Tourniquets are used in several surgical procedures (Fitzgibbons et al., 2012). Relative to total knee replacement (TKR) surgery their use facilitates a near bloodless field, improving visualization of crucial structures, and accelerating the surgical procedure (Smith and Hing, 2010; Estebe et al., 2011). However, the benefits of tourniquet application are not without negative post-surgical side-effects e.g., delayed wound healing, vascular injury, muscular damage, and greater post-operative pain (Estebe et al., 2011; Fitzgibbons et al., 2012). Tourniquet mediated side-effects are not restricted to solely tissue damage (via direct tourniquet tissue compression), but also through ischemic reperfusion (IR) mediated metabolic disruption (Fitzgibbons et al., 2012). Reperfusion of occluded tissue (i.e., the leg after TKR specific tourniquet use) may lead to ischemic injury with activated leukocytes increasing reactive oxygen species (ROS) and/or free-radical formation, consequently resulting in transient elevations in oxidative stress (OS, Grace, 1994). Although ROS are essential for normal cellular signaling (Ray et al., 2012) and demonstrate an association with hormesis (Radak et al., 2008), a rapid increase in ROS can disrupt redox balance, resulting in OS induced protein denaturation and lipid oxidation (Halliwell and Chirico, 1993; Hawkins and Davies, 2001).

Reduced glutathione (GSH) is the most abundant endogenous antioxidant and can acquiesce increases in OS via its oxidation (e.g., cysteine originated reducing equivalent donation of H⁺+ e⁻ to unstable molecules, such as, ROS; i.e., ROS scavenging) to oxidized glutathione (GSSG). The GSH:GSSG ratio can indicate cellular redox balance, with increases in the latter indicative of increased OS and a pro-oxidant state (Fisher-Wellman and Bloomer, 2009). During knee prosthesis implantation surgery, following ~85 min tourniquet application (thigh), both local (the occluded leg), and systemic (the arm) blood borne free radical generators hypoxanthine and xanthine oxidase increased, whilst GSH:GSSG only demonstrated significant pro-oxidant values locally, all of which were seen 5 min post-reperfusion (Karg et al., 1997). Elevated OS following IR of tissues is associated with inflammation and impaired wound healing (Soneja et al., 2005), and postulated to induce greater post-operative pain (Orban et al., 2006), whilst acquiescing OS reduces oxidant mediated apoptosis (Primeau et al., 2002) and post-operative pain (Waikakul et al., 1999). Interestingly, short non-lethal cycles of IR [ischemic preconditioning (IPC)] can prime the intended tissue and bestow protection for future IR stress (Murry et al., 1986; Saita et al., 2002). This method of IR has been shown to reduce knee surgery mediated increases in OS following tourniquet use when compared to controls (Koca et al., 2011). Furthermore, IPC in vivo has been shown as effective in reducing post-operative knee surgery pain and/or corresponding length of hospital stay (Memtsoudis et al., 2010).

Inhaled hypoxic air preconditioning (HPC) can infer cellular tolerance to subsequent hypoxia-mediated OS in vivo (Taylor et al., 2012), inducing similar protective effects to those conveyed by IPC (Bushell et al., 2002a,b; Berger et al., 2010; Samaja and Milano, 2015; Verges et al., 2015; Chacaroun et al., 2017), with the simplicity of the intervention (i.e., inhaling hypoxic air) advantageous compared to IPC. At present HPC, unlike IPC, has not been utilized prior to TKR to alleviate IR stress in vivo, despite similar protective effects. Characterization of occlusion mediated blood and muscle IR stress responses are heavily biased to OS markers. Given cells can initiate protective mechanisms during OS complimentary to GSH reduction, particularly increasing transcription and translation of a highly conserved cytoprotective family of proteins, known as heat shock proteins (HSPs) (Kalmar and Greensmith, 2009; Morton et al., 2009b), characterization of both OS and HSP responses to TKR like occlusion IR stress appears mechanistically warranted. Indeed, in vivo HPC mediated increases in basal HSP72 and hemeoxygenase-1 (HSP32) were both associated with resisting subsequent hypoxia/reoxygenation (HReox, Samaja and Milano, 2015) induced OS upon systemic HReox; attributed to restoring the function of OS mediated denatured proteins (principally HSP72, Taylor et al., 2010a, 2012) and degradation of ROS-producing heme molecules (principally HSP32, Gozzelino et al., 2010; Taylor et al., 2012). Readers are directed to multiple reviews for a detailed overview of the transcription factors and functional roles of HSP72 and HSP32 (Kregel, 2002; Morton et al., 2009b; Gozzelino et al., 2010; Henstridge et al., 2014).

