Bernardo Diniz Coutinho, Universidade Federal do Ceará, Brazil Chao-Qun Yan, Dongzhimen Hospital, Beijing University of Chinese Medicine, China

#### \*Correspondence:

J. Mikhail Kellawan<EMAIL_ADDRESS>
Received: 27 June 2019 Accepted: 19 August 2019 Published: 13 September 2019

#### Citation:

Sun J, Ashley J and Kellawan JM (2019) Can Acupuncture Treatment of Hypertension Improve Brain Health? A Mini Review. Front. Aging Neurosci. 11:240. doi: 10.3389/fnagi.2019.00240

Hypertension independently increases the development of cerebrovascular disease and neurodegenerative disease (Baumgart et al., 2015; Benjamin et al., 2019). Although a direct mechanistic link between hypertension and poor brain health is unknown, having hypertension predicts lower cognitive function (Kilander et al., 1998) and increased risk for dementia with age (Sharp et al., 2011). Many hypothesize that the etiology stems from poor blood pressure (BP) and cerebrovascular regulation. This dysfunction fosters hypoperfusion, stressing the brain through a nutrient mismatch, subtly causing damage over many years (de la Torre, 2012). Animal models indicate that increased systolic BP (SBP) and cerebrovasculature remodeling in hypertension ultimately reduce brain blood flow, leading to behavioral and cognitive impairments (Pires et al., 2013; Wiesmann et al., 2017). In hypertensive adults, a diminished cerebral perfusion may accelerate the development of Alzheimer's via decreased oxygen delivery in ischemia-sensitive brain regions like the hippocampus, inducing neurodegeneration and subsequent cognitive decline (de la Torre, 2000).

Treatment of hypertension has shown benefits in neurodegenerative disease development and overall brain health. Most observational studies have suggested that improved SBP control reduces the risk of Alzheimer's and other dementias (Qiu et al., 2005; Hughes and Sink, 2016). Additionally, treating hypertension is among the most effective strategies to prevent a stroke (Meschia et al., 2014). Each year, approximately 795,000 US adults suffer a new or recurrent stroke (Benjamin et al., 2019). Therefore, effective treatment of hypertension has tremendous far-reaching impacts on brain health.

However, pharmacological treatment of hypertension faces challenges such as unwanted side effects, limited adherence, and difficulty individualizing treatment for such a diverse population. Up to 97% of patients taking antihypertensive medications experience adverse side effects (Toyoshima et al., 1997; Bardage and Isacson, 2000), which can reduce future adherence (Tedla and Bautista, 2016). Moreover, 25% of patients do not fill their initial antihypertensive prescription (Holland et al., 2008; Franklin et al., 2012; Berra et al., 2016). As a result, four out of five patients do not sufficiently adhere to antihypertensive treatment, failing to control their BP (Petrilla et al., 2005; Gwadry-Sridhar et al., 2013). Additionally, only two out of the six classes of antihypertensive drugs are independently associated with decreased risk of dementia (van Middelaar et al., 2017). These circumstances result in patients maintaining an elevated dementia and stroke risk. Therefore, there is a dire need to identify adequate antihypertensive treatments to improve cerebral blood flow (CBF) that circumvents the limitations presented.

Acupuncture is regarded as a promising complementary and integrative antihypertensive approach that does not share many of the limitations of medical interventions. It is a practice of traditional East Asia medicine in which specific points on the body are stimulated, most often by inserting disposable thin stainless-steel needles through the skin. Acupuncture has documented benefits easing various types of pain (e.g., low back, neck, osteoarthritis, headache, etc.) and conditions including cardiovascular diseases (World Health Organization, 2002; McDonald and Janz, 2017). Western cultures have become more welcoming of acupuncture; a 2017 clinical practice guideline from the American College of Physicians included acupuncture among the nondrug treatment options for management of both acute and chronic back low-back pain (Qaseem et al., 2017). However, acupuncture's effects on the cardiovascular system are still under-researched, preventing its utilization as a therapeutic option in the Western world.

While detailed clinical and mechanistic experimental evidence is lacking, acupuncture has been observed to reduce BP (Li et al., 2015; Liu et al., 2015) and improve endothelial function in hypertensive adults (Park et al., 2010). Further, acupuncture has been shown to have specific cerebrovascular effects in healthy adults, highlighting possible neuroprotective properties (Byeon et al., 2011; Hyun et al., 2014; Im et al., 2014). However, it is yet to be determined if the literature is in support of using antihypertensive acupuncture prescriptions to improve brain health via enhanced cerebrovascular control. Therefore, our review is aimed at evaluating the acupuncture literature; as a treatment for hypertension, its effects on cerebrovascular control, the evidence for specific acupuncture hypertension treatment improving brain health outcomes, and the mechanisms by which acupuncture can affect BP and cerebrovascular control.

# EVIDENCE SUPPORTING ACUPUNCTURE AS A TREATMENT OF HYPERTENSION

Recent randomized control trials (RCTs) indicate that acupuncture is effective at lowering BP in humans. One of the largest RCTs involving more than 400 mostly non-hypertensive adults found reductions in SBP (122–113 mmHg) and DBP (68–65 mmHg) following 6 weeks of biweekly acupuncture compared to no change in sham and auricular acupuncture (Abdi et al., 2017). However, it should be noted that the auricular acupuncture and the two sham groups had lower initial SBP (110, 116, and 111 mmHg, respectively). This leads to speculation that acupuncture only impacts individuals with higher BP, specifically higher SBP. Therefore, the hypotensive effect of acupuncture may be best represented in hypertensive individuals.

Four weeks of acupuncture treatment (20-minutes/treatment, 2 treatments/week) reduced SBP and DBP ∼ 7mmHg in hypertensive adults. After 8 weeks SBP and DBP remained lower (∼6.5 and ∼4.9mmHg, respectively) than initial values. However, 4 weeks after treatment was ceased, SBP and DBP remained ∼5 mmHg lower, with only DBP being significantly different. The time-matched control did not have a change in BP at any time point (Liu et al., 2015). Similarly, Li et al. (2015) compared 8 weeks of acupuncture in hypertensive adults at acupoints shown to have a BP-lowering effect vs. control acupoints (sites that do not alter BP). Acupuncture reduced SBP by 6 mmHg and DBP by 4 mmHg. Subsets of the subjects participated in crossover assessment with the active treatment lowering SBP ∼7 mmHg and DBP ∼4 mmHg compared to control. A further subset of subjects stopped all treatments and were assessed at 1 and 2 months posttreatment. At 1 month following cessation of treatment, SBP remained lower and DBP reverted to pre-treatment values. Two months after treatment, BP was not different from pre-treatment values. From these studies, it is clear that acupuncture can have an effect on hypertension, and this effect can be sustained for up to 1 month, with diminishing effects thereafter.

The promising results presented above highlight the growing research interest in acupuncture and are only a fraction of the large volume of research that is currently being generated. This volume has further spurred many recent systematic reviews on acupuncture and hypertension. Interestingly, few determine that acupuncture alone can manage BP in hypertensive adults (Wang et al., 2013). Rather, the consensus is that acupuncture, when combined with pharmacological therapy, lowers BP than either treatment alone (Li et al., 2014; Zhao et al., 2015, 2019; Chen et al., 2018; Yang et al., 2018; Niu et al., 2019). Therefore, acupuncture may best be used as an adjunctive treatment for hypertension.

Although the recent results seem promising, it is not unanimous. The above listed reviews were meticulous in limiting bias and incorporating the most robust research, yet there are still methodological concerns with antihypertensive acupuncture RCTs. There is enormous heterogeneity of study design among the RCTs, with no consensus for how to properly provide control/placebo for acupuncture. Control types used in the RCTs include a time-matched group, a sham acupuncture group, and an inactive acupoint acupuncture group. The use of sham acupuncture has been argued as the best means for control (Yang et al., 2018), mainly because the use of ''inactive'' acupoints raises concerns of possible spillover effects. Meaning, stimulation of an acupoint believed to have no effect on cardiovascular parameters could have a minor effect, limiting statistical determination of active acupoint stimulation. Additionally, many RCTs contain small sample sizes and do not always utilize a crossover design. For instance, in Li et al. (2015), post hoc power analysis determined that at least five more participants per group would be needed to achieve the appropriate power. These limitations cause skepticism about the effectiveness, efficacy, and safety of acupuncture treatment in hypertension.

Overall, while it seems that acupuncture's effect on lowering BP is minimal, as an adjunctive treatment to conventional therapies, acupuncture is a promising avenue in the quest to control hypertension and limit its damaging effects throughout the body. Although meaningful issues are present in most of the RCTs examined, acupuncture appears to be effective in lowering BP in hypertensive adults. However, the lack of mechanistic integrative human research elucidating a link between acupuncture and BP regulation and experimental design concerns justify the need to critically evaluate acupuncture as a reliable treatment option.

# THE EFFECT OF ACUPUNCTURE TREATMENT ON CEREBRAL HEMODYNAMICS

Attempts to determine acupuncture's effect on brain health outcomes have steadily increased over the years. However, true assessment of these outcomes requires longitudinal studies that have yet to be undertaken. Assessing brain hemodynamics and outcomes is more immediate and has been researched using various imaging modalities [e.g., transcranial Doppler (TCD), near-infrared spectroscopy (NIRS), functional magnetic resonance imaging (fMRI), etc.] that have various strengths and weakness that can complicate interpretation. However, each provides valuable information of the brain environment.

Lower CBF, measured using TCD, is associated with cognitive decline and neurodegenerative disease (Ruitenberg et al., 2005). Therefore, it is theorized that treatments that improve or restore CBF may attenuate or possibly prevent the onset of these conditions. Interestingly, TCD studies present evidence that acupuncture can improve CBF and that there is acupointcerebral vessel specificity. Acupuncture of GB20 point has been shown to improve CBF regulation in posterior (Vertebral and Basilar) arteries but not the middle cerebral arteries (MCAs; Yuan et al., 1998; Im et al., 2014). Similarly, in healthy adults, a single 20-min acupuncture treatment at acupoint ST36 improved flow and CO2 reactivity in the basilar artery but only reactivity in the MCA (Hyun et al., 2014), whereas 20-min acupuncture of GV20 increased CBF and CO2 reactivity in both middle and anterior cerebral arteries (basilar not measured; Byeon et al., 2011).

As previously stated, the effects of acupuncture are best exemplified using diseased individuals. Applying acupuncture to stroke patients at acupoints LV3, LV4, SJ5, and GB34 significantly increased CBF (in MCAs). This is accompanied by a decreased SBP; however, the sham acupuncture group also saw a decreased DBP (Ratmansky et al., 2016). Using NIRS, acupuncture intervention on stroke patients showed a significant increase in regional cerebral blood volume or oxyhemoglobin parameters (Li et al., 2011). A systemic review of NIRS studies indicates that acupuncture varies wildly in healthy adults but seems to be more appropriate for disordered populations such as stroke patients (Lo et al., 2015).

Experiments using fMRI have been completed to determine acupuncture's effect on specific brain regions and the brain network. Acupuncture (Ll11 and ST36) in both healthy and stroke patients improved brain activity in various areas of the brain (Cho et al., 2013). Further, increased activity and connectivity across hemispheres and various portions of the brain following acupuncture has also been observed in patients with mild cognitive impairment (Feng et al., 2012), Alzheimer's disease (Wang et al., 2014), and stroke (Chen et al., 2014).

Single-photon emission computerized tomography (SPECT) and position emission tomography–computed tomography (PET-CT) allow for a depiction of regional brain perfusion. In healthy subjects, acupuncture (Ll4 and Ll11) increased both regional CBF and glucose metabolism in both frontal regions (An et al., 2009) and specifically at important regions regarding the limbic system, middle cingulum, and medial orbitofrontal gyrus (Jung et al., 2011).

The literature advocates for acupuncture having positive effects on brain hemodynamics in an acupoint-specific manner. However, many of these studies have: (a) been conducted in healthy populations and (b) suffered from similar methodological concerns raised in our discussion of acupuncture on BP regulation. What remains unclear is a mechanistic link between acupoints and brain-specific responses as well as if there is a connection between acupoints used in the treatment of hypertension and cerebral responses. Garnering further understanding of these acupoints and if acupuncture treatment of hypertension actually improves cerebrovascular responses could have a significant biomedical impact as it would provide further information on the mechanistic links between hypertension and poor brain health, as well as offer a greater number of treatment options.

# EFFECTS OF ACUPUNCTURE TECHNIQUES AIMED AT TREATING HYPERTENSION ON BRAIN HEALTH OUTCOMES

As alluded to earlier, hypertension is a strong risk factor in the development of cerebrovascular diseases and neurodegenerative diseases. As such, few studies have focused on acupuncture's effect on stroke via regulating hypertension. Work in hypertensive animals support hypertensive-acupuncture treatment that prevents stroke through several pathways related to the nervous system, oxidative stress, the endocrine system, cardiovascular function, and hemorheology (Zheng et al., 2018). However, the human data are unclear. Meta-analyses have challenged the efficacy of acupuncture for the treatment of hypertension as a risk factor for stroke (Sibbritt et al., 2018). However, controlling BP via acupuncture as an additive treatment is an effective secondary prevention of stroke (Du et al., 2017). In stroke patients complicated with hypertension, antihypertensive acupuncture improved the National Institutes of Health stroke scale and Barthel index, both of which are indicative of stroke-related neurologic deficit. Further, the treatment reduced morning BP (esp. DBP) and improved SBP and DBP load (Guo and Shi, 2019), therefore suggesting antihypertensive acupuncture as a post-stroke treatment option rather than just a preventative measure.

