Older Post Hip Fracture Females Biology Essay

4.0 Introduction

Advancing age is characterized by decrements in physical function and performance, with a gradual decline in muscle mass and strength (Binder et al., 2004). It is now recognized that the preservation of physical function is critically important to promote independent living and quality of life (Brown et al., 2000; Ensrud et al., 2008; Cawthon et al., 2009). Annually 75,000 cases of hip fractures occur in the United Kingdom, with estimates suggesting that by 2015 this will increase to 91,500 (Turkington, Mc Donald, Elliott & Beringer, 2012). Hip fractures therefore represent a major public health concern in the modern era. Falls are cited as the cause for 95% of hip fractures, lifestyle interventions such as exercise can decrease the risk of falls through improvements in physical function, balance and muscle strength (Dontas & Yiannakopoulos, 2007). The period following hip fracture surgery is of critical importance for physical rehabilitation including exercise, as 22-75% of post hip fracture females are unable to return to their pre-fracture functional status (Binder et al., 2004; Osnes, Lofthus & Meyer., 2004; Oliveira, Narciso, Santos et al., 2008). Exercise has the potential to decrease incidence of hip fractures, and prevent associated complications (Lord et al., 2010; Sahota, Morgan & Moran, 2012).

It is commonly accepted that individuals suffering from a hip fracture can experience an increase in inflammation up to a year following a fracture episode, which can result in poorer prognosis (Joseph, Kenny, Taxel, Lorenzo, Duque & Kuchel, 2005; Sun et al., 2011). Coupled with this disturbances in the intracellular redox balance with advancing age have also been shown to result in a state of low grade inflammation (Meng et al., 2010). Age associated inflammation and hip fractures have been implicated with declining physical function, whereby inflammatory cytokines are known to exert catabolic effects on muscle resulting in the loss of muscle mass, strength and power (Newman et al., 2007; Nacri & Mafulli, 2010). Inflammation is associated with both increased mortality and morbidity in older populations (Ceseari et al., 2004; Schaap et al., 2006). Declines in circulating hormones such as estrogens, testosterone and IGF-1 reported during the same period of life (Kuchel et al., 2001), represent additional challenges for the normal functioning of skeletal muscle. Research has suggested that the combination of reductions in growth factors and hormones alongside higher levels of inflammation and oxidative stress are central contributors towards the age-associated loss of muscle mass and strength (Nacri & Mafulli, 2010). Interactions between these inflammatory and hormonal changes occur through a number of interlinked signalling pathways, ultimately affecting the metabolism as well as the mass and structure of bone (Meng et al., 2010; Ogawa et al., 2010).

In order to maximise hip fracture recovery it is important to establish the kinetics of inflammatory cytokines in individuals following a hip fracture. The intricate relationship between hormones, cytokine function and immunity has been shown to be further complicated for those who have experienced a hip fracture. Increases in circulating cytokines may predict or contribute to the progression of loss in muscle mass and strength commonly termed sarcopenia, which represents the age associated loss of muscle mass and strength. Exercise represents the only common modality which can target and potentially attenuate age associated changes in muscle mass, muscle strength and bone health, with minimal negative side effects (Carter et al., 2001). Examination of the acute response to exercise, provides useful information into the regulatory events and mechanisms which orchestrate more long term changes and adaptations (Gleeson et al., 2011). Research has shown that a multitude of transient cellular and biochemical events occur during their period following completion of exercise and for the following 48 hours (Pederson et al., 2006). However research findings in this area have been conflicting, due largely to variations in study design and exercise interventions utilised. Enhancing knowledge into the response to environmental stimuli on an acute basis has practical relevance, permitting a greater understanding of the underlying mechanisms which can prove beneficial not only in terms of exercise prescription but can also act as a tool in determining the efficacy of chronic exercise periods for older individuals (Thompson et al., 2000: Hawley et al., 2006). As it is important to remember that if the applied stimulus is not of a magnitude capable of disrupting cellular homeostasis training adaptations may not occur (Zanchi et al., 2012)

The aim of this study was to examine the acute effects of resistance exercise on inflammation and muscle strength in a hip fracture sample. It was predicted that inflammation would be comparatively higher within the hip fracture sample than healthy controls, and that this would pose a negative effect on measures of muscle strength and physical performance.