Contextually, the cost of TKR surgery is ~$7,500 (Dakin et al., 2012), with the majority of this cost associated with long duration post-operative patient hospitalization (Smith et al., 2008). IPC has some efficacy for positively influencing variables associated with post-operative length of stay, however its administration typically occurs within the operating theater, and thus is not economically viable regarding time (theater availability is finite and often pressurized, operating list congestion, etc.). Therefore, a pre-operative (ideally on the ward) easily administrable preconditioning intervention (i.e., HPC) with positive physiological effects akin to IPC would be advantageous clinically (improved surgical outcome) and financially (reduced post-operative length of stay without increased time within theater). Logistically, HPC may be facilitated by supplementary low O₂ gas inhalation in an equivalent manner to that of supplementary O₂ or medical Nitrous oxide (Berkowitz et al., 1976; Greif et al., 2000), or via inhalation of nitrogen rich gas administered through small portable generators utilized in athletic training (Millet et al., 2010) or by creating rooms of low O₂ concentration via nitrogen gas (Mekjavic et al., 2016; Simpson et al., 2016). Given acute exercise elicits significant OS across populations and modalities (Fatouros et al., 2004; Vincent et al., 2005; Hillman et al., 2011; Taylor et al., 2016), with this increase impacting muscle force production (Powers and Jackson, 2008), the benefits of HPC and IPC as a preconditioning strategies to ameliorate the cellular disruptions to contractile function in this paradigm also warrant further investigation. Physical activity (Debevec et al., 2017) and exercise training reduce OS (Miyazaki et al., 2001; Vinetti et al., 2015), with training induced elevations in intramuscular HSP content (Liu et al., 1999; Morton et al., 2009a) a potential pathway for these benefits (Oksala et al., 2014). As such HPC (and IPC) may provide an alternative strategy for individuals undertaking exercise for health, or athletic performance to reduce the potentially negative impact of acute OS (Braakhuis and Hopkins, 2015; Vinetti et al., 2015).

Therefore, the aim of this study was to examine the effects of interventional HPC and IPC prior to subsequent TKR like tourniquet induced IR stress, in comparison to a control condition using an ecologically valid model. It is hypothesized that a bout of either HPC or IPC would prime the HSP system, providing resistance to the physiological stresses induced via tourniquet ischemia. Furthermore, tourniquet mediated stress would be monitored via associated redox markers (GSH/GSSG, protein oxidation) post-occlusion.

A significant difference was observed in age [F(_{2, 15)} = 4.36, p = 0.032] between IPC_I and CON (p = 0.032). No other significant differences (p ≥ 0.34) were noted in participant demographics (Table 1).

A significant main effect displayed a decrease in hemoglobin saturation [F_{(8, 40)} = 17.331, p < 0.001] between baseline and all subsequent time points (p < 0.05) in the HPC_I intervention. However, there was no significant main effect [F_{(8, 40)} = 1.130, p = 0.365] in HR noted by the same exposure (Figure 2).