Studies investigating the possible underlying molecular mechanisms of acupuncture in the treatment of neurodegenerative diseases, specifically dementia, have found that acupuncture can alter neurotrophin regulation (Hwang et al., 2010; Lee et al., 2012; Lin et al., 2016), reduce oxidative damage (Liu et al., 2006), and modulate apoptotic signaling (Chen et al., 2012; Xue et al., 2014) in a manner that promotes positive health outcomes. However, there are few studies that have investigated the effect of acupuncture on dementia directly related to hypertension despite the strong human epidemiological evidence linking the two. Such a study in spontaneous hypertensive rats found that acupuncture at DU20 (also referred to as GV20) and ST36 acupoints reduced BP, increased microvessel dilation, increased CBF, attenuated neuron injury, and restored cognitive impairment (Tian et al., 2013). In hypertensive humans, acupuncture at LR3 activated anterior cingulated gyrus (measured with fMRI) to lower BP through modulation of parasympathetic nervous activity. Additionally, through anterior cingulated gyrus activation, the connection with the surrounding areas was strengthened to improve cognitive impairment caused by long-term hypertension (Sun et al., 2014).

Currently, the evidence of acupuncture's favorable effect on brain health outcomes directly via regulating hypertension is still scant. However, the data are promising and justify more experimental and clinical studies into acupuncture's reliability as a possible treatment/prevention strategy for brain health outcomes related to hypertension.

# POTENTIAL MECHANISM(S) OF ACUPUNCTURE IMPROVING HYPERTENSION AND VASCULAR CONTROL

There are many different proposed mechanisms linking acupuncture to positive BP and vascular outcomes; however, most fail at drawing a logical link from acupuncture stimulus to observed outcome. Acupuncture involves sticking needles just below the skin surface, twisting the needle, and expecting a change in a corresponding system such as the cardiovascular system (**Figure 1**). Accumulating evidence indicates that the hypotensive effect is mediated by a reduction in sympathetic outflow (Sato et al., 1993; Michikami et al., 2006) and an increase in endorphin release (Guo and Longhurst, 2007). Increases in various endorphins has independently been linked to reduced BP in hypertension (Ba˛dzy´nska et al., 2016; Li et al., 2016). This seems to be a paradox, as previous research shows venipuncture increases catecholamine release, synonymous with a hypertensive effect (Frankenhaeuser et al., 1976). The difference must be attributed to the depth of the needle (venous vs. just below the skin surface) and the twisting of the needle, which promotes ''de qi,'' a key component of acupuncture. Langevin et al. (2001) describe that, during de qi, the connective tissue is wrapped around the needle, promoting tension. The authors speculate that this tension could be activating various sensory organs within the skin, increasing afferent nerve firing. Zhou et al. (2005a) specifically looked at responses to acupoints that reduced BP and observed increased afferent sensory neuronal firing. When afferent nerves are severed, the hypotensive effect and sympathetic nerve inhibition are eliminated (Sato et al., 1993). In healthy humans, the data are not definitive. Stimulation of acupoints PC6 and HT7 have been argued to elicit hypotensive effects through alterations in sympathetic (Jung et al., 2006) and parasympathetic nerve activity (Jung et al., 2007). Conversely, in patients with post-stroke insomnia, intradermal acupuncture at PC6 and HT7 greatly decreased the number of non-dippers possibly by lowering LF/HF ratio (Lee et al., 2009). Taken

together, acupuncture works through a feedback loop where afferent nerves initiate a correction in autonomic nerve outflow, properly regulating BP. Insight into the mechanisms by which acupuncture is reducing sympathetic outflow come from elegant animal studies investigating the activity in the rostral ventrolateral medulla (RVLM) where sympathetic nerve activity is controlled. In normotensive rats, acupuncture (PC6) may alter the baroreflex. PC6 stimulation increased afferent neuron firing, which was associated with a decrease in RVLM activity, resulting in a reduction of sympathetic outflow and a lowering of BP (Zhou et al., 2005b). Similarly, Wang et al. (2018) performed a highly controlled study proving that acupuncture (LR3) reduces oxidative stress in the RVLM and that the observed antihypertensive effect was directly tied to NADPH oxidase activity and the REDOX environment of the RVLM in spontaneously hypertensive rats. Again, linking reduced BP and sympathetic outflow via RVLM. Alternatively, in humans, acupuncture increases firing in the gracile nucleus, frontal lobe, cerebellum insula, hypothalamus, and many other areas of the brain related to maintenance of BP (Chen and Ma, 2003; Chen et al., 2013; Zheng et al., 2016).

While the cited research has provided a general mechanistic outline for acupuncture to modulate BP, these data are far from conclusive. However, the promising results provide the foundation for future research to delineate physiological mechanistic responses to acupuncture. Until these mechanisms are well understood and verifiable, mass adoption of acupuncture as an additive treatment for hypertension cannot be recommended.

# LIMITATIONS

This review is not without some limitations. First, this review prioritized human RCTs. Thus, the volume of studies reviewed and the sample size within each reviewed article are limited. However, we contest that RCTs provide the best evidence of the effectiveness of acupuncture (Sibbald and Roland, 1998). Further, this choice limits review of articles using animal models especially outside our ''mechanisms'' section. Thus, some well-designed studies were omitted. However, these restrictions allow for the most rigorous and translational review regarding acupuncture's effect on BP and cerebrovascular control. Finally, mostly English language articles were cited. Acupuncture literature is written in many other languages and we have included several non-English articles (Li et al., 2011; Sun et al., 2014; Zheng et al., 2018; Guo and Shi, 2019). However, there may be a few relevant articles that our study team was unable to access.

# DISCUSSION

Evaluation of the literature suggests that acupuncture, at best, can be used as a co-treatment option for hypertension. Further, acupuncture has been found to improve cerebrovascular control and brain activation in regions consistent with positive health outcomes via the maintenance of BP and cognition. The mechanisms that link acupuncture to positive health outcomes are still poorly understood, but they appear to be linked to improvements in autonomic cardiovascular control. Regardless, the evidence is in support of safe and efficacious use of acupuncture in human hypertension. Yet, there is a profound need for tightly controlled mechanistic human research to determine the validity and the physiological underpinnings of acupuncture before wider adoption of acupuncture as a treatment option for improved cardiovascular and brain health can be recommended.

# REFERENCES

# AUTHOR CONTRIBUTIONS

JS, JA, and JK contributed equally to the writing and editing of the manuscript.

# FUNDING

Open access fees were provided from the University of Oklahoma Libraries Open Access Fund.

of stroke: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 45, 3754–3832. doi: 10.1161/STR.0000000000000046

review and meta-analysis. PLoS One 10:e0127019. doi: 10.1371/journal.pone. 0127019

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Sun, Ashley and Kellawan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effect of a Moderate-Intensity Aerobic Training on Joint Biomarkers and Functional Adaptations in Rats Subjected to Induced Knee Osteoarthritis

#### Edited by:

Leonardo Alexandre Peyré-Tartaruga, Federal University of Rio Grande do Sul, Brazil

#### Reviewed by:

Júlio Cezar De Oliveira, Federal University of Mato Grosso, Brazil Marli Maria Knorst, Federal University of Rio Grande do Sul, Brazil

#### \*Correspondence:

Ana Cristina Rodrigues Lacerda<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 14 June 2019 Accepted: 29 August 2019 Published: 18 September 2019

#### Citation:

Martins JB, Mendonça VA, Aguiar GC, Fonseca SF, Santos JM, Tossige-Gomes R, Melo DS, Oliveira MX, Leite HR, Camargos ACR, Ferreira AJ, Coimbra CC, Poortmans J, Oliveira VC, Silva SB, Domingues TE, Bernardo-Filho M and Lacerda ACR (2019) Effect of a Moderate-Intensity Aerobic Training on Joint Biomarkers and Functional Adaptations in Rats Subjected to Induced Knee Osteoarthritis. Front. Physiol. 10:1168. doi: 10.3389/fphys.2019.01168 Jeanne Brenda Martins1,2, Vanessa Amaral Mendonça1,2,3, Grazielle Cordeiro Aguiar4 , Sueli Ferreira da Fonseca1,2, Jousielle Márcia dos Santos1,3, Rosalina Tossige-Gomes1,2 , Dirceu de Sousa Melo1,2, Murilo Xavier Oliveira1,3, Hércules Ribeiro Leite1,2 , Ana Cristina Resende Camargos5 , Anderson José Ferreira4 , Cândido Celso Coimbra4 , Jacques Poortmans6 , Vinícius Cunha Oliveira1,3, Sara Barros Silva1,3 , Talita Emanuela Domingues1,2, Mário Bernardo-Filho7 and Ana Cristina Rodrigues Lacerda1,2,3 \*

1 Centro Integrado de Pós-Graduação e Pesquisa em Saúde (CIPq-Saúde), Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina, Brazil, 2 Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Sociedade Brasileira de Fisiologia (SBFis), Diamantina, Brazil, 3 Programa de Pós-Graduação em Reabilitação e Desempenho Funcional (PPGReab), Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina, Brazil, 4 Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil, 5 Programa de Pós-Graduação em Ciências da Reabilitação, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 6 Faculty of Motor Sciences, Université Libre de Bruxelles, Brussels, Belgium, 7 Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil

Background: Knee osteoarthritis (kOA) is a common chronic disease that induces changes in redox status and inflammatory biomarkers, cell death, and motor impairment. Aerobic training can be a non-pharmacological alternative to prevent the progression of the disease.

Objective: To evaluate the effects of an 8 weeks moderate-intensity treadmill aerobic training program on redox status and inflammatory biomarkers and motor performance in kOA-like changes induced by monosodium iodoacetate (MIA) in rats.

Methods: Twenty-seven rats were randomly divided into three groups: SHAM; induced kOA (OA); and induced kOA + aerobic training (OAE). Motor performance was evaluated by the number of falls on rotarod test, the total time of displacement and the number of failures on a 100 cm footbridge. Data for cytokines and histology were investigated locally, whereas plasma was used for redox status biomarkers.

Results: The OA group, compared to the SHAM group, increased 1.13 times the total time of displacement, 6.05 times the number of failures, 2.40 times the number of falls. There was also an increase in cytokine and in thiobarbituric acid reactive substances (TBARS) (IL1β: 5.55-fold, TNF: 2.84-fold, IL10: 1.27-fold, IL6: 1.50-fold, TBARS: 1.14 fold), and a reduction of 6.83% in the total antioxidant capacity (FRAP), and of 35% in the number of chondrocytes. The aerobic training improved the motor performance in all

**82**

joint function tests matching to SHAM scores. Also, it reduced inflammatory biomarkers and TBARS level at values close to those of the SHAM group, with no change in FRAP level. The number of falls was explained by IL1β and TNF (58%), and the number of failures and the total time of displacement were also explained by TNF (29 and 21%, respectively).

Conclusion: All findings indicate the efficacy of moderate-intensity aerobic training to regulate inflammatory biomarkers associated with improved motor performance in induced kOA-like changes, thus preventing the loss of chondrocytes.

Keywords: osteoarthritis, aerobic training, exercise, biomarkers, joint function

# INTRODUCTION

Chondrocytes are responsible for tissue maintenance which impact on joint function and performance (Sophia Fox et al., 2009; Akkiraju and Nohe, 2015; Charlier et al., 2016). Current literature has reported exercise-induced chondroprotection in knee osteoarthritis (kOA) (Loeser, 2010; Golightly et al., 2012; Geneen et al., 2017). A potential explanation is the mechanical signal transduction (O'Hara et al., 1990; Urban, 1994), preserving the cartilage proteoglycans, and promoting chondrocytes modulation (Little and Ghosh, 1997; Loeser et al., 2012). Low-magnitude mechanical stress seems to suppress the pathway of interleukin-1 beta (IL1β) and tumor necrosis factor (TNF) release (Roman-Blas et al., 2009; Leong et al., 2010; Li et al., 2013; Assis et al., 2016). The IL1β and TNF are inflammatory mediators involved in joint degeneration caused by kOA (Blasioli and Kaplan, 2013; Mabey and Honsawek, 2015). The modulation of these cytokines in the joint would regulate the synthesis of proteoglycans and collagen, thereby attenuating the swelling process (Knobloch et al., 2008; Iijima et al., 2015).

The literature points out redox imbalance and the related increase on reactive oxygen species (ROS), swelling process and necrosis of chondrocytes as the pathophysiology of kOA (Altindag et al., 2007; Rose et al., 2012; Hui et al., 2016). Moreover, redox imbalance and the increased inflammatory biomarkers may cause cartilage damage, neuroinflammatory disease progression, and joint disability (Kim et al., 2010; Watari et al., 2011; Attur et al., 2013, 2015; Reed et al., 2014).

