Edited by: Ovidiu Constantin Baltatu, Anhembi Morumbi University, Brazil
Reviewed by: Igor B. Mekjavic, Jožef Stefan Institute, Slovenia; Jacek Kot, Gdańsk Medical University, Poland
*Correspondence: Weigang Xu
This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology
†These authors have contributed equally to this work.
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Skin lesions are visual clinical manifestations of decompression sickness (DCS). Comprehensive knowledge of skin lesions would give simple but strong clinical evidence to help diagnose DCS. The aim of this study was to systematically depict skin lesions and explore their pathophysiological basis in a swine DCS model. Thirteen Bama swine underwent simulated diving in a hyperbaric animal chamber with the profile of 40 msw-35 min exposure, followed by decompression in 11 min. After decompression, chronological changes in the appearance of skin lesions, skin ultrasound, temperature, tissue nitric oxide (NO) levels, and histopathology were studied. Meanwhile bubbles and central nervous system (CNS) function were monitored. All animals developed skin lesions and two died abruptly possibly due to cardiopulmonary failure. A staging approach was developed to divide the appearance into six consecutive stages, which could help diagnosing the progress of skin lesions. Bubbles were only seen in right but not left heart chambers. There were strong correlations between bubble load, lesion area, latency to lesion appearance and existence of cutaneous lesions (
Since decompression sickness (DCS) was first identified among caisson workers, “skin bends” have been recognized as a symptom of mild DCS (DCS Type I) (Mitchell et al.,
The pathogenesis of skin lesions still has not been clarified. It is widely accepted that rapid decompression generates sub-cutaneous bubbles which leads to a cascade of reactions and sometimes ultimately produces cutis marmorata (CM) (Hugon,
Comprehensive knowledge of skin lesions appearing with DCS could assist diagnosing DCS. Based on the swine DCS model of a prior experiment, the purpose of this study was to explore the time course of the appearance of skin lesions and the basic causes, toward increased understanding of the underlying pathogenesis.
Ethical clearance for this study was obtained from the Ethics Committee for Animal Experiment of the Second Military Medical University. The experiment protocol was carried out in accordance with internationally accepted humane standards (Russell and Burch,
Thirteen Bama swine were received by the animal husbandry facility of the university. They were males, castrated 2 weeks after they were born to avoid the influence of hormonal effects. The animals were allowed to acclimatize individually to general laboratory temperature of 23 ± 1°C and humidity of 50–65% and provided standard laboratory swine meal, in portions of 2% of their body weight daily and water was made available
The animals were singly utilized in the experiment. Four days before hyperbaric exposure, they were trained to walk on a treadmill once a day for 2 days. Two days before hyperbaric exposure, evoked potentials were measured and normal skin samples were collected. One day prior to hyperbaric exposure, a thorough skin examination was performed to find contusions, abrasions and lacerations and the hair of all chosen sites was trimmed. Structure and blood flow of heart were measured. One hour before the exposure, skin temperature, and cutaneous ultrasound (for structure and blood flow) were recorded at trimmed sites.
The swine were placed one at a time in a 1,000 L animal compression chamber (DCW150, Yangyuan, Shanghai, China) with view ports to observe the animal inside. The chamber was pressurized with air to 40 msw and maintained for 35 min before decompression. Compression was performed in 5 min which began at 5 msw/min to minimize the possible discomfort to the swine. Chamber oxygen (O2), carbon dioxide (CO2), temperature, and relative humidity were continuously monitored and maintained at 21%, <0.3%, 22–24°C, and 65–75%, respectively. Decompression was conducted in linear segments of 5 msw/min from 40 to 30 msw, 4 msw/min from 30 to 20 msw, 3.3 msw/min from 20 to 10 msw, and 2.9 msw/min from 10 to 0 msw.
After surfacing, the skin was thoroughly examined for up to 36 h. The observations were continuous in the first 6 h and periodic with 1 h in next 18 h. In the last 12, 2–3 h interval of observations was arranged. The appearance, evolution, locations, and dimensions of lesions were recorded on a swine-shape figure (Figure
Body skin map of a swine.
Based on our previous experimental results, several sites were selected as main observational regions including the four limbs, neck, chest, abdomen, waist, and back. Skin temperature was recorded using infrared thermometer (JXB182, Berrcom, China) with 0.1°C resolution. Skin thickness from the keratin cell layer to the dermis layer was measured using an Apogee 1000 ultrasound unit and a 14 MHz transducer (L8L38C, SIUI, Shantou, China) in color Doppler mode to image skin vessels. Both measurements were repeated during the evolution of lesions.