Significant group × time interaction effects (F = 3.058, p = 0.048) were observed in muscle Hsp72 relative gene expression. There was an increase between time-points PrT and 15PoT (95% CI −3.771, −0.124; p = 0.035) in CON displaying a large effect size (1.44). Also, between PrT and 120PoT, a pronounced 116% increase (95% CI −3.779, −0.400; p = 0.014) was noted in IPC_I producing a large ES (1.59). Furthermore, when compared to CON at 15PoT muscle Hsp72 was lower in HPC_I (95% CI 0.634, 3.934; p = 0.007) and IPC_I (95% CI 0.675, 4.114; p = 0.006), both demonstrating large effect sizes (1.90 and 2.19, respectively) (Figure 3).

There were no significant (p > 0.05) group × time interaction effects for gastrocnemius Hsp32 (F = 0.147, p = 0.961) (Figure 3) leukocyte Hsp72 (F = 1.195, p = 0.347), leukocyte Hsp32 (F = 1.406, p = 0.244), PC (F = 0.681, p = 0.707), or GSH/GSSG (F = 1.959, p = 0.105) (see Table 3).

In agreement with the presented hypothesis it is a novel experimental finding that both IPC_I and HPC_I primed the localized (gastrocnemius) HSP system producing a blunted response in Hsp72 mRNA post (15PoT) 30 min TKR like femoral blood flow occlusion (termed simply occlusion from this point forward) compared to CON (Figure 3). However, changes in local Hsp32 were not seen (Figure 3), contrary to the stated hypothesis, this lack of response may be indicative of minimal intramuscular OS within the gastrocnemius following 30 min of occlusion. This amelioration in Hsp72 response (15PoT) was seen without systemic changes in OS (PC and GSH:GSSG; Table 3) and leukocyte Hsp32, in agreement with the stated hypothesis.

HSPs are up-regulated in response to a variety of stressors relevant to IR and HReox mediated in vivo stress, including OS, hypoxia, and ischemia (Kalmar and Greensmith, 2009; Morton et al., 2009b; Gibson et al., 2017). Elevated localized Hsp72 mRNA in CON at 15PoT (+84 ± 78%) indicates that the occlusion stressor was sufficient to potentiate initiation of the heat shock response (Theodorakis et al., 1999; Noble et al., 2008), whilst amelioration of this response (indicative of early phase IPC tissue protection; Bushell et al., 2002a,b; Loukogeorgakis et al., 2005) was seen when occlusion was preceded by IPC_I (+19 ± 35%) or (HPC_I +7 ± 33%; Figure 3). This change in Hsp72 mRNA within CON was not confounded by the larger basal gene expression of one participant in this group at this time point. Amelioration of Hsp72 mRNA is not novel regarding efficacy of IPC to blunt the respective gene response post-reperfusion of occluded distal lower limb skeletal muscle (rat tibialis anterior) (Bushell et al., 2002a,b) however, blunting of the Hsp72 mRNA stress response in vivo via HPC_I within the presented paradigm is. It is ecologically relevant that the blunted response in HPC_Iis contained within a time frame whereby surgery may be performed i.e., up to 120PoTwith the time course of responses comparable to those observed in similar Hsp72 mRNA experiments (Febbraio et al., 2002; Tuttle et al., 2017). Mechanistically, this intervention mediated reduction in Hsp72 mRNA cannot be attributed to systemic reduction in OS (PC or GSH:GSSG; see Table 3) nor increased degradation of ROS-producing heme molecules by Hsp32 mRNA, as these outcome variables were unchanged within and between all conditions and time points (Table 3, Figure 3). However, the absence of localized measures of OS precludes inferences relative to a local to systemic difference in OS (although others have shown such a difference post knee surgery which utilized a tourniquet; Karg et al., 1997) mediating the local to systemic differential Hsp72 mRNA response at 15PoT. Given the high affinity for changes in OS to induce a HSP32 and Hsp32 mRNA heat shock response (Gozzelino et al., 2010; Taylor et al., 2012), and the absence of changes in Hsp32 mRNA locally (and systemically), it is likely local Hsp72 mRNA amelioration via the interventions at 15PoT is mechanistically distinct from OS—at least within the occlusion paradigm utilized within the present design.

