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 (IGT and/or IFG). A detailed search of MEDLINE (1966–2006) and EMBASE (1980–2006) 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% (range 42–63%); 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 [1]. Prediabetes is the collective term for people with IGT and/or IFG [2]. Prediabetes is associated with an increased risk of development of type 2 diabetes [3] and cardiovascular disease [4–6]. 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 [7–9]. 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 [10–14]. 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 (1966 to February week 4, 2006) and EMBASE (1980 to week 8, 2006) 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 (MeSH) 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’ (subheading ‘prevention and control’), 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 (T. Yates) 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 (T. Yates and K. Khunti) 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 (age ≥18 years) diagnosed with prediabetes. Prediabetes was defined as IGT and/or IFG using one of the sets of criteria previously recommended by the WHO [15, 16] or the American Diabetes Association (ADA) [17, 18]. 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 (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).
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 (2-h plasma glucose for IGT, or fasting glucose for IFG).
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 (interquartile range). Results reporting the SD or the 95% CI were converted to SEM using the formulas  (where t is the t distribution value for a 95% CI), 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 (see Fig. 1). 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 (men/women)Inclusion criteriaType of interventionType of dietary interventionType of exercise interventionMethod of physical activity measurementLindström et al. 2003 [21]Finland/Finnish Diabetes Prevention StudyRCT3 years522 (172/350)IGT (WHO criteria, 1985), 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 [12]USA/Diabetes Prevention Research GroupRCTAverage follow-up 2.8 (range 1.8–4.6) years2,161 (680/1481)aIGT (ADA criteria, 1997), age ≥25 years, BMI ≥24 kg/m2 (≥22 kg/m2 if Asian), 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 [10]China/The Da Qing IGT and Diabetes Study RCT6 years530 (283/247)IGT (WHO criteria, 1985), 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 [24]Sweden/The 6-year Malmö feasibility studyNon-randomised controlled trial5 years260 (260/0)IGT (2-h post-challenge glucose values 7–11 mmol/l, and fasting plasma glucose <7.8 mmol/l)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 [19]EnglandRCT2 years69 (39/30)IGT (WHO criteria, 1985)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 [22]Netherlands/Study on lifestyle-intervention and impaired glucose tolerance MaastrichtRCT2 years114 (64/50)IGT (2-h post-challenge glucose values 7.8–12.5 mmol/l, and fasting plasma glucose <7.8 mmol/l), 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 [23]SwedenRCT1 year186 (69/117)IGT (WHO criteria, 1985), 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 [20]USARCT2 years62 (29/33)IGT (WHO criteria, 1998)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 (l/min)Change in VO2max (l/min)aBaseline self-reported physical activity levelsChange in self-reported leisure time physical activityaBaseline body mass (kg)Change in body mass (kg)aBaseline 2-h plasma glucose (mmol/l)Change in 2-h plasma glucose from baseline (mmol/l)a Baseline fasting glucose (mmol/l)Change in fasting plasma glucose (mmol/l)aRelative risk of diabetes in the intervention group vs the C: group (95% CIs)Lindström et al. 2003 [21]N/AN/AL: 156 (62 to 288) min/weekL: 61 (−33 to 168) min/weekb`L: 86.7 ± 0.8Results at 3 yearsL: 8.9 ± 0.1Change at 1 yearL: 6.1 ± 0.05Change at 1 year0.4 (0.3–0.7)C: 169 (65 to 352) min/weekC: 6 (−91 to 104) min/weekC: 85.5 ± 0.9L: −3.5 ± 0.3bC: 8.9 ± 0.1L: −0.9 ± 0.1bC: 6.2 ± 0.04L: −0.2 ± 0.04C: −0.9 ± 0.4C: −0.3 ± 0.1C: 0.0 ± 0.04Change at 3 yearsChange at 3 yearsL: −0.5 ± 0.2L: 0.0 ± 0.05C: −0.1 ± 0.2C: 0.1 ± 0.05Knowler et al. 2002 [12]N/AN/AL: 15.5 ± 0.7 MET-h/weekL: 6 MET-h/weekb,cL: 94.1 ± 0.6L: −5.6bL: 9.1 ± 0.03NRL: 5.9 ± 0.01NR0.4 (0.3–0.5)C: 17.0 ± 0.9 MET-h/weekC: 1 MET-h/weekcC: 94.3 ± 0.6C: −0.1C: 9.1 ± 0.03C: 5.9 ± 0.02Pan et al. 1997 [10]N/AN/AE: 3.4 ± 0.2 unitsdE: 0.6 unitsdNRD&E: −2.5E: 8.8 ± 0.1E: 1.7E: 5.6 ± 0.1E: 1.3E: 0.5 (0.2–0.9)D&E: 3.1 ± 0.2 unitsdD&E: 0.8 unitsdE: −0.4D&E: 9.1 ± 0.1D&E 1.7D&E 5.7 ± 0.1D&E: 1.5D&E: 0.6 (0.3–0.9)C: 2.4 ± 0.2 unitsdC: 0.1 unitsdC: −1.0C: 9.0 ± 0.1C: 4.0C: 5.5 ± 0.1C: 2.1Eriksson and Lindgärde 1991 [24]L: 2.46 ± 0.04L: 0.20bN/AN/AL: 82cL: −3.3bL: 8.2 ± 01L: −1.1bNRNR0.4 (0.2–0.7)C: 2.29 ± 0.1C: −0.05C: 83cC: 0.2C: 8.3 ± 0.1C: 0.1cOldroyd et al. 2006 [19]N/AN/ANRNRL: 85.3 ± 2.9L: −1.8 ± 1.1bL: 9.2 ± 0.1Change at 1 yearL: 6.1 ± 0.1Change at 1 yearN/AC: 85.5 ± 2.5C: 1.5 ± 0.5C: 9.2 ± 0.2L: −0.6 ± 0.3C: 6.2 ± 0.2L: 0.0 ± 0.1C: 0.2 ± 0.3C: 0.1 ± 0.2Change at 2 yearsChange at 2 yearsL: 0.2 ± 0.3L: 0.3 ± 0.1C: −0.5 ± 0.4C: 0.1 ± 0.2Mensink et al. 2003 [22]L: 2.15 ± 0.1L: 0.09 ± 1.90bN/AN/AL: 86 ± 1.9L: −2.4 ± 0.7bL: 8.9 ± 0.3Change at 1 year L: 5.9 ± 0.1Change at 1 yearN/AC: 2.13 ± 0.1C: −0.03 ± 2.77C: 83.7 ± 1.5C: 0.1 ± 0.5C: 8.6 ± 0.2L: −0.9 ± 0.3bC: 5.8 ± 0.1L: −0.1 ± 0.1C: 0.3 ± 0.3C: 0.1 ± 0.1Change at 2 yearsChange at 2 yearsL: −0.6 ± 0.3bL: 0.2 ± 0.1C: 0.8 ± 0.4C: 0.5 ± 0.1Lindahl et al. 1999 [23]L: 2.12 ± 0.1eL: 0.21 ± 0.1b,eN/AN/AL: 86.4 ± 1.1L: −5.4 ± 0.5bL: 7.5L: −0.7 ± 0.2L: 5.4L: −0.5 ± 0.1N/AC: 1.89 ± 0.1eC: −0.02 ± 0.1eC: 83.6 ± 1.1C: −0.5 ± 0.3C: 8.0C: −0.3 ± 0.3C: 6.1C: −0.3 ± 0.1Carr et al. 2005 [20]E: 1.93 ± 0.1E: 0.16 ± 0.04bN/AN/AE: 66.5 ± 2.9E: −1.8 ± 0.5bE: 9.2 ± 0.2Change at 6 monthsE: 5.3 ± 0.1Change at 6 monthsN/AC: 2.02 ± 0.1C: −0.04 ± 0.03C: 69.7 ± 2.6C: 0.6 ± 0.5C: 9.1 ± 0.2E: −0.7C: 5.4 ± 0.1E: 0.0C: −0.1C: 0.1Change at 2 yearsChange at 2 yearsE: −0.6E: 0.0C: 0.0C: 0.1Data are presented as means±SD or medians (interquartile range)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 [10, 12, 19–23]. The non-randomised trial identified control participants by using individuals who, for various (unstated) reasons, were not enrolled in the intervention programme [24].