4.1- Methods

4.1.1- Participant characteristics

Following approval from the Office for Research and Ethics Committee in Northern Ireland (ORECNI) and the South Eastern Health and Social Care Trust, ten (n=10) females who experienced a hip fracture were recruited from the Ulster Hospital, Dundonald (Table 1 for participant characteristics). Thirteen healthy females (n=13) were also recruited from the University of Ulster Alumni database, AGE NI, local walking groups and leisure centres via emails and a recruitment poster. Inclusion criteria required individuals to be community dwelling and to gain a score of 25 or above out of 30 in a Mini Mental state examination (MMSE, Folstein, 1975), as a marker of ability to provide fully informed consent. Individuals with cardiopulmonary disease or neuromuscular impairment, angina or congestive heart failure, or those on hormone therapy were excluded from participating in the study. All procedures and potential risks were fully explained prior to exercise. Each participant was provided with an information sheet and informed consent form to sign (previously outlined in methods section 3.4, See Appendix 2).

Table 1- Age and physical characteristics at baseline

Dependent Variable

Hip fracture

n=10

Healthy

n=13

Age (yrs)

72 ± 4

71 ± 5

Stature (m)

1.61 ± 0.02

1.63 ± 0.06

Body Mass (kg)

63.7 ± 4.2

64.53 ± 7.78

BMI (kg/m2)

24.6 ±1.5

24.23 ± 3.09

4.1.2- Experimental Design

During their first visit all participants completed a bout of resistance exercise. Prior to this visit participants were asked to refrain from exercise and alcohol consumption for 48 hours. All testing procedures were conducted between 9.00 and 11.00 am following a standard 10 hr overnight fast to control for biological variation.

4.1.3- Anthropometric measurements

Upon arrival at the laboratory, measurements of stature and body mass were recorded for each participant as outlined in Methods section 8.1.

4.1.4- Acute Exercise protocol

Following completion of maximal leg strength testing (Methods section 9.2), a bout of acute resistance exercise was completed using the Concept 2 Dyno Strength Trainer (Concept 2 Ltd, Wilford, Notts, UK). Each participant completed two sets of ten repetitions of knee extension/flexion at 80% of 1 RM with 2 minutes rest period between sets. This 1RM was calculated manually and resistance was gauged by adjusting the dampers to a level approximately 80% of their measured 1RM.

4.1.5- Collection and analysis of blood samples

Venous blood was collected at baseline (prior to acute exercise) and immediately after completing acute exercise. Blood samples were drawn aseptically from an antecubital forearm vein using the vacutainer method (Methods section 11.0).

4.1.6- Blood analysis

Blood was analysed for TNFα, IL-10 and IL-6 via enzyme linked immunosorbent assay (ELISA) using the microplate reader manufacturers details. Detailed descriptions of all biochemical and ELISA assays can be found in methods section 12.0.

4.1.7- Statistical analysis.

Statistical analysis was performed using the SPSS social statistics package (Version 19.0, Surrey, UK). Data were analyzed using parametric statistics following mathematical confirmation of a normal distribution using Shapiro-Wilks tests. The linear relationship between the two continuous variables of 1 Repetition Maximum (1RM) and (Interleukin 6 (IL-6), and 1 Repetition Maximum (1RM)) and Tumor Necrosis Factor (TNFα) was established using Pearson’s product moment correlation. Muscle strength characteristics including both 1RM and acute resistance scores between healthy and hip fracture participants were compared using independent sample t-tests. Pre and post acute resistance exercise data in healthy and hip fracture participants were compared using a two-way split plot [A x (B)] mixed analysis of variance (ANOVA) which incorporated one between (group: Hip vs. Healthy) and one within (Time: Pre vs. Post) subjects factor. Where significant interaction effects (Time x Group) were noted, within subjects factors were analyzed using Bonferroni-corrected paired samples t-tests. Between subjects differences were analyzed using a one-way ANOVA with a posteriori Tukey Honestly Significant Difference (HSD) test. The alpha level was established at P<0 .05 (95% confidence interval) and all values are reported as a mean ± standard deviation (SD).