Several interventions are used to improve motor performance in patients with k**OA** (Golightly et al., 2012; Geneen et al., 2017), and a low-cost aerobic training may be an alternative (Hunter and Eckstein, 2009; Semanik et al., 2012). Aerobic training improves blood soluble TNF receptors level and brainderived neurotrophic factor (BDNF) plasma level in people with kOA (Gomes et al., 2012; Simão et al., 2014). Moreover, BDNF seems to impact on chondrocyte differentiation, changing it from proliferative to differentiation program (Hutchison, 2012).

Beyond insufficient information about the modulatory effect of the aerobic training in the knee joint degeneration parameters and possible relationships between joint parameters and motor performance, the literature still presents gaps regarding the effect of aerobic training in the joint preservation on kOA-like changes. Many studies detail mechanotransduction mechanisms but still remain questions about joint function. Moreover, few studies address joint function tests to establish a new therapeutic approach in clinical practice. The current study aimed to investigate the effects of moderate-intensity aerobic training in the inflammatory and redox biomarkers modulation of kOA-like changes, and its possible link to motor performance in rats. Therefore, because experimental kOA induces joint swelling process, we hypothesized that moderate-intensity aerobic training would attenuate the swelling process, favoring the redox balance and preserving chondrocytes in rats with kOA -like changes. Improved modulation in joint biomarkers levels might explain an effect on motor performance.

# MATERIALS AND METHODS

Male Wistar rats were used in the current study that was part of a Masters in Physiology at the Universidade Federal dos Vales do Jequitinhonha e Mucuri (Martins, 2017). The project was approved by the local Ethics Committee (protocol 005/2015).

Twenty-seven rats were randomized into three groups: sham group (SHAM), n = 9; induced kOA group (OA), n = 9; and induced kOA + aerobic training (OAE), n = 9. The rats had available water and food (i.e., Nuvilab CR1, Nuvital Nutrientes S/A, Brazil) as they desire, and their environment was controlled (i.e., humidity of 60% and temperature of 22◦C).

# Induced Osteoarthritis

Twelve-week-old male Wistar rats were anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.). Then, kOA was induced on the right knee joint at 90◦ flexion by direct infiltration of monosodium iodoacetate (MIA) (1.2 mg diluted in 50 µL saline solution). We used a 29G X 1/2 BD Ultra-FineTM insulin needle (Guzman et al., 2003; Cifuentes et al., 2010; Takahashi et al., 2018). The SHAM group received an infiltration containing 50 µL of saline solution (0.9% NaCl).

# Aerobic Training

To perform the aerobic training, a motorized treadmill (Insight R , SP, Ribeirão Preto, Brazil) with six individual lanes and with no inclination was used in the study. OAE group started the familiarization to the treadmill (10 min/day for 5 days) 24 h after the kOA-induction procedure. Then, 24 h after the familiarization period, initiated the training program, which consisted of treadmill running at the velocity of 16 m/min,

3 days per week during 8 weeks (Cifuentes et al., 2010), and the duration of running sessions was increased from 30 to 50 min at the fourth week. The workload of all groups was analyzed at the end of the training program during a treadmill incremental test (initial speed of 10 m min−1 , 1% slope, with no electrical stimulus, an increase of 2 m min−1 every 3 min) (Balthazar et al., 2009; Primola-Gomes et al., 2009). The workload (W; J) was calculated as: W = body weight (kg) × total time to fatigue (min) × treadmill speed (m min−1 ) × sin θ (treadmill inclination) × 10 (Lacerda et al., 2006).

# Evaluation of Motor Performance – Forced Locomotion (Rotarod Test)

In the rotarod test (Scienlabor, Brazil), the rats are stimulated to walk around a circle drum surface. The rotarod test measures balance, coordination, physical performance, and motor-planning by calculating the number of falls during determined speed. The time that a given rat stays on this circle rod represents the joint function. We used a protocol adapted from Piel et al. (2014) to quantify the number of falls of rats during 3-min period keeping a fixed speed of 8 revolutions per minute (rpm) (Piel et al., 2014).

# Locomotion Test on a Footbridge

Rats locomoted on a footbridge to investigate joint function when moving a short distance. The footbridge had a length of 100 cm delimited by 3 mm thick aluminum filets. Image and time to complete a single pass during animals' locomotion were recorded. Records were later analyzed by a blinded investigator to quantify the total time of displacement and the number of times each rat stepped out of the space between the filets (number of failures).

# Euthanasia

Animals were euthanized individually by decapitation and their right knee joints were analyzed. Approximately 12 mL of blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA), then centrifuged at 500 × g for 10 min and the serum aliquoted and frozen in a −80◦C freezer for further analysis.

# Joint Lavage

For later analysis, the joint lavage (JAL) supernatants were stored at −80◦C. Immediately after recovering JAL, we removed the joint capsule and stored it in a freezer at −80◦C. Moreover, we homogenized the sample in phosphate buffer and also frozen it at −80◦C for future analysis. IL1β, TNF, and interleukin-10 (IL10) knee joint biomarkers were analyzed according to the manufacturer's instructions by ELISA kits (DuoSet, R&D Systems, United States).

# Macerated Joint Capsule

The capsule was placed into a beaker with 750 µL of cytokine extraction solution, and a tissue homogenizer (Tecnal, TE-103) was used to obtain the macerate. Then, the capsule macerate was processed at 8 rpm speed for 2 min, the volume centrifuged at 3500 xg at 4◦C for 10 min and stored in a freezer. BDNF and interleukin-6 (IL6) levels were analyzed according to the manufacturer's instructions by ELISA.

# Measurements of Redox Status

The reaction of the thiobarbituric acid with malondialdehyde (MDA) was used to determine lipid peroxidation by thiobarbituric acid reactive substances (TBARS) plasma levels (Ohkawa et al., 1979). The ferric reducing ability of plasma (FRAP), i.e., the reduction of ferric-tripyridyltriazine [Fe(III)-TPTZ] complex to ferrous-tripyridyltriazine [Fe(II)- TPTZ] (Benzie and Strain, 1996) was used to determine the total antioxidant capacity. The Bradford method using bovine serum albumin was used as a standard to determine the samples protein levels (Bradford, 1976).

# Histology

The right knee joints were placed in 4% neutral-buffered formalin for 24 h. After that, tissues were placed in 10% EDTA at pH 7.4 for decalcification (Jimson et al., 2014). Sagittal sections were prepared from knee joints. Two slides of the femur compromised by MIA induction or SHAM were prepared for histological analyses. Three cuts in each slide. Tissues were placed in formalin, dehydrated in a graded series of ethanol and xylol, embedded in paraffin, cut into 6 µm serial sections, and stained with hematoxylin-eosin.

# Quantification of Chondrocytes

To investigate the number of active cells, the middle third of the joint was used. For cell counting, the nuclei stained by hematoxylin present in the superficial, intermediate and transitional areas were considered. The analysis was performed on the articular facet of the right femur. The image was captured by a microscope with a 40 × magnification. The software Image J was used for cell counting. Two micrographs were taken in series and a blinded investigator analyzed them on different days (ICC = 0.99). For statistical analysis, the average number of cells was established and used.

# Statistical Analysis

We used the SPSS statistical package, version 22.0 (Inc., United States) and Graph Pad Prism, version 5.0 (Inc., United States). Data are expressed as mean ± standard error (S.E.M). Normality of data was assessed using the Shapiro– Wilk test. For comparisons, we used the one-way ANOVA with Tukey's post hoc tests for parametric data (Body mass, IL6 and number of chondrocytes) and Kruskal-Wallis with Dunn's post hoc test for non-parametric data (IL1β, TNF, IL10, TBARS, FRAP, number of falls, total time of displacement, and number of failures). Effect Size (d) was checked in G∗Power <IP_ADDRESS> program. Effect size conventions for test family (F tests) and one-way ANOVA: d = 0.10 (small); d = 0.25 (medium); d = 0.40 (large). The Spearman correlation investigated associations between two intra-articular biomarkers of joint damage (IL1β, TNF) and joint function tests: number of falls, total time of displacement and number of failures.

To determine intra-examiner reliability for the evaluated outcomes, the intraclass correlation coefficient (ICC) adopting a 95% confidence interval was determined. Multiple linear stepwise regression models confirmed the association between

selected biomarkers and joint function, adjusting by Bonferroni at α = 0.017. Graphs were built using the GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, United States).

# RESULTS

Twenty-seven animals were available for this study, recovered uneventfully from surgery and exercise procedure. By the end of the study, the rats were about 6 months old. No significant differences were observed in body weight between the three groups at the surgery and at the 8th-week post-surgery (**Figure 1**).

Aerobic training increased the total workload of OAE rats by 64% as compared to sham and OA groups (SHAM: 23.8 ± 8.0 J, OA: 23.7 ± 11.0 J, OAE: 69.2 ± 15.9 J, p = 0.0002, d = 0.85). Levels of IL1β, TNF, IL10 in the joint washed and the level of IL6 in the joint capsule increased respectively by 5.55, 2.84, 1.27, and 1.50-fold in the OA group. In OAE group compared with OA group, the aerobic training modulated levels of these joint cytokines close to those in the SHAM group (IL1β: p < 0.0001, d = 1.18; TNF: p = 0.0001, d = 1.00; IL10: p < 0.0001, d = 1.32; IL6: p = 0.0001, d = 1.68) (**Figure 2**).

The BDNF level in the OAE group was 41% higher than in the OA group (p = 0.05; d = 0.86). The induction of kOA-like changes increased by 1.14 times the TBARS plasma level and reduced the FRAP plasma level by 6.83% (p = 0.02) in the OA group compared with SHAM group. In OAE group compared with OA group, the aerobic training returned the plasma level of TBARS close to those in the SHAM group, without changing the total antioxidant capacity (TBARS: p = 0.001; d = 0.86; P = 0.95; FRAP: p = 0.05; d = 0.62) (**Figure 3**).

The analysis of the joint function tests showed that the kOAinduction (OA group) increased by 1.13 times the total time of displacement on a 100 cm footbridge and increased by 6.05 times the number of failures in the course when compared with SHAM group. The number of falls during the rotarod test increased 2.40 times in the OA group compared with SHAM. Therefore, in OAE group compared with OA group, the aerobic training had a positive effect on the disease, since it improved the performance in the three parameters evaluated, matching the

as Mean ± S.E.M. #Difference between OA vs. OAE.

SHAM score (Number of falls: p = 0.002; d = 1.05; Number of failures: p = 0.0002 d = 0.26; Total time of displacement: p = 0.005; d = 0.72) (**Figure 4**).

Number of falls (rs = 0.69; p = 0.0003) and number of failures (rs = 0.66; p = 0.0002) were associated with the IL1β. Functional performance measures: number of falls in the rotarod test (rs = 0.73; p < 0.0001); total time of displacement (rs = 0.63; p = 0.0005); and number of failures (rs = 0.52; p = 0.005) were associated with the TNF. Multiple linear stepwise regression models of IL1β and TNF explained 58% of the variability in the number of falls (IL1 β: p = 0.001; TNF: p = 0.002). TNF only explained 29% of the variability of the total time of displacement (p = 0.02) and 21% of the variability of the number of failures (p = 0.02). The increase of 1 pg/mL in intra-articular IL1β level leads to an increase of 0.40 points in the number of falls. The increase of 1 pg/mL in the intra-articular TNF level leads to an increase of 0.51 points in the number of falls; 0.57 s in the total time of displacement; and 0.49 points in the number of failures (**Table 1**).

Quantification of hematoxylin-stained nuclei showed that the induction of kOA reduced the number of cells (chondrocytes) alive by approximately 35% compared with the SHAM group. Aerobic training (OAE group) prevented chondrocyte cell death since the number of active cells in the trained group was 51.35% higher than in the OA group (p < 0.0001) (**Figure 5**).

# DISCUSSION

Overall, the current study showed an association between worse joint function and high levels of joint degeneration biomarkers, as indicated by high levels of TNF and IL1β. Moreover, aerobic training could reverse local inflammatory biomarkers and decrease systemic MDA level with an improvement in gait tasks, motor, and physical performance. Histological data of the femoral joint also confirmed the beneficial effect of the proposed exercise to the kOA-induced.

Scientific evidence suggests the involvement of inflammatory biomarkers and redox status parameters for the advancement and progression of kOA (Regan et al., 2005; Koike et al., 2015). In patients with kOA, chondrocytes and synovial cells stimulate the production of inflammatory cytokines, i.e., IL1β and TNF (Altindag et al., 2007; Kim et al., 2010; Wojdasiewicz et al., 2014; Kunisch et al., 2016). Kim et al. (2010) evidenced that IL1β and TNF are responsible for mitochondrial DNA damage in kOA, promoting the development of ROS and chondrocyte death (Kim et al., 2010).

