For a long time, it has been recognized that trauma induces a general increase in microvascular permeability, which may cause hypovolemia and shock even in the absence of hemorrhage (1-4). In clinical practice, this condition necessitates treatment with intravenous administration of fluids to restore blood volume and to improve tissue perfusion. However, administration of intravenous fluids has side effects such as edema formation, which can increase oxygen diffusion distances and increase tissue pressure. It is likely that treatments aimed at reducing the trauma-induced loss of fluid from the circulation and/or optimization of fluid therapy can improve outcome. For this purpose, there is a need for experimental models in which a trauma-induced nonhemorrhagic hypovolemia can be produced in a standardized and reproducible fashion.
To the best of our knowledge, there is only 1 model published in which changes in plasma volume (PV) and permeability were analyzed after a nonhemorrhagic trauma (5). However, the trauma in that study was unspecific and included several intra-abdominal organs, and concerns about the standardization of the trauma can be raised. Furthermore, no attempt was made to evaluate if the trauma induced a local or general increase in permeability. Although a clinical trauma often involves several organs, there is an advantage of limiting the experimental trauma to a single organ for best reproducibility to facilitate the interpretation of the observed hemodynamic alterations, and it allows a standardized injury. Skeletal muscle tissue is a suitable organ to study in this respect because it is the largest internal organ of the body and frequently suffers traumatic injuries.
The aim of the present study was to establish a standardized and reproducible nonhemorrhagic trauma model for the analysis of trauma-induced changes in permeability and PV and for the investigation of underlying mechanisms and potential treatment strategies. For this purpose, the abdominal rectus muscle of the rat was traumatized in a standardized fashion, and the effects of the trauma on PV and transcapillary escape rate (TER) of albumin were evaluated. The plasma concentrations of various cytokines were measured to evaluate if the trauma induced a systemic inflammatory response.
Material and anesthesia
The study was approved by the Ethics Committee for Animal Research at Lund University, Sweden (application no. M8-08), and the animals were treated in accordance with the guidelines of the National Institutes of Health for Care and Use of Laboratory animals. Adult male Sprague-Dawley rats (n = 60) weighing 355 ± 14 g (mean ± SD) were used. Anesthesia was induced by placing the animals in a covered glass container with a continuous supply of 5% isoflurane in air (Forene, Abbot Stockholm, Sweden). After induction, the animals were removed from the container, and anesthesia was maintained with 1.6% to 1.8% isoflurane in air delivered via a mask. After tracheostomy, the animals were connected to a ventilator (Ugo Basile; Biological Research Apparatus, Comerio, Italy) and ventilated in a volume-controlled mode using a positive end-expiratory pressure of 4 cm water. Body temperature, measured rectally, was kept at 37.1 to 37.3°C via a feedback-controlled heating pad. End-tidal PCO2 was monitored continuously and kept between 4.8 and 5.5 kPa (Capstar-100, CWE, Ardmore, Pa). Left femoral artery was cannulated for measurement of MAP and to obtain blood samples for measurement of arterial blood gases, electrolytes, and hematocrit ([Hct] I-STAT; Abbot, Abbot Park, Ill).
The left femoral vein was cannulated and used for injections and kept open with a continuous saline infusion of 0.5 μL/min. The internal jugular vein was cannulated in some animals for measurement of central venous pressure. Urine was collected in a glass vial from the end of the preparation until the end of the experiment. After the experiment, animals were killed with an intravenous injection of potassium chloride.
After a longitudinal midline skin incision over the abdominal wall with diathermia, a laparotomy was performed by an incision along the linea alba. This was followed by the standardized trauma of the rectus muscle at 12 different locations, 6 on each side of the midline, extending approximately 4 cm laterally using a medium-sized anatomic forceps (Fig. 1). The trauma was induced by closing the forceps for 3 to 5 s, 3 times at each of the 12 locations. To reduce evaporation, we kept the time of exposure to the atmosphere of the wound area at a minimum. For this reason, the trauma was performed in 2 steps. First, half of the laparotomy (approximately 4 cm in length) was performed, and the trauma was induced on the corresponding part of the muscle, after which the abdominal opening was closed with surgical clips. After that, the other half of the trauma was performed in the same way. The abdomen was kept open to the atmosphere for 5 min at the most. Careful inspection revealed no signs of hemorrhage after trauma in any of the animals. The skin was closed with clips. Sham trauma animals were not subjected to any surgical trauma but only to anesthesia, cannulation, and tracheostomy.
In additional experiments, all surgical procedures except the muscle trauma itself were performed in an attempt to separate the effects of the skeletal muscle trauma from those of the rest of the surgical procedures (skin dissection and laparotomy).