4.2- Results

All participants completed the acute resistance exercise bout during their first visit to the laboratory, the exercise stimulus was well tolerated with minimal signs of discomfort reported. Additionally blood samples were collected during all time points, with the exception of one failed attempt during post acute exercise for one of the post hip fracture group.

4.2.1- Interleukin 6 (IL-6)

A significant interaction effect was observed (group x time, P < 0.05) in IL-6. Acute resistance exercise significantly increased IL-6 in healthy females when compared to pre exercise values (P < 0.05). Contrastingly reductions were experienced in IL-6 for post hip fracture females over the same time period. Table 2 demonstrates changes in IL-6 between pre resistance values (hip fracture pre v healthy pre, P < 0.05).

4.2.2- Interleukin 10 (IL-10)

There was a time x group interaction following acute resistance exercise for IL-10 (P < 0.05). IL-10 increased by 26.5% within post hip fracture females (4.39± 0.79 vs. 5.56 ± 2.05, P < 0.05), as displayed in Table 2. However further post hoc tests did not show any significant changes between IL-10 pre and post acute resistance for either group (P > 0.05).

4.2.3- Tumor Necrosis Factor (TNFα)

A significant interaction effect was observed (group x time, P< 0.05) in TNFα, whilst there were main effects observed for time (pre vs. post pooled data, P<0.05) and group (hip fracture vs. healthy pooled data, P <0.05). Differences were observed within groups following acute resistance (hip fracture pre v hip fracture post, P< 0.05) and (healthy pre v healthy post, P < 0.05). TNFα concentration was comparatively higher for post hip fracture females during both time points (hip fracture pre vs. healthy pre, P < 0.05) and (hip post vs. healthy post, P< 0.05).

Table 2- Cytokine measures pre and post-acute resistance exercise between post hip fracture (n=10) and healthy (n=13) older females.

Hip fracture

Healthy

Pre Acute

Post Acute

Pre Acute

Post Acute

IL-6 (pg/ml)

10.8 ± 6.6

7.7 ± 5.4

4.7 ± 1.1*

7.6 ± 2.1†

IL-10 (pg/ml)

6.42± 2.23

4.02± 2.18†

4.39±0.79

5.56 ±2.05

TNFá (pg/ml)

54.71± 28.62

45.81 ± 25.76†

6.29 ±0.41*

8.71±1.92 †*

* Indicates a difference between group as a function of condition (hip vs. healthy pre vs pre, P< 0.05).

† Within group difference as a function of time (P < 0.05).

4.2.4- Muscle Strength (1RM/Acute Resistance)

As displayed in figure 1, both one repetition maximum (1RM) and acute resistance scores were significantly lower for the post hip fracture group, (P < 0.05). Hip fracture scores were 25% lower than healthy controls.

Figure 1- Muscle strength during acute resistance exercise among post hip fracture (n=10) and healthy (n=13) older females.

*Denotes difference between groups pre and post acute exercise phases (hip fracture v healthy acute resistance score, P<0.05) and (hip fracture v healthy one repetition maximum, P<0.05).

4.3 Discussion

The major finding of this study was that the acute inflammatory response to resistance exercise was significantly different for the post hip fracture sample. Concentrations of pro and anti inflammatory markers were significantly different between groups. Post hip fracture individuals documented elevated levels of all cytokines at baseline, however completion of an acute bout of resistance exercise significantly depressed TNFα and IL-10, documenting slight reductions in IL-6. This was in direct contrast to observed patterns of activation within the healthy group, following acute resistance exercise IL-6 and TNFα significantly increased, additionally demonstrating modest improvements within IL-10. Completion of acute resistance exercise resulted in opposing patterns of activation in terms of the acute inflammatory response between groups. Healthy females demonstrated significantly higher levels of muscle strength in both 1 Repetition Maximum and acute resistance repetitions scores.