The compression forces of low magnitude during physical exercise seem to promote physiological control. Such control modulates the synthesis of collagen and proteoglycans possibly inhibited in swelling joints (Quinn et al., 1998; Fehrenbacher et al., 2003; Park et al., 2004). This could indicate the greater maintenance of proteoglycan in the joint cartilage of rats exposed to moderate exercise (Galois et al., 2003; Cifuentes et al., 2010; Li et al., 2013; Mohammadi et al., 2013). Thus, the authors suggest that the positive effects are linked to the decrease of chondrocyte necrosis in the experimental group, lowering metabolites of cell

death and induction of inflammatory factors expression (Galois et al., 2003). The high level of ROS would act on the expression of cytokines, making worse the swelling. The ROS may initiate the cartilage degeneration and advancement of lipid peroxidation in chondrocytes (Yudoh et al., 2005; Ostalowska et al., 2006; Kim et al., 2010; Watari et al., 2011).

In kOA, inflammatory cytokines alter the joint biochemical balance inducing chondrocyte necrosis. The production and secretion of catabolic cytokines augment ROS production inducing a redox imbalance. Free radicals interacting with chondrocyte DNA acts directly by altering cellular components, i.e., proteoglycans, collagens, and protein oxidation, favoring cartilage degeneration, which may compromise the thickness of the synovial fluid, and the synthesis of other components (Henrotin et al., 2003; Rose et al., 2012; Reed et al., 2014). Physical and functional damages occur due to tissue structural change. Catabolic cytokines (IL1β and TNF), and regulatory cytokine (IL6) are probably the main factors in this process, facilitating paths of degeneration by activating paths such as matrix metalloproteases (Rojas-Ortega et al., 2015; Assis et al., 2016). The augmented inflammatory profile marks the loss of extracellular matrix integrity, developing an oxidative injury, and, lastly, the chondrocytes death. The death of these chondrocytes seems related to the compromised joint function since this cellular type is responsible for the mobilization of essential components that assure the main functions of load distribution and reduction of friction during static or dynamic exercises that guarantee the joint function. Mohammadi et al. (2013) assessed histological data of depth ratio of lesions demonstrating that 4 weeks of moderate exercise almost treated kOA symptoms in rats (Mohammadi et al., 2013). Moreover, the level of MDA increased in induced kOA dogs. This intensification revealed degeneration of the type II collagen (Goranov, 2007), implying a relationship between redox imbalance and cartilage degeneration. Oxidative injury can result in cell death, triggering particles and oxidized molecules release, cellular degeneration, and increased inflammation.

kOA subjects have greater ROS plasma level and lower antioxidant supplies (Abruzzo et al., 2013; Germanou et al., 2013). The redox imbalance can play a critical role in the cartilage degeneration (Henrotin et al., 2003; Reed et al., 2014). Thus, in both human and animal, kOA transporters have a high level of systemic biomarkers which means cellular damage led by ROS. As a consequence, cell-matrix may be compromised. Inhibition of this course can successfully avoid degeneration of articular cartilage and neo-formation of type II collagen (Poole et al., 2002).

The transduction of mechanical signals of dynamic pressure in chondrocytes may favor the pathways that counteract tissue catabolism. Li et al. (2013) assessed anabolic responses on bovine cartilage in vitro cells culture inducing matrix biosynthesis with different compression ranges (10, 20, and 30%) and demonstrated that moderate dynamic compression can exert an "anti-catabolic" effect, and suppress the expression of TNF, IL6, and soluble IL6 receptors (Li et al., 2013). The catabolism control appears to relate to a range of compression frequency, amplitude, and to the low-to-moderate intensity load, showing the importance of a voltage amplitude threshold for the regulation of inflammatory

paths and cell survival (Cifuentes et al., 2010; Beckett et al., 2012; Rojas-Ortega et al., 2015; Rios et al., 2018). Moreover, the exercise load influences BDNF production and release

β, Standardized regression coefficient; R2 , Adjusted R square. IL1β, Interleukin-1 beta; TNF, Tumor necrosis factor.

(Nofuji et al., 2008; Yarrow et al., 2010; Szuhany et al., 2015). BDNF is an important growth factor expressed in joint chondrocytes and in epiphyseal plaques of kOA subjects. BDNF causes the growth and, mainly, differentiation (Hutchison, 2012) of chondrocytes, inducing the proliferation pathway that can act as a restoration mediator. Furthermore, BDNF level has been also systemically increased in patients with kOA (Simão et al., 2014).

 In the current study, we decided to use an aerobic training similar to the study of Cifuentes et al. (2010) that investigated the effects of impact exercise on the kOA-induced cartilage aspects in rats. This protocol was chosen because the data of that investigation demonstrated that aerobic training contributed to the preservation of some joint cartilage parameters in experimental kOA. Moreover, the overload to the intensity and inclination of exercise sessions were not applied, once it could directly affect the dynamic compression of knee joint cartilage cells, leading to misinterpretations of our results. Thus, active mechanotransduction induces changes in oxygen tension and subsequent positive effects on matrix synthesis and cell growth (Urban, 1994; Park et al., 2004). These effects are explained by the displacement of growth factors or cellular cytokines by shifting cellular metabolism (Tilwani et al., 2017). Finally, our results showed that the proposed training offered an inflammatory control, confirmed by the modulation effect in biomarkers levels (IL1β and TNF in the joint wash; IL10 and IL6 in the knee joint).

The positive effects of aerobic training are attributed to the ability of the suppression of signals transduction paths of inflammatory and catabolic mediators together with the stimulation of anabolic paths. In vitro studies have verified that mild to moderate mechanical stress inhibits swelling by suppression of IL1β, TNF and the transcription of various joint degeneration inflammatory biomarkers (Fehrenbacher et al., 2003; Rose et al., 2012; Yamabe et al., 2013). Experimental kOA studies showed a positive effect of aerobic training, whereas highintensity training had a deleterious effect (Beckett et al., 2012; Ni et al., 2012; Rojas-Ortega et al., 2015; Li et al., 2017). These data seem to determine the role of aerobic exercise appropriate dose (intensity, frequency, and duration) in modulating chondrocyte response (Ni et al., 2013; Na et al., 2014; Hill et al., 2017). In rats without previous kOA induction, high-intensity exercises in many treadmill angles were not able to induce knee damage (Beckett et al., 2012; Rios et al., 2018). Thus, biochemical responses appear to be sensitive to the force only in the injury. Our data once again is in accordance to this premise since the proposed protocol was effective in dropping the joint IL1β and TNF in the trained group, as well as in reducing the TBARS systemic level, augmenting knee joint function and physical performance. In the present study, we identified an increased chondrocytes number in the OAE group. It is already known that chondrocytes are responsible for tissue maintenance which impact on joint function and motor performance (Sophia Fox et al., 2009; Akkiraju and Nohe, 2015). Thus, we speculate that the largest number of chondrocytes and the lower joint

degradation cytokines level (IL1β and TNF) (Kim et al., 2010; Li et al., 2013, 2015; Rojas-Ortega et al., 2015) might have preserved the proteoglycans and collagen joint which lead to better motor performance of the OAE group compared to the OA group. Based on the results, exercise prevented the increase of inflammatory biomarkers, and, consequently, prevented the loss of chondrocytes (the only marker evaluated). We cannot extrapolate our results to the whole joint, because the number of chondrocytes was the only analysis. This theory needs to be better clarified ahead. Our study is innovative as it points out the benefits of a therapeutic approach to an experimental OA model. Assessing the effect on physical performance of the animals it was demonstrated that the dosecontrolled impact is achieved by ways not fully elucidated in this study. The modulation of inflammation, due to the known effects of aerobic training, has affected the redox status balance, lowering oxidative damage, improving motor performance in functional tasks.

A limitation of this study was that we did not determine kOA-like changes according to the OARSI score. However, it was not possible because we used a single hematoxylin and eosin (H&E) staining for analysis. Thus, immunohistochemistry assessments on cartilage tissue would be useful in future studies.

# CONCLUSION

To conclude, a moderate-intensity aerobic treadmill training appears to modulate chondrocytes via activation of anabolic paths, swelling control by IL1β and TNF levels modulation, systemic TBARS level lowering and positive regulation in joint BDNF level, resulting in physical and motor performance improvements. As a perspective, the efficacy of the training protocol used here should be investigated in older animals.

# DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

# REFERENCES

# ETHICS STATEMENT

This animal study was reviewed and approved by the Commission on Ethics in Animal Use of the Universidade Federal dos Vales do Jequitinhonha e Mucuri (protocol 005/2015). Written informed consent was obtained from the owners for the participation of their animals in this study.

# AUTHOR CONTRIBUTIONS

JM, VM, MO, HL, AC, CC, and AL conceived and designed the study. JM, VM, GA, SF, JS, RT-G, DS, MO, HL, AC, AF, CC, JP, and AL contributed to analysis and interpretation of the data. JM, VM, SF, SS, JS, RT-G, DS, TD, and AL drafted the article. JM, VM, SF, MO, HL, AC, AF, CC, JP, MB-F, and AL critically revised the article for important intellectual content. JM, VM, GA, SF, JS, RT-G, DS, MO, HL, AC, AF, CC, JP, VO, MB-F, and AL approved the final article. JM, SF, MO, HL, AC, JP, and AL statistically expertised the study. VM, MO, HL, AC, AF, CC, and AL provisioned the study materials. VM, GA, RT-G, DS, MO, AF, CC, and AL contributed to administrative, technical, or logistic support.

# FUNDING

This study was supported in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Numbers 408721/2016-2 and 303746/2018-1), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (Numbers APQ-02503-16, APQ-01239-14, and APQ-03119-18), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (finance code 001).

# ACKNOWLEDGMENTS

We acknowledge Martins (2017) who provided data from her master's degree conducted in the Universidade Federal dos Vales do Jequitinhonha e Mucuri. Besides, the assistance of the Brazilian Society of Physiology is gratefully acknowledged.

knee osteoarthritis in rats. Osteoarthr. Cartil. 24, 169–177. doi: 10.1016/j.joca. 2015.07.020

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Martins, Mendonça, Aguiar, Fonseca, Santos, Tossige-Gomes, Melo, Oliveira, Leite, Camargos, Ferreira, Coimbra, Poortmans, Oliveira, Silva, Domingues, Bernardo-Filho and Lacerda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Randomized Clinical Trial Comparing Three Different Exercise Strategies for Optimizing Aerobic Capacity and Skeletal Muscle Performance in Older Adults: Protocol for the DART Study

#### Edited by:

Emanuele Marzetti, Agostino Gemelli University Polyclinic, Italy

#### Reviewed by:

Renato Gorga Bandeira De Mello, Federal University of Rio Grande Do Sul, Brazil Hélio José Coelho Júnior, Campinas State University, Brazil

#### \*Correspondence:

Dallin Tavoian<EMAIL_ADDRESS>Brian C. Clark<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Geriatric Medicine, a section of the journal Frontiers in Medicine

Received: 08 July 2019 Accepted: 07 October 2019 Published: 22 October 2019

#### Citation:

Tavoian D, Russ DW, Law TD, Simon JE, Chase PJ, Guseman EH and Clark BC (2019) A Randomized Clinical Trial Comparing Three Different Exercise Strategies for Optimizing Aerobic Capacity and Skeletal Muscle Performance in Older Adults: Protocol for the DART Study. Front. Med. 6:236. doi: 10.3389/fmed.2019.00236 Dallin Tavoian1 \*, David W. Russ 1,2, Timothy D. Law1 , Janet E. Simon3 , Paul J. Chase4 , Emily Hill Guseman5,6 and Brian C. Clark 1,7 \*

1 Ohio Musculoskeletal and Neurological Institute, Ohio University, Athens, OH, United States, 2 Laboratory for Integrative Muscle Biology, Division of Physical Therapy, Ohio University, Athens, OH, United States, 3 Division of Athletic Training, School of Applied Health Sciences and Wellness, Ohio University, Athens, OH, United States, 4 Division of Exercise Physiology, School of Applied Health Sciences and Wellness, Ohio University, Athens, OH, United States, 5 Diabetes Institute, Ohio University, Athens, OH, United States, 6 Department of Family Medicine, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, United States, 7 Department of Biomedical Sciences, Ohio University, Athens, OH, United States

Background: Age-related declines in physical function lead to decreased independence and higher healthcare costs. Individuals who meet the endurance and resistance exercise recommendations can improve their physical function and overall fitness, even into their ninth decade. However, most older adults do not exercise regularly, and the majority of those who do only perform one type of exercise, and in doing so are not getting the benefits of endurance or resistance exercise. Herein we present the study protocol for a randomized clinical trial that will investigate the potential for high-intensity interval training (HIIT) to improve maximal oxygen consumption, muscular power, and muscle volume (primary outcomes), as well as body composition, 6-min walk distance, and muscular strength and endurance (secondary outcomes).

Methods and Analysis: This is a single-site, single-blinded, randomized clinical trial. A minimum of 24 and maximum of 30 subjects aged 60–75 that are generally healthy but insufficiently active will be randomized. After completion of baseline assessments, participants will be randomized in a 1:1:1 ratio to participate in one of three 12-week exercise programs: stationary bicycle HIIT, stationary bicycle moderate-intensity continuous training (MICT), or resistance training. Repeat assessments will be taken immediately post intervention.

Discussion: This study will examine the potential for stationary bicycle HIIT to result in both cardiorespiratory and muscular adaptations in older adults. The results will provide important insights into the effectiveness of interval training, and potentially support a shift from volume-driven to intensity-driven exercise strategies for older adults.

