Diabetes-related foot disease is a frequent complication of type 2 diabetes mellitus (T2DM), with up to 25 % of diabetic patients eventually developing foot ulceration [1–3]. The physical, emotional and financial costs for patients, their carers and the community are substantial. The utilisation of health care services and associated expenses are high in diabetic patients with ulcers [4]. In the US, it has been shown that the costs of foot ulceration caused by diabetes and related complications amount to between $9 and $13 billion per annum using conservative measures [1, 5]. Responsible for a large proportion of hospital admissions and health care costs in diabetes [2], such figures are likely to increase. The prevalence of T2DM is escalating worldwide [2, 6]. Although international data are scarce, an improved survival rate of diabetic patients has been reported in wealthier nations in particular [7, 8]. A potential consequence of this may be a higher prevalence of diabetic ulcers, especially as duration of diabetes is a major risk factor for the development of lower extremity ulceration [9].

Arguably the most feared complication of diabetic foot ulceration is the need for amputation. A UK study observed this occurred in 19 % of cases over a 5 year period in patients with a diabetic ulcer present for 2 weeks [10]. In a 2016 systematic review, amputation rates associated with diabetic ulceration ranged from 5 to 35 % [11]. Rates have improved in some higher income countries by 40 to 60 % [12], however data from countries of lower income and poorer access to health care (yet a higher prevalence of diabetes) are notably lacking [8, 12]. Further, some population groups even within wealthy nations are clearly at much higher risk of amputation [13–15]. In Australia, the Indigenous diabetic community is at particularly high risk. A 2000–2008 analysis revealed in some regions this population experienced up to 27 times higher risk of minor amputation and 38 times higher risk of major amputation [13]. The benefits of improved access to primary health foot care, in addition to multidisciplinary foot clinics, cannot be overemphasised. They are highlighted by the impressive 72 % reduction, following adjustment of variables, in lower extremity amputation rates identified in a 15 year longitudinal observational study of a West Australian city [16]. Despite this, from 2002 to 2012 there was a 30 % overall increase in diabetes-related amputations in well-resourced Australia [17], with one of the highest rates of amputation amongst developed nations [18]. The need to improve statistics worldwide for diabetic ulcer prevalence, complication rate and cost is undeniable, and new therapeutic options must be considered. This article will assess the potential for repeat remote ischaemic conditioning (RRIC) as a novel adjuvant treatment in diabetic lower extremity ulceration.

Utilising PubMed and Scopus databases in January 2016, publications relating to all forms of local and remote ischaemic conditioning were searched. Articles with an emphasis on neural mechanisms, diabetes or comorbidity in ischaemic conditioning, RIC or RRIC were identified in particular. The papers retrieved were examined, and further relevant references obtained by reviewing cited articles and cross-referencing. Alerts utilising Scopus and PubMed were established in these search areas over a 7 month period ending August 2016, enabling access to the most current research. Further searches were performed as required for other material covered in this article, including diabetic lower extremity ulceration, diabetic neuropathy and diabetic mobilopathy.

Sustained hyperglycaemia and associated abnormal metabolic pathways in T2DM lead to peripheral neuropathy, micro- and macro- vascular peripheral arterial disease, mechanical changes and subsequent foot trauma (e.g. Charcot neuroarthropathy), high plantar pressure, increased susceptibility to infection, skin changes resulting from autonomic neuropathy, increased pro-inflammatory cytokines, decreased neovascularisation and tissue regeneration, decreased neuropeptides involved in angiogenesis, and an altered extracellular matrix [2, 10, 19–22]. Diabetic neuropathy and bone marrow microangiopathy have been shown to prevent mobilisation of haematopoietic stem cells (stem cell “mobilopathy”), crucial in the response to ischaemia [23, 24], and can result in abnormal neurovascular regulation [25]. Decreased production of stromal cell-derived factor-1α in diabetic wounds impairs homing of endothelial progenitor cells (EPCs) to the ulcer region [26]. Diabetes also impairs the release of vascular endothelial growth factor (VEGF), normally upregulated in response to hypoxia, with resultant abnormal neovascularisation [27, 28]. All of these factors may contribute to the development of lower extremity ulceration or prevent healing. Poor foot self-care, age, and the presence of other microvascular complications are also important risk factors [29].

