyle and control groups onlyb1 unit = 30 min of mild exercise , or 20 min of moderate exercise , or 10 min of strenuous exercise , or 5 min of very strenuous exerciseTable 2Baseline values and change in main outcomesAuthors , yearBaseline VO2max -LRB- l/min -RRB- Change in VO2max -LRB- l/min -RRB- aBaseline self-reported physical activity levelsChange in self-reported leisure time physical activityaBaseline body mass -LRB- kg -RRB- Change in body mass -LRB- kg -RRB- aBaseline 2-h plasma glucose -LRB- mmol/l -RRB- Change in 2-h plasma glucose from baseline -LRB- mmol/l -RRB- a Baseline fasting glucose -LRB- mmol/l -RRB- Change in fasting plasma glucose -LRB- mmol/l -RRB- aRelative risk of diabetes in the intervention group vs the C : group -LRB- 95 % CIs -RRB- Lindström et al. 2003 -LSB- 21 -RSB- N/AN/AL : 156 -LRB- 62 to 288 -RRB- min/weekL : 61 -LRB- − 33 to 168 -RRB- min/weekb ` L : 86.7 ± 0.8 Results at 3 yearsL : 8.9 ± 0.1 Change at 1 yearL : 6.1 ± 0.05 Change at 1 year0 .4 -LRB- 0.3 -- 0.7 -RRB- C : 169 -LRB- 65 to 352 -RRB- min/weekC : 6 -LRB- − 91 to 104 -RRB- min/weekC : 85.5 ± 0.9 L : − 3.5 ± 0.3 bC : 8.9 ± 0.1 L : − 0.9 ± 0.1 bC : 6.2 ± 0.04 L : − 0.2 ± 0.04 C : − 0.9 ± 0.4 C : − 0.3 ± 0.1 C : 0.0 ± 0.04 Change at 3 yearsChange at 3 yearsL : − 0.5 ± 0.2 L : 0.0 ± 0.05 C : − 0.1 ± 0.2 C : 0.1 ± 0.05 Knowler et al. 2002 -LSB- 12 -RSB- N/AN/AL : 15.5 ± 0.7 MET-h/weekL : 6 MET-h/weekb , cL : 94.1 ± 0.6 L : − 5.6 bL : 9.1 ± 0.03 NRL : 5.9 ± 0.01 NR0 .4 -LRB- 0.3 -- 0.5 -RRB- C : 17.0 ± 0.9 MET-h/weekC : 1 MET-h/weekcC : 94.3 ± 0.6 C : − 0.1 C : 9.1 ± 0.03 C : 5.9 ± 0.02 Pan et al. 1997 -LSB- 10 -RSB- N/AN/AE : 3.4 ± 0.2 unitsdE : 0.6 unitsdNRD & E : − 2.5 E : 8.8 ± 0.1 E : 1.7 E : 5.6 ± 0.1 E : 1.3 E : 0.5 -LRB- 0.2 -- 0.9 -RRB- D&E : 3.1 ± 0.2 unitsdD & E : 0.8 unitsdE : − 0.4 D&E : 9.1 ± 0.1 D&E 1.7 D&E 5.7 ± 0.1 D&E : 1.5 D&E : 0.6 -LRB- 0.3 -- 0.9 -RRB- C : 2.4 ± 0.2 unitsdC : 0.1 unitsdC : − 1.0 C : 9.0 ± 0.1 C : 4.0 C : 5.5 ± 0.1 C : 2.1 Eriksson and Lindgärde 1991 -LSB- 24 -RSB- L : 2.46 ± 0.04 L : 0.20 bN/AN/AL : 82cL : − 3.3 bL : 8.2 ± 01L : − 1.1 bNRNR0 .4 -LRB- 0.2 -- 0.7 -RRB- C : 2.29 ± 0.1 C : − 0.05 C : 83cC : 0.2 C : 8.3 ± 0.1 C : 0.1 cOldroyd et al. 2006 -LSB- 19 -RSB- N/AN/ANRNRL : 85.3 ± 2.9 L : − 1.8 ± 1.1 bL : 9.2 ± 0.1 Change at 1 yearL : 6.1 ± 0.1 Change at 1 yearN/AC : 85.5 ± 2.5 C : 1.5 ± 0.5 C : 9.2 ± 0.2 L : − 0.6 ± 0.3 C : 6.2 ± 0.2 L : 0.0 ± 0.1 C : 0.2 ± 0.3 C : 0.1 ± 0.2 Change at 2 yearsChange at 2 yearsL : 0.2 ± 0.3 L : 0.3 ± 0.1 C : − 0.5 ± 0.4 C : 0.1 ± 0.2 Mensink et al. 2003 -LSB- 22 -RSB- L : 2.15 ± 0.1 L : 0.09 ± 1.90 bN/AN/AL : 86 ± 1.9 L : − 2.4 ± 0.7 bL : 8.9 ± 0.3 Change at 1 year L : 5.9 ± 0.1 Change at 1 yearN/AC : 2.13 ± 0.1 C : − 0.03 ± 2.77 C : 83.7 ± 1.5 C : 0.1 ± 0.5 C : 8.6 ± 0.2 L : − 0.9 ± 0.3 bC : 5.8 ± 0.1 L : − 0.1 ± 0.1 C : 0.3 ± 0.3 C : 0.1 ± 0.1 Change at 2 yearsChange at 2 yearsL : − 0.6 ± 0.3 bL : 0.2 ± 0.1 C : 0.8 ± 0.4 C : 0.5 ± 0.1 Lindahl et al. 1999 -LSB- 23 -RSB- L : 2.12 ± 0.1 eL : 0.21 ± 0.1 b , eN/AN/AL : 86.4 ± 1.1 L : − 5.4 ± 0.5 bL : 7.5 L : − 0.7 ± 0.2 L : 5.4 L : − 0.5 ± 0.1 N/AC : 1.89 ± 0.1 eC : − 0.02 ± 0.1 eC : 83.6 ± 1.1 C : − 0.5 ± 0.3 C : 8.0 C : − 0.3 ± 0.3 C : 6.1 C : − 0.3 ± 0.1 Carr et al. 2005 -LSB- 20 -RSB- E : 1.93 ± 0.1 E : 0.16 ± 0.04 bN/AN/AE : 66.5 ± 2.9 E : − 1.8 ± 0.5 bE : 9.2 ± 0.2 Change at 6 monthsE : 5.3 ± 0.1 Change at 6 monthsN/AC : 2.02 ± 0.1 C : − 0.04 ± 0.03 C : 69.7 ± 2.6 C : 0.6 ± 0.5 C : 9.1 ± 0.2 E : − 0.7 C : 5.4 ± 0.1 E : 0.0 C : − 0.1 C : 0.1 Change at 2 yearsChange at 2 yearsE : − 0.6 E : 0.0 C : 0.0 C : 0.1 Data are presented as means ± SD or medians -LRB- interquartile range -RRB- aChange from baseline values reflect results at final follow-up , unless stated otherwisebp < 0.05 vs C : cValue estimated from graphd1 unit = 20 min of moderate E : or 10 min of strenuous E : eFitness measurement taken in a randomly selected subgroup , n = 45C control , D diet , E exercise , L lifestyle , MET-h/week metabolic equivalent per week , NR not reported , N/A not applicable Study design Seven of the eight trials involved randomisation of subjects to a treatment group or control group -LSB- 10 , 12 , 19 -- 23 -RSB- . The non-randomised trial identified control participants by using individuals who , for various -LRB- unstated -RRB- reasons , were not enrolled in the intervention programme -LSB- 24 -RSB- . Sample size Sample size ranged from 62 to 2,161 . Two studies reported a power calculation based on the expected difference in the incidence of diabetes between groups -LSB- 12 , 21 -RSB- , and one reported a power calculation based on the expected difference between groups in the proportion of individuals with IGT at the end of the study -LSB- 19 -RSB- . In the latter study , Oldroyd et al. calculated that a total of 100 participants were required to detect a 0.6 mmol/l difference in fasting glucose and a 20 % difference in the number of individuals with IGT , allowing for a 90 % power at a significance of 0.05 . Three studies had sample sizes of fewer than 100 participants at follow-up -LSB- 19 , 20 , 22 -RSB- . Inclusion criteria All studies examined in this review included individuals with IGT and excluded those with isolated IFG -LSB- 10 , 12 , 19 -- 24 -RSB- . Sex Except for one trial that involved only men -LRB- n = 188 -RRB- -LSB- 24 -RSB- , all trials included both men and women . In the included studies a total of 40 % of participants were men . Intervention conditions Seven of the eight included studies used a multi-component lifestyle intervention -LSB- 10 , 12 , 19 , 21 -- 24 -RSB- , and one used a structured gym-based exercise training intervention -LSB- 20 -RSB- . Six of the lifestyle intervention studies were based on encouraging individuals to increase their physical activity to approximately 150 min of exercise of moderate to vigorous intensity per week whilst also encouraging weight loss through a healthy energy-restricted diet -LSB- 10 , 12 , 19 , 21 , 22 , 24 -RSB- . Participants in all six studies received regular encouragement and counselling from a trained dietician at least once every 3 months throughout the duration of the intervention . Two of the six studies also provided participants with the option of attending supervised exercise classes for some or all of the study duration -LSB- 21 , 24 -RSB- and one provided discounted access to local gyms -LSB- 19 -RSB- . One study determined the effect of diet and exercise separately and in combination -LSB- 10 -RSB- . One lifestyle intervention included an initial 1-month stay at a wellness centre where individuals were provided with healthy dietary options and encouraged to take part in 2.5 h/day of light to moderate intensity exercise using the leisure facilities provided -LSB- 23 -RSB- . After the stay at the wellness centre , participants were encouraged to make plans about how they could incorporate healthier habits into everyday life and then received no further contact until follow-up . The structured exercise intervention study used a training protocol of 180 min per week of aerobic exercise at 70 % of heart rate