Heart structure was evaluated using transthoracic echocardiography (TTE) (P3f14C, SIUI, Shantou, China). A left ventricular long axis view was obtained, and the probe position was then adjusted to obtain an apical four chamber view. In the latter view an examination (incorporating observation of gross structure and use of color flow Doppler) was made for structural abnormalities in the atrial and ventricular septum. Although no formal bubble contrast studies were conducted, the real time repeat observations of venous and arterial bubbles after decompression served as an additional check for the presence of right to left shunt.
Bubbles in heart chambers were detected extrathoracically by the same machine and detector described above. Detection was repeated at 30, 60, 90 min, 2, 3, 4, and 6 h following surfacing, each lasting for 2 min. Left ventricular long axis view was found as the datum plane first, in which left atrium (LA), left ventricle (LV), and aorta (AO) can be seen clearly. Then the probe was adjusted to the aortic root short axis as the final view for detection. In this view, right ventricular outflow tract (RVOT), pulmonary artery (PA), and AO were presented. No detection was performed after 6 h due to the necessary oxygen breathing during anesthesia for evoked potential detection. Bubbles in ultrasound images were scored by Eftedal-Brubakk grading scale (Eftedal and Brubakk,
The two training sessions before hyperbaric exposure were defined as complete when each animal walked comfortably on a treadmill (YS-C600, KunYu, Henan, China) for 5 min at 1.6 km/h. At 2 and 6 h post-dive, motor function were tested on the same treadmill. The speed was gradually increased to 1.6 km/h at 0.4 km/h increments over 30 s, and the animal walked for 5 min at the highest speed, which earned a score of 5. If the animals could not stand or the endpoint on the treadmill was not achieved, the 5-point Tarlov score was adopted to grade motor function (Mahon et al.,
Evoked potential detection was performed 2 days prior to, and 6 h after, the hyperbaric exposure. Animals fasted for 12 h before anesthesia, which was induced by intramuscular injection of 0.05 mg/kg atropine and 0.1 ml/kg Sumianxin and maintained with inhaled isoflurane (6%) via endotracheal intubation and an anesthetic machine (WATO EX-20 Vet, Mindray, Shenzhen, China). Sensory evoked potential (SEP) was measured using electromyography and evoked potential instruments (NDI-094, Haishen, Shanghai, China). Stimulating electrodes were placed into the right and left ankles to stimulate tibial nerves and electroneurographic signals were collected from first lumbar vertebra and head by receiving electrodes, which were defined as spinal somatosensory evoked potential (SSEP) and cortical somatosensory evoked potential (CSEP), respectively.
After each evoked potential detection with the animal still under anesthesia, skin samples were collected from selected areas and the center of stage IV lesions (described in Results)/adjacent non-affected sites. Biopsy sites were cleaned thoroughly with povidone iodine and saline. Full thickness skin biopsies were obtained, 3 × 1 cm spindle shaped. Excess fat from the biopsy was removed and 0.5 g of skin tissue separated from each specimen was stored at −80°C prior to NO determination using a total nitric oxide assay kit (Beyotime, Shanghai, China). The rest of the skin was fixed in 10% formaldehyde solution for 48 h before being mounted in paraffin, longitudinally sectioned, stained with Haematoxylin and Eosin (HE), and observed and photographed using a microscope (Eclipse55i, Nikon, Japan).
All data are presented as mean ±
Eleven swine survived the experiments, and all developed skin lesions. The lesions showed obvious temporal patterns of change. The erythema, homogeneous purple-red color of the lesion gradually progressed into patterns of marbling or scattered lesions and completely disappeared without leaving any residual macroscopic skin changes. We therefore proposed a graduated system for DCS skin lesions. Not all lesions displayed each feature in the sequence, but typical lesion development can be described using stages shown in the caption of Figure
Typical appearance of skin lesions in a swine DCS model. Swine were subjected to a simulated air dive to 40 m for 35 min with a decompression in 11 min, skin appearance were observed and recorded. The hair had been removed prior to the experiment.
Each swine had at least two lesions located in different sites along the body. All stages appeared in most of the lesions. In each lesion, stage III was the most serious appearance and the area was measured. 10–20% lesions in each animal were mild, the lesions faded quickly within 1 h after Stage I and II. Hence, the mild lesions did not show Stage III manifestation and were not counted in the total lesion area. Occurrence of Stage III lesions in the body sites are listed in Table
Occurrence of skin lesions in the body sites in a swine DCS model.
