Abstract
This study was performed to investigate the effects of high-voltage electrical burns (HEB) on the pulmonary microcirculation in rabbits. Total of 120 rabbits were randomly divided into control and HEB group using a random number table. HEB model was developed with a voltage regulator and experimental transformer. Laser Doppler perfusion imager was utilized to monitor and quantify the blood perfusion in pulmonary microcirculation. The microvascular morphologic changes of the lung were observed using light microscopy and transmission electron microscope (TEM). The lung wet/dry weight ratio and the PaO2 were determined. The values of blood perfusion in rabbit pulmonary microcirculation in the HEB group were decreased at 5 min, but increased at 1 h after burn (P < 0.01) and then decreased gradually. Light microscopy reveals microthrombus formation in pulmonary venules and bleeding in venous capillaries in HEB group. We found the number of microvilli in the capillary endothelial cells decreased, the rough endoplasmic reticulum expanded and severe degranulation occurred, the mitochondrial cristae fused or disappeared, and severe edema surrounded the capillary endothelial cells by TEM. The values of lung wet/dry weight ratio were higher and the PaO2 were lower than that of before burn group (P < 0.01). These results demonstrated that microcirculatory disorders play a major role in the development of progressive lung damage after high-voltage electrical burns.
Abbreviations
Control
Electrical burn groups
High-voltage electrical burns
Lisca Laser Doppler perfusion imager
Partial pressure of oxygen in arterial blood
Transmission electron microscope
Wet weight/dry weight ratio
Introduction
Electrical burns are among the most devastating of thermal injuries in our daily lives. High voltage electrical injuries can result in extensive deep tissue damage and are associated with multiple complications, long term morbidity and a high mortality rate. It was reported that electrical burn is still a major risk factor for amputations and the majority of amputations were caused by high-voltage electrical injury [16]. Extensive damage to the deeper soft-tissue (muscles, blood vessels and nerves, etc) may be present beneath the intact and unburned skin and sometimes they pose a major threat to life [7, 14]. Epidemiological studies in china demonstrated high voltage was directly correlated to severity clinical complications, and amputation. The percentage of myocardial impairment was 79.3% among patients who suffered with electrical current through heart tissue [15]. Along with the development of electric power, electrical injuries are becoming a growing concern of the world wide, especially in developing countries, and children are a major group susceptible to electrical injuries [13]. Electrical injuries induce serious tissue damage, need long hospital stay, and result in high rate of permanent disability and economic hardship for the afflicted families.
Over the recent decades, an improved understanding of the pathophysiology and the advances which had been made in burn management, have contributed to the dramatic rise in the survival and the reduced morbidity which result from high-voltage electrical injuries [7]. However, the influences of HEB on microcirculation of main organs in body remain unclear. We have reported previously that HEB cause abnormal rheological property of leukocytes and platelets and lead to microcirculation disturbance in heart, pancreas, bulbar conjunctiva and mesentery [19–23]. The lung is one of the main target organs for injury after HEB [4]. Patients present with progressively worsening dyspnea and anoxia as signs of progressive lung parenchyma damage [10]. Research shows that microcirculatory disorders play an important role in the initial and progressive development of lung damage [3]. In present study, we aimed to investigate the changes in the pulmonary microcirculation and other influencing factors after HEB, to explain the mechanisms of the microcirculatory disorder.
Methods
Main instruments and reagents
TC-30-20KVA voltage regulator and YDJ–10KVA experimental transformer (Electrical Appliance Co. Ltd., Wuhan, China); transmission electron microscope (Hitachi H600 TEM; Hitachi, Japan); 15 type Brad projection microscope system (BVPM; Bradford Research Institute, Chula Vista, CA, USA); laser Doppler perfusion imager (LISCA II PIM, Perimed, Jarfalla, Sweden); microcirculation image analysis system (Optical Instrument Factory, Xuzhou, China); blood gas analyzer (ITC Irma Trupoint, Diamond Diagnostics, Holliston, MA, USA); sodium sulfide powder (Yongda Chemical Reagents Development Center, Tianjin, China).