Measurement of PV
Plasma volume was determined by measurement of the increase in radioactivity per milliliter of plasma after an intravenous injection of a known amount of activity of human 125I-albumin (GE Health Care, Bio-Science, Kjeller, Norway). The increase in radioactivity was calculated by subtracting the activity in a blood sample taken just before the injection from that taken 5 min after the injection. Through this technique, the PV measurement was independent of the remaining radioactivity from previous radioactive injections. To determine the exact dose injected, we subtracted the remaining radioactivity in the emptied vial, syringe, and needle from the total radioactivity in the prepared dose. As discussed previously, this is a reliable technique, giving reproducible results, and possible sources of error are small (6, 7).
Measurement of skeletal muscle water content
To evaluate to what extent the amount of a PV loss can be referred to edema in the traumatized muscle, we measured and compared with total PV reduction the increase in water content of the muscle after the trauma. Muscle edema in the rectus muscle was estimated by determination of water content 3 h after the trauma or the sham procedure. For this purpose, the traumatized muscle (measuring approximately 6.5 × 4.0 cm on each side) and the corresponding part of the muscle in the sham animals was resected, weighted, and put in an oven at a temperature of 100°C for 1 week. The water content in the tissue was measured with a wet-dry tissue technique as follows: [(wet tissue weight − dry tissue weight) / wet tissue weight] × 100. By subtracting the increase in tissue water content (mL/kg body weight) in the traumatized muscle from the PV loss (mL/kg body weight) induced by the muscle trauma, the fluid loss in noninjured parts of the body can be calculated (for details of the calculation, see Results).
Measurement of TER for albumin
Transcapillary escape rate for albumin after trauma was determined by measurement of the reduction in the radioactivity per time unit after injection of a bolus dose of 125I-albumin. For this purpose, blood samples of 250 μL were taken in heparinized vials at 5, 15, 30, 45, and 60 min after the 125I-albumin injection. After centrifugation at 8000 rpm, radioactivity in a PV of 100 μL was measured with a gamma counter (Wizard 1480; LKB-Wallace, Turku, Finland). The amount of unbound radioactivity in the injected 125I-albumin in the PV and TER groups was measured regularly after precipitation with trichloroacetic acid and was found to be less than 1% in all cases.
The plasma concentrations of IFN-γ, IL-4, IL-6, IL-10, and TNF-α were measured from arterial blood samples collected 1 and 3 h after the trauma or the sham procedure. Cytokine levels were determined with a flow cytometer using cytometric bead array kits specific for respective cytokines according to the instructions provided by manufacturer (BD Biosciences, Franklin Lakes, NJ).
The study consisted of 3 main groups: a PV group, a TER group, and a cytokine group. The experimental protocols for these groups are illustrated in Figure 2. For all 3 groups, preparation including anesthesia, cannulation, and tracheostomy lasted for about 40 min. The animals were undisturbed during the next 15 min to assure hemodynamic stability. This was followed by the experimental trauma or sham trauma, lasting for about 25 min from start of the surgical preparation until the skin was closed (see above).
In the PV group, the PV was measured before the trauma and 3 h after the trauma, and the data were compared with those from sham animals (n = 7 per group). The water content of the rectus muscle in the trauma and the sham group was determined at the end of the experiment as described earlier. On a post hoc basis, experiments were performed to investigate to what extent the skin preparation and the laparotomy per se contributed to the observed PV decrease after trauma. These experiments followed the same protocol, except that no rectus muscle trauma was performed (n = 4 per group).
In the TER group, the 125I-albumin was injected 30 min after completion of the trauma, followed by the TER measurement lasting for 1 h as described earlier. The TER data were compared with corresponding data from sham animals (n = 7 per group). The TER measurement started 30 min after the trauma because it takes some time for the increase in capillary permeability to develop, and we wanted to make the TER measurement in the middle of the experimental period. Change in central venous pressure was measured via the right internal jugular vein to evaluate if the change in venous pressure could affect the TER measurement via a change in hydrostatic capillary pressure (see Discussion). On a post hoc basis, experiments were performed to investigate to what extent the skin preparation and the laparotomy per se contributed to the observed increase in TER. These experiments followed the same protocol, except that no rectus muscle trauma was performed (n = 4 per group).
The cytokine concentrations were measured in the cytokine group at 1 and 3 h after the trauma to be compared with the corresponding values in the sham animals (n = 8 per group).
The results are presented as mean ± SD. Statistical comparisons between 2 groups were performed with the Student t test, when the data were normally distributed, and with the Mann-Whitney rank sum test, when the normality test failed. Physiological data were analyzed with the Kruskal-Wallis test followed by the Dunn multiple comparison test. P values below 0.05 were considered significant. Sigma Plot 11 software was used for the analysis.