reserve -LSB- 20 -RSB- . Exercise training was supervised for the first 6 months and both groups were encouraged to eat a healthy energy-balanced diet , with those in the exercise training group also being encouraged to eat a diet with a high percentage of energy from carbohydrate -LSB- 20 -RSB- . Outcomes Four studies included the incidence of diabetes as the main outcome -LSB- 10 , 12 , 21 , 24 -RSB- , and four used 2-h plasma glucose levels as a direct measure of glucose control -LSB- 19 , 20 , 22 , 23 -RSB- . All the studies using the incidence of diabetes as their main outcome were based on a multi-component lifestyle intervention -LRB- see intervention conditions -RRB- . Incidence of diabetes and physical activity All four of the intervention studies that measured the incidence of diabetes as their primary outcome found a significant reduction in the incidence of type 2 diabetes in the intervention group . Diabetes incidence was reduced by 42 -- 63 % in this group compared with the control group -LRB- see Table 2 -RRB- . The study that investigated the effect of diet and physical activity both separately and in combination found a greater reduction in the incidence of diabetes -LRB- 46 % reduced risk -RRB- in the physical activity-only group than in either the combined physical activity and diet group -LRB- 42 % reduced risk -RRB- or the diet-only group -LRB- 13 % reduced risk -RRB- , although the difference between groups was not statistically significant -LSB- 10 -RSB- . Three of these four studies relied on self-reported measures of physical activity -LSB- 10 , 12 , 21 -RSB- , and of these , only the Diabetes Prevention Program -LRB- DPP -RRB- -LSB- 12 -RSB- and the Finnish Diabetes Prevention Study -LRB- FDPS -RRB- -LSB- 21 -RSB- reported using a validated physical activity questionnaire . All three of the studies relying on self-reported physical activity levels reported non-significant to small changes in physical activity levels in the intervention group . For example , the DPP reported a mean increase in energy expenditure due to leisure time physical activity of around six metabolic equivalent hours per week -LSB- 12 -RSB- , which is approximately equivalent to walking at a moderate pace for 15 min/day -LSB- 25 -RSB- . The FDPS reported no significant change in total physical activity levels compared with the control group and an increase of 9 min/day in moderate to vigorous physical activity -LSB- 21 -RSB- , and the Da Qing IGT and Diabetes Study reported no significant change in physical activity levels compared with the control group -LSB- 10 -RSB- . The Malmö Feasibility Study , which used an objective outcome measure -LRB- cardiovascular fitness -RRB- , reported an 8 % increase in maximal oxygen uptake -LSB- 24 -RSB- . 2-h post-challenge plasma glucose and physical activity Three of the studies that used the incidence of diabetes as their primary outcome measure also measured 2-h plasma glucose before and after the intervention -LSB- 10 , 21 , 24 -RSB- . The FDPS reported a 0.9 mmol/l decrease in 2-h plasma glucose after 1 year , but no significant change after 3 years -LSB- 21 -RSB- ; the Da Qing IGT and Diabetes Study found that 2-h plasma glucose increased in all groups , but the increase in the control group was over twice that in either of the intervention groups -LSB- 10 -RSB- ; and the Malmö Feasibility Study reported a 1.1 mmol/l reduction in 2-h plasma glucose in the intervention group -LSB- 24 -RSB- . Of the three lifestyle intervention studies that used 2-h plasma glucose levels rather than the incidence of diabetes as the primary indictor of improved glucose tolerance -LSB- 19 , 22 , 23 -RSB- , only one reported a significant difference between the groups in terms of 2-h plasma glucose at follow-up -LSB- 22 -RSB- . Two of the studies used a measure of cardiovascular fitness as an indicator of physical activity levels -LSB- 22 , 23 -RSB- , and one -LSB- 19 -RSB- used distance walked in a shuttle test -LSB- 26 -RSB- as a measure of physical activity . Two studies found a small to moderate increase in cardiovascular fitness -LRB- < 10 % increase compared with baseline value -RRB- -LSB- 22 , 23 -RSB- , and the study using the shuttle test reported no change in the distance walked during the test -LSB- 19 -RSB- . Similarly , the moderate increases in cardiovascular fitness observed in the structured exercise training study were not associated with significant improvements in 2-h plasma glucose compared with the control group -LSB- 20 -RSB- . Fasting glucose None of the included studies reported a significant change in fasting glucose in the intervention group compared with the control group at follow-up . One study did not report fasting glucose values -LSB- 24 -RSB- . Discussion Eight controlled trials in individuals with IGT were included in this review . Four studies measured the incidence of diabetes as a primary outcome measure , and found that the risk of diabetes was reduced by approximately 50 % -LRB- range 42 -- 63 % -RRB- in individuals who were encouraged to reduce their body mass through changes in diet and physical activity -LSB- 10 , 12 , 21 , 24 -RSB- . Although the promotion of physical activity was an important component of these studies , the effect of exercise independent of other factors on the risk of diabetes in individuals with IGT is still unclear . All but one -LSB- 10 -RSB- of the studies included in this review reported significant weight loss among participants . Given that weight loss is known to improve many of the factors associated with IGT , including insulin sensitivity and glycaemic control -LSB- 27 -RSB- , and considering only modest increases in physical activity were found in these studies , the success of these interventions is likely to be largely explained by weight loss . The apparent success of the exercise-only intervention in the Da Qing IGT and Diabetes Study -LSB- 10 -RSB- is likely to be attributable , at least in part , to the significantly higher levels of physical activity at baseline in the exercise intervention group compared with the control group . The separation of physical activity and weight loss may seem an overcorrection given that increased physical activity may encourage weight loss through increased energy expenditure ; however , several meta-analyses of controlled trials investigating the effect of physical activity on glycaemic control in individuals with diabetes found that exercise training was not associated with weight loss -LSB- 28 , 29 -RSB- . Furthermore it is increasingly recognised that at least 60 min/day of moderate intensity exercise should be undertaken for the effective management of body mass -LSB- 30 -RSB- , an amount that none of the interventions included in this review achieved . Three of the four studies that investigated the effect of a lifestyle intervention in individuals with IGT on the incidence of type 2 diabetes -LSB- 10 -- 12 -RSB- relied on self-reported measures of physical activity . Given the limitations of subjective measures of physical activity , particularly when