Hind limb | 10/11 |
Waist and back | 8/11 |
Abdomen | 8/11 |
Chest | 7/11 |
Fore limb | 6/11 |
Neck | 5/11 |
Skin lesions appeared quickly following surfacing (13 ± 12 min) and almost simultaneously in different body sites. The speed of developing into stages varied among animals and lesions. Stage I and II usually existed for a relatively short time only, while stages III–VI took longer to evolve. Some lesions, especially in dorsal skin, remained at stage IV even after 24 h from surfacing. The mean duration for each stage is shown in Figure
Skin lesion durations of each stage in a swine DCS model. Eleven swine following a simulated air dive to 40 m for 35 min with 11 min decompression. A total 44 lesions were observed. Durations of each stage are shown
The latency to stage III lesion can be taken as a parameter to reflect lesion severity as it correlates well with lesion area. As shown in Figure
From the apical four chamber view, all swine had complete structure of ventricular septal and interatrial septum, and no patent foramen ovale and no abnormal blood flow were observed.
Figure
Bubble formation and correlation with cutaneous lesions in a swine DCS model. Ultrasound bubble detection was performed on 11 swine after a simulated dive to 40 m-35 min with a rapid decompression. An aortic root short axis view was chosen, which shows aorta (AO), right ventricular outflow tract (RVOT), pulmonary artery (PA) and right PA (RPA)
There were significant correlations between bubble load and maximum lesion area ratio, latency to stage III lesion or total duration of stage III and IV. As shown in Figures
Grading of skin lesions according to lesion area (
<30% | 4 | 56.3 ± 12.5 | 2.3 ± 0.9 |
30%~50% | 4 | 31.3 ± 13.2 | 2.9 ± 0.5 |
>50% | 3 | 15.0 ± 5.0 | 3.6 ± 0.5 |
Swine skin thickness from the squamous keratin layer to the dermis varied across body areas including the thinnest abdomen (1.4 ± 0.1 mm) and thickest waist and back (3.7 ± 0.2 mm). Figure
Lesion skin thickness in a swine DCS model. Thickness of skin was determined from the squamous keratin layer to the dermis
Generally, vessels could not be detected by color Doppler scanning in skin layer and were also hardly to be found in subcutaneous tissue. In lesion skin, especially in Stage III, in the lateral neck subcutaneous tissue, vessel images were not grossly apparent (Figure
Subcutaneous blood flow in a swine DCS model. The swine was subjected to a simulated air dive to 40 m for 35 min with an 11 min decompression. Skin blood flow was detected by color Doppler scanning on normal skin in the lateral neck before the dive and repeated on a lesion in the same location. Only two small vessels in subcutaneous tissue were found in normal skin
Surface temperature was slightly different in different body areas. However, changes showed no statistically significant variation during the six stages on all lesion surfaces (
As shown in Figure
Skin tissue NO levels in a swine DCS model. Tissues were sampled in the lesion and non-affected skin from 11 swine after a simulated air dive to 40 m for 35 min with an 11 min decompression. Normal skin samples were collected pre-dive. NO was detected by ELISA. Values are presented as mean ±
Congestion was the most common finding in skin lesions, with red blood cells (RBCs) were observed clogging in the dermal capillaries. Other changes included dilatation, hemorrhage, and neutrophil infiltrates (Figures not present). In non-affected skin from the decompressed swine, only dilatation was occasionally observed. There was no abnormal findings in normal control skin. A summary of histologic findings is shown in Table
Summary of histologic findings.