The standard principles of management of diabetic ulcers, ideally co-ordinated by a multidisciplinary team, are debridement (utilising various methods), infection control, pressure offloading, optimising glycaemic control, application of dressings and surgical intervention (revascularisation or orthopaedic), addressed in recent comprehensive reviews of these treatments [2, 22, 30–34]. Newer advanced or potential adjunctive therapies include hyperbaric oxygen therapy [2, 22, 30–34], bioengineered skin [2, 22, 30–32, 34], negative pressure wound therapy [2, 22, 30–34], growth factors [2, 22, 30–34], electrophysical therapy [2, 22, 30–32], and stem cells [22, 30, 33]. NorLeu3-angiotensin (1–7), substance P and extracellular matrix proteins have also been studied [reviewed in 22]. Many of these newer therapies are either expensive, difficult to implement, lack evidence for substantial benefit, or lack consensus on which method of administration is most effective for each therapy [31]. Even though extensive research has been undertaken investigating a wide range of treatment options and optimal management, chronic ulcers are still all too frequent. The average ulcer takes approximately 3 months to heal, and at least a half of diabetic ulcers recur within 3 years [3]. Further research into treatment options is therefore needed.

In a study by Jones et al. [90] using a mouse model, and in a study by Gross et al. [91] in dogs, the intriguing phenomenon of remote preconditioning of trauma was identified. The animals in both studies were subjected to a shallow wound being administered prior to inducing myocardial infarction, and this procedure was found to be cardioprotective, reducing infarct sizes by 80 and 59 % respectively. This protection could be blocked by administration of local anaesthesia at the wound site [90]. The application of topical capsaicin also had a similar effect in decreasing myocardial infarction size [90, 92]. These findings have been termed nociceptive-induced remote conditioning [66]. Redington et al. took the further step to demonstrate that electroacupuncture also was cardioprotective, with many similarities to RIPC and previous nociceptive conditioning studies [93].

Considering the impressive findings of the nociceptive-inducing cardioprotection studies, we propose that this may also be a substantial confounding factor in many translational RIPC studies, resulting in misleading conclusions. An example would be human studies in RIPC used prior to coronary artery bypass graft (CABG) surgery. The control groups do not receive the RIPC protocol in these trials, but both groups necessarily require a large central sternotomy wound. Applying the conclusions from the nociceptive-induced remote myocardial conditioning studies described above, one could argue that the central sternotomy wound in itself could provide conditioning, to which RIPC may not contribute significant additional benefit. The impact, location and timing of any co-administered local, regional and/or general anaesthesia could also potentially alter nociceptive-induced conditioning, varying with the protocol used. The same concepts may apply to any medical or surgical procedure or treatment involving trauma or nociception. Logically, it could therefore be anticipated that study groups receiving non-invasive treatment for relatively painless conditions would benefit most from RIC, with no co-conditioning from pain or trauma. The promising early use of RIC for ischaemic stroke is one such example [87].

To ignore the potential for nociceptive co-conditioning in a study may lead to incorrect conclusions regarding the response to RIPC. We suspect it would be important to evaluate previous and future study designs carefully, to establish whether pain or trauma could be a confounding factor due to co-conditioning. To the authors’ knowledge, the lowest threshold or dose necessary to induce nociceptive conditioning has not yet been established. If superficial skin wounds [91] and electroacupuncture [93] are sufficient, it remains to be seen whether other very minor procedures, such as intramuscular injections, cannulations and capillary blood sampling, can trigger nociceptive conditioning. Should small, minor stimuli be sufficient, it is plausible that the earliest recorded therapeutic use of nociceptive conditioning in humans was in fact 100BC (or earlier) in China, when acupuncture practice was first clearly documented [94].

Clinical studies that show benefits, such as the application of RIC in CABG surgery and elective and primary percutaneous coronary intervention (PCI), have not always been reproducible [36, 48, 95–99]. The most frequent positive findings have been seen in the use of RIC intervention in primary PCI [47]. Results from animal RIC studies are typically more promising than those in humans [98, 100]. Conflicting findings may be due to differences between the animal models studied and humans [48, 98, 100], or to limitations in study designs [48]. In human studies, it is also very likely to be due to co-morbidity [48, 98, 101], co-medication and differing anaesthetic protocols [98, 101–105], heterogeneity in study designs [48, 62], and other factors that cannot be predicted due to an incomplete understanding of the mechanisms and interaction of neural and humoral pathways in RIC. As a consequence, confounding factors are likely to be unwittingly present in studies, which could result in misleading conclusions.