**92**

Clinical Trial Registration: This trial is registered with ClinicalTrials.gov (registration number: NCT03978572, date of registration June 7, 2019).

Keywords: aging, exercise, intervals, resistance, aerobic, power, VO2, muscle

# INTRODUCTION

Nearly half of US adults over age 60 report difficulty performing one or more activities of daily living essential to maintaining independence, a fraction that has remained stable over the last 20 years despite an overall increase in the mean age of the US population (1). Increasing numbers of older adults with disabilities will continue to drive up healthcare costs, making maintenance of health and independence a top priority for both middle-aged and older adults (2). Contributors to this functional decline include poor cardiorespiratory fitness and skeletal muscle impairments (3–5), which can partially be attributed to reduced physical activity with age (6–8). Exercise is effective at maintaining function in older adults, as evidenced by data indicating those who take part in supervised exercise programs demonstrate improvements in functional outcomes (9–11). The fact that adaptations have been seen in the oldest adults (90+ years) is particularly encouraging (12–14).

Unfortunately, <12% of older adults meet the exercise recommendations provided by the American College of Sports Medicine (ACSM) (1, 15). These recommendations include weekly accumulation of at least 150 min of moderate intensity aerobic activity or 75 min of vigorous aerobic (endurance) activity, and at least 2 days of resistance exercise to improve muscular endurance, power, and strength (15). In 2014, 36.5% of adults over the age of 65 met the aerobic exercise recommendations, while only 16.5% met the resistance exercise recommendations; only 11.7% met both aerobic and resistance recommendations simultaneously (1). Consequently, the majority of older adults who do exercise are either not getting the muscular adaptations necessary for maintaining independence, or they are not getting the necessary cardiorespiratory adaptations. However, certain types of exercise may be able to induce both cardiorespiratory and muscular adaptations in older adults.

Resistance exercise appears to have minor effects on cardiovascular disease (CVD) risk factors, though its benefits include the prevention of musculoskeletal injuries, muscle wasting, and impairments in physical function (16). On the other hand, aerobic exercise is effective at reducing CVD risk by improving heart, lung, and metabolic function, though it appears to have little effect on muscular properties (17). However, recent work indicates that the absence of muscular adaptations in response to aerobic training may be related to exercise intensity and mode of exercise (18, 19). The most popular form of physical activity in older adults is walking, and recommendations for older adults promote walking as the primary means to increase physical activity levels (15, 20, 21). While walking may challenge older adults and result in cardiovascular improvements, the relatively low intensity of muscular contractions would be unlikely to elicit hypertrophy or functional adaptations of the leg muscles (22). Even higher intensity running does not seem to result in increased muscle size or strength (19, 23), and running may blunt the muscular benefits of resistance training when performed concurrently (24). In contrast, stationary bicycle training can increase muscular strength and size (25–27), and does not seem to interfere with the muscular adaptations to resistance training (24). Bicycle high-intensity interval training (HIIT) in particular may be the ideal form of aerobic exercise able to elicit muscular adaptations. Young adults performing bicycle HIIT demonstrate improved muscle strength (28) and power (29, 30), along with enhanced cardiorespiratory function (31).

HIIT has gained popularity in recent years, with most research in middle-aged and older cardiac rehabilitation patients (32, 33). Compared to traditional aerobic training, HIIT typically has a greater effect on VO2max (9.1% greater increase on average) and other CVD risk factors (e.g., cholesterol, blood pressure) (33). While its success in rehabilitation is encouraging, the effectiveness of HIIT as a general exercise strategy for older adults has not been adequately investigated, with few studies reporting physiological adaptations to HIIT in older adults (33–44). Furthermore, limitations make it difficult to generalize these studies to a healthy aging population. Specifically, these studies have had one or more of the following methodological confounds:

The lack of investigations that directly compare unique exercise strategies against established strategies represents a critical barrier to progress in the field of healthy aging. With this in mind, the DART Study (Dual-benefits of Aerobic and Resistance Training) is a phase 1B proof-of-concept, proof-of mechanism clinical trial that seeks to determine if bicycle HIIT is a more efficient standalone strategy to improve cardiovascular and lower extremity muscular function than established resistance or aerobic exercise training programs. We will test this "dualbenefits hypothesis" in generally healthy older adults that have yet to develop mobility limitations, but who, without intervention, could very likely develop mobility limitations in the future due to inactivity and normal time course of aging.

# METHODS AND ANALYSIS

## Design

This is a single-blinded (outcomes assessor), single-site, randomized control trial. It is a three (group) by two (time)

high-intensity interval training; Hx, history; MICT, moderate-intensity continuous training; MRI, magnetic resonance imaging; RT, resistance training; SBP, systolic blood pressure; SPPB, short physical performance battery; TRFD, twitch-evoked rate of force development; VRFD, voluntary rate of force development.

repeated measures factorial design. The overall study is illustrated in **Figure 1**.

# Study Subjects

A minimum of 24 (and up to 30) subjects aged 60–75 years of age that are generally healthy but sedentary will be recruited, enrolled, and randomized in this study (n = 8–10 per group). Potential subjects will be recruited from the local community by way of flyers, community events, or emails to individuals who have previously participated in studies with the Ohio Musculoskeletal and Neurological Institute at Ohio University in Athens, Ohio, USA. All interested individuals will complete a pre-screening phone interview, and all that are not ruled ineligible will be invited for in-person screening. Written informed consent will be obtained from each subject in accordance with the Declaration of Helsinki. Ethical Approval for this study has been obtained from the Ohio University Institutional Review Board (protocol number 18-F-55).

Eligible subjects will be insufficiently active. We define "insufficiently active" as not meeting either the endurance or resistance exercise recommendations for older adults, as set forth by the American College of Sports Medicine (ACSM), for three consecutive months. The current ACSM recommendations for endurance training are 150–300 min per week of moderateintensity activity (perceived exertion of 5–6 on 0–10 scale), or 75–150 min per week of vigorous-intensity activities (perceived exertion of 7–8 on 0–10 scale) (15). The recommendations for resistance training are at least 2 days per week of progressive weight training activities that use the major muscle groups (perceived exertion of 5–8 on 0–10 scale) (15). Additionally, the subjects must not be highly active outside of a structured exercise program (i.e., consistent hard physical labor). They must also have a 6-min walk distance within a normal range for adults aged 60–75. We calculated a normal 6-min walk range by averaging 6-min walk values from multiple studies, plus or minus one standard deviation (45–48). The calculated 6-min walk range for females was 400–675 meters, and for males was 450–725 meters. **Table 1** describes inclusion and exclusion criteria in detail. These criteria are designed to recruit a generally healthy, but insufficiently active population, and exclude individuals with poor health and physical function where there could be concerns about a subject's ability to appropriately perform the testing and exercise prescription.

# Randomization

Subjects will be randomized in a 1:1:1 ratio to receive a 12-week exercise program consisting of either resistance training (RT), moderate-intensity continuous training (MICT) on a stationary bicycle, or HIIT on a stationary bicycle. Due to the small sample size in each arm, permutedblock randomization will be used via computer-generated random numbers to ensure equal sample size. Specifically, we will create blocks of three with each treatment permuted within each block. The study subjects will be enrolled and assigned to their respective interventions by an unblinded project manager.

## Implementation

The allocation sequence will be generated by a biostatistician. Subjects will be enrolled in the study and assigned to their intervention group by the project manager.

# Blinding

Due to the nature of the intervention neither subjects nor staff can be blinded to allocation. The outcomes assessor and data analyst will be blinded after study completion by having the subjects' demographic and intervention group information coded.

# Sample Size

In this proof-of-concept, proof-of-mechanism trial, we report sample size estimates based on previous recommended literature (49). A sample size of ∼8 subjects per group will detect a moderate effect. Sample size calculation was based on expected effect sizes for the HIIT cycle, MICT cycle, and RT groups for the primary outcomes of V02max and thigh muscle CSA. Consistent with our "dual benefits hypothesis" we assumed, based on the literature, an 8% increase in thigh CSA for both the HIIT and RT groups (50) and 2% increase for the MICT group (51) and common SD across all groups of 4%. With the assumption of a 2-sided test and alpha level of significance equal to 0.05 an n = 7/group yields power of 0.83. With respect to VO2max we assumed a 32% increase in the HIIT group, a 15% increase in the MICT group, and a 10% increase in the RT group and common SD across all groups of 14% (52). With the assumption of a 2-sided test and alpha level of significance equal to 0.05 an n = 8/group yields power of 0.80. In line with other proofof-concept, proof-of-mechanism trials, no statistical control for type-I error from multiple comparisons will be considered, and p-values will be interpreted with care, as descriptive weights of evidence rather than as confirmatory claims. Lastly, this proof of concept, proof of mechanism trial is needed to test the complex interventions proposed, and the effect sizes calculated from this trial could be used to estimate sample size for a future large-scale clinical trial. Accordingly, we plan to enroll an n = 8–10 subjects per group.

# Study Timeline

This study will have a screening/baseline assessment period of 21 days (maximum) with three sessions spaced at least 48 h apart, a 12-week exercise training period, and a post-intervention assessment period of 10 days (maximum) with two sessions spaced at least 48 h apart. Subjects will visit Ohio University's Clinical and Translational Research Unit facilities prior to the intervention for baseline assessments. During Visit 1 we will obtain informed consent and conduct a full medical history screening and a short physical performance battery (SPPB) (53) to determine if candidates meet the inclusion/exclusion criteria. Subjects who meet the criteria will be enrolled in the study and complete a series of clinical and physiological outcome measures over the three baseline visits. Upon completion of baseline assessments, subjects will be randomized into one of the

#### TABLE 1 | Inclusion and exclusion criteria.

Inclusion Criteria:

three exercise groups for the 12-week exercise intervention. All exercises will be performed on site and supervised by an exercise professional 3 days per week. All baseline assessments will be repeated upon completion of the exercise intervention. A table of events for the study is illustrated in **Table 2**.

# Outcome Measures Primary Outcomes

#### **Knee extensor isokinetic power**

Maximal isokinetic power will be measured from the knee extensors. Peak torque will be recorded from the non-dominant

#### TABLE 2 | Schedule of events for all groups.

aExercise training sessions will be performed three times per week with at least 1 day between sessions, and no more than two exercise sessions on consecutive days in the same week. bPost-intervention functional task testing will be performed immediately prior to the final exercise training session (session 36).

cBaseline testing will be completed within 21 days of Visit 1, with at least 2 days between testing sessions.

dPost-intervention testing will be completed within 10 days of the final exercise session, with at least 2 days between testing sessions. 4SST, 4-square step test; DXA, dual-energy X-ray Absorptiometry; GXT, graded exercise test; MMSE, mini-mental state examination; MRI, magnetic resonance imaging; MVC, maximal voluntary contraction; PASE, physical activity scale for the elderly; SEE, self-efficacy for exercise scale; SPPB, short physical performance battery; VA, voluntary activation.

leg using a Biodex System 4 Dynamometer (Biodex Medical Systems, Inc., Shirley, NY). The subject's leg will be immobilized against the lever arm with the distal end of the lever arm secured three inches superior to the medial malleolus. The axis of the lever arm will be centered at the lateral knee joint and knee range of motion will be obtained by having the subject extend their knee as far as possible against the lever arm. The maximal knee extension angle will be recorded, and the knee extension limit will be set 10◦ less than the maximal knee extension angle. The subject will then relax to allow the leg to return to neutral position and the knee flexion limit will be set at 80◦ of knee flexion. The speed of the lever arm will be set at 60◦ /s for both extension and flexion. The subject will then extend the knee with maximal effort until they reach the knee extension limit, and then immediately flex the knee with maximal effort until they reach the knee flexion limit. The time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). The subject will complete six isokinetic trials, with 30 s rest between trials. The average of the three highest peak torque values for both extension and flexion will be recorded at baseline

and post-intervention, and percent change from baseline will be used for analysis.