Congestion | 11/11 | 0/11 | 0/11 |
Dilation | 9/11 | 3/11 | 0/11 |
Neutrophil infiltrates | 5/11 | 0/11 | 0/11 |
Hemorrhage | 3/11 | 0/11 | 0/11 |
Assessment of motor function at 2 and 6 h after decompression yielded a score of 5 in all survivors. Pre-dive and post-dive SSEP and CSEP were compared, with no significant changes (
Sensory evoked potential in a swine DCS model. Sensory evoked potential (SEP) was tested pre-dive and 6 h post-dive in 11 swine following a simulated air dive to 40 m for 35 min with an 11 min decompression. First lumbar vertebra and head were selected to collect electroneurographic signals from right and left ankles, which were defined as spinal somatosensory evoked potential (SSEP, channels 1 and 3) and cortical somatosensory evoked potential (CSEP, channels 2 and 4)
Swine have a number of anatomical and physiological similarities with humans that make them potentially a better model for procedures and studies than other large animal species (Smith and Swindle,
DCS is caused by bubbles that are formed as a result of reduction in surrounding pressure (Vann et al.,
The pathogenesis of cutaneous DCS remains controversial and three main perspectives exist: autochthonous bubbles, arterial bubbles embolizing the skin, and arterial bubbles embolizing the brain causing the skin symptoms via autonomic nervous system (Lambertsen,
It has long been assumed that bubble formation in the skin tissue or skin capillaries may be the initiating mechanism but it is still in the absence of any substantial evidence (Buttolph et al.,
The observed increase in NO in lesion tissue in this study may contribute to the vasodilatation in subcutaneous tissue, which was possibly the consequence of local bubbles diffused into capillaries, stimulating the endothelium (Palmer et al.,
Bubbles will expand or coalesce in the skin capillaries and may obstruct blood flow before distributing into the venous system (Lambertsen,
Arterial bubbles embolizing in the subcutaneous capillary plexus was also proposed as the possible etiology of cutis marmorata (Wilmshurst et al.,
One theory on the origin of skin DCS suggests that marbling is the result of skin blood vessel dilation and constriction abnormally regulated by the autonomic nervous system, which is driven by neurons located in the rostral ventromedial medulla of the brainstem close to the formatio reticularis (Germonpre et al.,
However, the lack of evidence in this DCS model still cannot rule out the arterial bubbles or CNS injury hypothesis. Small amount of bubbles could most possibly hide the detection, and CNS dysfunction indeed can cause cutaneous signs (Blogg et al.,
The evolution of skin symptoms in previous swine studies remains to be fully described. In this study six stages of skin lesions, generalized from the appearance and the evolution of lesions, are described for the first time, which could help clearly identify the progress of the symptoms. Initially symptoms showed as stageI and II, some lasted up to 1 h, but some evolved to stage III within minutes. The lesions culminated in stage III, the most serious and largest symptom, and lasted for around 100 min. Then symptoms subsided slowly for several hours from Stage IV to VI until the lesions disappeared. Though the staging system is for swine DCS model, it may still serve to some extent as a reference for divers.
To limit the number of animals involved, the exposure profile was selected so that mortality was anticipated to be as low as possible and yet skin symptoms as severe as possible. Milder symptoms might not be induced by the current profile; hence, the grading of the manifestation may not fit all kinds of DCS skin lesions.
Although bubble scores were highest at the first detection and decreased gradually thereafter. The peak of bubbles most possibly appeared before the first detection. In future studies detection should start earlier and finish later. However, in this study earlier start of bubble detection would have stimulated the animal and increased the risk of mortality substantially (experience from our previous experiment) using the current chamber system. A new chamber under construction with a faster delivery system designed specifically for large animals will permit detection within 5 min following decompression. Bubble detection was not continued beyond 6 h due to the necessary oxygen breathing during anesthesia for SEP determination at 6 h following decompression, right after the last bubble detection. Motor evoked potential evaluation was not performed in this study to avoid invasive interventions to the skull or cortex, which may affect the other experiment procedures. Based on a series of inflammatory reactions caused by local bubble stimuli, we speculated that temperature might change in lesion skin, but we observed no positive confirmation of this hypothesis. Inflammatory reactions caused by local bubbles were slighter than common infection or trauma, for which fever is not usually significant.
In conclusion, this is the first study to systematically elucidate the clinical appearance and give strong evidence to support autochthonous bubbles as the etiology of skin lesions in a swine DCS model. Swine offer a good, or possibly even the best, animal model for the study of cutaneous DCS. Skin symptoms are easier to induce in swine than in human beings, together with the correlation with bubbles, may help assess the general decompression stress. Although the possibility cannot be ruled out, CNS dysfunction and arterial bubbles involvement was not observed.
WX, LQ, and DA designed and LQ, DA, HY, YW, and QZ conducted the experiments. All authors listed contributed to data analyses and interpretation of the results. WX, LQ, and DA wrote the manuscript. WX, LQ, and DA prepared all the figures and the table. LQ and DA contributed equally to this work. All authors reviewed the manuscript and agreed to be accountable for the content of the work.
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.
The authors thank Jingtao Song and Cheng Xu for their excellent technical assistance during the experiments and Dr. Peter Buzzacott for language polishing. The opinions and assertions contained herein belong to the authors and are not to be construed as official or reflecting the views of the Second Military Medical University.