measuring non-structured forms of moderate physical activity such as walking activity -LSB- 31 -RSB- , these lifestyle intervention studies provide uncertain information about the effect of physical activity in individuals with IGT . Results from the lifestyle intervention studies that relied on changes in 2-h plasma glucose rather than the incidence of diabetes as the primary measure of glucose control were inconclusive -LSB- 19 , 20 , 23 -RSB- . Two of the three studies were unsuccessful at improving glucose tolerance -LSB- 19 , 23 -RSB- . Similarly , the one study that used an aerobic exercise training protocol found no improvements in glucose tolerance as measured by 2-h plasma glucose -LSB- 20 -RSB- . However , it did find a significant improvement in insulin sensitivity at both 6 and 24 months . This suggests that , although the intervention goal of 3 h/week of moderate intensity exercise was enough to improve insulin sensitivity , it was not long enough and/or of sufficient intensity to elicit the necessary magnitude of change in insulin sensitivity for this to be translated into a significant reduction in 2-h plasma glucose . Overall , non-significant results were seen in all but two of the studies that measured 2-h plasma glucose before and after the intervention . However , despite the link between 2-h plasma glucose and diabetes risk , it does not follow that the risk of diabetes was unchanged in these studies , as demonstrated by the FDPS , which reported a non-significant change in 2-h plasma glucose over the course of the intervention but a > 50 % reduction in the risk of diabetes -LSB- 21 -RSB- . One reason for this discrepancy is likely to be the poor repeatability of 2-h plasma glucose values -LSB- 32 -RSB- , and given the relatively small sample sizes in most of these studies , it is possible that improvements in glucose tolerance were not detected using 2-h plasma glucose . The glucose AUC has been identified as a more reliable measure of glucose tolerance than 2-h plasma glucose -LSB- 33 -RSB- , and is therefore a more sensitive measure of glucose tolerance . One study included in this review measured both 2-h plasma glucose and glucose AUC at baseline and follow-up -LSB- 20 -RSB- . It reported that , although 2-h plasma glucose did not change significantly at any of the follow-up time points , there was a significant reduction in the glucose AUC at 6 months , and a trend towards significance at 24 months . Given the failure of the lifestyle interventions to substantially increase physical activity levels , and the inconclusive result of the structured exercise training study , the role of physical activity independent of other lifestyle changes in the treatment of prediabetes remains equivocal . However , statistical analysis of the independent effects of physical activity , which has been carried out on some of the lifestyle intervention studies included in this review , show interesting results . For example , the conclusion of the Malmö Feasibility Study that cardiovascular fitness and weight loss were equally correlated to improved glucose tolerance is supported by data from the Study on Lifestyle Intervention and Impaired Glucose Tolerance Maastricht -LRB- SLIM -RRB- -LSB- 34 -RSB- , and a recent analysis of data from the FDPS found a 49 % difference in the risk of diabetes , after adjustment for changes to body mass and diet , when comparing those in the highest and lowest tertiles of moderate to vigorous leisure time physical activity change -LSB- 35 -RSB- . Thus , although the overall evidence for the independent effect of physical activity in the management of prediabetes is equivocal , encouraging evidence is starting to emerge in support of the importance of exercise . Given the limitations of the studies included in this review it is not possible to make any recommendations as to the intensity and duration of exercise needed to improve glucose tolerance and/or reduce the risk of diabetes in individuals with IGT , independently of other lifestyle changes . The equivocal nature of the evidence is reflected in the advice given by the ADA , which recommends that individuals with IGT should include 150 min/week of moderate to vigorous intensity exercise as part of a weight management programme -LSB- 36 -RSB- . However , the aforementioned analysis of the change in physical activity in the FDPS found that a difference of 246 min/week in median values between those in the lowest and the highest tertiles of moderate to vigorous physical activity change was associated with a significant reduction in the risk of diabetes , after adjusting for changes in diet and body mass . However , the difference of 120 min/week in median values between the lowest and middle tertiles was not associated with a reduced risk of diabetes -LSB- 35 -RSB- . Although this result was obtained by analysing the pooled cohort , and therefore provides little information about the effectiveness of the intervention itself , it does suggest that 150 min/week of moderate to vigorous intensity exercise is unlikely to be enough to significantly reduce the risk of type 2 diabetes in individuals with IGT , independently of other lifestyle changes . However , given that this analysis relied on self-reported physical activity levels , further rigorous studies are needed to confirm this . All studies included in this review selected individuals using IGT as an inclusion criteria . Therefore , any conclusions from this review can only be applied to individuals with IGT and it is impossible to determine whether or not exercise may be effective in treating individuals with isolated IFG . However as individuals with isolated IFG account for a minority of individuals with prediabetes -LSB- 37 -RSB- , conclusions about the effect of exercise on IGT drawn from this review will apply to the majority of individuals with prediabetes . In summary , the majority of studies identified for this review used interventions that encouraged dietary and physical activity to initiate and maintain weight loss in individuals with IGT . Analysis of these studies found that the independent effect of physical activity in reducing the risk of type 2 diabetes in individuals with prediabetes is equivocal . Furthermore , given the limited evidence , no definite conclusion can be drawn either as to the amount of physical activity needed to reduce the risk of diabetes in individuals with prediabetes or the effectiveness of a single-component physical activity intervention compared with more conventional multi-component interventions . Thus , more evidence from rigorously designed randomised controlled trials with objective measures of physical activity is needed . As the majority of studies promoting lifestyle changes included in this review failed to substantially increase physical activity levels , strategies for effecting increased physical activity in this population also need to be researched thoroughly . Further investigation is also needed into whether exercise is equally effective in treating the different phenotypes of IGT and IFG .