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 [12, 21], 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 [19]. 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 [19, 20, 22].
Inclusion criteria All studies examined in this review included individuals with IGT and excluded those with isolated IFG [10, 12, 19–24].
Sex Except for one trial that involved only men (n = 188) [24], 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 [10, 12, 19, 21–24], and one used a structured gym-based exercise training intervention [20].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 [10, 12, 19, 21, 22, 24]. 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 [21, 24] and one provided discounted access to local gyms [19]. One study determined the effect of diet and exercise separately and in combination [10].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 [23]. 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 [20]. 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 [20].
Outcomes Four studies included the incidence of diabetes as the main outcome [10, 12, 21, 24], and four used 2-h plasma glucose levels as a direct measure of glucose control [19, 20, 22, 23]. All the studies using the incidence of diabetes as their main outcome were based on a multi-component lifestyle intervention (see intervention conditions).
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 (see Table 2). 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 (46% reduced risk) in the physical activity-only group than in either the combined physical activity and diet group (42% reduced risk) or the diet-only group (13% reduced risk), although the difference between groups was not statistically significant [10]. Three of these four studies relied on self-reported measures of physical activity [10, 12, 21], and of these, only the Diabetes Prevention Program (DPP) [12] and the Finnish Diabetes Prevention Study (FDPS) [21] 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 [12], which is approximately equivalent to walking at a moderate pace for 15 min/day [25]. 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 [21], and the Da Qing IGT and Diabetes Study reported no significant change in physical activity levels compared with the control group [10]. The Malmö Feasibility Study, which used an objective outcome measure (cardiovascular fitness), reported an 8% increase in maximal oxygen uptake [24].
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 [10, 21, 24]. The FDPS reported a 0.9 mmol/l decrease in 2-h plasma glucose after 1 year, but no significant change after 3 years [21]; 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 [10]; and the Malmö Feasibility Study reported a 1.1 mmol/l reduction in 2-h plasma glucose in the intervention group [24]. 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 [19, 22, 23], only one reported a significant difference between the groups in terms of 2-h plasma glucose at follow-up [22]. Two of the studies used a measure of cardiovascular fitness as an indicator of physical activity levels [22, 23], and one [19] used distance walked in a shuttle test [26] as a measure of physical activity. Two studies found a small to moderate increase in cardiovascular fitness (<10% increase compared with baseline value) [22, 23], and the study using the shuttle test reported no change in the distance walked during the test [19]. 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 [20].
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 [24].
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% (range 42–63%) in individuals who were encouraged to reduce their body mass through changes in diet and physical activity [10, 12, 21, 24]. 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 [10] 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 [27], 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 [10] 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 [28, 29]. 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 [30], 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 [10–12] 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 [31], 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 [19, 20, 23]. Two of the three studies were unsuccessful at improving glucose tolerance [19, 23]. Similarly, the one study that used an aerobic exercise training protocol found no improvements in glucose tolerance as measured by 2-h plasma glucose [20]. 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 [21]. One reason for this discrepancy is likely to be the poor repeatability of 2-h plasma glucose values [32], 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 [33], 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 [20]. 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 (SLIM) [34], 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 [35]. 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 [36]. 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 [35]. 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 [37], 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.

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


Closed reduction and percutaneus Kirschner wire fixation for the treatment of dislocated calcaneal fractures: surgical technique, complications, clinical and radiological results after 2–10 years

Introduction To reduce complications, a minimally invasive technique for the treatment of dislocated intraarticular fractures of the calcaneus was used. Therefore previously described closed reduction and internal fixation techniques were combined and modified.
Introduction
A fracture of the calcaneus allowed to heal in improper anatomical position leads to static and dynamic malfunctions of the whole foot with consequent limited load bearing capacity and walking ability [4, 29]. The associated pain leads to a significant impairment in quality of life. The goal of therapy for calcaneal fractures is the elimination of pain and restoration of walking ability for patients with normal foot shape and the ability to wear normal footwear.
At present, there are multiple operative procedures for the treatment of calcaneal fractures. The procedures can be differentiated by approach, implant type and whether the treatment is one- or two-stages. Recently, open procedures using internal fixation have been favored for surgical therapy of the calcaneus [2, 5, 9, 15, 17, 23, 24, 26]. A possible complication of an open procedure is the disturbance of wound healing with skin and soft tissue necrosis [2] and the possibility of cutaneous flaps [5, 8, 9, 15, 17]. In addition to posttraumatic arthritis in the lower ankle and adjoining joints, there are reports of osteitis of the calcaneus [2, 5, 17]. Advanced osteitis of the calcaneus can require a calcanectomy [15] or amputation of the lower leg [9, 17].
In an effort to reduce the complications that can occur with an open procedure, we have combined and modified previously described closed reduction and internal fixation techniques [14, 27, 28] to create a minimally invasive technique.
The aim of this study was to evaluate the clinical and radiographic results of our minimally invasive surgical treatment of intraarticular fractures of the calcaneus at 2–10 years postoperatively. We then compared our results to the results of both open and minimally invasive surgical techniques found in the literature.
Material and methods
Patients
A total of 88 patients with 92 closed, dislocated and intraarticular fractures of the calcaneus were consecutively treated with a minimally-invasive technique developed at our institution by modifying and combining the procedures of Westhues [27], Poigenfürst and Buch [14], and Wondrák [28]. All the surgeries were performed without the use of bone graft. The average age at time of calcaneal fracture was 46.1 years (range 18–82 years) and most patients were male (71.6%). The cause of fracture was a fall from varying heights in 75 (85.2%) of cases and a motor vehicle accident in 13 (14.8%) of cases. From this group of patients, 63 (71.6%) patients with 67 fractures were available for retrospective examination with an average follow-up time of 5.7 years (range 2–10 years). Twenty-five patients were unavailable for follow-up examination; 12 patients who satisfied with the result of the surgery did not wish to participate in the study due to age or unwillingness to travel to the hospital, 7 patients were at an unknown address and 6 patients deceased.