The results of this study demonstrate opposing inflammatory responses between groups following acute resistance exercise. Results from the present study document significantly higher IL-6 and TNFα in the post hip fracture group, considering that research has shown IL-6 and TNFα to be major contributors towards the promotion of a pro-inflammatory environment (Maggio et al., 2006; Peterson et al., 2006), this may pose a negative impact towards the normal functioning of hip fracture individuals. TNFα has been shown to be detrimental towards physical functioning through the inhibition of myogenic differentiation and the promotion of protein catabolism via reducing the process of translation initiation (Petersen, Plomgaard, Fischer & Ibfelt, 2009; Ogawa et al., 2010). Additionally IL-6 plays a focal role in the reduction of muscle mass and strength, through mechanisms related to force generability (Visser et al. 2002; Dirks & Leeuwenburgh, 2006; Schaap et al., 2009). Normally in homeostatic conditions, cytokines and acute phase markers initiate and regulate the acute phase inflammatory response, however advancing age can often disrupt this balance towards a pro inflammatory state (Evans et al., 2010). Despite the observed changes in inflammatory milieu as we age, research has documented that a single bout of resistance exercise at a moderate intensity can decrease overall inflammation in elderly women (Philips, Flynn, Mc Farlin, Stewart & Timmerman, 2010). The results from the current study compliment findings of others, healthy females demonstrated a significant increase in IL-10 following acute resistance exercise, which can act towards reducing the inflammatory redox environment (Pederson et al., 2006; Gleeson et al., 2011).

The observed changes noted in the cytokine response to acute resistance training for healthy females are consistent with previous research within the area (Pederson & Febbario, 2008; Zanchi et al., 2012). Whereby completion of acute resistance exercise, consequentiality caused initial increases in IL-10, TNF and IL-6 (Haddad, Zaldivar, Copper & Adams, 2005; Hamada et al., 2005; Haddad & Adams, 2006). However post hip fracture group did not follow the same pattern of activation, emphasising how the effect which acute resistance exercise poses on cytokine levels within older females is diverging, remaining largely inconclusive reiterating the need for future research within specified populations (Peake, Gatta & Cameron-Smith, 2010).

In the current study IL-10 was higher in post hip fracture females during pre exercise, although completion of acute resistance bout decreased IL-10. Increased IL-10 is beneficial in older populations, through compromising the capacity of effector T cells to sustain an inflammatory response (Ouyang, Rutz, Crellin, Valdez & Hymowitz, 2011; Sun et al., 2011). Current study findings suggest that TNFα reductions in post hip fracture females occurred through an alternative mechanism other than increased IL-10, suggesting that a form of immune dysregualtion may exist within the post hip fracture group. Determining IL-10 concentrations in post hip fracture females over time may provide new insights into both their recovery and respective catabolic state, warranting further investigation. Anti-inflammatory properties of IL-6 may be an additional mechanism to explain the differences in IL-10 between groups (Ogawa et al., 2010). An emerging finding from the literature thereby demonstrates both pro and anti-inflammatory IL-6 characteristics (Opal & DePalo, 2000), whreby IL-6 can demonsrate a postive relationship with the inflammatory response (Shanti et al., 2008). IL-6 concentrations significantly differed between groups. Healthy controls experienced elevations in IL-6 and IL-10 respectively post exercise, while post hip fracture females experienced reductions in IL-6 and also IL-10 during the same time period, suggesting that increased IL-6 may be responsible for enhancing IL-10 post exercise. It has been postulated that IL-6 when released from contracting muscle during exercise exerts pro inflammatory actions towards TNFα through increasing IL10 and IL1 receptor antagonist (IL1RA) (Pederson & Fischer, 2007; Lira, Rosa, Yamashita et al., 2009). Additionally post hip fracture females documented elevated IL-6 at baseline in comparison to healthy controls, this is in agreement with research conducted by Sun and Colleagues, (2011) which demonstrated heightened pro inflammatory cytokines in post hip fracture females. Although it is plausible that higher IL-6 within the post hip fracture group at baseline may have been an attempt to lower extremely high TNFα (Pederson et al., 2006: Evans et al., 2010).