O O O B I O O O O B I I O O O O O B I O O O O O O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I I O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B O O O O B O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I I O O O B O O O O O O O O O O O O O O O O O B I O O O O O B I I O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B I I O O O B O O O B I I O O O B O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O B O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I I O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I I O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O B I O O O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O B O B O

exercise
ifg
impaired fasting glucose
igt
impaired glucose tolerance
physical activity
prediabetes
prevention
type 2 diabetes


Bone

Degeneration

and

Recovery

after

Early

and

Late

Bisphosphonate

Treatment

of

Ovariectomized

Wistar

Rats

Assessed

by

In

Vivo

Micro-Computed

Tomography

Bisphosphonates

are

antiresorptive

drugs

commonly

used

to

treat

osteoporosis

It

is

not

clear

however

what

the

influence

of

the

time

point

of

treatment

is

Recently

developed

in

vivo

micro-computed

tomographic

-LRB-

CT

-RRB-

scanners

offer

the

possibility

to

study

such

effects

on

bone

microstructure

in

rats

The

aim

of

this

study

was

to

determine

the

influence

of

early

and

late

zoledronic

acid

treatment

on

bone

in

ovariectomized

rats

using

in

vivo

micro-CT

Twenty-nine

female

Wistar

rats

were

divided

into

the

following

groups

ovariectomy

-LRB-

OVX

n

-RRB-

OVX

and

zoledronic

acid

-LRB-

ZOL

-RRB-

at

week

-LRB-

n

-RRB-

OVX

and

ZOL

at

week

-LRB-

n

-RRB-

and

sham

-LRB-

n

-RRB-

CT

scans

were

made

of

the

proximal

tibia

at

weeks

and

and

bone

structural

parameters

were

determined

in

the

metaphysis

Two

fluorescent

labels

were

administered

to

calculate

dynamic

histomorphometric

parameters

At

week

all

groups

were

significantly

different

from

each

other

in

bone

volume

fraction

-LRB-

BV/TV

-RRB-

connectivity

density

and

trabecular

number

-LRB-

Tb.N

-RRB-

except

for

the

early

ZOL

and

control

groups

which

were

not

significantly

different

for

any

structural

parameter

After

ZOL

treatment

at

week

BV/TV

structure

model

index

Tb.N

and

trabecular

thickness

significantly

improved

in

the

late

ZOL

group

The

OVX

and

ZOL

groups

showed

respectively

higher

and

lower

bone

formation

rates

than

the

control

group

Early

ZOL

treatment

inhibited

all

bone

microstructural

changes

seen

after

OVX

Late

ZOL

treatment

significantly

improved

bone

microstructure

although

the

structure

did

not

recover

to

original

levels

Early

ZOL

treatment

resulted

in

a

significantly

better

microstructure

than

late

treatment

However

late

treatment

was

still

significantly

better

than

no

treatment

Postmenopausal

osteoporosis

affects

millions

of

women

worldwide

and

results

in

loss

of

bone

mass

and

bone

microstructural

changes

which

lead

to

reduced

bone

strength

It

remains

unknown

how

exactly

the

microstructure

is

affected

over

the

time

course

of

osteoporosis

development

and

to

what

extent

this

structure

can

be

recovered

by

drug

treatment

Since

the

assessment

of

bone

structure

in

humans

is

still

limited

to

a

few

peripheral

sites

the

ovariectomized

rat

is

used

in

many

studies

as

an

animal

model

for

osteoporosis

in

cross-sectional

evaluations

Numerous

cross-sectional

studies

have

determined

the

loss

of

trabecular

bone

in

the

proximal

tibia

in

rats

following

ovariectomy

-LRB-

OVX

-RRB-

at

different

time

points

by

measuring

bone

structural

parameters

-LSB-

-RSB-

OVX

has

been

shown

to

decrease

bone

volume

fraction

connectivity

and

trabecular

number

and

to

increase

trabecular

separation

and

structure

model

index

which

indicates

that

trabecular

bone

changes

from

plate-like

to

more

rod-like

in

the

proximal

tibial

metaphysis

of

the

female

rat

However

conflicting

results

were

found

for

the

effects

of

OVX

on

trabecular

thickness

showing

increases

decreases

or

no

response

at

all

-LSB-

-RSB-

These

cross-sectional

studies

are

limited

to

comparing

averages

per

group

only

which

provides

a

reduced

sensitivity

for

detection

of

differences

In

addition

no

information

is

obtained

regarding

the

specific

locations

of

bone

changes

Recently

however

in

vivo

micro-computed

tomographic

-LRB-

CT

-RRB-

scanners

became

available

which

can

monitor

the

process

of

bone

loss

and

microstructural

changes

in

small

living

animals

So

far

two

studies

have

been

reported

in

which

this

equipment

was

used

to

monitor

changes

in

rat

bones

after

OVX

Interestingly

these

studies

reported

respectively

significant

increases

-LSB-

-RSB-

and

decreases

-LSB-

-RSB-

in

trabecular

thickness

after

OVX

indicating

that

the

response

may

vary

While

the

process

of

bone

degeneration

after

OVX

is

not

fully

understood

even

less

information

is

available

for

the

process

of

recovery

after

drug

treatment

The

most

widely

used

drugs

for

treatment

of

diseases

associated

with

increased

bone

resorption

such

as

postmenopausal

osteoporosis

are

now

bisphoshonates

Many

cross-sectional

studies

have

determined

the

effects

of

bisphosphonates

in

rats

either

as

a

preventive

treatment

before

development

of

OVX-induced

bone

loss

or

as

a

recovering

treatment

after

development

of

bone

loss

Although

the

results

are

difficult

to

compare

directly

due

to

differences

in

experimental

design

preventive

treatment

in

animals

may

lead

to

the

same

amount

of

final

bone

mass

as

recovering

treatment

The

associated

microstructure

in

terms

of

number

and

thickness

of

trabeculae

however

can

be

different

While

the

influence

of

the

time

point

of

treatment

is

highly

relevant

clinically

no

studies

were

found

that

directly

compared

the

effects

of

a

preventive

and

a

recovering

treatment

on

final

bone

mass

and

associated

microstructure

In

addition

the

fact

that

only

results

of

cross-sectional

studies

have

been

reported

complicates

comparison

of

the

final

and

original

bone

structure

and

provides

little

or

no

information

on

the

changes

in

structure

over

time

Here

also

we

expect

that

longitudinal

in

vivo

micro-CT

imaging

will

potentially

provide

more

information

about

the

specific

changes

in

the

bone

both

spatially

and

temporally

For

the

present

study

we

had

two

goals

-LRB-

-RRB-

to

determine

the

process

of

bone

loss

and

bone

microstructural

changes

in

ovariectomized

rats

and

-LRB-

-RRB-

to

determine

the

effects

of

early

and

late

treatment

of

osteoporotic

rats

with

a

bisphosphonate

We

used

an

in

vivo

high-resolution

micro-CT

scanner

to

image

the

proximal

tibia

of

female

adult

Wistar

rats

For

the

treatment

zoledronic

acid

-LRB-

ZOL

-RRB-

was

used

since

this

is

a

third-generation

bisphosphonate

that

proved

a

potent

osteoclast

inhibitor

It

has

shown

to

be

effective

in

cancer

patients

with

bone

metastases

and

is

currently

being

tested

in

clinical

trials

as

a

promising

therapeutic

agent

for

osteoporotic

patients

-LSB-

-RSB-

Methods

Animals

Thirty-six

female

retired

breeding

Wistar

rats

were

obtained

from

Harlan

Laboratories

-LRB-

Horst

The

Netherlands

-RRB-

and

allowed

to

acclimate

for

days

before

the

start

of

the

experiment

The

rats

were

maintained

with

a

cycle

of

hours

light

and

hours

darkness

and

allowed

to

eat

and

drink

ad

libitum

The

experiment

was

approved

by

the

Animals

Ethics

Committee

of

the

University

of

Maastricht

The

Netherlands

The

rats

were

divided

into

four

groups

based

on

weight

control

-LRB-

n

-RRB-

OVX

-LRB-

n

-RRB-

OVX

and

early

ZOL

-LRB-

n

-RRB-

OVX

and

late

ZOL

-LRB-

n

-RRB-

All

rats

were

ovariectomized

at

week

and

the

control

group

underwent

sham

OVX

Success

of

OVX

was

confirmed

at

necropsy

by

determining

atrophy

of

the

uterine

horns

ZOL

-LRB-

kindly

donated

by

Novartis

Pharmaceutical

Basel

Switzerland

-RRB-

was

dissolved

in

a

saline

vehicle

The

early

ZOL

group

was

administered

ZOL

at

a

single

dose

of

μg/kg

body

weight

s.c.

at

OVX

-LRB-

comparable

to

the

phase

III

clinical

trial

dose

used

in

osteoporotic

patients

-RRB-

The

late

ZOL

group

was

administered

the

same

single

dose

weeks

after

OVX

Eleven

and

days

before

necropsy

the

fluorescent

labels

calcein

-LRB-

Fluka

Buchs

Switzerland

-RRB-

and

alizarin

-LRB-

Merck

Dietikon

Switzerland

-RRB-

were

administered

s.c.