Experimental procedure
Using a repeated measures design, 120 healthy adult rabbits (purchased from the Experimental Animal Center of Hebei Medical University), weighing 2.0–2.6 kg, were divided into two groups using a random number table, consisting of a control group and an electrical injury group. We assigned 60 rabbits to each group and measurements were taken 15 min before HEB and 5 min, 1 h, 2 h, 4 h, and 8 h after HEB with 10 rabbits assessed at each phase. The day before the first test day, the rabbits underwent depilation of the left fore and right hind legs and chest with 80 g/L sodium sulfide. Rabbits were induced anesthesia and the electrical burn models were developed. Following the preparation of electrical burn model, we created a pulmonary microcirculation observation window, assessed the microcirculation, and collected lung specimens and other samples for measurement.
Preparation of animal model
High-voltage electric burn animal model
The animal models were prepared using the method we developed previously [23]. Briefly, rabbits were placed in the supine position and secured their limbs to the work station, and were induced anesthesia with an intraperitoneal injection of 10 g/L sodium pentobarbital at a dose of 40 mg/kg. The anesthetized animals received intermittent positive-pressure ventilation (IPPV) using a small animal breathing machine (tidal volume 8 mL/kg, frequency 35 per min). Two electrodes were fixed to the left fore and right hind legs of the animals. An electric shock was given to the rabbits using voltage regulator and experimental transformer with the following parameters: alternating current output voltage, 10 kV; current strength, 1.85 ± 0.25 A; frequency, 50 Hz; electrical injury time, 3 seconds. The burn area of the entrance and exit were the same (2×2 cm2) and the wound-depth reached bone, but hypovolemic shock did not occur in all animals. For the control group, all the procedures were identical with the electrical injury group, but no current was passed through the wire. The animals were placed on a homeothermic table to maintain the body temperature at 37°C. During the different experimental period from injury to creating the pulmonary microcirculation observation window, the animals were induced anesthesia continuely throughout the experiment by supplementing 10 g/L sodium pentobarbital at a halfdosage.
Pulmonary microcirculation observation window
To assess the effects of high-voltage electrical burns on the pulmonary microcirculation in rabbits, we firstly prepared a pulmonary microcirculation observation window in HEB animal model and utilized LDPI to monitor and quantify the blood perfusion in pulmonary microcirculation in lower lobe of left lung at each time point. We used iodophors to sterilize the neck and chest of the rabbits, and made a 2 cm horizontal incision in the ventral neck skin in the lower 1/3 of the neck. We then dissected and exposed the trachea; made a T-shaped tracheal incision; inserted and secured the endotracheal tube; connected the ventilator; and initiated artificial respiration at 30 breaths/min. Next, we made a 5 cm longitudinal incision parallel to and 2 cm away from the sternum between left ribs 2–6. We then dissected into the chest cavity with careful hemostasis to form a 3×5 cm2 elliptical observation window in the chest wall defect. This exposed the lungs, which allowed the placement of moist saline gauze over the lungs and subsequent assessment of the microcirculation.
Specimen collection and measurements
Quantification of the blood perfusion in pulmonary microcirculation
We followed Zhang et al’s method [20] to detect the blood perfusion in pulmonary microcirculation. The vertical distance between the laser sensor and the surface of the left lower lung lobe was 20 cm, and the laser scanning area was 1.5×1.5 cm2, with the relative number “V” as the unit ofmeasurement.
Morphology of pulmonary micrangium
We collected the lower lobe of left lung of two rabbits in each group at each time point. One part of the specimen was fixed in 4% formaldehyde. The other part of tissues was cut into fragments (1 mm×1 mm×1 mm), fixed in 2.5% glutaraldehyde made up in 0.1 M phosphate buffer (pH 7.2) for more than 2 h and postfixed in 1.0% osmium tetroxide (pH7.3∼7.4) for 2 h. For optical and transmission electron microscopy, a slice thickness of 5 μm and 50 nm was used, respectively, and then observed the microvascular structural changes.