Examination
All the medical records, radiographs, pre- and post-operative computed tomography scans were available for the entire study group. Traumatic soft tissue damage was determined using the classification method of Tscherne and Oestern [25]. In addition to radiographic evaluation using the calcaneus lateral, calcaneus axial and Broden views at 20° and 40° [3], all the patients obtained a bilateral calcaneal preoperative CT scan for fracture classification and surgical planning. Fractures were evaluated by the classification scale of Sanders et al. [17, 18]. The duration of surgery, as well as the time the X-ray image intensifier used, were noted as operative data. For postoperative evaluation of the reduction, radiographs of the calcaneus in lateral, axial, Broden 20° and 40° [3] views were obtained. At the last follow-up evaluation, a CT scan of both calcanei was obtained to examine the geometry of the calcaneus and to evaluate arthritic changes in the lower ankle joint.
For a reconstruction of the calcaneal posterior facet to be considered satisfactory a joint deviation of ≤2 mm had to be seen radiographically on the postoperative radiographs. For the evaluation of the clinical results, the Zwipp score was used [30].
Statistical analysis
For statistical analysis the Fisher’s exact test and the Chi-square test were used.
Perioperative treatment and surgical technique
After a calcaneal fracture was diagnosed, the lower leg was evaluated. If the soft tissue was in good condition, primary surgery performed. In cases of severe swelling with potential soft tissue damage NSAIDs, local cryotherapy and active movement exercises determined the course of therapy.
Once the soft tissue was in good condition, surgery was performed. The patient was placed prone on the traction table under general anaesthesia or spinal anaesthesia without arrest of blood supply. After application of the calcaneus wire for traction in the dorso-cranial plane, the varus or valgus malaligment of the back foot was corrected with the wire positioned orthogonally to the longitudinal axis of the calcaneus. To achieve correct placement, the surgeon pulled at the traction bow with the knee bent along the longitudinal axis of the calcaneus. The subsequent traction in the plantar plane was performed with the leg stretched. Once placed in the proper orientation, the traction bow was connected to the retaining jig of the traction table (traction of 20 kg). The surgeon then held the heel of the patient with both hands and applied compression both medially and laterally with his thenar muscle to reduce the main medial and lateral fragments.
With the use of the X-ray image intensifier, the restoration of Böhler’s angle and the reduction of the posterior facet were verified. A Steinmann pin was put with the pointed end into a universal chuck and the other end into the dorso-lateral calcaneus beneath the posterior facet with a stab incision (Fig. 1a). For reduction of the posterior facet, the traction wire was used as a hypomochlion (Fig. 1b). The anatomical restoration of the posterior facet was verified with use of the Brodén radiographic views (20° and 40°). The reduction of the calcaneus was verified by examining the lateral radiographic view (Fig. 2a). The fragments were fixed with percutaneus Kirschner wires, which were arranged conically into the talus and/or cuboid (Fig. 2b). All of the main fragments to be reduced were held in place by the Kirschner wires and the arch of foot was held until the bone healed [28]. The wires were bent above the skin level, the wire traction removed and the stab incision closed with sutures.
Fig. 1The Steinmann pin is placed into the dorso-lateral calcaneus above the traction wire (left side). For the reduction of the posterior facet, the traction wire is used as a hypomochlion (right side). The hematome is drained though the stab incisionFig. 2Intraoperative lateral radiograph of a dislocated calcaneal fracture before reduction (left side). After reduction the fragments are fixed with percutaneus Kirschner wires (right side)
Postoperatively, a dorsal lower leg splint was applied for immobilization and the lower portion of the operative side was elevated. On the first postoperative day, the patient was mobilized on crutches with no weight bearing on the operative side. Depending on pain levels, the patient began with active dorsal extension and plantar flexion in the upper ankle joint. The radiographic controls, (upper ankle joint anterior and lateral, calcaneus axial Brodén views [25]) were performed immediately postoperative, and again 2 and 8 weeks postoperative. After the fracture was healed, the Kirschner wires were removed. Partial weight bearing began after the eighth postoperative week at 20 kg and was increased up to full weight bearing by the twelfth postoperative week (Fig. 3).
Fig. 3Clinical example. a Preoperative lateral radiograph of an intraarticular, displaced calcaneal fracture (left side). The preoperative CT scan shows a Sanders Type II AC fracture on the coronal reconstruction (right side). b Lateral radiograph taken 2.5 years postoperatively. The patient had a Zwipp score of 166 points (left side). Anatomical reconstruction of the posterior facet was achieved (right side). This coronal CT image shows the anatomical reconstruction of the posterior facet with no joint deviation
Results
Fifty-five of the 88 (62.5%) patients suffered exclusively from calcanal fractures. Thirty-three of the 88 (37.5%) patients had multiple injuries. Twenty of the 33 (61%) patients with multiple injuries had local co-injuries such as fractures of the upper ankle joint, tarsus and front-foot. In 83 of the 92 calcaneal fractures, the soft tissue injury was graded as 1° or 2°; in 9 fractures, the soft tissue injury was graded as 3°. In eight cases, including three patients with multiple injuries, surgery was performed immediately on the day of the trauma.
Length of surgery averaged 61 min (range 20–175 min) and screening time averaged 115 s (range 20–454 s). To obtain proper retention of the fracture, between 4 and 7 Kirschner wires were used. The Kirschner wires were removed with or without local anaesthesia at an average of 10 weeks (range 7–15 weeks) as an outpatient procedure. Full weight bearing was achieved at an average of 15 weeks postoperative.
Sanders classification
Sanders type II, III and IV fractures were diagnosed (Table 1). All the patients had a joint deviation of at least 2 mm. Patients with type I non-dislocated fractures were treated conservatively and were therefore not included in this study. Reconstruction of the posterior facet was verified radiographically in 13 of 15 (86.7%) type II fractures, in 47 of 52 (90.4%) type III fractures and in 16 of 25 (64%) type IV fractures immediately postoperatively (Table 1).
Table 1Patients with immediate postoperative reconstruction of the posterior facet by Sanders fracture classificationSanders fracture classification (N = 92)Radiological reconstruction of posterior facet achievedType II15 (16.3%)13 (86.7%)Type III52 (56.5%)47 (90.4%)Type IV25 (27.2%)16 (64.0%)
The Zwipp score of 67 patients at the last follow-up evaluation
The Zwipp score of the 67 patients available for follow-up averaged 130/200 points (range 48–186 points) which is considered a good result [30]. Regardless of fracture type, 41 (61.2%) of the 67 patients had a very good or good result, 24 (35.8%) patients had a satisfactory result and 2 (3%) patients had a bad result. The worst clinical results occurred with type IV fractures (Table 2). Thirty (44.8%) patients considered their treatment outcome very good or good, 28 (41.8%) as satisfactory and sufficient and 9 (13.4%) as insufficient.