in

a

saline

vehicle

to

all

rats

at

doses

of

mg/kg

and

mg/kg

body

weight

respectively

to

evaluate

bone

formation

dynamics

in

the

proximal

tibial

metaphysis

Rats

were

killed

at

weeks

by

exsanguination

Micro-CT

Scanning

Directly

after

the

operation

a

micro-CT

scan

-LRB-

kV

μA

projections

per

degrees

ms

integration

time

-RRB-

with

an

isotropic

resolution

of

μm

was

made

of

the

proximal

tibia

using

an

in

vivo

micro-CT

scanner

-LRB-

vivaCT

Scanco

Medical

Bruettisellen

Switzerland

-RRB-

The

CT

scanner

was

calibrated

and

a

beam-hardening

correction

algorithm

was

applied

to

all

scans

Follow-up

in

vivo

CT

scans

were

made

after

and

weeks

to

monitor

bone

structure

One

CT

scan

took

minutes

during

which

the

animal

was

anesthetized

with

isoflurane

and

the

scanned

leg

was

placed

in

a

custom-made

leg-fixating

device

The

design

of

the

rat

holder

was

such

that

the

left

leg

was

not

exposed

to

radiation

while

scanning

the

right

leg

Radiation

damage

to

the

scanned

bone

was

not

expected

to

occur

based

on

a

previous

study

in

which

CT

scans

with

the

same

radiation

dose

caused

no

detected

bone

damage

-LSB-

-RSB-

In

that

study

we

also

showed

that

the

reproducibility

of

all

structural

parameters

was

high

with

a

coefficient

of

variation

of

about

Image

processing

included

gaussian

filtering

and

segmentation

and

was

described

elsewhere

in

detail

-LSB-

-RSB-

In

brief

the

same

filtering

and

segmentation

values

were

used

for

every

measurement

of

each

animal

-LRB-

sigma

support

threshold

density

g

hydroxyapatite/cc

equivalent

to

of

maximal

gray-scale

value

-RRB-

From

every

baseline

and

follow-up

CT

scan

the

metaphyseal

trabecular

bone

was

manually

selected

and

bone

structural

parameters

-LRB-

bone

volume

fraction

-LSB-

BV/TV

-RSB-

connectivity

density

-LSB-

Conn.

D

-RSB-

structure

model

index

-LSB-

SMI

-RSB-

trabecular

number

-LSB-

Tb.N

-RSB-

trabecular

thickness

-LSB-

Tb.Th

-RSB-

and

trabecular

separation

-LSB-

Tb.Sp

-RSB-

-RRB-

were

automatically

determined

The

average

attenuation

coefficient

of

the

trabecular

bone

tissue

was

determined

for

all

measurements

using

a

protocol

provided

by

the

manufacturer

of

the

micro-CT

scanner

With

this

protocol

the

gray

levels

of

voxels

near

the

trabecular

surfaces

are

not

included

to

ensure

that

the

measurements

are

not

affected

by

partial

volume

effects

Also

cortical

thickness

of

the

metaphysis

was

analyzed

The

cortical

bone

starting

from

the

tibia

fibula

junction

was

manually

selected

for

each

measurement

resulting

in

a

region

approximately

mm

long

The

same

filtering

and

segmentation

values

were

used

as

for

the

trabecular

analysis

Cortical

thickness

was

automatically

determined

from

the

selected

region

When

two

CT

scans

of

the

same

animal

are

made

at

different

time

points

the

position

of

the

animal

in

the

scanner

will

not

be

exactly

the

same

and

therefore

the

CT

scans

will

also

differ

In

order

to

detect

bone

structural

changes

on

a

micro

level

one

CT

scan

needs

to

be

translated

and

rotated

to

match

the

other

We

developed

image

registration

software

that

registers

two

scans

based

on

minimizing

the

correlation

coefficient

-LSB-

-RSB-

Every

follow-up

scan

is

registered

with

the

first

scan

thereby

revealing

any

possible

changes

in

the

microstructure

and

minimizing

errors

in

the

region

of

interest

for

the

structural

parameters

Mechanical

Testing

After

sacrifice

all

left

tibiae

were

dissected

and

used

for

two

different

mechanical

tests

First

a

standard

three-point

bending

test

was

applied

The

tibiae

were

placed

on

the

lateral

surface

on

two

rounded

supporting

bars

with

a

distance

of

mm

A

preload

of

N

was

applied

-LRB-

Zwick

Ulm

Germany

-RRB-

at

the

medial

surface

of

the

diaphysis

by

lowering

a

third

rounded

bar

A

constant

displacement

rate

of

mm/minute

was

applied

until

failure

Displacement

was

measured

from

the

actuator

displacement

transducer

of

the

testing

machine

Stiffness

ultimate

force

and

ultimate

displacement

were

calculated

for

each

sample

To

assess

the

mechanical

properties

of

the

proximal

tibia

a

proximal

tibia

compression

test

was

performed

similar

to

the

method

of

Hogan

et

al.

-LSB-

-RSB-

A

slice

of

the

proximal

metaphysis

including

both

cortical

and

trabecular

bone

was

sawed

below

the

growth

plate

using

a

diamond

saw

-LRB-

Struers

Ballerup

Denmark

-RRB-

This

slice

was

axially

compressed

-LRB-

-RRB-

during

five

preconditioning

cycles

between

and

N

After

the

last

preconditioning

cycle

axial

compressive

load

was

applied

at

mm/minute

until

failure

while

axial

displacement

was

measured

using

an

extensometer

Stiffness

ultimate

force

and

ultimate

displacement

were

determined

for

all

samples

Histomorphometry

At

necropsy

all

right

tibiae

were

fixed

in

ethanol

and

embedded

in

methylmethacrylate

-LRB-

Technovit

Heraeus

Kulzer

Wehrheim

Germany

-RRB-

Thin

sections

-LRB-

μm

-RRB-

were

cut

to

determine

fluorochrome-based

dynamic

histomorphometric

parameters

of

bone

formation

using

an

Axiophot

photomicroscope

-LRB-

Zeiss

Oberkochen

Germany

-RRB-

linked

to

a

camera

-LRB-

CF

MC

Kappa

Gleichen

Germany

-RRB-

and

a

QUANTIMET

image

analysis

system

was

used

to

calculate

the

amount

of

mineralized

surface

per

bone

surface

-LRB-

MS/BS

percentage

-RRB-

corrected

mineral

apposition

rate

-LRB-

MAR

-RRB-

-LRB-

μm/day

-RRB-

and

bone

formation

rate

-LRB-

BFR

-RRB-

per

bone

surface

-LRB-

μm/day

-RRB-

Calculations

of

the

dynamic

parameters

were

performed

as

recommended

previously

-LSB-

-RSB-

Statistics

Since

this

experiment

concerned

repeated

measures

in

multiple

groups

several

statistical

tests

were

performed

to

reveal

changes

in

parameter

values

over

time

and

between

groups

For

all

structural

parameters

the

percentage

change

at

different

time

intervals

was

calculated

for

each

animal

individually

A

one-way

analyses

of

variance

-LRB-

ANOVA

-RRB-

with

repeated

measures

was

performed

on

the

percentage

change

for

all

structural

parameters

and

all

groups

Furthermore

a

one-way

ANOVA

with

a

Bonferroni

post-hoc

test

was

used

to

determine

differences

between

the

groups

at

all

time

points

for

each

structural

parameter

Also

a

one-way

ANOVA

with

a

Bonferroni

post-hoc

test

was

used

to

determine

differences

between

the

groups

in

cortical

thickness

histomorphometric

results

and

mechanical

results

For

each

group

a

paired

Student

's

t-test

was

performed

on

the

absolute

values

of

the

structural

parameters

of

each

time

point

compared

to

baseline

values

to

determine

the

earliest

point

at

which

a

detectable

change

was

found

A

paired

Student

's

t-test

was

used

to

determine

late

ZOL

treatment

effects

within

this

group

Finally

a

paired

Student

's

t-test

was

performed

on

the

absolute

values

between

each

measurement

for

each

group

and

structural

parameter

P

was

considered

significant

Results

Ovariectomy

Based

on

the

uterine

weight

we

determined

seven

unsuccessful

OVXs

The

rats

concerned

were

removed

from

further

analyses

which

left

nine

rats

in

the

control

group

five

in

the

OVX

group

seven

in

the

OVX

and

late

ZOL

group

and

eight

in

the

OVX

and

early

ZOL

group

For

each

animal

we

determined

the

percentage

change

in

structural

parameters

compared

to

the

values

at

week

Figure

shows

the

average

percentage

change

and

upper

standard

deviation

for

all

groups

for

BV/TV

Conn.