Lung wet weight/dry weight (W/D) ratio
The right lungs were isolated from both groups of rabbits and the surface moisture was blotted using filter paper. We then recorded the wet weight of the lung tissue (W), placed the right lung tissue in an 80°C oven for 48 h for complete dehydration, and then measured the dry weight (D). The lung tissue W/D ratios were then calculated.
Blood gas analysis
Blood samples (1 ml) were collected from the left ventricle, and the PaO2 was measured using an automatic blood gas analyzer.
Statistical analysis
All data were presented as mean ± SD. The inter- or intra- group variation was analyzed by the repeated measures one-way ANOVA and the difference between each time point was assessed by Paired-sample t test using the statistical software, SPSS 16.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was used to indicate the existence of significant difference.
Results
Quantification of the blood perfusion in pulmonary microcirculation
As shown in Table 1 and Fig. 1, the values of blood perfusion in pulmonary microcirculation at each phase after HEB in the electrical burn group were significantly lower than that in the control group (P < 0.01) and that of baseline (P < 0.01), but it increased obviousely at 1 h after HEB compared with that at 5 min after HEB (P < 0.01), and declined gradually, thereafter. There was a significant difference in the pulmonary microcirculation blood perfusion between 1 h and 8 h after HEB (P < 0.01). Accordingly, the color of lung becomes pale from pink immediately after electrical shock and gradually recovers to pink from the external appearance.
Pulmonary microvascular morphology
Microstructural changes
The pulmonary histomorphology in the EB group showed no difference compared with the control group before HEB. Both groups had similar arteriole, venule, and capillary structural integrity, no red blood cell aggregation or leukocyte adhesion, and no bleeding from blood vessels (Fig. 2a). The venule lumens showed leukocyte adhesion 5 min after HEB (Fig. 2b) and red cell aggregation increased in the capillaries 1 h after HEB (Fig. 2c). Capillaries showed red cell aggregation and leukocyte adhesion 2 h after HEB (Fig. 2d). At 4 h after injury, the alveolar walls ruptured causing capillary bleeding into the alveolar space (Fig. 2e). Microthrombosis developed and extensive alveolar interstitial hemorrhage appeared in the venules 8 h after HEB (Fig. 2f).
Ultrastructural changes
The pulmonary histomorphology in the EB group did not differ from the control group before HEB. Capillaries were composed of one or two endothelial cells; the endothelial cell nuclei were clear; basal membranes had normal integrity; microvilli were visible; there was a greater number of pinocytosis vesicles; mitochondrial cristae and membranes had normal integrity; and Golgi complexes were normal (Fig. 3a). At 5 min after HEB, the number of microvilli in the capillary endothelial cells was decreased; mitochondrial membrane fusion was found and the cristae disappeared; severe rough endoplasmic reticulum degranulation occurred; and the number of pinocytosis vesicles decreased (Fig. 3b). At 1 h after HEB, the number of microvilli in capillary endothelial cells was noticeably decreased; the mitochondrial cristae disappeared; and severe rough endoplasmic reticulum degranulation occurred (Fig. 3c). At 2 h after HEB, capillary effusion increased; the microvilli number in capillary endothelial cell decreased; mitochondrial membrane fusion were observed and cristae disappeared; and severe rough endoplasmic reticulum degranulation occurred (Fig. 3d). At 4 h after HEB, the mitochondria in capillary endothelial cell showed marked swelling and the mitochondrial crests were irregularly arranged; parts of the crests disappeared; and rough endoplasmic reticulum expansion and severe degranulation occurred (Fig. 3e). At 8 h after HEB, marked peri-capillary endothelial cell edema occurred; the microvilli of capillary endothelial cell disappeared; severe cell fusion, mitochondrial swelling, disappearance of the cristae and membranes, and vacuolated, rough endoplasmic reticulum expansion with severe degranulation occurred; and the part of inside and outside layer nuclear membrane fused and became unclear (Fig. 3f).
Lung W/D ratios
As shown in Table 2, the lung weights (W/D ratios) in control group were not altered at each time point after sham stimulation. The EB group had significantly higher ratios than that in the control group at 2 h, 4 h and 8 h after HEB (P < 0.01), but no significant difference was found at 5 min and 1 h after HEB compared with 15 min brfore HEB (P > 0.05). There was a significant difference in the lung W/D ratios between 2 h and 8 h after HEB (P < 0.01).