As ischaemia plays an important role in the pathophysiology of many complications of T2DM, and the diabetic state appears particularly vulnerable to ischaemia–reperfusion injury [106], it seems intuitive that patients with T2DM would benefit from ischaemic conditioning. However, mixed and neutral findings have been reported in preclinical and clinical studies in diabetes [48, 107–110], with particularly poor results in preclinical postconditioning studies [48]. The majority of human studies in diabetes have assessed cardioprotection in acute myocardial infarction (AMI) following ischaemic conditioning, with disappointing results [98]. Conversely, evidence for the preservation of RIPC cardioprotection in human diabetics undergoing percutaneous coronary intervention was found in a 2014 meta-analysis [111].

The number of human ischaemic conditioning studies specifically in T2DM is, however, very small, particularly with the gold-standard endpoint of myocardial infarction size, and most of these do not assess remote ischaemic conditioning efficacy [98, 100, 107]. The first early window of protection in diabetes has also received considerably more study than the second window. In studies performed with favourable cardioprotective outcomes for ischaemic (pre- and post-) conditioning in diabetes, a common finding is that an increased ischaemic stimulus is often required [112–115].

A more recent method of ischaemic conditioning is repeat ischaemic conditioning, typically administered remotely. Although often termed repeat, regular or intermittent remote ischaemic preconditioning, intervention studies utilising this method are generally applied to a chronic condition (such as heart failure [188], endothelial dysfunction [189], stroke recurrence [43] or chronic wounds [44]) or following an acute ischaemic episode (such as in a lower limb [190], post-myocardial infarction [191] or cerebrovascular accident [43]). Depending on the context, the chronicity of pathological ischaemia in the cohort, and endpoints studied, this would more accurately represent combinations of pre, per and post-conditioning, reflected in the term repeat remote ischaemic conditioning (RRIC). Neural plasticity effects of RRIC have also been examined in an intriguing small study of healthy adult participants, demonstrating that RRIC aided motor learning and retention [192]. Although few in number and with a broad variety of endpoints, it would appear that both preclinical and clinical RRIC studies cited above have more consistently yielded significant beneficial results, particularly in comparison to clinical single-dose RIC and local ischaemic conditioning studies. Importantly, early evidence shows efficacy of RRIC in humans is not lost with advanced age. A study of 58 patients aged 80–95 years found twice daily RRIC for 180 days was effective in preventing stroke recurrence compared to sham control, without any safety concerns [193]. In contrast, RIC studies examining the impact of ageing, most assessing cardioprotection in animals, have revealed that efficacy predominantly declines with increasing age [48].

As we have demonstrated above, many factors potentially limit the benefits from ischaemic conditioning and RIC in diabetic patients. It is therefore unexpected that a recent study showed diabetic patients with refractory lower leg ulcers had significant improvement following repeat RIC [44]. In Shaked et al.’s double-blinded randomised study with sham control, 22 patients received 3 fortnightly cycles of RRIC to both arms, in addition to standard wound care. At the completion of the 6 week study, 9/22 (41 %) of the study group had complete healing of their ulcer compared to 0/12 of the control group [44]. Questionably, only one participant in each group was reported as having “neuropathy”, even though this was not listed in the exclusion criteria for the study. 46 % of the study group at commencement were documented as having local infection in the ulcer and 69 % in the control group. There were some further limitations with the study due to other important variables being unmatched between groups.

Despite the weaknesses and limitations of Shaked et al.'s small study, the wound healing rate of 41 % of refractory ulcers in just 6 weeks in a cohort of patients with very poorly controlled diabetes, after a mere 3 fortnightly remote ischaemic conditioning cycles, is not to be dismissed. Further, 14/22 patients in the intervention group reached 75 % healing at 6 weeks compared with 3/12 in the control group. 55 % of patients in the study group with ulcer infection at enrolment had completely healed wounds, compared to none in the control group with infection [44]. The findings could indicate the potential for RRIC providing significant benefit in this group, which is easily and cost-effectively administered. Should these results be reproducible in larger trials with a more satisfactorily matched control group, RRIC could revolutionize treatment of chronic diabetic foot ulcers. If this were the case, it may also point to the potential for RRIC to be effective in other chronic diabetic complications or causes of ulceration. Given the findings of improved endothelial function and increased NO, EPCs and VEGF levels in RRIC studies (albeit with predominantly healthy cohorts) discussed above, the effects of RRIC in diabetic nephropathy, neuropathy, coronary artery disease, peripheral arterial disease (PAD) and erectile dysfunction would be interesting to study. Endothelial progenitor cell therapy is being researched with some success as a potential treatment for stroke [199], another frequent complication of diabetes where RRIC may prove effective.

