An evaluation of dermal microcirculatory occlusion under repeated mechanical loads: Implication of lymphatic impairment in pressure ulcers

Pressure ulcers are caused by prolonged mechanical loads deforming the underlying soft tissues. However, the mechanical loads for microcirculatory occlusion are unknown. The present study was designed to characterize the simultaneous response of microvascular and lymphatic structures under repeated mechanical loading.

The sustained external pressure and shear forces experienced by immobile individuals in the lying and sitting postures can result in internal tissue deformations, causing two major damage mechanisms. 4 First, ischemic damage from the occlusion of blood and lymph vessels, which occurs at relatively low internal tissue strains. The resulting deficit of vital nutrients and accumulation of toxic metabolites lead to tissue damage, a process that can take several hours to develop. 5 Alternatively, direct cell damage resulting from high internal strains can occur within tens of minutes. Seminal research in the field identified an association between tissue damage and both the magnitude and duration of tissue deformations, 6 which reflects that tissue damage is possible at high deformations for short periods of loading and at lower pressures applied for prolonged periods. 7 This relationship inevitably depends on the health status of the individual and their respective tissue tolerance, which is influenced by age, comorbidities, and nutritional status. 8 To date, an international team of bioengineers have investigated the etiology of pressure ulcers creating a framework of understanding for the mechanisms, which lead to skin and subdermal tissue damage. 8 Such research encompasses a hierarchical approach, involving cell models, tissue-engineered constructs, and evaluations of specific subpopulations at risk of developing PUs. 9 Several studies have assessed the effects of large strains in subdermal tissues involving muscle and fat, by evaluating the biomechanical and physiological response of the tissues employing imaging and biomarker analysis. 10,11 However, this approach does not account for the damage mechanisms associated with the majority of PUs involving small tissue strains in superficial dermal tissues. Indeed, in this case, researchers have used biophysical and imaging techniques to assess the response of superficial skin tissues under representative loads. [12][13][14][15] These studies revealed that due to microvascular compromise, tissue ischemia can occur in both able-bodied and patient cohorts during periods of lying and sitting postures. In addition, specialized imaging techniques adopted in the host laboratory have enabled the quantification of dermal lymphatic vessel occlusion under applied loads. 12,13 With the advent of state-of-the-art imaging and biophysical modalities, it is now possible to simultaneously assess microvascular and lymphatic changes during periods of loading and subsequent recovery. Therefore, the present study aimed to determine thresholds of dermal vessel occlusion and recovery following representative loading using a customized biophysical and imaging experimental design protocol.

| Participants
Participants between the ages of 18 and 65 years were recruited via poster advertisement. Ethics Approval for the study was granted by the Local Institutional Committee at the University of Southampton (REC ID: 19378). Each participant was provided with an information sheet, which detailed any risks associated with the protocol.
Exclusion criteria included a history of skin damage or disease and contraindications for fluorophore injections.

| Methodology
The experiments were performed in an environmentally controlled laboratory, with an ambient temperature of 22 ± 1°C and relative humidity of 42 ± 6%. For each participant, the dominant arm was positioned on a foam-based structure at the level of the heart, designed to ensure that the arm remained as still as possible during testing ( Figure 1). This position ensured minimal contribution of passive mechanisms to lymphatic clearance and reduced potential movement artefacts during imaging.
F I G U R E 1 A, Test setup involving the dominant arm of participants resting on a foam surface. The indenter has a specially designed interface to house the transcutaneous gas electrode, and the lymphatic imaging was conducted proximal to the loading site. B, Image of delineated dermal lymphatic image using ICG injection and near-infrared imaging Indocyanine green (ICG) injections of 50 µL 0.05%w/v at a shallow intradermal depth were administrated to delineate dermal lymphatic vessels. Single injections were delivered into the two interdigital spaces between the thumb and the second finger by a registered practitioner (PW), constituting a total microdose of 0.05 mg. To encourage rapid uptake of ICG into lymphatic vessels, each participant was instructed to clench their fists 20 times following injection. Dermal lymphatic video sequences were captured using a Near-Infrared Fluoroscopy Lymphatic Imaging (NIRFLI) system, with a commercial camera (Fluobeam R 800) and associated software (Fluobeam v3.1.1, Fluoptics, France). The system incorporates an integrated laser (780 nm) and CCD sensor, with appropriate filters to isolate fluorescence of ICG (peak 830 nm). Frame acquisition was captured at frequencies of be-