D

SMI

Tb.N

Tb.Th

and

Tb.Sp

The

OVX

group

without

treatment

showed

large

changes

in

structural

parameters

indicating

the

development

of

OVX-induced

bone

loss

-LRB-

Figs.

and

-RRB-

Paired

Student

's

t-test

indicated

significant

changes

within

weeks

after

OVX

for

all

structural

parameters

BV/TV

Conn.

D

and

Tb.N

decreased

during

the

experiment

and

SMI

and

Tb.Sp

increased

Both

Tb.Th

and

Tb.N

decreased

within

weeks

indicating

that

both

thinning

and

complete

resorption

of

trabeculae

started

directly

after

OVX

The

initial

rapid

loss

of

bone

and

connectivity

was

largely

accompanied

by

trabecular

thinning

while

secondary

slower

loss

of

bone

was

concomitant

with

a

decrease

in

Tb.N

While

BV/TV

Conn.

D

SMI

Tb.N

and

Tb.Sp

showed

continuous

changes

in

the

same

direction

throughout

the

experiment

Tb.Th

initially

decreased

significantly

until

weeks

after

OVX

and

then

increased

again

until

after

weeks

Tb.Th

was

significantly

higher

than

in

the

control

group

Fig.

percentage

change

in

structural

parameters

in

the

metaphyseal

proximal

tibia

and

upper

standard

deviation

for

all

groups

at

all

time

points

Brackets

indicate

P

at

week

slice

of

an

unprocessed

CT

scan

of

the

same

rat

in

the

OVX

group

taken

at

weeks

-LRB-

a

-RRB-

-LRB-

b

-RRB-

-LRB-

c

-RRB-

-LRB-

d

-RRB-

-LRB-

e

-RRB-

and

-LRB-

f

-RRB-

Images

show

typical

trabecular

bone

loss

due

to

OVX

in

the

metaphysis

Green

line

shows

the

analyzed

metaphyseal

bone

The

absolute

average

values

and

standard

deviations

for

all

structural

parameters

of

all

measurements

are

shown

in

Table

At

the

starting

point

some

differences

in

the

values

between

the

groups

were

present

This

was

due

to

the

fact

that

some

animals

which

mostly

had

a

relatively

low

BV/TV

were

removed

from

the

study

because

they

did

not

respond

to

the

OVX

However

since

we

compared

the

relative

changes

in

each

animal

the

results

were

most

likely

not

affected

by

the

difference

in

absolute

values

Table

values

and

standard

deviation

of

all

structural

parameters

of

all

groups

at

the

start

of

the

experiment

GroupBV/TV

-LRB-

-RRB-

Conn.

D

-LRB-

-RRB-

SMI

-LRB-

-RRB-

Tb.N

-LRB-

-RRB-

Tb.Th

-LRB-

μm

-RRB-

Tb.Sp

-LRB-

μm

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

OVX

late

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

OVX

early

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

ZOL

Treatment

The

early

ZOL

treatment

and

control

groups

were

not

significantly

different

at

all

time

points

for

all

structural

parameters

Up

until

weeks

after

OVX

when

ZOL

was

administered

to

rats

in

the

late

treatment

group

the

OVX

group

and

the

late

treatment

group

were

not

significantly

different

for

all

structural

parameters

BV/TV

SMI

Tb.Th

and

Tb.N

were

significantly

improved

in

the

late

ZOL

group

weeks

after

the

single

ZOL

injection

-LRB-

Table

-RRB-

In

addition

Tb.Sp

significantly

improved

within

weeks

after

injection

while

Conn.

D

showed

no

significant

response

Between

weeks

and

all

structural

parameters

stayed

constant

Table

values

of

paired

Student

's

t-test

in

the

late

ZOL

group

after

ZOL

treatment

Structural

parameterP

for

weeks

for

weeks

Significant

-LRB-

P

-RRB-

At

week

BV/TV

Conn.

D

and

Tb.N

were

significantly

different

between

all

groups

except

for

the

early

treatment

and

control

groups

-LRB-

Table

-RRB-

Tb.Th

was

only

significantly

different

between

the

control

and

OVX

groups

Although

substantial

differences

in

Tb.Sp

and

SMI

were

seen

between

all

groups

except

for

the

early

treatment

and

control

groups

significant

differences

were

only

found

between

the

OVX

and

control

groups

and

between

the

OVX

and

early

treatment

groups

Table

values

of

ANOVA

plus

Bonferroni

post-hoc

test

comparing

all

groups

at

all

time

points

for

all

structural

parametersGroup

ΔBV/TVΔConn

vs.

vs.

vs.

40.9990.9990.9990.9990.3220.9990.9990.9990.9990.9990.9990.9990.9990.9990.9992

vs.

vs.

vs.

Group

vs.

vs.

vs.

40.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9990.9992

vs.

0.9990.9990.9990.9990.1520.9990.9990.1280.001

vs.

vs.

0.1090.9990.9990.2790.0840.1870.6670.999

Groups

control

OVX

late

ZOL

early

ZOL

Significant

-LRB-

P

-RRB-

CT-derived

mineralization

values

in

all

groups

did

not

change

over

time

and

did

not

differ

between

groups

at

all

time

points

Aging

An

ANOVA

with

repeated

measures

on

all

structural

parameters

of

the

control

group

was

performed

to

determine

if

there

was

a

significant

effect

of

aging

BV/TV

and

Tb.N

were

found

to

significantly

decrease

and

Tb.Sp

was

found

to

significantly

increase

due

to

aging

All

other

structural

parameters

did

not

change

significantly

in

weeks

An

ANOVA

with

repeated

measures

was

performed

on

the

percentage

change

in

structural

parameters

comparing

the

control

and

early

treatment

groups

to

determine

any

significant

differences

BV/TV

and

Tb.N

showed

a

significant

effect

of

age

treatment

and

age

treatment

and

Tb.Sp

showed

a

significant

effect

of

age

and

age

treatment

While

BV/TV

decreased

significantly

over

time

in

both

the

early

treatment

and

control

groups

it

was

found

to

decrease

at

a

higher

rate

in

the

latter

Tb.N

significantly

decreased

over

time

in

the

control

group

however

it

did

not

significantly

change

in

the

early

treatment

group

as

a

net

result

of

OVX

aging

and

ZOL

treatment

Tb.Sp

significantly

increased

in

the

control

group

due

to

aging

while

it

did

not

in

the

early

treatment

group

Cortical

Thickness

OVX

initially

led

to

a

decrease

in

metaphyseal

cortical

thickness

and

at

weeks

both

the

OVX

and

the

late

ZOL

groups

were

significantly

lower

than

the

early

ZOL

group

-LRB-

Fig.

-RRB-

However

after

weeks

it

increased

again

in

the

OVX

group

and

at

weeks

cortical

thickness

was

significantly

higher

than

in

the

control

group

Early

ZOL

treatment

inhibited

the

changes

in

cortical

thickness

The

late

ZOL

group

just

like

the

OVX

group

showed

an

increase

in

cortical

thickness

between

and

weeks

although

this

was

less

pronounced

Fig.

percentage

change

in

cortical

thickness

in

the

metaphyseal

proximal

tibia

and

upper

standard

deviation

for

all

groups

at

all

time

points

At

week

the

OVX

and

the

late

ZOL

groups

were

significantly

lower

than

the

early

ZOL

group

and

at

week

the

OVX

group

was

significantly

higher

than

the

control

group

Mechanical

Testing

Experimental

test

results

were

obtained

for

seven

-LRB-

control

-RRB-

five

-LRB-

OVX

-RRB-

six

-LRB-

OVX

and

late

ZOL

-RRB-

and

seven

-LRB-

OVX

and

early

ZOL

-RRB-

tibiae

No

significant

differences

were

found

between

all

parameters

determined

in

the

three-point

bending

test

-LRB-

Fig.