Table 2Clinical and radiological results at last follow-up evaluation by Sanders fracture classificationSanders fracture classificationVery good or good resultArthritis in lower ankle jointNormal Böhler angle achievedOverall6741 (61.2%)33 (49.3%)47 (70.1%)Type II 6 (8.9%)4 (66.7)1 (16.7%)5 (83.3%)Type III 39 (58.2%)29 (74.4%)12 (30.8%)31 (79.5)Type IV 22 (32.8%)8 (36.4%)20 (90.9%)11 (50.0%)
Thirty-seven (55.3%) of the 67 patients had no pain while full weight bearing or could walk at least 4 h without pain at last follow-up evaluation. Nine (13.4%) patients had constant pain. Thirty-three (49.3%) patients had a range of motion in the upper ankle joint identical to that of the non-affected side. Thirty-four (50.7%) patients had a restricted range of motion of up to 15° and more than half (58.2%) of the patients had achieved more than 75% of their total range of motion in the lower ankle joint. Forty-three (64.2%) of 67 patients were able to wear normal shoes while 5 (7.4%) used shoes with an unroll aid. Nineteen (28.3%) patients had obtained orthopaedic shoes on their own at last follow-up.
Two (3%) of 67 patients had arthritis of the upper ankle joint at last follow-up. Independent of fracture type 33 (50.2%) patients had arthritic changes in the lower ankle joint and 14 (20.89%) cases had arthritic changes in the calcaneocuboidal joint (Table 2). Böhler’s angle was restored in 70.1% (47 of 67) of the cases (Table 2). A total of 85.1% (57 of 67) cases had a reduction in calcaneal height to within 10% when compared to the non-operative side. Additionally, a reduction in the length of the calcaneus occurred within 10 in 94% (63 of 67) of cases when compared to the non-operative side. Thirty-eight (56.8%) of the 67 patients had a widening of the calcaneus to within 10% compared to the opposite side.
Complications
Of the 92 surgically treated calcaneus fractures, 76 (82.6%) healed without complications. In nine (9.8%) cases, superficial skin infections, perforations of the Kirschner wires and bone dystrophy occurred and healed without any further complications. Significant complications occurred in six (6.5%) cases: three cases had osteitis of the calcaneus, one case had dislocation of the fracture requiring revision surgery and two cases had peroneal tendon impingement. Three cases of osteitis healed through conservative therapy with oral antibiotics and the two patients with impingement of the peroneal tendon refused any operative intervention.
Using our method, disturbance of wound healing with skin and soft tissue necrosis requiring operative intervention was not observed. Additionally, no lower leg amputations and no total or partial calcanectomies had to be performed.
Statistical analysis
Using the Chi-square test a statistical significant correlation was found between the fracture type and the incidence of subtalar arthritis (p < 0.0268). Using the same test there was no statistical significant correlation found between the fracture type and quality of reduction (p > 0.6522), between fracture type and clinical result (p > 0.3204) and between fracture type and Böhler’s angle (p > 0.5488). However, a statistical significant correlation was found between the clinical result and subtalar arthritis (p < 0.0013) and between the incidence of subtalar arthritis and quality of reduction when using the Fisher`s exact test (p < 0.0003).
Discussion
To correct deformations of the calcaneus, spare the soft tissue and lower the complication rate, indirect and less invasive reduction and fixation techniques to treat calcaneal fractures have been developed [7, 10, 12–14, 16, 19, 21, 22]. Besides techniques with percutaneous reduction and internal K-wire fixation [7, 14, 16, 21] percutaneous reduction techniques with external fixation are described in the literature [12, 13, 19, 22]. In our department we have combined and modified previously described closed reduction and internal fixation techniques [14, 27, 28] to create a minimally invasive technique. This method used the lower joint surface of the talus as a guide for remodeling the posterior facet and reconstruction of the calcaneus.
A multiple of different evaluation scores makes a comparison of the clinical results with published results difficult [11, 17, 20, 30]. Using the Zwipp score, 61.3% of the cases (41 of 67) treated by our modified method had good or very good results regardless of fracture type. In 38.7% (26 of 67) of cases, a satisfactory or bad result was achieved. After open reduction and internal fixation with a plate, Rammelt et al. [15] and Boack et al. [2] reported good and very good results in 65 and 66% of cases, respectively with the use of the ±200 Zwipp score. Boack et al. [2] presented satisfactory or bad clinical results in 34% of cases also using the Zwipp score.
Sanders et al. [18] showed that under application of a lateral approach followed by internal fixation with a plate, the worst clinical results occurred with type IV fractures. In this study the worst clinical results were achieved in the treatment of type IV fractures although within our cases no statistical significant correlation between fracture type and clinical result could be demonstrated (Table 2).
Patient satisfaction is an essential criterion for the successful treatment of calcaneus fractures. With a comparable follow-up period of 5.4 years, Thermann et al. [23] reported that 48.3% of patients viewed their treatment outcome as good or very good after open reduction of their calcaneus fractures, 37.3% of patients judged their treatment outcome as satisfactory and sufficient and 14.4% judged their outcome as insufficient. Our treatment method had comparable results with 44.8% of patients considering their treatment outcome very good or good, 41.8% as satisfactory and sufficient and 13.4% as insufficient.
At last follow-up evaluation, 64.2% of patients in this study were able to wear normal shoes while 28.4% required orthopaedic shoes. Comparably, in the study by Therman et al. [23], 68.7% of the patients wore normal shoes and 16.8% required orthopaedic shoes.
Overall, satisfactory mobility of the joints adjoining the calcaneus was achieved with our surgical technique. In approximately half of our patients, complete range of motion in the upper ankle joint was achieved and in the lower ankle joint, approximately 60% of patients had no or low movement restriction. Similar mobility in the upper and lower ankle joints has been reported in the literature after open reduction and osteosynthesis with a plate [5, 23]. However, Buch [6] reported a worse range of motion in the upper and lower ankle joint after performing a percutaneus wire osteosynthesis in 100 calcaneal fractures with a varus or valgus deviation of the back foot occurring in half of the cases. In this study, malpositions of the back foot of more than 5° were diagnosed in 6 (9%) cases of the 67 cases. Clinically relevant changes in the axis of the front foot were not observed.
The radiological evaluation of the joints adjoining the calcaneus showed arthritic changes in the upper ankle joint in two of our cases. Half of our cases showed arthritic changes in the lower ankle joint and in approximately a fifth of our evaluated cases, arthritic changes were found in the calcaneocuboidal joint. In our study, 66.7% of type-II fractures and 98% of type-III/IV fractures showed arthritic changes in the lower ankle joint. These results are similar to those presented by Thermann et al. [23], who showed arthritic changes in the lower ankle joint in 65.2% of type-II fractures and in 81.7% of type III/IV fractures.
Regardless of fracture type, anatomical restoration of the joint surface or a joint deviation of up to 2 mm of the posterior facet was achieved postoperatively in 82.1% of cases. A total of 86.7% of type-II fractures and 72.8% of type-III/IV fractures achieved anatomical restoration of the posterior facet. These results are comparable to those presented in the literature with open reduction and internal fixation with plates or screws [2, 17, 23].
In this study, the average length of the surgery was 61 min. This is considerably less than the average length of surgeries utilizing open reduction and internal fixation of 139 and 168 min [2, 20].
In 9.8% of cases, complications such as superficial skin infections, perforation of the Kirschner wires and bone dystrophy healed with conservative therapy or after removal of the Kirschner wires. This rather insignificant rate of complications is similar to those described in the literature after operative treatment of calcaneal fractures utilizing a minimally invasive technique [7, 16, 21].