| Loading protocol
To investigate the effects of repeated loading on lymphatic activity and transcutaneous gas tensions, two distinct loading regimes were employed. The first involved incremental repeating loading with pressures of 30, 60 and 90 mmHg applied to the test site with the 38-mm-diameter indenter ( Figure 1). Each load was maintained for a 20-minute period followed by 20 minutes of unloading (recovery phase). During these loading and unloading periods, video sequences with the NIRFLI were recorded. In addition, transcutaneous tissue gas tensions (T c PO 2 and T c PCO 2 ) were continuously monitored at 0.5 Hz, with changes in loading conditions identified at each interval.
On a separate day separated by a minimum of 48 hours, six participants returned to the laboratory for a further test session.
This involved three repeated loads each at a constant pressure of 30 mmHg applied to the identical test site. Identical time periods, each of 20 minutes, were employed for loading and unloading, with continuous monitoring of transcutaneous gas data and NIRFLI capture at each test condition. This was performed to assess the accumulative effects of repetitive loading, in comparison with that of the incremental pressures, previously described.

| Data and statistical analysis
Robust parameters of lymphatic function from the imaging sequences were identified using a customized software application (MATLAB, The MathWorks), described in previous publications. 13,14 To review briefly, the features were established using a droplet morphometry and velocimetry (DMV) tracking approach.
Here, image subtraction, binary conversion, and centroid tracking provided the basis to identify and measure each transient lymph packet event captured within the 20-minute video sequences.
Lymph packets are related to contractile propulsion events, associated with the lymph "pump", 13

| Participants
A total of twelve participants (7 male and 5 female) were recruited

| Associations between lymphatic occlusion and transcutaneous changes
The effects of the incremental loading regime on both the perfusion, in the form of the oxygen debt parameter, and the lymphatic activity for two participants are illustrated in Figure 6. There is a clear increase in oxygen debt with a corresponding reduction in lymphatic activity during the periods of incremental loading. At each unloading cycle, full recovery of perfusion and an associated increase in lymphatic activity were observed ( Figure 6A). These temporal trends were apparent in the vast majority of participants (n = 10). In a small number of participants (n = 2), the responses were less evident with minimal changes in both lymphatic activity and perfusion during the loading cycles ( Figure 6B).