Measurement of PaO2
From Table 3, the results showed that the PaO2 values at each time point after HEB in the EB group were significantly lower than that in the control group and that of baseline (P < 0.01). It decreased markedly at 5 min after HEB, increased at 1 h after HEB, then decreased continuously (P < 0.01). Although the values of PaO2 increased at 8 h post burn compared with that at 2 h after HEB (P < 0.01), but it still remained lower level than that of baseline (P < 0.01).
Discussion
The pulmonary alveolar epithelium is responsible for gas exchange and oxygen transport, whereby oxygen from the air sacs of the lung is exchanged with carbon dioxide in the blood through the alveolar microcirculation. The alveolar microcirculation has a special morphological structure and hemodynamic characteristics, which is necessary to ensure the timely release of carbon dioxide in the blood into the alveoli, and oxygen absorption into the bloodstream within the alveolus [5]. Abnormal structure and function of the alveolar microcirculation has a direct influence on gas exchange and can be life-threatening. We have reported that high voltage current through the body, causing microcirculatory disorders in the bulbar conjunctiva and heart, and leading to obstruction of material exchange between tissues and blood flow [21, 23]. Then the question is whether high voltage current damages alveolar microcirculation, thus reducing the blood gas exchange? In present study, a high-voltage electric burn animal model was used to investigate the effects of high voltage current on the pulmonary microcirculation perfusion, histomorphology, W/D ratios and partial pressure of oxygen in arterial blood. The present study will provide a experimental basis for further understanding the mechanism of progressive damage in pulmonary microcirculation induced by high-voltage electrical burn.
Often high-voltage burns were associated with multiple visceral injuries including cardiac arrest, extradural hematoma, hypoxic encephalopathy, pulmonary hemorrhage and and postelectrocution acute respiratory distress syndrome [12]. We have reported previously that HEB cause abnormal rheological property of leukocytes and platelets and lead to microcirculation disturbance in heart, pancreas, bulbar conjunctiva and mesentery [19–23]. To observe the changes of the pulmonary microcirculation induced by HVB, the LDPI, which not only reflects the number of moving blood cells, but also the speed of blood cells flowing in the measured tissues [6], were utilized to monitor and quantify the blood perfusion in pulmonary microcirculation. In this study, the results of blood perfusion in rabbit pulmonary microcirculation in the electrical burn group showed a decreasing (5 min after HEB) - increasing (1 h after HEB) - decreasing (2–8 h after HEB) pattern, which is similar to our previous reports for other organs or tissue microcirculation perfusion after HEB [19, 23]. Early decrease of pulmonary microcirculation perfusion induced by HEB are associated with the body’s stress response. When high voltage current pass through the body, the body enters an intense stress state. The current stimulates vascular smooth muscle excitation-contraction coupling, and together with stimulation of the sympathetic nervous system, large amounts of catecholamines are released immediately after electric shock [2, 17]. The release of catecholamines from injured tissues resulted in pulmonary vascular constriction and led to markedly decreased pulmonary microcirculation perfusion flow rate. At 1 h after HEB, the increase of blood perfusion in pulmonary microcirculation might result from “autotransfusion”. With the removing of high-voltage current, the vasodilation and blood redistribution play a role in the process of “autotransfusion”, enabling higher pulmonary microcirculation perfusion. So we think the early pulmonary microcirculation disturbances after HEB are associated with the body’s acute stress response.