The rate of significant complications in this study was 6.5%. In two cases, an impingement of the peroneal tendon occurred. One case had a dislocation of the fragments requiring revision surgery. Three cases had infections of the calcaneus and were related to a traumatic soft tissue injury that healed with conservative therapy. With open reduction, skin and soft tissue necrosis [2] with possible cutaneous flaps have been reported in scattered cases [5, 8, 9, 15, 17]. Folk et al. [9] demonstrated that in 25% of cases, wound complications required an additional surgery after open reduction of calcaneus fractures 84% of the time. Abidi et al. [1] reported wound healing disturbances in 33% of cases following open reduction and internal fixation of calcaneal fractures. There have also been reports in the literature of partial or total calcanectomies [15], including amputations, as a result of calcaneal osteitis after open reduction [9, 17]. Sanders et al. [17] considered a lower leg amputation if a patient suffered continual osteitis. In the current study no disturbances of wound healing, skin or soft tissue necrosis that required microsurgical intervention were observed.
In this study no statistical significant correlation could be found between fracture type and the ability to restore Böhler`s angle. However, regardless of fracture type Stulik et al. [21] showed similar results using a comparable minimally invasive technique while Thermann et al. [23] had worse results following open reduction with internal fixation.
In our study, there was a slight reduction in the height and length of the operated calcaneus when compared to the non-operated side. With our closed reduction and internal fixation technique, more than half of the cases had a widening of the calcaneus of more than 10% when compared to the opposite side, which is an unsatisfactory result. At the same time only two patients had an impingement of the peroneal tendon that the patients considered tolerable.
In summary, we presented a minimally invasive technique for the treatment of intraarticular, dislocated calcaneus fractures and were able to produce results comparable to open techniques with a lower rate of serious complications. In the majority of cases, an almost identical Böhler angle and geometry of the calcaneus was achieved when compared to the opposite side at the time of last follow-up. Simple removal of the Kirschner wires and shorter surgery time decrease patient stress and must be recognized as an advantage of this minimally invasive technique. Thus, we feel that our minimally invasive technique is a viable alternative for the treatment of intraarticular, dislocated calcaneal fractures.

dislocated calcaneal fracture
minimal-invasive


Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.
Introduction
Spinal cord injury (SCI) occurs in an instant, but its devastating effects last a lifetime at huge personal and economic cost [1]. The spinal cord conveys both afferent sensory and efferent motor information, so disruption of spinal cord function results not only in motor paralysis but also sensory and autonomic impairment distal to the injury [2, 3]. Sensory dysfunction contributes to the generation of pressure sores that, like bladder impairment, are a major source of morbidity and even mortality in those with spinal cord injury [4].
Restoration of function in longstanding spinal cord injuries is poor, although some limited functional restoration has been reported [5]. Therefore, there is much interest in reducing the extent of the initial damage from traumatic spinal cord injury in the acute phase. Trauma often results in primary damage resulting from mechanical disruption of the nerve axons in the spinal cord that is not amenable to neuroprotective therapy. However, secondary pathological changes involving cascades of biochemical, molecular, and cellular changes can produce even more extensive damage, and these changes are potentially susceptible to therapeutic intervention with neuroprotective agents [3, 6]. Thus, pathological changes occur from the moment of injury and continue for years afterwards and have been divided into three phases: an acute phase, a phase of secondary tissue loss, and a chronic phase [3, 6].
In the acute phase, which starts at the moment of injury and extends over the first few days, numerous pathological processes begin. Mechanical injury induces an immediate change in neuronal tracts at the moment of impact, blood flow is reduced, creating substantial ischemic necrosis [7, 8] and a cascade of pathophysiological processes rapidly follows mechanical trauma to the spinal cord, resulting in secondary neuronal damage that can significantly exacerbate the original injury [9]. Traumatic injury to the spinal cord also leads to a strong inflammatory response, with the recruitment of peripherally derived inflammatory cells, including macrophages [10]. Damage to the spinal cord also results in extensive cell proliferation in and around the epicenter, many of which are microglia and macrophages. This acute inflammatory response at the site of the initial lesion is at least partly responsible for secondary spinal cord pathology [11–13]. The inflammatory cells (particularly macrophages/microglia) can mediate tissue damage by producing a variety of cytotoxic factors including interleukins [14], tumor necrosis factor-alpha (TNF-alpha) [15], and reactive nitrogen species [13, 16–18]. Neuronal and oligodendroglial cell loss is apparent in the lesion epicenter, and rostral and caudal to it within 4 h of injury [19]. From days to years after the initial trauma, apoptotic cell death continues, and scarring and demyelination accompany Wallerian degeneration. All these processes contribute to motor and sensory functional deficits [20, 21].
Many pharmacological agents have been reported to reduce secondary injury and to be neuroprotective in a variety of animal models; these include anti-inflammatory [22–24] and antiapoptotic agents [24, 25] and agents that elevate cyclic adenosine monophosphate (cAMP) [26, 27]. None has yet proved effective in ameliorating the effects of acute spinal cord injury in clinical trials in humans.
There is increasing evidence that the non-adenine-based purine guanosine acts as an intercellular signaling molecule. It is released from cells and has several diverse effects on cells in vivo and in vitro, particularly trophic effects modulating cellular growth, differentiation, and survival [28, 29]. Guanosine has a number of effects on various cell types that make it a good candidate to test as a neuroprotective agent in acute spinal cord injury since it might potentially interact with several steps of the biochemical and cellular cascade. It is neuroprotective [28–32] and stimulates nerve regeneration [5, 33]. It also protects several cell types against apoptosis induced by a variety of agents such as staurosporine [34] and beta-amyloid [35] and has been reported to increase intracellular cAMP [36, 37]. Therefore, in the present study, we assessed whether guanosine might ameliorate tissue damage and enhance functional outcome after acute spinal cord injury.
Materials and methods
Animals
Adult female Wistar rats (280–300 g body weight, Charles River) were maintained in a temperature-controlled vivarium on a 12:12-h light-dark cycle with food and tap water freely available. Rats were handled daily for 2 weeks before surgery.
Spinal cord injury induction and experimental design
Spinal cords were surgically exposed and compressed with modified coverslip forceps to produce a moderate traumatic spinal cord injury [5, 38]. Before surgery, rats were given buprenorphine (0.03 mg/kg body weight, subcutaneously) for pain relief. They were then anesthetized with isoflurane (3–5%): O2 (1 l/min) and a laminectomy was performed at T11/T12 to expose the spinal cord that was then crushed with modified coverslip forceps [5, 38–40]. The forceps were closed slowly (over 2 s) compressing a 5-mm length of the spinal cord to a thickness of 1.4 mm for 15 s. The wound was closed by suturing the muscles and fat pad, and by clipping the skin with stainless steel clips. Postoperatively, the rats were kept quiet and warm.
To evaluate the effect of guanosine on acute phase SCI pathophysiology, we studied two groups of rats (n = 24) with SCI. After surgery and prior to treatment, behavioral tests were done for each rat. Two of the total 24 animals were excluded from the study because of incorrect injury (BBB score at the surgery day before treatment below 4 or above 7; in our experience, about 91% of rats with this degree of crush were within the range of 5 to 6 on the BBB scale at first day after injury [38]). The 22 rats were then randomly divided into two groups. Starting 4 h after surgery, rats received either daily intraperitoneal (i.p.) injections of 8 mg/kg guanosine or the same volume of saline containing 0.001 N NaOH [5] for 2 weeks. On day 7 after injury one animal in the saline group was euthanized because of a severe bladder infection.