IPC has been cited to diminish circulatory redox disturbances associated with tourniquet-induced IR stress following TKR surgery (Koca et al., 2011). This is in disagreement with the current study, whereby it was noted that systemic OS markers remained unchanged within and between conditions at all times points when assessing lipid peroxidation (malondialdehyde concentrations) via measurement of thiobarbituric acid-reactive substances (Koca et al., 2011). This particular method of assessing malondialdehyde lacks specificity (Powers et al., 2010b), as such, the changes noted may be due to methodical inaccuracies rather than experimental effect. Additionally, systemic OS may not be a reliable surrogate marker for an intracellular response as aforementioned. It is however plausible that the 30 min occlusion utilized by the present study did not induce sufficient stress to observe changes in systemic OS markers and leukocyte Hsp mRNA (Table 3). This occlusion duration is lower than that commonly used during TKR surgery (mean ± SD; 79.9 ± 12.7 min; Cheng et al., 2003). Therefore, it is expected that the OS would be greater during surgery thus the occlusion stressor in the present study is not completely externally valid. However, our pilot testing revealed a longer occlusion period was not tolerable in vivo by non-anesthetized humans thus precluding its implementation. In an exercise setting, variants of our IPC (de Groot et al., 2010; Bailey et al., 2012; Cruz et al., 2015; Kido et al., 2015; James et al., 2016; Sabino-Carvalho et al., 2016), and HPC (Lee et al., 2014; Turner et al., 2016; Chacaroun et al., 2017), have been implemented to induce positive physiological responses. These data reflecting positive responses in clinical paradigms (Landry et al., 1982; Tomai et al., 1999; Otani, 2008; Mateika et al., 2014; Verges et al., 2015; Baillieul et al., 2017). Our data present a novel contribution to the area demonstrating the potential facilitative role HSPs have in response to OS.

Early phase IPC protection is temporally aligned to a 1–2 h window (occlusion occurred within this window) post IPC, whilst late phase IPC protection commences ~24 h post IPC and has a window of effect between ~24 and ~72 h post IPC. This late phase protection is dependent on the induction of protective proteins (Loukogeorgakis et al., 2005) including HSP72 (Bushell et al., 2002a,b; Marber et al., 1993, 1995). HSP72 is thought to refold sub-lethally damaged proteins and diminish their interactions with viable proteins during repeated IR bouts (Marber et al., 1993), consequently conveying cellular protection. The cumulative IR signal in IPC_I [i.e., five distinct IR stressor bouts; intervention (four IR cycles) and occlusion (one 30 min IR cycle)] compared to HPC_I [occlusion (one 30 min IR cycle) preceded by interventional HReox] is greater. Therefore, intervention activated IR preconditioning specific biological processes may have been exacerbated (particularly IR mediated cyclical increases in bradykinin and adenosine; Loukogeorgakis et al., 2005) by the subsequent occlusion IR stress and may underpin the enhanced gastrocnemius Hsp72 mRNA transcription seen at 120PoT in IPC_I, a response absent in HPC_I. This could be an early transcriptional level initiation of late phase IPC, within HPC_I, to which HSP72 protein translation is central (Loukogeorgakis et al., 2005). Therefore, this enhanced Hsp72 mRNA transcription at 120PoT could be interpreted as a priming mechanism for late phase IPC HSP72 translation within IPC_I, a response absent within HPC due to a lack of sufficient IR signal. In support of this postulation it has been eloquently shown elsewhere that remote limb IPC within humans results in cumulative up-regulation of peptides, with the magnitude of increase dependent on the number of discrete IR bouts (Hepponstall et al., 2012). Future research should examine the accumulative and temporally ordered effects (in line with recent attempts within a rat model; Kocman et al., 2015) of IPC (IR) and HPC (HReox) conveyed preconditioning in vivo within humans. A specific emphasis should be placed on adenosine, bradykinin, extracellular signaling proteins (ERK1/2, AKT; Winter et al., 2016) and relevant gene transcription and translation relative to the temporally ordered protective effects (i.e., early and late phase IPC tissue protection; Loukogeorgakis et al., 2005). The lack of such an approach is a limitation of the present study.