Data for sodium (Na+) and potassium (K+) concentrations, Hct, pH, PaCO2, PaO2, and base excess (BE) for the TER and the PV groups are summarized in Table 1. There were no differences in these parameters at baseline between the groups. There was a trend toward an increase in Hct 1.5 h after trauma, which reached statistical significance 3 h after trauma. Sodium concentration was unchanged during the experiments in the sham group and was slightly decreased 3 h after trauma. Potassium increased in the traumatized animals, whereas no change could be detected in the sham group animals. Base excess and pH decreased in the traumatized animals, and a decrease in BE compared with baseline could be detected 3 h after the sham procedure in the sham group animals. There was no difference in PaO2 and PaCO2 during the experiments between the trauma and the sham groups. In the trauma and the sham groups, the urine production was 0.9 ± 0.5 and 1.0 ± 0.2 mL/kg per hour, respectively, and did not differ between the groups.
A summary of the blood pressure values for the PV group and the TER group at baseline, just after completion of the trauma, 30, 60, 90, 120, and 180 min after the trauma is presented in Table 2. The mean values for the PV group and the TER group are presented together up to 90 min after the trauma. Only the PV group values are presented after 90 min because the TER experiment was terminated at that point of time. There was a significant reduction in blood pressure at the end of the experiments compared with baseline in both groups (P < 0.05), but blood pressure did not differ significantly between the trauma and the sham groups.
In the traumatized animals, PV decreased from 41.8 ± 0.6 mL/kg at baseline to 31.4 ± 2.2 mL/kg at the end of the experiments (n = 7; P < 0.05). In the sham animals, PV was 41.4 ± 2.6 mL/kg at baseline and 42.0 ± 2.4 mL/kg at the end of the experiment (n = 7; Fig. 3). The PVs in the animals exposed only to skin incision and laparotomy were 41.0 ± 2.7 mL/kg at baseline and 39.2 ± 3.3 mL/kg at the end of the experiment (n = 4).
Skeletal muscle edema
The relative water content in the traumatized muscle was 79.5% ± 0.6% as compared with 73.4% ± 2.1% in the sham animals, giving a difference of about 6% between the groups (P < 0.01; n = 7 per group). With a mean weight of the analyzed rectus muscle per rat of 9.5 ± 0.9 g for nontraumatized tissue, the 6% correspond to a mean increase in water content of the traumatized muscle of 0.6 ± 0.1 mL. This corresponds to a fluid loss from the intravascular compartment of approximately 1.6 mL/kg body weight.
TER for albumin
In the traumatized rats, TER was 18.5% ± 2.3% compared with 13.9% ± 2.5% per hour in the sham group (n = 7 per group; P < 0.05; Fig. 4). A regression line with an R2 value above 0.9 for all measurements confirmed that there was a good agreement between the measured values and the slope of the curve. The corresponding TER in the experiments exposed only to skin incision and laparotomy was 14.2% ± 3.1% per hour (n = 4).
For the purpose of evaluating a possible effect of venous pressure for the TER results, central venous pressure in the trauma and the sham animals was measured in the TER group. Mean central venous pressure was 2.8 ± 1.0 and 2.6 ± 0.7 mmHg before start of trauma and sham trauma, respectively; 2.3 ± 0.3 and 2.6 ± 0.2 mmHg 30 min after trauma and sham trauma, respectively; and 2.4 ± 0.4 and 2.7 ± 0.7 mmHg at the end of trauma and sham trauma, respectively. There was no difference in central venous pressure between the trauma group and the sham group at any point in time.
The concentrations of the cytokines IFN-γ, IL-4, IL-6, IL-10, and TNF-α at 1 and 3 h after trauma or sham trauma are presented in Figure 5. A significant increase in IL-6 and IL-10 could be detected 1 h after trauma (n = 8 per group). One 1-h value in the sham group was excluded due to an analytical error.
The present study on the rat aimed at designing an experimental trauma model that can be used for the evaluation of changes in PV and microvascular permeability after a nonhemorrhagic trauma. The results showed that a blunt trauma to the abdominal rectus muscle induced a decrease in PV, coinciding with an increase in Hct, and an increase in TER for albumin compared with sham injured animals. The trauma also induced an increase in water content of the traumatized muscle and an increase in plasma concentrations for IL-6 and IL-10. The increase in K+ after trauma was most likely caused by release from the damaged tissue. We have no reasonable explanation for the unexpected decrease in Na+ concentration at 3 h after the trauma (Table 1). There was no difference between the sham and the trauma groups regarding MAP, urine output, or central venous pressure.