# **Maximal oxygen uptake (VO2max)**

VO2max will be obtained with a ParvoMedics TrueOne 2400 metabolic measuring system with a Hans Rudolf 3813 (Shawnee Mission, KS, USA) pneumotachometer to measure ventilation. The TrueOne 2400 is a mixing chamber system that uses a paramagnetic oxygen analyzer (range 0–25%) and an infrared, single beam, single wavelength carbon dioxide analyzer (range 0–10%). Prior to each test the system will be allowed to heat up for 30 min, and then will be calibrated according to the manufacturer's recommendations. This consists of a room air auto-calibration and a gas calibration with a single gas tank (16.00% O2, 4.008% CO2). Additionally, the flow meter will be calibrated with a 3.000-liter Hans Rudolf 5530 series syringe, with a 5-stroke calibration using different flow rates for each stroke. Ten ECG electrodes will be placed on the subject's body according to Mason-Likar procedures (54, 55). Prior to placement, the areas will be shaved with a disposable razor, wiped with an alcohol pad, and then lightly abraded with finegrit sandpaper. The subject will be fitted with a Hans Rudolf Oro-Nasal reusable facemask (Shawnee Mission, KS, USA) with a 2-way non-rebreathing valve connected to the metabolic cart with large-bore, low-resistance tubing. The testing protocol will be explained to the subject, as well as how to communicate with investigators while wearing the facemask (i.e., hand signals). Resting VO2 values will be collected from the subject after 3 min of rest in a seated position on the cycle ergometer. Resting blood pressure will be taken immediately after resting VO2 is obtained. The subject will begin cycling on a magnetically braked cycle Lode Corival CPET ergometer (Lode B.V., Groningen, NL) at 60–80 RPMs (depending on subject comfort level) with a starting power output of 15 watts (W) for 1 min, collecting VO2 values every 20 s. Power output will be increased by 15 W every minute until subject can no longer continue the test or criteria have been met (**Table 3**). With 20 s remaining in each stage, the subject will indicate their RPE on a 6–20 Borg scale (56). With 10 s left in each stage, heart rate will be recorded from ECG readouts. Blood pressure will be taken at the beginning of even numbered stages. Subjects will be verbally encouraged throughout the test, and airflow will be provided through the use of a rotating fan. After test termination, the subject will perform a 5-min cycling cool down at 30 W. Blood pressure will be taken every 5 min for 20 min after the completion of the graded exercise test (GXT). Maximal heart rate will be determined from ECG readings during the final stage of the GXT for all subjects. Absolute VO2max measured in L/min will be recorded at baseline and post-intervention, and change from baseline will be used for analysis. Additionally, VO2max relative to body mass measured in mL/kg/min will be recorded at baseline and post-intervention, and change from baseline will be used for analysis.

#### **Quadriceps muscle volume**

Quadriceps muscle volume will be obtained via magnetic resonance imaging (MRI) scans performed with a 0.25-Tesla Musculoskeletal MRI system (Esaote G-Scan Brio, Genoa, Italy) to acquire contiguous transverse T-1 weighted spin echo image slices in the thigh region with a slice thickness of 10 mm and an inter-slice distance of 10 mm. The isocenter will be positioned at mid-thigh, midway between the patella and the inguinal crease, and the subjects will be supine. Images will be transferred to a computer for calculation of quadriceps anatomical crosssectional area (CSA). Beginning with the slide with the first discernable visual of the rectus femoris and including the subsequent four proximal slides, quadriceps muscle area will be traced using a polygon tool, excluding bone, as well as fat tissue surrounding the muscles (MIPAV version 7.3.0). Intramuscular fat will then be subtracted by applying a shading correction to each slide, determining average voxel density and standard deviation voxel density from a sample of the lightest area of fat tissue, computing a cutoff value at three standard deviations darker than the sample voxel density, and excluding all pixels with a voxel density at or below the computed value. Pre- and post-intervention slides will be displayed simultaneously, and slides will be visually compared to ensure that tracing patterns are identical and that the same structures are excluded (i.e., neurovascular bundle, intermuscular fat) on both slides before CSA values are recorded. This process will be completed for each analyzed slide, resulting in five measures of quadriceps CSA with intermuscular and intramuscular fat excluded for both pre and post time points. Muscle volume will then be calculated using the Cavalieri method [MV = T (A1 + A2 + A3 + A4 + A5)], where MV = muscle volume, T = the known distance between slices, and A = area (57). Quadriceps muscle volume will be recorded at baseline and post-intervention, and percent change from baseline will be used for analysis.

### Secondary Outcomes

### **Knee extensor isometric strength**

Maximal isometric force production will be measured via three maximal voluntary contractions (MVCs) of the knee extensors while the subject is positioned in the Biodex Dynamometer as described above, with the lever arm immobilized at 90◦ of knee flexion. The subject will be instructed to gradually increase force for the first second, and then exert maximal effort for ∼3–4 s. The subject will perform three MVCs with a 30–60 s rest period between contractions. Verbal encouragement will be provided during each trial. The time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). The trial with the highest value will be recorded at baseline and post-intervention, and percent change from baseline will be used for analysis.

#### **Knee extensor isokinetic fatigue**

Fatigue resistance of the knee extensors will be measured with the subject positioned in the Biodex dynamometer as described above. Subjects will be asked to perform a series of isokinetic leg extensions at 120◦ /s (the flexion component will be passive at a speed of 240◦ /s). First, study subjects will perform three isokinetic extensions recorded as pre-fatigue peak torque values. Next, study subjects will be given 3 min of rest before beginning the fatigue portion of the test. For the fatigue test, subjects will perform 120 consecutive maximal isokinetic leg extension TABLE 3 | Criteria for test termination during graded exercise testing.

ACSM, American College of Sports Medicine; RPE, rating of perceived exertion; RPM, rotations per minute; VO2, volume of oxygen consumption.

contractions (test time ∼4-min). Peak force for each of the 120 contractions will be summed to calculate total work output and will be recorded at baseline and post-intervention, and change from baseline will be used for analysis. The subject will be verbally encouraged throughout the test. Lastly, post-fatigue peak torque will be assed at 2, 5, and 10 min after the completion of the fatigue test. For all fatigue measurement, the time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). Average torque of three contractions at each of the post-fatigue timepoints will be recorded and expressed relative to the pre-fatigue torque at baseline and post-intervention, and percent change from baseline will be used for analysis.

### **Six-minute walk distance**

A 6-min walk distance test will be performed in a 30-meter hallway marked off with cones at either end, and distance marked every three meters. The subject will start at the end of the hallway (starting cone) and be instructed to walk as quickly as they can for 6 min. They will walk toward the end of the hallway, around the second cone, and then back toward the starting cone. The subject will complete as many laps as possible within 6 min, rounding the cones each lap. Subjects will be given feedback on elapsed/remaining time every 30 s, and encouraged to continue walking as quickly as possible. Distance covered in 6 min will be recorded to the nearest meter at baseline and post-intervention, and change from baseline will be used for analysis.

## **Body composition**

Total body fat mass will be obtained via whole-body dual-energy X-ray absorptiometry (DXA) scans (Hologic Discovery QDR model Series, Waltham, MA, USA) using the system's software package (Hologic APEX, Version 4.0.2). Subjects will be scanned at the same time of day pre- and post-intervention and will be encouraged to maintain a similar sleeping and eating schedule for both scans. Subjects will be advised to report to the laboratory in a hydrated state and will be given scrubs to wear during the scan. They will also be given the opportunity to use the restroom prior to the scan. Care will be taken to follow The International Society for Clinical Densitometry guidelines for positioning during the scan (58). Total body fat mass will be recorded at baseline and post-intervention, and percent change from baseline will be used for analysis.

### Other Outcomes

### **Knee extensor voluntary rate of force development (VRFD)**

VRFD of the knee extensors will be measured with the subject positioned in the Biodex Dynamometer as described above with the lever arm set to 90◦ of knee flexion. The subject will extend the knee into the immobilized lever arm with maximal effort over ∼500 ms, then immediately relax. The process will be explained to the subject so they understand that they are to produce as much force as possible, as quickly as possible, and then to relax (e.g., pretend you are kicking a tire as hard as you can). The time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). The trial with the highest VRFD (slope of force tracing between 10 and 90% of maximal force) will be calculated at baseline and post-intervention, and change from baseline will be used for analysis.

## **Twitch-evoked rate of force development (TRFD) and voluntary activation**

TRFD and voluntary activation of the knee extensors will be obtained with the subject positioned in the Biodex dynamometer as described above with the lever arm set to at 90◦ of knee flexion. A surface stimulating electrode will be placed on the distal motor point of the vastus medialis and another on the proximal motor point of the vastus lateralis. Single pulses of incrementally increasing current will be delivered via a Digitimer DS7AH constant current stimulator (Digitimer Ltd., Hertfordshire, UK) until twitch force plateaus. The time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). TRFD (slope of the twitch response between 10 and 90% of maximal force) at maximal stimulation intensity will be calculated at baseline and postintervention, and change from baseline will be used for analysis. Subjects will then perform two MVCs separated by 2 min, and during the MVC a doublet will be delivered at maximal intensity (intensity at which twitch force plateaus), followed by a second doublet delivered to resting muscle. Voluntary activation is expressed as [1–(doublet force during MVC/doublet force during relaxation)] ∗ 100. The time-series torque signal will be collected at 500 Hz by a Biopac MP150 system (Biopac Systems Inc., Santa Barbara, CA, USA). The subject will perform two voluntary activation trials, and the average of the two measures will be recorded at baseline and post-intervention, and change from baseline will be used for analysis.

#### **4-square step test (4SST)**

The 4SST is a test that challenges motor planning and initiation as well as motor sequencing and recall (59, 60). A four-foot by four-foot square will be marked with athletic tape and split into quadrants (**Figure 2**). The subjects will start in square 1, facing square 2. The subject then steps forward into square 2, laterally to square 3, backwards to square 4, laterally to square 1, laterally to square 4, forwards to square 3, laterally to square 2, and backwards to square 1, facing the same direction throughout the entire sequence. Subjects must place both feet in the specified quadrant before they can move into the next square, and must have at least one foot on the ground at all times. The test will be timed with a stop watch to the nearest 0.01 s. When the subject is ready the investigator will say "ready, set, go." The timer will begin at "Go" and stopped when the subject has placed both feet back in square 1 after completing a clockwise and counterclockwise cycle. The subject will perform two trials with 30 s rest between trials, and the fastest will be recorded at baseline and post-intervention. Change from baseline will be used for analysis.

### **Stair climb power**

Stair climb power will be calculated as [power = force x velocity] where force = body mass in kilograms x acceleration due to gravity, and velocity = cumulative stair height/stair climb time. The subject will be weighed to the nearest pound, and free weights equivalent to 20% of their body weight will be distributed evenly into two canvas bags. The bags will be set aside for later weighted stair climb testing. The subject will stand at the bottom of a flight of eight stairs (each stair 7′′ in height) with feet together. The subject will then be instructed to safely climb the stairs as quickly as possible without skipping any stairs, maintaining at least one foot on a stair at all times. The subject will also be instructed that they may use the handrails if necessary for safety purposes. The test will be timed with a stop watch to the nearest 0.01 s. When the subject is ready the investigator will say "ready, set, go." The timer will begin at "Go" and stopped when the subject's first foot makes contact with the eighth stair. The subject will perform two trials, and the fastest will be recorded at baseline and post-intervention. Stair climb power will be calculated as described above, and change from baseline will be used for analysis. The subject will repeat the test while carrying the weighted bags equivalent to 20% of their body weight, one bag in each hand. The subject will perform two trials and the

fastest will be recorded at baseline and post-intervention. Stair climb power will be calculated as described above, and change from baseline will be used for analysis. 30–60 s rest will be allowed between each trial.

### **Grip strength**

Maximal grip strength will be obtained with a Jamar hydraulic grip strength dynamometer (Performance Health, LLC, Akron, OH USA). Handle position of the dynamometer will be standardized at position II (61, 62). Hand dominance will be determined by asking the subject if they were right-handed or left-handed. The dominant hand will be tested first. The subject will be seated with the shoulder in neutral position and the elbow unsupported and flexed to 90◦ with the forearm and wrist in neutral position (61). The subject will squeeze the dynamometer handle as hard as possible for 3 s. Maximal force will be recorded in kg. The subject will perform three trials with each hand, and 15 s rest will be allowed between trials. Average force of the three trials for each hand will be recorded at baseline and postintervention, and change from baseline will be used for analysis.

# Exercise Intervention

Each subject will perform their prescribed exercise three times per week for 12 weeks. Time between visits will generally be 48–72 h, but may range from 24 to 96 h to meet the demands of the subject's schedule. Subjects may be allowed to exercise on consecutive days one time per week, but must have at least 48 h before a third exercise session is completed (for example, Monday, Tuesday, Thursday is acceptable, but Monday, Tuesday, Wednesday is not). Prior to each exercise session an exercise supervisor will perform a brief medical safety check. If the subject reports changes in health status that are concerning, has abnormal vital signs (blood pressure readings that exceed 170 mmHg for systolic blood pressure or 100 mmHg for diastolic blood pressure), or has changed medications/dosage, they will not be allowed to continue exercising until the study physician has reviewed the changes and clears the subject. After the medical safety check, the subject will perform a 5-min warm-up on the stationary bike at a low intensity (i.e., output 50% or less of their maximal output during the GXT). After the subject has completed the prescribed exercise for the day they will perform a 5-min cooldown at a low intensity on the stationary bicycle. For the RT and MICT exercise protocols we will employ a pragmatic trial design that follow the recommendations set forth by the ACSM, but that are not necessarily matched by time. There are currently no recommendations for HIIT in older adults, and the exercise sessions are shorter in duration due to the high-intensity nature.