Shaked et al.’s study involved administration of remote conditioning. As local ischaemic preconditioning (IPC) appears to rely upon humoral and not neural mechanisms, this could mean local IPC may be relatively unaffected by the presence of DAN or DPSN. We propose repeat local IPC in the limb affected by the ulcer could possibly prove even more effective than remote ischaemic conditioning on this basis, assuming it is as safe and well tolerated as remote conditioning. Comparative studies between repeat remote and local conditioning interventions in diabetic patients with foot ulcers could be worth performing to test this theory. It is also interesting to consider Shaked et al.’s diabetic ulcer study had a control group where standard care did not involve significant pain or trauma, thus making co-conditioning from nociception less likely to confound the outcome.

Shaked et al.’s study excluded those with moderate-to-severe PAD, where ankle brachial index was <0.7. It is important to note that even in the absence of large vessel peripheral arterial disease, diabetic patients frequently have small vessel disease and tissue hypoxia in the lower limbs. Shaked et al. write that “peripheral neuropathy induces increased pressure points on the diabetic foot, which in turn lead to bouts of cutaneous IR injury” [44, p 194], from which RRIC appears likely to provide protection.

Diabetes suppresses angiogenesis in wound healing [200]. Injection of CD34+ cells into diabetic wounds has been found to improve diabetic ulcer wound healing and significantly enhanced wound revascularisation within 7 days [167]. In addition, serum CD34+/CD45-dim levels measured soon after the development of neuropathic diabetic ulcers have shown to be predictive of subsequent wound healing [201]. It is unfortunate EPCs/CD34+ cells and other factors such as VEGF were not measured in Shaked et al.’s study, but it is plausible that RRIC may have increased VEGF and/or NO, and thus EPCs (potentially overcoming mobilopathy), in the intervention group, consequently improving the impaired neovascularisation and wound healing. Both study cohorts were likely to have a high prevalence of CAN and (potentially undetected) DPSN. Neural mechanisms have not been identified nor studied in RRIC. Should the findings of Shaked et al. be replicated with more appropriately matched control and intervention groups, this may suggest that RRIC mechanisms are not as inhibited by DPSN as RIPC in diabetes. The issues regarding the beneficial effects of local ischaemic conditioning and RIC being decreased in the diabetic state, particularly relevant to the cardiomyocyte, may be less problematic with a different endpoint or target organ, such as wound healing. Similarly, it appears likely that the mechanisms in RRIC are significantly different to RIC, and coexisting neuropathy or co-medication may not result in such negative effects. Considering the evidence suggesting that RRIC leads to a state favouring neovascularisation, raised NO and EPCs, and improved microcirculatory function, one is tempted to postulate that RRIC may even slightly reverse the effects of diabetic neuropathy [202], or microangiopathy- and neuropathy-linked mobilopathy [23], hence simultaneously improving the response to each subsequently applied dose of RIC. Intermittent hypoxia is known to induce neuroplasticity and improve recovery of neurological function, including in motoneurons [192].

Raising levels of EPCs in diabetic patients is highly desirable; lower levels being associated with adverse cardiovascular outcomes [143, 203]. Current methods to increase EPCs include administration of granulocyte-colony stimulating factor, which has serious cardiovascular side effects and is often ineffective in diabetics [23, 169], or by culturing and re-administering EPCs [83]. RRIC would offer a safer, more cost-effective and easily-administered alternative, with automated RRIC devices already designed and manufactured for self-administration at home.

It is interesting to note that smoking [204] and hyperlipidaemia [205] are associated with poor bone marrow release of EPCs, in addition to a poorer response to RIPC [98, 206]. Statins [207], independent of effects on serum lipid levels, and sitagliptin [151, 159, 208] have cardioprotective properties in diabetes, and statins enhance cardioprotection in RIC [157, 206]. Studies have demonstrated that statins [209, 210] and sitagliptin [211] augment mobilisation of EPCs from bone marrow. Many of these facts may be interlinked, particularly in relation to the late phase of RIC and possibly RRIC. The effects of statins, smoking and anti-diabetic medication on RRIC are yet to be determined.