Motor and sensory functional recovery assessment
All rats were handled daily for 2 weeks preoperatively to acclimatize them to the handling and behavioral testing. After spinal cord compression, the locomotor behavior segmental reflexes and spinothalamic senses of the rats were assessed immediately prior to treatment (day 0) and on days 1, 3, 5, 7, 14, 21, and 28 after the injury. Five tests were used: an open field walking task [5, 41, 42], hind limb placing response [38, 40], foot orienting response [38, 40, 43], an inclined plane test [38, 44, 45], and a hot plate test [46]. Behavioral analyses were performed by individuals who were blinded with respect to treatment groups.
An open field walking testing (OFWT) was used to assess the locomotor functional recovery of the hind limb. It was conducted in a child’s circular plastic swimming pool (1.3 m in diameter [5, 38]). Cagemates (two animals) were placed in the center of the open field. They were observed for 5-min periods and scored for general locomotor ability using the standard BBB scale. The rats were rated on a scale of 0 to 21, 0 being no function and 21 being normal. If the animal stopped moving for a minute, it was placed again in the center of the open field; otherwise it was left undisturbed for the duration of the 5-min test period.
Reflexes in the hind limbs were assessed with hind limb placing response (HLPR) and foot orienting response (FOR). They were each scored on a scale of 0 to 2, 0 indicating no function and 2 indicating full function [38, 40, 43]. Half-scores were assigned if the behavioral response appeared intermediate. Hind limbs were scored separately for each measure. To assess HLPR, we grasped the hind foot between the thumb and forefinger, pulled backwards, and then released the foot. The placement of the foot on the table surface was then scored [38, 40]. The FOR followed Gruner’s [40] protocol modified from the previous descriptions of this reflex [43]. When a rat is raised and lowered by the tail, it shows a characteristic behavior of the hind legs. A normal rat spreads the toes of its hind leg wide apart and generally holds them apart for several seconds. After spinal cord injury, this response is sometimes lost completely, or reduced in magnitude.
The inclined plane test (IPT) measured the ability of the rats to maintain their position for 5 s on an inclined plane, covered by a rubber mat containing horizontal ridges (1 mm deep, spaced 3 mm apart, self-made) [44]. The rats were observed as the angle of the surface was increased from 5 to 90° at 5° intervals. The angle at which the rat could no longer stay in position was the outcome measure.
Hind limb sensory function was tested by ability to perceive a standard application of a controlled stimulus to the trunk below the level of the lesion using a standard testing apparatus hot plate (IITC Life Science Inc., Woodland Hills, CA, USA) [46]. The time taken for the rat to withdraw its hind paws from contact with the hot plate was noted. A cutoff time was about 20 s after which the animals were removed to prevent thermal injury [46].
Recovery of lower urinary tract function
Normal lower urinary tract (LUT) function involves both spinal and supraspinal circuitry that controls urine storage and release [47]. Incomplete SCI results in an initial loss and later partial or complete recovery of LUT function depending on the severity of injury [48, 49].
After SCI the rats were not capable initially of spontaneous micturition, and their bladders were manually expressed twice daily. The volume of expressed urine was measured each time, and the data were used to estimate the initiation of LUT function after SCI.
Histological analysis
Tissue processing and immunohistochemical staining Twenty-eight days after SCI, animals were killed for histological and immunohistochemical analysis. Rats were deeply anesthetized with sodium pentobarbital (50–60 mg/kg b/w, i.p.) and perfused transcardially; first with 100 ml 0.05 M phosphate-buffed saline (PBS) containing 0.1% heparin, followed by 300–500 ml of 4% paraformaldehyde (PFA). The T9 to L1 segments of the spinal cords were removed and incubated in the same fixative solution overnight at 4°C and then cryoprotected in PBS solution containing 30% sucrose. A segment of each cord, extending from 5 mm rostral to 5 mm caudal to the lesion site was embedded in medium (Tissue-Tek® O.C.T. compound, Sakura Finetek USA, Inc., Torrrance, CA, USA). Serial sections were cut at 20-μm intervals on a cryostat and mounted onto slides (ColorFrost/Plus; Fisher, Pittsburgh, PA, USA) for histochemical staining. Some cords were transverse-sectioned for immunohistochemical analysis using the antibodies described in the section below. Longitudinal sections were cut for Luxol fast blue staining, a lipophilic dye used commonly to stain myelin [38].Details of these immunohistochemical procedures have been described previously [5, 38]. Briefly, the cryostat sections were thawed, air-dried, and then incubated in hydrogen peroxide to reduce endogenous peroxidase activity, before being rinsed in PBS. The sections were then incubated in 1% sodium borohydride for 15 min. After thorough washing with PBS, the sections were treated with PBS/5% normal goat serum with 0.3% Triton X-100 at room temperature for 30 min. Overnight incubation with primary antibody was performed in humidified boxes at 4°C. Macrophages and reactive microglia were detected with a mouse monoclonal antibody against ED-1 (MCA 341R, 1:500; Serotec, Hornby, ON, Canada) and reactive astrocytes (astrogliosis) were determined with rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies (Zymed® Lab-SA System Kit, dilution was 1:600, Invitrogen Canada Inc., Burlington, ON, Canada). The following day, sections were rinsed with PBS and incubated with either rhodamine-conjugated or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Sections were then rinsed, coverslipped, and examined under a confocal microscope.To determine the spared axons and myelin, double fluorescent immunolabeling was performed, combining mouse monoclonal antibodies against neurofilament (RT-97, 1:10; Developmental Hybridoma Bank) and rabbit anti-myelin basic protein (MBP) polyclonal antibodies (1:50; Chemicon Int., Temecula, CA, USA). For double immunolabeling, sections were developed using a mixture of FITC-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG in 1% normal goat serum and 0.25% Triton X-100; 1:200 (Invitrogen Canada Inc., Burlington, ON, Canada) for 2 h. Sections were then rinsed, coverslipped, and examined under a confocal microscope. An investigator who was blinded to the treatment groups conducted histological analysis.
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling assays and quantification For detection of apoptotic cells, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stain was performed using the “In situ Cell Death Detection Kit-Fluorescein” (Roche Molecular Biochemicals, Chemicon Int., Temecula, CA, USA), according to the manufacturer’s instructions. After fixation, tissue sections were incubated in TdT-mediated dUTP-biotin nick end labeling (TUNEL) reaction mixture containing TdT buffer with TdT and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature (37°C) for 30 min with fluorescence-conjugated antibody. The results were analyzed using confocal fluorescence microscopy. TUNEL-positive cells in the lesion site in the spinal cord were quantified by counting positively stained cells. Sections (5–7 per animal) taken from the penumbra of the lesion and spaced about 100 μm apart were analyzed for each animal (n = 3–5 animals per group). Apoptotic cell death was determined by counting the total number of TUNEL-positive nuclei through entire cross sections. Low power sections were digitized and manually outlined using an image analysis system. Any cavities present in the sections were excluded from analysis. Data are expressed as cells per section [5].