### Exercise Interventions

## **RT**

Subjects will perform 10 resistance exercises for the major muscle groups (**Table 4**). The training program is lower extremityfocused, with 70% of exercises isolating the lower extremities. For the first 2 weeks subjects will perform 1–2 sets of 15 repetitions for each of the ten exercises, using a weight that elicits a rating of perceived exertion (RPE) at the end of the final repetitions of the respective sets of 5–6 (0–10 scale) as reported by the subject. Rest between sets/exercises will range from 30 to 60 s. Weeks 3–4 subjects will perform 2–3 sets of 12–20 repetitions at an RPE of 5–8 with 60–90 s rest between sets. Weeks 5–8 subjects will perform 3–4 sets of 10–20 repetitions at an RPE of 6–8 with 60–90 s rest between sets. Weeks 9–12 subjects will perform 3–5 sets of 6–20 repetitions at an RPE of 7–8 with 60–90 s rest between sets. Contraction velocity for each exercise will be moderate (180–240◦ /sec), with duration lasting ∼2 s for concentric actions and 2 s for eccentric actions (63). This protocol meets the ACSM resistance training recommendations for older adults (15), and progresses from entry-level to a more demanding protocol. Duration is expected to last ∼45–75 min. Subjects will occasionally be asked their perceived effort level on 1–10 scale after individual exercise sets to ensure that they are exercising at the prescribed intensity.

## **MICT**

The MICT will be performed on a stationary bicycle (Peloton Interactive, Inc. New York City, NY, USA) interfaced with a computer monitor that plays selected pre-recorded "spin classes." Heart rate and power output are displayed in real time. The goal of MICT is to maintain an output that elicits the prescribed heart rate throughout the exercise session. Heart rate reserve (HRR) will be calculated by subtracting the subject's resting heart rate (obtained during medical history check at visit 1) from their maximal heart rate (obtained during the GXT). The progression of the 12-week program will go as follows. For the first week subjects will cycle for 20–30 min at 50–60% of their HRR. Week 2 subjects will cycle for 20–30 min at 55–65% of their HRR. Weeks 3–4 subjects will cycle for 30 min at 60–70% of their TABLE 4 | Resistance training group exercises.

The five Daily Exercises are performed during each exercise session. The remaining five exercises each session will be chosen by the exercise supervisor from the list of Rotating Exercises.

HRR. Weeks 5–8 subjects will cycle for 30–45 min at 65–75% HRR, and in weeks 9–12 subjects will cycle for 45 min at 70–75% HRR. These ranges are based on recommendations from a metaanalysis describing a dose-response relationship between exercise intensity and VO2max adaptations in older adults (64), An exercise supervisor will oversee each exercise session. The target output (in watts) will be determined during the first exercise session by having the subject cycle for 5 min at a cadence of 75 RPMs with a resistance that produces 50 W. Once a consistent heart rate is established (using a chest-strap heart rate monitor), we will increase or decrease the resistance until the target heart rate is maintained. The target output for each subsequent session will be the highest average output from the previous week. If there was no increase from the previous week, target average output will be manually increased by 3%. If the subject is maintaining the target output but not achieving the target heart rate, output will be increased incrementally by 2–5 W until the heart rate is maintained in the target range. During each exercise session the subject will follow the cadence recommendations of the spin class instructor (ranging 50–100 RPMs) while the in-person exercise supervisor modifies the resistance to ensure the output is maintained within the target range. At times the subject may be cycling at the target output but have a heart rate that exceeds the target. In these cases heart rate range takes precedence over output range, and output will be decreased until the target heart rate is maintained. This may occur when cycling at higher cadences (e.g., 90–100 RPMs). At the end of each exercise session subjects will be asked their perceived effort level for the entire session on a 1–10 scale and a Borg 6–20 scale. See **Table 5** for exercise duration of each session.

# **HIIT**

Subjects in the HIIT group will use the same stationary bicycle setup as in the MICT group. The progression of the 12-week program will go as follows. Subjects will cycle continuously for 20–30 min at 50–60% of their HRR for the first week, and 20– 30 min at 55–65% of HRR for the second week, similar to the

#### TABLE 5 | MICT and HIIT exercise groups cycling duration.

Duration (in minutes) of cycling for the moderate-intensity continuous training (MICT) and the high-intensity interval training (HIIT) groups for exercise sessions 1–36.

MICT group. During the subsequent weeks, subjects will perform bouts of higher intensity cycling (target intensity of 80–100% of their HRR) interspersed with low- intensity rest periods (target intensity of 40–60% of their HRR). Here, the duration of the bout (or interval) will range from as little as 15 s and upwards of 1 min, and rest periods will be matched in a work/rest ratio of 2:1, 1:1, or 1:2 (1:1 on average). During weeks 3–4 the intensity will be at 80– 95% of their HRR and the overall duration will last 15–20 min. Weeks 5–8 the intensity will be at 80–100% of their HRR with a duration of 15–30 min. During weeks 9–12 intensity will be at 85–100% of their HRR and duration will last 20–30 min. When the subject begins week three, the target average output will be determined by multiplying the subject's best average output from the first 2 weeks by 1.2. The subject will cycle at the target average output for the first 2–3 min of each session, at 150– 300% of their target average output during the high-intensity intervals, and at 25–50% of their target average output during their low-intensity intervals in order to achieve the target heart rate ranges. The target average output for each subsequent week of exercise sessions will be the highest average output from the previous week. If there was no increase from the previous week, target average output will be manually increased by 3%. Subjects will complete 15, 20, or 30-min sessions throughout the study (**Table 5**) and will begin each session by cycling at the target average output for 2–4 min, followed by several high- and lowintensity intervals. Subjects will cycle at a high intensity for ∼6 total min during 15 min sessions, ∼8 total min during 20 min sessions, and ∼10 total min during 30 min sessions, maintaining an average work/rest ratio of 1:1. The subject will follow the cadence recommendations (ranging 50–100 RPMs) of the spin class instructor while the in-person exercise supervisor modifies the resistance to ensure the output is maintained within the target ranges. It is unlikely that the subject's heart rate will reach 40– 60% of HRR during the rest intervals, however, an output that is 25–50% of the target average output should elicit a heart rate that is 40–60% of HRR under normal conditions. Therefore, rest intervals will be long enough that the subject's heart rate will decrease by 5–20 beats per minute prior to the start of the next high-intensity interval. At the end of each exercise session subjects will be asked their perceived effort level for the entire session on a 1–10 scale and a Borg 6–20 scale. See **Table 5** for exercise duration of each session.

### Intervention Discontinuation

Participation in the study will be discontinued if the subject fails to follow the study requirements, experiences serious side effects (e.g., heart attack, exercise related injury), changes the dosage of anti-hypertensive medication or starts taking an exclusionary medication during the course of the study (**Table 1**), or requests to be removed from the study.

# Concomitant Exercise and Diet

It is possible that some subjects may be participating in some low-volume physical activity prior to study enrollment and still be eligible for the study (e.g., yoga, yard work). To control for this, all subjects will be encouraged to continue normal activity outside of the study throughout their enrollment in the study. Similarly, study subjects will be asked to maintain their normal diet throughout the study.

## Adherence

Successful adherence will be defined as a study subject who achieves at least an 80% adherence rate (i.e., attends 29 of 36 exercise sessions).

# Data Management

Data will be entered into the database immediately after each measure is performed by the investigator collecting the measure, and then again at the end of the study by the outcomes assessor. Any incongruous measures will be re-analyzed separately by the outcomes assessor and another blinded investigator. Demographic and intervention group data will be coded and the code will be stored in a locked cabinet unavailable to the outcomes assessor.

# Statistical Methods

The three intervention arms (HIIT, MICT, and RT) will be compared against each other for all primary analyses. For total fat mass and the primary and secondary knee extensor-related outcomes (isokinetic power, muscle volume, MVC, and isokinetic fatigue), we will compute a percentage change from baseline to 12 weeks. For absolute and relative VO2max and 6-min walk distance we will compute absolute change from baseline to 12 weeks. We will test differences in group means using one-way ANOVA with Sidak option in SPSS v. 25.0. Tukey post hoc tests will be performed if significant differences exist, alpha level set at 0.05. Additionally, as this is a proof-concept, proof of mechanism trial we will calculate effect sizes between groups using the corrected effect size Hedge's g with 95% confidence intervals for small sample sizes (65).

# Harms

In our study, adverse events (AEs) will be defined as an unexpected medical problem that happens during the course of the study related or unrelated to the intervention or assessments. All AEs occurring after informed consent is signed and until study completion will be recorded. An AE that meets the criteria for a serious adverse event (SAE) will be reported to the Ohio University IRB as a SAE. A SAE is defined as an unexpected medical problem that is believed by the investigators to be causally related to the study intervention or assessments and results in any of the following: a life-threatening condition, severe or permanent disability, prolonged hospitalization, or death. Prior to each exercise session the exercise supervisor will perform a brief medical safety check and will ask the subject if they experienced any health issues since the last exercise session. Any issues will be recorded and reviewed by the project manager. The issue will then be recorded as an AE, and will be noted if the project manager believes the issue is related to the intervention (e.g., muscle soreness).

# ANTICIPATED RESULTS

# The Dual-Benefits Hypothesis

The HIIT group will see improvements in endurance measures (VO2max, 6-min walk distance, isokinetic fatigue) equal to the MICT group and greater than the RT group, and improvements in muscular characteristics (isokinetic power, MVC, thigh muscle volume, VRFD, TRFD, voluntary activation) equal to the RT group and greater than the MICT group.

# Other Hypotheses

All groups will exhibit similar changes in the 4-square step test and stair climb.

The RT group will see greater improvements in grip strength than either MICT or HIIT groups.

The MICT group will see greater reductions in fat mass than either HIIT or RT groups.

# DISCUSSION

High-intensity interval training has become a popular exercise strategy for young adults for the equivalent or greater health benefits to traditional aerobic training with only a fraction of the time commitment (30). HIIT may also be a highly beneficial exercise strategy for older adults with potential to elicit both muscular and cardiorespiratory adaptations. However, there have been few studies assessing the effects of HIIT in older adults, with the majority being performed in individuals with overt health conditions (33, 35). Although a preliminary study, this is, to our knowledge, the first randomized controlled trial to compare muscular and cardiorespiratory adaptations of bicycle HIIT to adaptations seen in response to traditional bicycle aerobic training or resistance training in generally health older adults. Schjerve et al. (52) compared the effects of HIIT, MICT, and RT in middle-aged, obese adults, reporting large improvements in VO2max but no strength gains in the HIIT group. However, this study used a walking protocol for both MICT and HIIT groups, and we postulate the lack of effect on strength was due to the lower levels of muscular activity and force associated with this protocol [e.g., there is evidence that >60% of maximal muscle force must be produced during training to see increases in muscle size and/or function (22)]. HIIT has already been demonstrated to improve cardiorespiratory fitness equal to, or greater than, MICT in older adults (33, 38). Therefore, we propose in this study to determine if HIIT can produce muscular adaptations similar to those seen in response to resistance training. We will be using a cycling protocol for the HIIT group, as improvements in strength and power have been demonstrated in response to HIIT cycling protocols in young adults (28, 30).

The largest potential problem that could arise throughout the course of the study relates to the possibility of AEs. The risk of cardiovascular events in adults participating in HIIT programs is low, as reported by Rognmo et al. (66), who found that of the 4,800+ cardiac rehabilitation patients participating in either HIIT or MICT at a cardiac rehabilitation center, three suffered from cardiac arrest during training (one fatal during MICT, two non-fatal during HIIT). The rate of complications to the number of patient exercise hours was 1 per 23,182 h for HIIT (66). The target population for the current study will include insufficiently active older adults, but those with certain known heart conditions will be excluded from the study, and any subjects who display specific irregular cardiac responses during the baseline GXT (**Table 3**) will also be excluded. As such, risk of cardiac events is expected to be lower for our population than has been reported in cardiac rehabilitation patients. The American College of Sports Medicine recognizes the risks associated with exercise, but states that the health benefits of exercise outweigh the risks (15).

This study will assess the effectiveness of a high-intensity interval cycling exercise protocol in older adults to produce both cardiorespiratory and muscular adaptations. If shown to be effective it will pave the way for a paradigm shift from volume- to intensity-driven exercise recommendations for older adults, with the ultimate goal of improving health and reducing age-related physical dysfunction.

# ETHICS STATEMENT

# Research Ethics Approval

This study has been approved by the Ohio University Institutional Review Board (approval number: 18-F-55).

# Protocol Amendments

Any modifications to the protocol which may impact the conduct of the study, potential benefits to the subject, or affect subjects safety (including changes to study objectives, study design, patient population, sample sizes, study procedures, or significant administrative changes) will require a formal amendment to the protocol. Such amendments will be agreed upon by the principal investigators, and approved by the Ohio University Institutional Review Board prior to implementation.

# Consent

Informed consent will be obtained from each subject after the study has been fully described to the subject, and the consenting investigator believes that the subject fully understands the study requirements and can make an informed decision.

# Confidentiality

Each subject will be assigned a number and experimental data will be recorded using that number. Any identifiable personal information will be kept in a locked cabinet in OMNI, and only the PI will have key access to the information.

# Access to Data

The principal investigators will have direct access to all data sets. Data dispersed to other project team members will be blinded of any identifying subject information.

# Ancillary and Post-trial Care

There are no provisions for ancillary or post-trial care.

# Dissemination Policy

The investigators will provide the subjects with estimates of when the trial will end and when data will be published. The American Heart Association Predoctoral Fellowship requires that all journal articles be made freely available in PubMed Central within 12 months of publication. Eligibility for authorship of manuscripts resulting from this study include (1) substantial contributions to the conception or design of the project, or

# REFERENCES

the acquisition, analysis, or interpretation of the data, AND (2) drafting or revising the manuscript, AND (3) final approval of the manuscript. There is no intention to use professional writers.