Post hip fracture females in the current study were recruited 3-6 months following fracture. During this period levels of catabolic markers are known to be high (Sun et al., 2011). It is possible that the underlying mechanisms modulating the cytokine response to exercise become defective with advancing age, compromising the ability of immune cells to mount a normal response towards changes within inflammatory milieu (Pederson et al., 2006; Peake et al., 2010). High baseline pro inflammatory cytokines were documented for post hip fracture females, suggesting that this relationship is further complicated by experiencing a fracture episode (Smith, 2007; Sedlar, Kudrnova, Trca., et al., 2008; Evans et al., 2010). When evaluating an acute bout of resistance exercise it has been recommended that an immediate sample time point, which was included in the current study should be used in addition to time points post exercise to provide heightened knowledge of the time course of cytokine kinetics (Ogawa et al., 2010; Peake et al., 2010). Hypothetically if experiencing a hip fracture results in an acute dysregualted and blunted inflammatory response, it is plausible that their levels of plasma cytokines may have taken longer to rise, however this information is not currently available as the protocol utilised for the purpose of this study included a single post exercise sample immediately following exercise.

Post hip fracture females possessed significantly higher IL-6 and TNFα but completion of acute resistance decreased these, contrastingly performance of the acute resistance exercise phase resulted in elevations within healthy controls. Elevations in inflammatory markers can exist for up to a year after the fracture (Maggio et al., 2006; Visser et al., 2006; Sun et al., 2011). This is indicative of the normal acute response to exercise, largely supported in the research literature (Peake et al., 2010). Research has shown that circulating inflammatory biomarkers originate from a number of sources (Pederson & Fischer, 2007). Visceral adipose tissue has been highlighted as a central contributor, where higher levels of fat promote macrophage recruitment and both adipocytes and macrophages secrete numerous cytokines (adipokines), including IL-6 and TNFα (Mathur & Pedersen, 2008). Results from the present study demonstrated higher TNFα within the post hip fracture group, however no significant differences were found in terms of BMI between groups. This potentially suggested that higher TNFα in post hip fracture females are not explained by levels of body fatness. With advancing age, research has shown that the categorisation of individuals in terms of BMI does not always correlate towards enhanced levels of physical function (Newman et al., 2004). Although BMI has been shown to play a role within the age associated loss of muscle mass and strength, this relationship is not always straightforward. As an individual may report a normal BMI but this could be masked with higher fat mass, and a low level of muscle mass, correspondingly an individual with an BMI in the overweight region may report greater muscle mass coupled with higher fat mass (Maggio, Laurentani & Ceda, 2013). Although as DXA body composition was not conducted for the post hip fracture group, we are unable define precise regions of both muscle and fat mass which would have provided interesting information warranting use of DXA in future studies.

Significant differences were observed between groups for 1RM and acute resistance scores, post hip fracture females reported 25% lower scores in comparison to healthy females. It is postulated that IL-6 demonstrates strong links with functional recovery in the 12 months following a hip fracture, through mechanisms related to muscular strength (Miller et al., 2006). Research has consistently highlighted that heightened concentrations of IL-6 and TNFα are associated with lower muscle mass and strength, however the relationship appears to be stronger for that of muscle strength in comparison to that of muscle mass (Visser et al., 2000; Di Monaco, Vallero, Di Monaco et al., 2006; Miller et al., 2006). Current study findings demonstrate significant positive correlations between 1RM with TNF α and IL-6, although this relationship was only observed with healthy females. Throughout the 20 repetitions completed in the acute resistance phase, healthy females were able to sustain values of over 80% of 1RM, in many situations their scores maintained and improved their average % of their 1RM was 88%. Contrastingly some of the post hip fracture females struggled to maintain repetitions displaying signs of fatigue within this range, overall their average % of their 1RM was 81%. Lower extremity function has been highlighted in the research as a strong predictor of functional disability, current results suggest that females who have not experienced a hip fracture possess higher maximum strength scores. It has been previously reported that inflammation poses negative effects on muscle contractile properties (Mc Ardle et al., 2005). TNFα has been shown to decrease contractile force at the myofilament level post hip fracture females demonstrated significantly higher TNF scores, reactive oxygen species (ROS) and nitric oxide (NO) generation have been implicated as focal contributors, although this requires further clarification within human models (Mc Ardle et al., 2000). Due to the strong association between TNFα and muscle strength, it is possible that high concentrations experienced in post hip fracture group in the present study may accelerate the loss of muscle mass and strength, and potentially delay recovery (Narci & Mallufi, 2010).