Quantification For cell counting, 5–7 sections from each animal (n = 5 for each group) at the lesion site (every third section, 100 μm apart) were analyzed. OX-42-positive microglia and TUNEL-positive nuclei were counted through the entire cross section. Data are expressed as the number of immunostained cells per section (mean ± SEM).
Statistical analysis
All data are presented as mean ± SEM. The statistical significance of behavioral scores was analyzed by Kruskal-Wallis nonparametric analysis of variance followed by Mann-Whitney U tests. Histological data were evaluated by Student’s t-tests.
Results
Guanosine improves neurological function
Over the course of 4 weeks after spinal cord injury, control rats (which received saline treatment) recovered occasional weight-supported plantar steps, but no fore-hind limb coordination, and had a mean BBB locomotor score of 9.3 ± 0.6 (Fig. 1). In contrast, the guanosine-treated rats recovered to a BBB score of 14 ± 0.5 (Fig. 1); they exhibited consistent weight supporting with consistent fore-hind limb coordination. Hind limb locomotor function in guanosine-treated animals was significantly better than in rats that received saline treatment (P < 0.01).
Fig. 1Open field walking test (OFWT) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 21, whereas a score of 0 represents total paralysis. Hind limb locomotor function in guanosine-treated animals (n = 11) was significantly better than in rats that received saline treatment (n = 10; P < 0.01) from day 1 to 28 after injury
Uninjured rats have normal HLPR scores of 2 [38, 40]; they always place an extended hind limb briskly beneath the body in a proprioceptive placing response. Injured rats place their hind limb either partially, or unreliably, or not all, depending on the time since the injury and the treatment. In the present study, 4 weeks after injury, control rats with saline injection attained a 1.0 ± 0.1 HLPR score (Fig. 2) that was characterized by little or no attempt to place the foot, or leaving the foot extended with its dorsal surface down. In contrast, rats treated with guanosine reached a score of 1.7 ± 0.1 (Fig. 2), with the toes of their hind legs spread wide apart, a finding which is significantly better than rats that received saline treatment (P < 0.01). Uninjured rats have a normal FOR score of 2. Saline-treated animals had a FOR score of 1.0 ± 0.1 (Fig. 3). These rats extended their hind legs laterally with toe spread but turned their feet outward. When these rats were lowered, they did not orient their feet toward the surface. In contrast, the guanosine-treated rats recovered to a score of 1.7 ± 0.1 (Fig. 3), which is significantly different (P < 0.01) from the saline-treated rats.
Fig. 2Hind limb placing response (HLPR) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their hind limb placing responses (P < 0.01) from day 1 to 28 after injuryFig. 3Foot orienting response (FOR) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their foot orienting responses (P < 0.01) from day 1 to 28 after injury
Uninjured rats maintain their position on the inclined plane even at an angle of 90°. In the present study, saline-treated control rats recovered their ability to maintain their position at 69° ± 2 by 4 weeks after injury, whereas guanosine-treated rats were able to maintain their position to a mean incline of 79° ± 1 (Fig. 4) by 4 weeks (P < 0.05).
Fig. 4Inclined plane test (IPT) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better score in their inclined plane test (P < 0.05) from day 1 to 28 after injury
Both saline- and guanosine-treated rats were insensitive to the thermal stimulus using the hot plate test during the first 2 days after injury. There was a gradual recovery of sensory function thereafter with recovery being accelerated in guanosine-treated animals compared to controls (P < 0.05) (Fig. 5).
Fig. 5Post-lesional sensitivity of hind limbs touched by a hot plate from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals. Values are the means ± SEM of the average time of withdrawal of left and right hind paws during contact with a hot plate. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their sensory function (*P < 0.05) from day 2 to 14 after injury
Guanosine accelerated LUT functional recovery following SCI
In saline-treated rats the volume of manually expressed urine increased over the first 7 days after SCI and then decreased as spontaneous micturition was reestablished. On the basis of a previous study [48], the increase in volume of expressed urine during the first week after injury was interpreted as resulting from increased bladder size in the absence of spontaneous micturition. The subsequent decrease in manually expressed urine indicates the initiation of spontaneous micturition (Fig. 6). In guanosine-treated rats there was a significantly lower residual urine volume compared to the saline-treated animals (Fig. 6; P < 0.001). More importantly, the rats that received guanosine recovered their LUT function completely by 7 days after injury (Fig. 6).
Fig. 6Time course of recovery of lower urinary tract function (spontaneous voiding). Urinary bladders were expressed every 12 h, and the collected urine volume was measured. Means ± SEM of the volume for each group. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had significantly less urine collected with time after SCI (*P < 0.05; **P < 0.001). The rats that received guanosine had empty bladders by day 7 after injury
Guanosine attenuates macrophage and microglia activation, but not astrogliosis
After SCI, early inflammatory reactions consisting of neutrophil and macrophage invasion as well as activation of microglia and astrocytes were reported in earlier studies [50, 51]. In the present study, the activated macrophages and microglia were labeled by the ED-1 immunoreaction. Guanosine treatment decreased the number of the ED-1 immunopositive cells compared with saline treatment (Fig. 7a, b, e; P < 0.01). However, no differences were found between the saline and guanosine groups in the number of activated astroglia (astrogliosis) indicated by the GFAP immunostaining (Fig. 7c, d).
Fig. 7Fluorescent immunostaining using antibodies against a marker (ED-1) for macrophages and activated microglia in cross sections of cords from saline-treated (a) and guanosine-treated (b) animals at the lesion site 4 weeks after injury. There were fewer ED-1-immunolabeled cells in cords of guanosine-treated rats (b, e) compared to the cords of vehicle-treated rats (a, e; P < 0.01). GFAP-immunofluorescent staining of cross sections of spinal cords from vehicle-treated (c) and guanosine-treated (d) animals at the lesion site 4 weeks after injury showed no difference in immunostaining of GFAP between the cords of guanosine-treated rats (d) and vehicle-treated rats (c). Scale bar = 50 μm for all
Guanosine attenuates apoptotic cell death in lesion site of spinal cord
In the present study, apoptotic cell death induced by traumatic spinal cord injury was determined with TUNEL staining. TUNEL-positive nuclei were not uniformly distributed throughout the cross section of the spinal cord but were more numerous in the vicinity of the injury center. Quantification of TUNEL-positive nuclei showed that there were significantly fewer TUNEL-positive nuclei in the spinal cords of guanosine-treated rats than saline-treated control animals (Fig. 8; P < 0.001).
Fig. 8TUNEL-positive apoptotic cells in spinal cord lesions were quantified by counting the total number of TUNEL-positive nuclei through entire cross sections. Compared with the cords from saline-treated animals (a, c), guanosine-treated cords had significantly fewer TUNEL-positive cells (b, c; P < 0.001). Scale bar = 50 μm for both a and b
Guanosine increases axon and myelin sparing
Axons were labeled with antibodies against RT-97 (a neuronal cytoskeletal protein; Fig. 9a, b) and myelin was labeled with antibodies against MBP (a specific marker for myelin; Fig. 9c, d). The immunohistochemistry showed that there were more axons (red, Fig. 9b) and myelin (green, Fig. 9d) as well as myelinated axons (yellow, Fig. 9f) in the spinal cords from guanosine-treated animals than in the cords from saline-treated control rats (Fig. 9a, c, e), indicating that systemic administration of guanosine is associated with preservation of tissue including neuronal and glial elements.