The PV measurement dilution technique using 125I-albumin as tracer is well established for the measurement of PV, both in experimental and in clinical studies, showing reproducible results during normal as well as inflammatory states (6, 7). The fact that measured baseline PVs of 41 to 42 mL/kg were in the same range as those presented in the literature for the rat supports the reliability of the PV-measuring technique (8, 9). Because of the transcapillary escape of albumin during the 5-min period between tracer injection and blood sampling, the albumin-derived radioactivity measured in plasma may have been somewhat decreased, resulting in an overestimation of the PV. The overestimation, however, must be of about the same size for all groups, and it must be small because the blood sample was taken shortly after the tracer injection.
At 3 h after trauma, PV had decreased by about 10 mL/kg, and the increase in muscle water content during the same period was estimated at 1.6 mL/kg, suggesting that only about 15% of the PV loss can be explained by edema in the traumatized rectus muscle. During the experiment, great care was taken to minimize external fluid losses due to evaporation and bleeding from wound areas during and after surgery. The fact that no bleeding could be observed in the wounds and that Hct increased after trauma suggests that blood loss did not contribute to the observed decrease in PV. Furthermore, urine production in the sham and traumatized animals did not differ. Considering that evaporative losses are small and that only a minor part of the PV loss was localized to the traumatized muscle, the major part of the PV must have been lost to the extravascular space in nontraumatized parts of the body.
The method for measurement of TER for albumin in our study is well established, both in experimental and in human research (2, 10-12). It has been shown that cardiac surgery could increase TER by 100% to 300% from a baseline value of 5% per hour, but TER changes after accidental trauma have not been reported. Normal TER for the anesthetized rat is reported to be in the range of 11% to 14% per hour (10, 13, 14), and the TER value of 13.9% per hour in the sham group thus agrees with the normal values for TER in the rat and supports the reliability of our technique. The TER for albumin is influenced by both the microvascular permeability for albumin and the transcapillary hydrostatic pressure because transcapillary transport of macromolecules occurs by both convective and diffusive mechanisms (7).
Because there was no difference in arterial and central venous pressures between the trauma and the sham groups, it is unlikely that an increase in hydrostatic capillary pressure could explain the trauma-induced increase in TER. The increase in TER after trauma in the present study from 13.9% to 18.5% may therefore be explained mainly by an increase in microvascular permeability, which is likely to be an important mechanism for the observed loss of PV. Considering that plasma is lost to the whole body, it is likely that the increase in TER reflects an increase in permeability in organs other than the injured muscle. The hypothesis that an isolated trauma may increase permeability also in distant organs is supported by several studies, showing that, for example, brain trauma may increase permeability of both the lung and the intestines (15, 16).
By comparing the results showing an increase in TER for albumin from 13.9% to 18.5% per hour after trauma, with the TER value of 14.2% per hour when the rats were exposed only to a skin incision and a laparotomy, we concluded that the major part of the TER increase is induced by the rectus muscle trauma. As mentioned in the introduction, effects of a nonhemorrhagic intra-abdominal trauma on PV have been previously investigated in the rat (5). In that study, it was shown that the trauma decreased PV by about 3 mL/kg, whereas TER for albumin, in contrast to our study, was unchanged. Considering the small decrease in PV in that study, it is likely that the lack of effect on TER can be explained by a less severe trauma.
Several studies in both rodents and humans have shown that the serum levels of IL-6 increase after surgery, and in humans it has been shown that increases in IL-6 and IL-10 concentrations are correlated to severity of tissue injury, development of multiple organ failure, and mortality (17-22). Our results of an increase in both IL-6 and IL-10 suggest that our model mimics a clinical scenario in which an inflammatory response is triggered by the trauma. IL-6 has been suggested to increase endothelial permeability for albumin in vitro and may have contributed to the observed increase in TER after trauma (23). However, the mechanisms influencing microvascular permeability after a trauma are complex, and most likely the inflammatory response is modulated by many factors. Such factors include IL-10, which may counteract cytokine-induced edema formation and permeability-increasing complement factors, which are known to be activated by soft tissue trauma (24-26). Our finding of the absence of change in TNF-α is in line with several previous studies, showing that soft tissue trauma without major hemorrhage does not trigger the TNF-α production (17, 19, 27).
In conclusion, the present standardized nonhemorrhagic skeletal muscle trauma model in the rat confirms the hypothesis that there is a trauma-induced increase in microvascular permeability both in traumatized and in nontraumatized tissues, resulting in a decrease in PV. The model mimics several aspects of a clinical trauma and may be used for evaluation of the effects of different pharmacological and other therapeutic interventions aimed at the correction of hypovolemia.
The authors thank Peter Siesjö, MD, PhD, and Edward Visse, PhD, at the Department of Neurosurgery, Lund University Hospital, for generous help with the cytokine analysis. The authors also thank for her skilled technical assistance Mrs Helén Davidsson.
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