The absence of a measure of eccentric strength is a study limitation. The acute resistance exercise phase was conducted on the Concept 2 Dyno strength trainer in the current study, although advantageous in terms of usability and functionality one of its disadvantages is that it only utilises concentric contractions. Older individual have reported difficulties in the performance of certain activities involving a strong eccentric component such as sitting down or going down stairs (Lark et al., 2003), which may contribute towards an increased risk of falls (Perry et al., 2006). Research has suggested that eccentric muscle strength is well preserved with advancing age, with passive and active elements regulating muscle stiffness believed to be causative and contributing factors (Valour & Pousson, 2003; Ochala et al., 2004). Concentric strength however has been shown to be subject to marked decrements with advancing age (Roig, MacIntyre, Narici et al., 2010), examination of this measure may offer a greater insight into the levels of muscle strength, further justification as to why it was included in the present study. During concentric contractions muscle force is generated which is greater than a resistance force, sarcromeres change during concentric contractions via decreasing the distance between the Z-lines, I-bands, and H-zones, while A-band and filament lengths remain constant (Friden & Liebert, 2001; Elder, 2010). Previous research within the area has suggested that the cytokine response is directly related to muscle damage (Cusi et al., 2000). Research now suggests that muscle contraction either eccentrically or concentrically based, will elevate circulating levels of respective cytokines for a brief period only (Jonsdottir et al., 2000; Mac Donald et al., 2003; Pedersen, 2007; Pederson, Akerstron, Nielsen & Fischer, 2007; Pedersen & Febbrario, 2008). Boppart et al., (2000) documented increased JNK activity following both eccentric and concentric contraction, highlighting that eccentric exercise instead resulted in a greater magnitude of activation in comparison to concentric. As JNK has been shown to be an upstream activator of IL-6, it has been postulated that the type of contraction may not be the sole explanatory factor towards increased IL-6 in the blood, but that factors such as height, weight and exercise duration play a focal role in its activation (Mac Intyre, Sorichter, Mair, Berg & Mc Kenzie, 2001; Willoughby, Mc Farlin & Bois, 2003; Fischer, 2006).

Another limitation of the current study, is the relatively low participant numbers. Although representing an important clinical group within the population, future research would benefit from higher participant numbers especially in the post hip fracture group where there is large intra-individual variation. Original power calculations for the study indicated a sample size of 30 for each group, as recruitment for the current project is on a rolling basis it is hoped that the number of participants will increase. Post hip fracture females were recruited 3-6 months after the clinical event, it would provide both beneficial and interesting findings if it were possible to follow and assess over time, to ascertain if cytokine concentrations normalised themselves, or if they remain heightened. Despite lower than anticipated numbers, a number of significant improvements were noted. Research has shown that skeletal muscle possesses the ability to produce and secrete a variety of cytokines in response to muscular contraction (Febbario et al., 2004). It is believed that inflammatory markers activate a myriad of signalling pathways which mediate cellular growth and repair, advancing age appears to result in dysregualtion within these processes (Trenerry et al. 2000; Peake et al., 2010). It is generally assumed that acute exercise responses should differ from those elicited by chronic exercise training which justifies future examination. Currently knowledge surrounding how inflammation in skeletal muscle changes with age is incomplete. The causative mechanisms of inflammation within skeletal muscle must be established to promote a greater understanding.

4.4- Conclusion

In conclusion, post hip fracture females respond differently to acute resistance exercise in comparison to healthy females. A hip fracture episode clearly places increased physiological and biochemical demands on afflicted individuals. High levels of inflammation may potentially explain lower muscle strength. Increased knowledge into how clinical groups such as post hip fracture females react to acute exercise can potentially serve to better inform rehabilitation and intervention programmes. Ascertaining whether this acute response will differ when repeated over a period time, will further contribute towards the literature.