Fig. 9Immunostaining with antibodies against RT-97 for labeling axons (a, b) and against myelin basic protein (MBP) for central myelin (c, d) at the lesion site demonstrated the spared tissue. Cross sections from saline-treated (a) and guanosine-treated cords (b) demonstrate a neurofilament (NF) immunoreactivity surrounding the lesion site. c, d Cross sections from saline-treated (c) and guanosine-treated cords reveal myelin at the lesion site. e, f Merger of the two images demonstrates NF (red) and MBP (green) double fluorescent immunolabeling. There were more spared axons and more myelin in the cords around the lesion site from guanosine-treated animals compared to the saline-treated group. Scale bar = 50 μm for all
Discussion
These data are the first to demonstrate the ability of guanosine to act as a neuroprotective agent in vivo. These findings have potential clinical relevance. Previous studies have shown that after central nervous system injuries, the concentrations of guanosine are elevated around the injury and in CSF, sometimes for prolonged periods [32, 52]. Given the various trophic and antiapoptotic effects of guanosine in vitro [28, 34, 35], it seemed likely that increasing the concentration of guanosine in the central nervous system after injury in vivo might also be neuroprotective. We found that within 7 min of systemic administration, guanosine enters and progressively accumulates in the central nervous system (unpublished data), where it is converted to guanine. It remains to be determined whether guanosine, guanine, or both are the active neuroprotective agent. However, guanosine is more readily deliverable than guanine as a potential neuroprotectant.
The model of incomplete spinal cord injury that we have used is clinically relevant since about 50% of patients with spinal trauma have incomplete injury [53]. After incomplete spinal cord injury, both reflex and voluntary motor functions below the level of the injury are initially lost; partial recovery occurs over time [41, 54, 55]. The recovery of functions mediated by supraspinally controlled reflexes is slow and incomplete since these require the function of long tracts, many of which are irreversibly damaged by the injury [56, 57]. Recovery of locomotion and limb placement depends on ascending and descending spinal cord tracts, including cortico-, rubro-, reticulo-, vestibulo-, and raphespinal tracts [58]. The functional loss after spinal cord injury in rats involves interruption of descending serotonergic [59], reticulospinal, and other descending spinal tracts that facilitate segmental reflexes [40, 60]. The ascending spinothalamic tracts mediate the perception of pain and temperature below the level of the lesion. Since all of these motor and sensory functions were better in guanosine-treated animals than in control animals, it appears that guanosine preserved the function of multiple long tracts. This was associated with increased axonal survival and myelin preservation.
Normal micturition requires coordinated activation of the bladder smooth muscle (detrusor) and the striated muscle of the external urethral sphincter, controlled by spinal and supraspinal circuitry [47]. Thus after incomplete spinal cord injury, initially bladder function is lost, but later partially recovers, the extent of recovery depending on the degree of preservation of white matter (and hence the long tracts) at the injury site [48, 49]. Our data revealed that in guanosine-treated rats, residual urine volume was not only much less than in controls from the first day after the spinal cord injury, but that by 7 days after spinal cord injury micturition had returned to normal. Because guanosine began to exert a neuroprotective effect on both motor and bladder function soon after it was first administered, its neuroprotective effect on each may involve similar mechanisms.
The inherent complexity of the biological system coupled with the many potential trophic actions of guanosine make it difficult to determine the mechanisms by which guanosine produces its effects. Nevertheless, neuroprotective effects were observed by 24 h after spinal cord injury. As guanosine was administered 4 h post-injury, it must have affected processes that were important between 4 and 24 h post-injury. Within this time frame after traumatic injury of the spinal cord, both inflammatory responses and apoptosis are prominent. Activation and proliferation of microglia/macrophages play an important role in the secondary damage following spinal cord injury. Thus, cells at the center of the injury cascade are potential targets for neuroprotective treatments of acute SCI [21, 61]. As guanosine attenuated the activation and proliferation of microglia/macrophages following SCI, this may at least in part provide an explanation for its neuroprotective effect.
Guanosine has antiapoptotic effects in vitro [34, 35] and this is another potential mechanism through which guanosine might exert beneficial effects after spinal cord injury. Apoptosis after SCI has been described by many investigators [15, 62–67]. In these reports, early apoptosis of neural cells, including neurons, is followed by a delayed wave of predominantly oligodendroglial-programmed cell death in degenerating white matter tracts [63–67]. Studies of apoptosis in white matter after injury raise the possibility that glial apoptosis occurs, at least in part, as a consequence of axonal degeneration [68, 69]. However, the presence of activated microglia in contact with apoptotic oligodendrocytes after SCI indicates that this interaction may also activate cell death programs in the oligodendrocyte [70]. Secondary axonal degeneration may then follow [71, 72]. In the present study, guanosine significantly suppressed apoptosis in the injured spinal cords when it was systemically administered daily for 2 weeks beginning 4 h after injury. It seems reasonable to postulate that decreasing apoptosis may be an important mechanism through which guanosine improves neurological outcome after SCI.
Although the intracellular pathways through which guanosine suppresses apoptotic cell death following spinal cord injury are not known, a number of intracellular pathways which protect cells against apoptosis have been identified. These include the phosphatidylinositol 3-kinase (PI-3-K)/Akt/protein kinase B (PKB) pathway [73, 74] and the mitogen-activated protein (MAP) kinase pathway [75]. Our previous studies in vitro have shown that the antiapoptotic effect of guanosine is mediated by the activation of both PI-3-K/Akt/PKB and MAPK pathways [34, 35].
Guanosine can also increase intracellular cAMP in various cell types [36, 37], and increases in cAMP after spinal cord injury have been shown to increase recovery [26, 27]. However, it appears unlikely that increases in intracellular cAMP are responsible for the effects of guanosine since the principal effect of cAMP in enhancing recovery from spinal cord injury appears to be due to its ability to promote axonal regeneration. In contrast, the neuroprotective effects we observed early after administration of guanosine are unlikely to be explained on the basis of outgrowth of damaged axons. A much longer time frame could be required for this to occur.
A further possible mechanism by which guanosine might exert its neuroprotective effects is by stimulating cells to release and increase synthesis of various trophic factors, such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) [28]. Such trophic factors can contribute to tissue preservation after trauma [67]. The ability of guanosine to stimulate production and release of trophic factors may not only have early effects, but also may contribute to the reduced number of apoptotic cells observed 3 weeks after the injury.
This study is important not only because it is the first to demonstrate the neuroprotective effect of systemically administered guanosine in acute spinal cord injury, but also because it indicates some of the potential mechanisms whereby guanosine may exert its neuroprotective effects in vivo. This work provides a basis for further exploration of the purinergic mechanisms underlying the neuroprotective effects of exogenous guanosine. Furthermore, and of potential clinical importance, is that guanosine was effective when it was administered 4 h after the injury—a realistic time frame in which to initiate treatment after spinal cord injury in humans.

guanosine
apoptosis
inflammation
spinal cord injury
cell death
myelin
immunohistochemistry
glia
locomotor and sensory function