-RRB-

Fig.

ultimate

force

and

ultimate

displacement

determined

from

three-point

bending

tests

on

diaphyseal

tibia

-LRB-

a

-RRB-

and

axial

compression

tests

-LRB-

b

-RRB-

on

metaphyseal

bone

Groups

control

OVX

late

ZOL

early

ZOL

Significant

difference

between

groups

based

on

ANOVA

and

Bonferroni

post-hoc

test

-LRB-

P

-RRB-

For

the

results

from

the

proximal

tibia

compression

test

a

significant

difference

in

ultimate

displacement

was

found

between

the

OVX

group

and

the

early

ZOL

group

and

between

the

OVX

and

the

control

groups

-LRB-

Fig.

-RRB-

Although

not

significantly

determined

a

trend

was

observed

in

which

OVX

resulted

in

a

lower

stiffness

and

ultimate

force

while

late

ZOL

treatment

partially

recovered

these

values

and

early

ZOL

treatment

did

to

a

larger

extent

Histomorphometry

Histomorphometric

results

were

obtained

for

five

-LRB-

control

-RRB-

five

-LRB-

OVX

-RRB-

six

-LRB-

OVX

and

late

ZOL

-RRB-

and

five

-LRB-

OVX

and

early

ZOL

-RRB-

rats

The

OVX

group

showed

a

significantly

higher

mineralizing

surface

-LRB-

MS/BS

-RRB-

and

BFR

and

a

substantially

higher

mineral

apposition

rate

-LRB-

MAR

-RRB-

than

the

control

group

-LRB-

Fig.

-RRB-

Both

ZOL

groups

showed

a

significantly

lower

MS/BS

and

BFR

than

the

OVX

group

and

control

group

while

MAR

was

significantly

lower

than

only

the

OVX

group

Additionally

the

late

ZOL

group

had

a

significantly

lower

MS/BS

and

a

nonsignificantly

lower

BFR

than

the

early

ZOL

group

Fig.

bone

histomorphometric

parameters

for

all

groups

in

the

metaphyseal

proximal

tibia

Groups

control

OVX

late

ZOL

early

ZOL

Based

on

ANOVA

and

a

Bonferroni

test

asignificantly

different

from

group

bsignificantly

different

from

group

csignificantly

different

from

group

and

dsignificantly

different

from

group

-LRB-

P

-RRB-

Discussion

In

this

study

we

assessed

the

effects

of

OVX

and

of

early

and

late

ZOL

treatments

after

OVX

on

the

microstructure

of

metaphyseal

bone

in

the

proximal

tibia

of

aged

female

Wistar

rats

over

a

time

course

using

in

vivo

high-resolution

micro-CT

OVX

induced

significant

changes

in

all

structural

parameters

within

weeks

indicating

the

rapid

development

of

OVX-induced

bone

loss

An

initial

phase

of

rapid

decreases

in

BV/TV

and

Conn.

D

was

seen

followed

by

a

second

phase

in

which

changes

took

place

at

lower

rates

The

initial

rapid

loss

of

bone

and

connectivity

was

largely

caused

by

trabecular

thinning

while

the

secondary

slower

loss

of

bone

was

accompanied

by

a

decrease

in

Tb.N

OVX

induced

large

decreases

in

BV/TV

Conn.

D

and

Tb.N

and

large

increases

in

Tb.Sp

and

SMI

indicating

the

change

of

more

plate

to

rod-like

bone

which

agrees

with

the

literature

on

the

effects

of

OVX

in

rat

tibiae

-LSB-

-RSB-

A

different

response

was

however

seen

in

Tb.Th

which

initially

decreased

until

weeks

after

OVX

and

then

increased

until

at

the

end

point

of

the

experiment

it

was

higher

than

in

the

control

group

A

possible

explanation

for

this

response

could

be

that

due

to

increased

osteoclast

activity

initially

most

trabeculae

will

become

thinner

while

only

few

are

completely

resorbed

After

a

certain

period

of

resorption

the

thinner

trabeculae

will

be

perforated

and

completely

resorbed

Since

the

thicker

trabeculae

remain

present

the

average

Tb.Th

will

increase

Another

possible

explanation

would

be

that

mechanical

adaptation

takes

place

Since

much

bone

is

lost

after

OVX

and

the

number

and

thickness

of

trabeculae

decrease

all

load

bearing

goes

through

the

remaining

trabeculae

Therefore

the

stress

increases

in

these

trabeculae

which

stimulates

bone

formation

and

would

lead

to

increased

Tb.Th

It

is

also

possible

that

a

combination

of

these

mechanisms

actually

takes

place

In

the

two

studies

published

to

date

regarding

in

vivo

micro-CT

monitoring

of

the

effects

of

OVX

on

the

metaphyseal

proximal

tibia

in

rats

the

loss

of

trabecular

bone

was

similar

to

what

was

seen

here

-LSB-

-RSB-

However

Tb.Th

significantly

increased

in

one

study

and

decreased

in

the

other

directly

after

OVX

Although

the

same

rat

strain

was

used

and

the

same

anatomical

location

was

studied

it

could

be

that

differences

such

as

the

use

of

retired

breeders

vs.

virgin

rats

age

or

type

of

data

analysis

led

to

variable

results

Early

ZOL

injection

at

the

time

of

OVX

completely

prevented

all

changes

in

structural

parameters

and

thus

the

development

of

OVX-induced

bone

loss

Aging

caused

a

significant

decrease

in

BV/TV

Conn.