Prior research has shown that both acute hypoxic exposure (Taylor et al., 2010a) and IPC (Konstantinov et al., 2004) can stimulate leukocyte HSP72, which is in contrary to the data presented here (Table 3). Indeed in support of an association between OS and HSP responses antioxidant elevations are associated with leukocyte Hsp72 reductions (i.e., a lesser necessity for HSP72 transcription), thus OS necessitating HSP transcription could increase Hsp72 in leukocytes (Simar et al., 2012). Likewise antioxidant supplementation (500 IU.day⁻¹ RRR-a-tocopherol for 8 days) attenuates the change in leukocyte Hsp72 mRNA after exhaustive exercise (Niess et al., 2002). The extended 75 min hypoxic exposure (approximately the same 14.3% O₂ as used within the present study) utilized previously (Taylor et al., 2010a), would have induced a more substantial OS stimulus for the induction of Hsp72, compared to the 40 min exposure utilized within HPC_I in the present study. Given the very nature of IPC, and the lack of a change in circulating markers of OS, it is possible that the leukocytes were unable to experience the necessary fluctuations in OS that occurred locally within the skeletal muscle undergoing biopsy. As such it may be an experimental artifact associated with IPC alone that is responsible for the lack of Hsp72 mRNA responses in leukocytes rather than a mechanistic one. Utilizing a multi-gender sample and gene array analysis to assess Hsp72 (Konstantinov et al., 2004), with the former known to influence stress mediated changes in HSP (Paroo et al., 2002; Morton et al., 2009a) and the latter known to both over and underestimate gene transcript response (Feldman et al., 2002), may also explain some of the disparity in Hsp72 mRNA results.

Given that systemic OS was unchanged within and between all conditions (in line with previous data; Karg et al., 1997), it is likely that this absence of the potent OS stimuli (particularly ROS producing free-heme molecules) underpinned the lack of leukocyte Hsp32 mRNA response (Fehrenbach et al., 2003a) (Table 3). Although local OS markers were not obtained, absence of muscle Hsp32 mRNA change suggests only minimal (if any) disturbances in OS locally. To determine whether the 30 min occlusion was sufficiently stressful to induce intramuscular OS, future work should measure markers of this within the target tissue. To the authors' knowledge measurement of leukocyte and skeletal muscle Hsp32 mRNA within the presented in vivo occlusion paradigm is novel, with previous rodent model data lacking external validity to the presented TKR specific rationale (particularly time course). Rodent model data indicates that with five cycles of IPC (compared to the present study), muscle Hsp32 protein displays a 2-fold increase with glutathione remaining unchanged, albeit only assessed 48 h post IPC (Badhwar et al., 2004). The stable Hsp32 mRNA-values could be a fiber type specific response, with type I muscle fibers shown to readily express HSP32 in comparison to a blunted response in type II fibers (Vesely et al., 1999). The lateral head of the gastrocnemius consists of equal proportions of both fiber types (Edgerton et al., 1975). Therefore, the response observed in the present study may only be proportional to the percentage of type II fibers in the biopsied muscle. A lack of fiber type characterization within the gastrocnemius samples within the present study is an experimental limitation that should be addressed by future research designs.