The potential use of ischaemic conditioning in PAD has received some attention. A crossover study by Delagarde et al. examined claudication distance in 20 patients with intermittent claudication after administering 3 intermittent cuff inflations and deflations [212]. A treadmill test was undertaken 5 min after completing both the RIPC and the control sham procedure, which were 7 days apart. Results did not show any alteration in claudication distance. It was surmised by Delagarde et al. that patients with PAD suffer from chronic ischaemia and reperfusion, and thus are already maximally conditioned. Consequently, they proposed that this may be why RIPC offered no additional benefit [212]. It is important to consider that performing a treadmill test 5 min after one cycle of RIPC would not allow for second window of protection (nor RRIC) effects to be assessed other than, to some extent, in those participants who performed the RIPC protocol first and then control 7 days later. In addition, only 25 % of the participants in the study were recorded as being diabetic, and presence of neuropathy was not documented. Karakoyun et al. recently undertook a key study of leg ischaemia, with a comparison between repeat local ischaemic conditioning, repeat remote ischaemic conditioning, no conditioning (ischaemic group) and sham control groups in non-diabetic rats [190]. The right iliac artery and vein were ligated to create critical limb ischaemia in all except the sham group. In the repeat local ischaemic conditioning group, the ischaemic stimulus was applied to the right leg and in the RRIC group, to the left leg. Both the RRIC and repeat local ischaemic conditioning group received 3 cycles of 10 min of ischaemia followed by 10 minutes of reperfusion daily until sacrificed at days 1,7,14 and 30. The ischaemic group and sham group received no further intervention. The findings of Karakoyun et al.’s study were that RRIC resulted in increased angiogenesis in the ischaemic right limb, although with no improvement in blood flow. The repeat local ischaemic conditioning group demonstrated both improved angiogenesis and blood flow. These positive findings were significant when compared to the ischaemic and sham control groups. EPCs were also measured, and were particularly higher in the RRIC group [190].

The optimum dose and limb/s used for RRIC are not known [56, 87, 213]; considerable variation in study protocols exists without losing beneficial effects [57]. It is unknown if hyperconditioning may be problematic [213]. No adverse effects from administration of either RIC [95] or RRIC, other than transient minor bruising, have been reported, even with frequent and extended administration of RRIC to two limbs concurrently in a cohort with known vascular disease [43].

Despite RIC and RRIC being reported as very safe and well tolerated in virtually all studies to date [87], a concern of ours, shared by Heyman et al. is the potential for worsening or increased risk of proliferative diabetic retinopathy [214]. Diabetic foot ulcers occur particularly in patients with longstanding and/or poorly controlled diabetes, which are in turn both highly significant risk factors for diabetic retinopathy [215]. VEGF is a key factor in diabetic retinopathy progression [216]. Kimura et al. found VEGF levels to be increased after one month of 6 times daily RRIC [197]. In contrast, Czeiger et al. did not identify raised VEGF levels, despite increased EPC numbers following RIPC [83]. Further, increased EPC levels are associated with a decreased incidence of diabetic complications, including retinopathy [142, 143, 170, 217]. We recommend that future studies of ischaemic conditioning in diabetes should assess VEGF and EPC levels, particularly if studying repeat ischaemic conditioning over long periods in diabetic patients. It is possible that the presence or high risk of diabetic retinopathy may be found to be a future contraindication to RRIC, although we are not aware of any evidence proving such a link. Further information from studies is required to establish the relationship between frequency, dose and form of ischaemic conditioning, VEGF and EPC levels, and any progression of diabetic retinopathy, to guide recommendations.

The mechanisms associated with different forms of ischaemic conditioning have been discussed in the context of T2DM and diabetic lower extremity ulceration. Neural pathways may frequently be affected in diabetic cohorts with known or undiagnosed peripheral sensory neuropathy, CAN and/or GAN. Ischaemic conditioning studies performed on diabetic patients have shown varying and mixed results. Conclusions drawn from earlier studies with diabetic patients should be re-evaluated carefully, particularly in research protocols where such neuropathies were not screened for, disclosed or matched between study and control groups. We feel detailed investigation is required to assess the impact of diabetic neuropathies upon RIPC and repeat RIC in particular. Although so far there have been no reported concerns with safety in studies, further research and consideration are required to assess the levels of VEGF, and all alterations of humoral effectors as they become known in RRIC, and establish that these are not harmful in the longer term. Repeat RIC may be an effective new method to consider in the treatment of diabetic lower extremity ulceration and peripheral arterial disease that is inexpensive, easy to administer in any location, and well tolerated.