D

and

Tb.N

and

an

increase

in

Tb.Sp

in

the

control

group

which

agrees

with

the

literature

-LSB-

-RSB-

However

a

significant

decrease

was

merely

seen

in

BV/TV

in

the

early

ZOL

group

and

at

a

significantly

lower

rate

than

in

the

control

group

This

indicates

that

a

single

injection

of

ZOL

was

powerful

enough

to

inhibit

even

natural

deterioration

of

bone

volume

and

microstructure

due

to

aging

One

year

of

weekly

ZOL

injections

of

a

concentration

as

low

as

μg/kg

completely

suppressed

the

effects

of

OVX

in

vertebrae

of

Sprague-Dawley

rats

and

μg/kg

was

needed

to

retain

all

structural

parameters

and

mechanical

properties

in

the

femurs

-LSB-

-RSB-

Similarly

weeks

of

weekly

ZOL

injections

in

OVX

rhesus

monkeys

reduced

bone

loss

in

a

dose-dependent

manner

-LSB-

-RSB-

In

a

phase

II

study

both

a

yearly

and

a

quarterly

ZOL

dose

increased

spinal

and

femoral

bone

mineral

density

in

postmenopausal

women

-LSB-

-RSB-

In

the

current

study

we

have

shown

the

long-term

potent

inhibiting

effects

of

ZOL

on

both

BV/TV

and

the

microstructure

in

rats

While

the

preventive

effects

of

ZOL

have

previously

been

studied

in

rats

-LSB-

-RSB-

the

effect

of

ZOL

treatment

after

the

development

of

OVX-induced

bone

loss

on

the

microstructure

which

more

closely

resembles

the

clinical

situation

is

new

Late

ZOL

injection

after

weeks

of

bone

loss

development

inhibited

further

bone

loss

Possibly

as

a

result

of

the

filling

of

resorption

cavities

-LSB-

-RSB-

a

temporary

but

significant

increase

in

BV/TV

and

thickness

and

a

significant

decrease

in

SMI

were

seen

directly

after

ZOL

administration

Four

weeks

later

however

all

structural

parameters

stayed

constant

This

is

in

contrast

with

the

OVX

group

which

still

showed

deterioration

of

bone

structure

after

weeks

Overall

bisphosphonate

treatment

after

the

onset

of

osteoporosis

is

known

to

stabilize

or

increase

BV/TV

and

Tb.Th

in

both

humans

and

other

animals

which

agrees

with

the

data

presented

here

-LSB-

-RSB-

It

is

generally

thought

that

the

underlying

mechanism

for

an

increase

in

BV/TV

and

Tb.Th

is

the

inhibiting

effect

of

bisphosphonates

on

bone

resorption

preventing

further

thinning

of

trabeculae

and

the

reduction

of

remodeling

space

by

filling

of

resorption

cavities

leading

to

increased

bone

mass

-LSB-

-RSB-

Cortical

thickness

first

decreased

as

a

result

of

OVX

later

increased

again

and

finally

was

higher

than

that

of

the

control

group

Both

increases

and

decreases

in

metaphyseal

tibial

cortical

thickness

have

been

reported

in

cross-sectional

studies

which

may

be

due

to

different

analytic

methods

or

different

time

points

of

sacrifice

-LSB-

-RSB-

Early

ZOL

treatment

resulted

in

a

similar

cortical

thickness

as

seen

in

the

control

groups

which

was

fairly

constant

Late

ZOL

treatment

resulted

in

a

similar

pattern

as

in

the

OVX

group

although

less

pronounced

which

may

be

explained

by

the

inhibiting

effect

of

ZOL

on

bone

remodeling

rate

Cortical

thickness

profiles

in

all

groups

were

similar

to

those

for

Tb.Th

which

may

be

interpreted

as

an

indication

that

mechanical

adaptation

takes

place

in

both

cortical

and

trabecular

bone

after

sustained

bone

loss

No

significant

differences

were

found

in

mechanical

properties

determined

in

the

three-point

bending

test

between

the

OVX

and

control

groups

which

agrees

with

the

literature

-LSB-

-RSB-

Also

no

significant

influence

of

ZOL

treatment

was

found

on

the

mechanical

properties

determined

in

the

three-point

bending

test

which

may

be

expected

as

ZOL

inhibits

bone

resorption

and

does

not

increase

formation

In

the

proximal

tibia

compression

test

ultimate

displacement

was

found

to

be

higher

in

the

OVX

group

than

in

the

other

groups

ZOL

treatment

partially

recovered

this

increase

in

ultimate

displacement

Although

not

significantly

ultimate

force

and

stiffness

tended

to

be

lower

in

the

OVX

group

while

ZOL

treatment

inhibited

this

The

low

number

of

animals

per

group

and

variability

in

the

data

unfortunately

limit

the

interpretation

of

these

results

It

is

known

that

while

endosteal

bone

resorption

takes

place

during

aging

and

is

increased

as

a

result

of

OVX

periosteal

apposition

increases

as

well

-LSB-

-RSB-

which

was

also

seen

in

our

CT

scans

-LRB-

data

not

shown

-RRB-

thereby

compensating

for

the

loss

of

endocortical

and

trabecular

bone

-LSB-

-RSB-

Therefore

loss

of

trabecular

and

endocortical

bone

may

not

necessarily

mean

loss

of

strength

While

first-generation

bisphosphonates

such

as

etidronate

have

been

shown

to

directly

decrease

mineralization

of

bone

-LSB-

-RSB-

it

is

thought

that

ZOL

may

increase

mineralization

by

inhibiting

resorption

and

thereby

allowing

longer

mineralization

-LSB-

-RSB-

Furthermore

microdamage

could

accumulate

and

both

of

these

effects

could

alter

the

mechanical

properties

of

the

bone

tissue

Nonetheless

the

trends

in

the

mechanical

properties

resembled

the

trends

in

BV/TV

and

showed

that

ZOL

inhibited

the

changes

resulting

from

OVX

Bone-forming

activity

was

slightly

higher

in

the

OVX

compared

to

the

control

group

reflecting

an

increased

turnover

rate

Both

ZOL

groups

displayed

lower

bone-forming

parameters

compared

to

the

control

and

OVX

groups

probably

due

to

suppression

of

bone

formation

and

resorption

Additionally

the

late

ZOL

group

tended

to

show

slightly

less

bone-forming

activity

than

the

early

ZOL

group

possibly

due

to

the

later

time

point

of

treatment

These

results

agree

with

previous

reports

of

decreased

osteoblast

and

osteoclast

numbers

after

ZOL

injection

and

decreased

number

of

resorption

lacunae

-LSB-

-RSB-

CT-derived

estimates

of

mineralization

of

metaphyseal

trabecular

bone

were

however

not

significantly

increased

in

ZOL-treated

rats

This

would

indicate

that

ZOL

treatment

did

not

influence

mineralization

in

this

time

frame

As

previously

mentioned

at

times

substantial

differences

were

found

between

groups

however

due

to

a

relatively

high

standard

deviation

no

significant

differences

unfortunately

were

detected

This

high

standard

deviation

was

mostly

seen

in

the

OVX

and

late

treatment

group

This

was

most

likely

caused

by

the

fact

that

retired

breeders

were

used

which

are

known

to

show

a

variable

response

to

OVX

Due

to

an

unsuccessful

OVX

seven

animals

were

removed

from

the

study

This

decreased

the

number

of

animals

used

in

the

data

analysis

and

since

these

were

mostly

animals

with

a

relatively

low

BV/TV

the

average

BV/TV

at

the

start

of

the

study

was

higher

in

the

OVX

and

late

treatment

groups

Therefore

we

performed

statistical

tests

on

percentage

changes

in

structural

parameters

rather

than

on

the

absolute

values

The

trend

for

the

percentage

and

absolute

changes

looked

similar

and

the

P

values

were

similar

for

most

cases

However

when

ANOVA

was

performed

on

the

absolute

values

comparing

all

groups

at

week

less

significant

differences

in

structural

parameters

were

found

indicating

the

more

sensitive

approach

of

the

in

vivo

experimental

design

Another

limitation

to

this

study

was

the

fact

that

on

average

at

weeks

in

the

study

the

OVX

group

was

in

a

more

deteriorated

condition

than

the

late

treatment

group

while

there

was

no

demonstrable

explanation

for

this

Although

the

ANOVA

showed

no

significant

differences

between

the

groups

the

treatment

group

still

had

a

more

favorable

starting

point

when

ZOL

was

administered

However

because

after

ZOL

injection

significant

improvement

took

place

while

the

OVX

group

still

deteriorated

it

is

shown

that

this

will

not

have

affected

the

final

conclusions

The

method

of

in

vivo

CT

scans

combined

with

image

registration

software

presented

here

has

enabled

us

to

study

our

goals

in

a

more

efficient

way

in

terms

of

number

of

animals

needed

in

the

experiment

Follow-up

micro-CT

scans

were

made

of

the

same

animals

enabling

us

to

use

paired

statistics

and

relative

percentage

changes

in

structural

parameters

By

registering

all

CT

scans

we

were

able

to

select

the

same

part

of

the

metaphyseal

trabecular

bone

for

every

measurement

thereby

decreasing

the

variation

in

structural

parameters

due

to

a

varying

selected

region

of

interest

In

conclusion

OVX

induced

rapid

changes

in

trabecular

bone

mass

and

structure

A

single

injection

of

ZOL

at

OVX

completely

inhibited

these

changes

in

bone

mass

and

structure

seen

in

osteoporotic

rats

A

single

ZOL

injection

after

OVX-induced

bone

loss

improved

bone

mass

and

structure

per

group

and

compared

to

the

control

group

BV/TV

and

the

associated

bone

microstructure

at

the

end

point

of

this

study

were

however

significantly

less

favorable

than

in

the

early

treatment

group

O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O