HPC has been shown to blunt IR mediated tissue damage in animal models (Beguin et al., 2005; Berger et al., 2010), however, to the author's knowledge, no previous studies have performed HPC in humans prior to limb-tourniquet application and subsequent IR stress. HPC is thought to confer cellular protection through similar mechanisms to IPC, essentially hormesis from appropriate protein accumulation (the precise mechanism/stimuli for such accumulation are not robustly described in vivo). The present data provides provisional evidence that in vivo HPC prior to IR stress provides similar reductions in localized cellular stress as an established IPC model. Interestingly, IPC has preliminary in vivo evidence supporting its use to reduce post-operative pain following TKR surgery (Memtsoudis et al., 2010), although some equivocal evidence is also present (Memtsoudis et al., 2014). Practically, IPC involves close monitoring to ensure correct ischemic periods (typically within theater), yet the HPC_I protocol used here would not require this human resource as the inhalation of hypoxia is continuous, thus potentially allowing HPC to take place during surgical preparation (perhaps on the ward utilizing methods described earlier). It should be noted that previous work has utilized intermittent hypoxia as a preconditioning strategy, future experimental designs should make comparisons between continuous and hypoxic interventions as previously utilized (albeit with differing clinical applications) in animals (Beguin et al., 2005) and humans (Lyamina et al., 2011; Chacaroun et al., 2017), to determine which elicits the most desirable preconditioning responses. From a practical perspective an intermittent approach may be less practical to administer in clinical setting if using the current equipment i.e., it would require manually switching between hypoxic and normoxic air. However, further experimentation is required to demonstrate whether localized reductions in cellular stress (Hsp72 mRNA) from HPC_I (as also seen in IPC_I) can convey the same positive effects (reduced pain, accelerated wound healing, reduced length of hospital stay, as raised within the Introduction and Discussion sections) attributed to IPC, within externally valid models related to TKR like knee surgery which utilizes a tourniquet. Indeed, concerns could be raised to the safety of exposing elderly patients (population most likely to receive TKR surgery for example) to acute hypoxia. However, research has demonstrated that an elderly population with a high prevalence of cardiovascular disease tolerated hypobaric hypoxic remarkably well (Levine et al., 1997). Given that exercise and heating initiates increases in Hsp72 mRNA (Fehrenbach et al., 2001; Gibson et al., 2015a,b), in a temperature dependent manner (Gibson et al., 2016), passive heating [which has also been shown to increase Hsp72 mRNA; Horowitz et al., 1997; Maloyan et al., 1999] may also provide a viable preconditioning strategy that may be applied on the ward prior to surgery. In addition to the application presented (i.e., reperfusion) both HPC and IPC appear to initiate cellular responses (HSP increases) which are likely beneficial in across stimuli e.g., exercise, hypoxia, heat, though this is yet to be experimentally elucidated.

Although adequately powered it is important that the present data and postulations relative to the use of HPC are viewed relative to this sample size and preliminary nature of the experimental findings/design. It has been previously observed there can be high variability between participants with regards to basal and stress-mediated values of these measures, this variation based upon individual differences in physiological profile (Fehrenbach et al., 2000a,b, 2003a; Bruce et al., 2003) which require consideration relative to the presented data and discussion of the application of HPC vs. IPC prior to surgery. These reasons may explain the high basal Hsp72 mRNA content in one CON group participant in the present study. As such further work with populations displaying more diverse phenotypes are warranted. In additions to experimental limitations acknowledged throughout the discussion, given many antioxidants are procured naturally from the diet, all with varying half-lives, this could have influenced OS related outcome variables within the present study (Powers et al., 2010a). Although every effort was made to control this (standardized morning and afternoon meal), it is extremely challenging to control participants' diet over a long period of time, while ensuring continued participation. Therefore, the lack of change in OS markers could have partly been due to dietary variation.

In summary, it can be seen that a bout of HPC_I primed the HSP system thus bestowing localized cellular protection to tourniquet IR mediated stress induced via an ecologically valid TKR model. Furthermore, HPC_I provided similar levels of cellular protection to IPC_I, thus providing a novel framework for the use of HPC_I to convey cellular protection in light of a subsequent IR related stressor.

JB participated in the study conception, methodological optimization, data collection, sample and statistical analysis and manuscript drafting and revisions. OG participated in the statistical analysis and manuscript drafting and revisions. JT aided in data collection, sample and statistical analysis and manuscript revision. BC assisted with statistical analysis and manuscript revision. OP, JP, SG, CK, NG, FR, ZO, AS, and SN contributed to the study conception and optimization of muscle sample collection. LT participated in study conception, optimization of muscle sample collection, manuscript drafting and revision. All authors have read and approved the final manuscript.

The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.