Following the increase of pulmonary microcirculation perfusion at 1 h post burn, the values of blood perfusion in rabbit pulmonary microcirculation in electrical burn group were continuously decreasing during the 2–8 h after HEB. This was associated with the progressive damage of lung parenchyma [10]. From light microscope, we found leukocyte adhesion, red blood cell aggregation, and microthrombosis in alveolar capillary, as the main abnormal hemodynamic changes leading to decreased pulmonary microcirculation perfusion. We also saw alveolar capillary rupture after HEB, with blood cells in the lung interstitium and alveoli. Intraalveolar and interstitial hemorrhage caused thickening of alveolar walls, reduced alveolar capacity, and increased gas dispersion distance. This prevented alveolar gas exchange with the blood, causing pulmonary ventilation dysfunction. Scanning electron microscopy found that the high-voltage histopathological effects on alveolar capillary endothelial cell membranes, organelles, and cell connections, resulted in abnormal endothelial cell structure. It could be seen that the injury of capillary endothelial cells triggered the progressive damage of lung parenchyma. The lung W/D ratio increased at 2 h after HEB, and showed a rising trend, also reflecting a progressive rise in lung permeability. Regardless of the exact abnormality in lung tissue microflow, or microvascular structure and function damage, all changes can affect gas exchange, resulting in reduced respiratory function. The rabbit arterial blood PaO2 values declined significantly after HEB, further confirming that high-voltage electrical burns lead to lung dysfunction eventually.
Electric burn injury results in not only a local inflamatory response, but also a systemic inflamatory response. Following a major burn injury, heart rate and peripheral vascular resistance was increased significantly. This is due to the release of catecholamines from injured tissues, and the relative hypovolemia that occurs from fluid volume shifts [17]. It is reported that the injury of vascular endothelial cells triggers the increase of endothelin [9], thromboxane A2 [18], platelet activating factor and reactive oxygen species [11] during 2–8 h post burn. Our previous studies demonstrated that HEB caused the increase of platelet activating factor (PAF), thromboxane A2 (TXA2), prostacyclin (PGI2), P-selectin, E-selectin and L-selectin levels [20]. These increased vasoactive mediators resulted in pulmonary microvascular spasm. In addition, the blood flow was slowed and the pulmonary microcirculation blood perfusion was further decreased by the platelet adhesion, erythrocyte aggregation and deformation and microthrombosis in the venules [1].
It is notable that the levels of PaO2 levels were gradually rising from 1 hour post-burn, whereas the lung edema measured by W/D ratio was reaching maximum post-injury values during the same time period. As we all know, PaO2 is refers to the tension produced by oxygen molecules physically dissolved in arterial blood. It is affected by the gas exchange process between lungs and environment as well as the gas exchange between alveoli and pulmonary capillary blood. In this study, the levels of PaO2 decreased significantly at 1 h post burn due to the constriction of pulmonary vessels and bronchus, and are gradually rising from 1 hour post-burn in company with the vasodilation and blood redistribution. However, the lung edema measured by W/D ratio was a gradually process due to the damage of lung parenchyma, so the W/D ratio is reaching maximum post-injury values during the same time period.
Electro-thermal effect is one of the early factor that caused pulmonary microcirculation disorders after HEB. The breakdown of cell membrane by electroporation also play a role in microvascular endothelial cell damage [8], affecting the structure and function of the pulmonary capillaries. The present study explored the pulmonary microcirculation perfusion, microvascular histological morphology and respiratory function following HEB, confirming that HEB affects lung microcirculation, and suggesting that when treating HEB patients, it is important to prevent and control pulmonary microcirculation disorders.
Conclusion
This study aimed to investigate the changes in the pulmonary microcirculation and other influencing factors after HEB. Rabbit animal model of high-voltage electrical burn were used to examine the blood perfusion in pulmonary microcirculation and the microvascular morphologic changes. The lung wet/dry weight ratio (W/D) and the PaO2 also be detected. It is found that HEB decreased the blood perfusion in pulmonary microcirculation and arterial blood PaO2 values, but increased the lung tissue W/D ratios, affecting the structure and function of the pulmonary capillaries. These results indicated that the pulmonary microcirculation disorder plays an important role in the development of progressive injury after HEB. More future studies are needed to better understand the pathophysiology of HEB.
Conflict of interest statement
There are no any financial and personal relationships with other people or organizations while performing this research work and drafting the manuscript.
Footnotes
Acknowledgments
This project is funded by the Natural Science Fund of Hebei Province (C2011206080) and Hebei Provincial Construction Project of Science and Technology Conditions (139677131D), China.