# AUTHOR CONTRIBUTIONS

DT, BC, and DR conceived of the study and initiated the study design. PC and EG helped with implementation. TL provided medical oversight. JS provided statistical expertise. All authors contributed to refinement of the study protocol and approved the final manuscript.

# FUNDING

This work was supported by American Heart Association Grant #19PRE34380496/Dallin Tavoian/2019 (DT), the Osteopathic Heritage Foundations Graduate Fellowship Program at Ohio University Heritage College of Osteopathic Medicine (DT), and the John J. Kopchick Molecular and Cellular Biology/Translational Biomedical Sciences Research Fellowship Award (DT).

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**Conflict of Interest:** DT has received research funding from the American Heart Association, the Osteopathic Heritage Foundations, and the John J. Kopchick Fellowship Award from Ohio University. In the past 5-years BC has received research funding from the NIH, Regeneron Pharmaceuticals, Astellas Pharma Global Development, Inc., RTI Health Solutions, Biophytis, and the Osteopathic Heritage Foundations. In the past 5-years BC has received consulting fees from Regeneron Pharmaceuticals, Abbott Laboratories, and the Gerson Lehrman Group. Additionally, BC is co-founder with equity, and serves as the Chief of Aging Research, of AEIOU Scientific, LLC.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Tavoian, Russ, Law, Simon, Chase, Guseman and Clark. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Emerging Cognitive Intervention Technologies to Meet the Needs of an Aging Population: A Systematic Review

Fady Alnajjar 1,2 \* † , Sumayya Khalid1 , Alistair A. Vogan1 , Shingo Shimoda2 , Rui Nouchi 3 and Ryuta Kawashima3

*1 College of Information Technology, United Arab Emirates University, Al Ain, United Arab Emirates, 2 Intelligent Behavior Control Unit, CBS-TOYOTA Collaboration Center, RIKEN, Nagoya, Japan, 3 Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Japan*

Background: Cognitive training helps to promote healthy aging and ease activities of daily living for older adults. Recently, experiments have been conducted using robots to perform this cognitive training.

Methods: A review was conducted to examine the effects of computer-based cognitive interventions for older adults who were either healthy or experiencing mild cognitive impairment (MCI). A second study also examined the evolution of socially assistive robots (SAR) and their effectiveness at administering cognitive training for older adults.

# Edited by:

*Trentham Furness, Australian Catholic University, Australia*

#### Reviewed by:

*Lakshmi Rajagopal, Northwestern University, United States Corey Joseph, Monash Health, Australia*

#### \*Correspondence:

*Fady Alnajjar<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### †ORCID:

*Fady Alnajjar orcid.org/0000-0001-6102-3765*

Received: *09 July 2019* Accepted: *09 October 2019* Published: *24 October 2019*

#### Citation:

*Alnajjar F, Khalid S, Vogan AA, Shimoda S, Nouchi R and Kawashima R (2019) Emerging Cognitive Intervention Technologies to Meet the Needs of an Aging Population: A Systematic Review. Front. Aging Neurosci. 11:291. doi: 10.3389/fnagi.2019.00291* Results: Eighty-one studies published between 2009 and 2019 were identified for review, 56 of which focused on computerized cognitive training (CCT) while 25 examined the use of robotics. Twenty-four of the 56 CCT studies met the inclusion criteria. These were further classified into two groups: studies which used self-designed programs, and studies using commercially available ones. Of the 25 studies examining the use of robotics in cognitive intervention 7 met the inclusion criteria. Review shows that CCT improves cognitive function but that robots are more effective tools for improving cognition.

Conclusion: It can be concluded that CCT is beneficial for older adults and though there are drawbacks to this approach they are overcome by the introduction of robots into the training process. Culture, language, and socio-economic considerations vis-a-vis robot design and training methodology should be included in future research.

Keywords: cognitive impairment, cognitive training, computerized cognitive training, socially assistive robots, robotics for elderly

# INTRODUCTION

In recent decades, the world has witnessed measures of poverty drop while, on average, those of education, income, quality of life, and life expectation have risen significantly (Pinker, 2018). Indeed, on many measures there is reason for optimism. However, a quick study of the demographics indicates a rapidly aging global population1 . It is during this period that many begin

1United Nations: World Population Prospects The 2015 Revisions (2015). Available online at: http://esa.un.org/unpd/wpp/ Publications/Files/Key\_Findings\_WPP\_2015.pdf (accessed September 2015).

to experience the challenges of performing simple daily self-care and other independent living activities (McColl et al., 2013). This is because, in the later years, cognitive functions such as working memory have been found to diminish, while the prevalence of various diseases and disorders, including age-related dementia and Alzheimer's Disease, grows (Bozoki et al., 2013). In fact, dementia is one of the main reasons for the increased dependency of older people since it results in the deterioration of those specific cognitive functions needed in daily life (McColl et al., 2013). This deterioration is manifested in symptoms such as loss of memory, problems of orientation, depression, behavioral changes, and impaired communication skills. According to recent findings, each year over 9.9 million new cases of dementia are identified worldwide; this suggests a new case emerging every 3.2 s2 At this rate, by 2050, those experiencing dementia will have reached an alarming 131.5 million2 . The financial impact of this has the potential to be overwhelming. While the present economic worldwide cost of dementia is approximately 818 billion US dollars, in only a few years it is expected to climb to a trillion-dollar challenge2 . This, too, applies acutely to the United Arab Emirates (UAE). At present, the UAE possesses a relatively young population; however, in the coming years this is projected to change. According to the United Nations World Economic Situation and Prospects 2010 Report, by 2050 there will be a substantial rise in the population of those aged 60 or over, from 2.4% to an alarming 27%1 . For healthcare systems to fully-prepare themselves to meet this new reality significant innovations will need to be explored. Since no effective treatment or cure for dementia exists, an increased effort is being made to establish the efficacy of non-pharmaceutical strategies. One of these strategies is targeted cognitive training for older adults which may lead to prevention of dementia or delay of its onset (Brinke et al., 2018).

Cognitive training has been shown to maintain, or even improve, cognitive function for elderly (Kueider et al., 2012). In the past two decades this form of training has gained popularity. Studies have demonstrated its effectiveness in improving memory, attention and cognitive skills (Willis et al., 2006; Mowszowski et al., 2010; Kelly et al., 2014; Rebok et al., 2014). One randomized trial using cognitive training found diminishment in the decline of instrumental activities of daily living (IADL), thus leading to prevention and reduction of further risk of developing functional decline in elderly (Rebok et al., 2014).

Cognitive training involves a well-structured practice of complex mental exercises. Training can be given in multiple ways; it can be process-based, comprising repetitive training on specific tasks, or more strategic, individualized intervention based on memory formation strategies (Walton et al., 2015). However, there are hurdles to its widespread implementation. The traditional method of cognitive training requires a trained instructor, for example. This necessitates face-to-face interaction, which entails a meeting location, the coordination of schedules and travel time. Additionally, training can be very expensive since trainers usually charge by the hour; there is also the added cost of equipment and materials. Furthermore, not all elderly individuals are comfortable in traveling regularly to a meeting location. In fact, some older adults may be home-bound, live in an assisted living or nursing home facility, or may simply not be able to easily access transportation (Kueider et al., 2012). As a result, it becomes difficult to take part in these programs with regularity.

Recent informational technological advancements which potentially alleviate this problem have made their way into healthcare, and now play a significant role in cognitive training. Computer-based cognitive training (CCT) has been found to be easier to implement since it is cost-effective, can be accessed from anywhere and at any time, and can be performed from the comfort of the user's home (Kueider et al., 2012). Also, it can be customized according to specific needs of individuals. Moreover, CCT provides real-time performance assessment and feedback, and allows for the adjustment of application difficulty level accordingly. There are three approaches to impart CCT: (1) brain-training programs, (2) working memory training programs, and (3) video game training programs (Boot and Kramer, 2014). Computer and video games are designed to be fun and exciting. This serves to motivate users to maintain engagement throughout the training program. However, while there are many cognitive training products in the market, there is still a lack of evidence supporting their effectiveness at imparting cognitive training with significant improvement in cognitive function (Kueider et al., 2012). One study, a meta analytical review concluded that a commercially available computer-based training program for working memory skills was only able to have short-term specific training effects and did not generalize to "real world" cognitive skills, which raises question regarding the methodological approach or theoretical support for the current available training mechanisms (Melby-Lervåg et al., 2016).

To provide effective care or to improve the efficacy of the training program, it is important that the methods used should be comfortable for users. For example, the elderly may feel more at ease when training is conducted in their native tongue and when it is developed with an orientation to their individual culture. The consequence of this will be that training is more impactful and enjoyable since the user will be more greatly motivated to engage within each session. Furthermore, with increasing research being conducted on interventions for agerelated cognitive impairment, it is important to understand and distinguish the effectiveness of various methodologies used. The effectiveness of any individual methodology may be determined, for practical purposes, by the extent to which a transfer effect is produced. The transfer effect, in this case, would refer to the effect that the knowledge or abilities acquired in one area might have on the knowledge acquisition in other areas. From this understanding, the transfer effect can be bifurcated into "near" and "far" transfer effect, with the further distinction that the production of a range of "near" and "far" transfer effects might identify a quality methodology (Nouchi and Kawashima, 2014). While short-term cognitive training has been demonstrated to produce a limited and temporary effect, training conducted regularly and with vigor over an extended period of time can have a sustained meaningful impact (Tapus and Vieru, 2013).

2Dementia Statistics, Alzeihmer's Disease International. Available online at: https://www.alz.co.uk/research/statistics.

Frontiers in Aging Neuroscience | www.frontiersin.org

Unfortunately, the healthcare facilities are already under pressure with a shortage of staff and space. It is very challenging to provide a customized setup for each individual, according to their specific requirements (Tapus and Vieru, 2013).

Intelligent robotic systems have been designed for humanrobot interaction (HRI). HRI is a field of study dedicated to understanding, designing and evaluating robotic systems for use by or with humans. It's a communication link between human and robots3 As per Wikipedia the purpose of HRI is to model human expectations, regarding robotic interaction, to aid in robot design and algorithm development, which can allow more natural and effective interaction between human and robots.

Newly developed robotic systems have evolved considerably and can now be effectively used to provide that individualized care to the elderly, and from the comfort of the user's home (McColl et al., 2013). Additionally, socially interactive robot may have a tremendous impact on overall cognitive and social wellbeing. Developing a more human-like social robot with natural gestures and speech can engage a user and more fully support them as they carry out their exercises (Tapus and Vieru, 2013). Robots such as these could be programmed to help users choose from a variety of exercises and could motivate them along the way by giving applause, praises, or encouraging feedback during the training. Additionally, social assistive robots (SAR) have been shown to provide a companionship which improves user engagement in activities. This plays a powerful role in cognitive health (Tapus and Vieru, 2013). Furthermore, the specific needs of the user can be met through a customization of the social robot appearance; in particular, they have been designed to resemble pets, such as dogs or seals, producing positive benefits. Robots like Paro, ICat, Albo, and Pearl have been studied for their effect on the elderly, and a positive psychological and social impact has been demonstrated, such as improvement to both the mood and well-being of the users (Broekens et al., 2000). Unfortunately, most interventions have been limited to nursing homes or health facility. Few studies exist which examine the impact of assistive robots on the basic daily activities of the elderly in their own homes.

It seems however, a future socially assistive humanoid robot could help not only with the daily activities of the elderly and by providing company, but also by performing cognitive training with regularity and accuracy. When doing this it would be able to maintain users' scores and learn and adapt continuously to these individuals over time as cognition improved. This adaptive, user-friendly, reliable robot would provide an engaging and motivating customized therapy to users, establishing a lifeenriching human-robot relationship.

To make this possible, some system requirements that can be identified are two-way communication, safety, services and assistive functions, therapy and smart situation awareness (Gross et al., 2011). Furthermore, to ensure a good human robot relationship and interaction, we need to make the robot as human-like as possible. The main requirements would be for it to have more appealing, human-like interaction capabilities, demonstrate appropriate social behavior and be able to focus user attention in order to help achieve specific goals (Tapus et al., 2007).

There are a few areas of focus that need to be addressed when designing a humanoid robot: (1) physical appearance, (2) personality, (3) empathy, (4) engagement, (5) adaptation, and (6) transfer (Tapus et al., 2007).

A vast amount of literature exists which explores the effects of cognitive training (both computerized and non-computerized) on the elderly.

This review focuses on the effect of CCT on samples (healthy older adults and healthy older adults with MCI), to delay or prevent onset of dementia. For the purpose of this review, related articles published over the last 10 years were researched to answer the following questions: (1) To what degree has CCT been impactful as a tool for cognitive training of individuals experiencing age-related cognitive decline? (2) Can CCT delay or prevent the onset of dementia and which programs have been found to be most effective? (3) How have robots been used for cognitive training? (4) What challenges have been identified in robot development as they relate to cognitive training of the elderly?

# METHODS