| D ISCUSS I ON
This study represents for the first time a simultaneous evaluation of both dermal microvascular and lymphatic compromise as a direct result of mechanical loading of the skin. The combination of biophysical sensing and NIRFLI provided a unique opportunity to assess compromise to skin microcirculation, with a focus on two key mechanisms of pressure ulcer development, namely ischemia and impaired lymphatic drainage. 9 The results of this study on an able-bodied cohort revealed that even under relatively low pres- The results of the study have demonstrated that an applied pressure of 30 mmHg can compromise dermal lymphatic vessels. This pressure was smaller than the 60 mmHg used in a previous study, which resulted in impaired valve function and backflow events in some of the able-bodied participants. 13 In both studies, however, there was considerable variability in both the basal lymphatic activity levels and the individual responses to mechanical loading. In addition, each study revealed a full recovery of lymphatic activity during the unloaded recovery phases (Figures 2 and 4). The present study design includes the advantage of imaging during the loading period, which was not available during the previous study. 16 With reference to the seminal animal study, which used radioisotope tracers to monitor the clearance of deeper lymphatic vessels, critical uniaxial pressures of between 60 mmHg (8 kPa) and 75 mmHg (10 kPa) were reported. 17 However, direct comparisons between this and the present study must be treated with caution due to the differences in lymphatic anatomy and physiology between species under investigation. It is of note that lymphatic vessel function characterized in the present study may not be related to lymphatic capillary exchange occurring distally, although similar pressures were shown to cause distinct changes in interstitial flow, causing local halo patterns of ICG dispersion. 12 The changes in transcutaneous gas data revealed that moderate compromise was observed at 30 mmHg, representing a Category 2 response. Repeating this pressure resulted in a similar response. By contrast, when pressure was incrementally increased, changes in both T c PO 2 and T c PCO 2 were observed (Category 3), indicative of an ischemic state in over half the able-bodied cohort. Previous studies have shown that to achieve a 50% reduction in T c PO 2 from unloaded resting value in an able-bodied cohort, applied pressures ranged from 22 to 92 mmHg. 16 These findings highlight the individual nature of the tissue response, in terms of tolerance to mechanical-induced impairment of the microcirculation. This was also reported using biophysical measures to assess the physiological response to prolonged lying and sitting postures. 15,18,19 Here, a proportion of individuals, typically 15%-30% of the total, demonstrated ischemic responses during common postures, that is, supine, lateral lying and high sitting, with corresponding interface pressure values ranging between 30 and 90 mmHg. 9 This could be due to differences in soft tissue structure and geometry, underlying changes in physiology, such as preclinical changes in microcirculation or nutrition factors. 8 It is clear that the blood and lymphatic vessels demonstrate different sensitivities to mechanical-induced occlusion, which could be attributed to their unique anatomy and physiology. For example, the lumen of lymphatic vessels is wider and more irregular than in blood vessels, and under normal conditions, the lymphatic capillaries are maintained in a collapsed state. 20 By contrast, a relationship is known to exist between cutaneous mechanosensitivity and vasodilation, referred to as pressure-induced vasodilation (PIV). 21 When an external pressure is applied on the skin, the cutaneous microarteries vasodilate to prevent ischemia. The present study has shown that under relatively low pressures of 30 mmHg, microcirculation remained functional with only small changes in T c PO 2 , which often showed some recovery during loading indicative of PIV (Figures 3 and 5). However, at the higher loads of 60 and 90 mmHg, there appears to be greater compromise in a number of individuals, with elevated T c PCO 2 levels corresponding to complete microcirculation occlusion (Figure 3).
The changes in microcirculation values should be put into the context of normative capillary pressures, which range from 10.5 to 22.5 mmHg at the apex of the capillary loop. 22 Hence, our loading regimes were above that of the normative values. However, PIV by vascular smooth muscle tone appears to compensate for loads up to a threshold between 60 and 90 mmHg in most cases, a finding that has also been demonstrated in the microvascular response in animal models, for example, threshold of 70 mmHg in murine model. 23 Similar pressures have been reported to cause local ischemia and inflammation, 5,24 with an accumulation of metabolites associated with a change from aerobic to anaerobic cellular respiration. 5,25 The present study clearly examines the response of a relatively small cohort of young able-bodied participants, limiting its generalizability to cohorts of patients at risk of pressure ulcers, such as those with comorbidities including diabetes or the spinal cord injured.
Demographic factors such as body fat, blood pressure, and hydra- In clinical practice, carers and healthcare professionals advise vulnerable patients to regularly reposition to allow recovery of previously compromised tissues. 1 The present study has highlighted the importance of tissue recovery within an able-bodied cohort. However, individuals with microvascular compromise and reduced tissue tolerance may require an extended period to recover. 26 In addition, the pressure required to cause local ischemia and lymphatic occlusion appears to differ between individuals, even within an able-bodied cohort. This highlights the need for personalized care strategies, where patients are assessed and monitored depending on their individual tolerance to prolonged postures. Further studies are required on specific patient subpopulations who may have underlying comorbidities, which affect their microcirculation and the dermal tissue tolerance to applied loading.