Freise, Hendrik MD; Anthonsen, Sören; Fischer, Lars G. MD; Van Aken, Hugo K. MD; Sielenkämper, Andreas W. MD
Thoracic epidural anesthesia (TEA) is routinely used after major thoracic or abdominal surgery and in trauma patients (1,2). In comparison with general anesthesia alone, the use of TEA reduces cardiopulmonary mortality (3).
The use of TEA in intensive care medicine has become a subject of increasing interest (4–6). The aim is to transfer TEA’s favorable perioperative effects on the heart and the gut to the treatment of the critically ill. After cardiac and noncardiac surgery, TEA reduces the incidence of postoperative myocardial infarction and troponin release (7–9). It maintains intestinal mucosal perfusion during extended visceral surgery and promotes postoperative recovery of intestinal function (10–12). Both cardiac and splanchnic protection by TEA have been partly explained by a sympathetic block with baroreceptor reflex-driven increase in sympathetic activity outside the anesthetized regions (4,13). Especially in critically ill patients, however, the effects of TEA on regional blood flows and microcirculation, the immune system, neurohumoral responses, and hemostasis are almost unknown.
Experimental research in critical care medicine such as cecal ligation and puncture (CLP) sepsis or acute pancreatitis is extensively performed in prolonged small rodent models (14,15). However, there has been no technique of continuous TEA established for use in these models. In contrast to single-shot TEA, a model of continuous TEA would more closely resemble the clinical situation and would allow investigators to obtain valid data in combination with prolonged models of severe diseases. Experimental continuous TEA needs to be applicable without general anesthesia. Moreover, it must not compromise hemodynamic stability, respiration, and motor function. For this reason, the objective of this work was to evaluate a new model of continuous TEA in the awake and unrestricted rat.
Institutional approval for the experiments was obtained from the Animal Care Committee of the district government of Muenster. Thirteen male Sprague-Dawley rats (Harlan Winkelmann, Germany) weighing 250 to 300 g were used for the experiments. The animals were fed with standard rat chow and water ad libitum in a 12 h light-dark cycle. They were allowed to acclimate to laboratory conditions for 1 wk.
All surgical interventions were performed under inhaled anesthesia using 1.4 vol% isoflurane in 50%/50% oxygen/room air via a face mask. Spontaneous breathing was maintained. The ventral and dorsal aspect of the cervical region and both sides of the body from the cervicothoracic transition to the midabdominal region were shaved. The fields of incision were disinfected. The right jugular vein was dissected and cannulated with a PE 50 tube (0.96 mm outer diameter) advanced 30 mm to place its tip into the superior vena cava. A second catheter was introduced into the left carotid artery.
Epidural catheterization was performed using the microsurgical technique we previously described (16). Each rat was placed in prone position. The lumbar vertebral column was flexed by placing a metal cylinder transversely under the lower abdomen. The fourth lumbar spinal process was exposed and cut. A small hole was drilled through the cranial margin of the arch and the ligamentary structures of the intervertebral space L3-L4. A PE 10 tube (0.61 mm outer diameter) was introduced and advanced cephalad 70–75 mm. The catheter was sutured to the remnant of the spinous process to prevent dislocation. A repeated negative liquor aspiration test excluded subdural position of the catheter. One animal with a positive aspiration test was excluded from the study. After completion of the experimental protocol the position of the catheter was verified by autopsy.
All catheters were tunneled to the neck and protected by a metal swivel. This allowed continuous use of the catheters while the animal was awake and unrestricted movement in the cage. The dead space of the epidural catheter was filled with 28 μL saline solution or bupivacaine 0.5%. The animals were allowed to recover for 24 h.
After surgery, animals were housed individually in standard cages with free access to food and water that were withdrawn immediately before the beginning of measurements. Saline solution 2 mL/h was infused throughout the complete observational period. The room temperature was maintained at 21.2 ± 0.2°C. All measurements were done in the afternoon.
To assess the effects of continuous epidural anesthesia, rats were randomly assigned to two groups: Control rats (CON, n = 5) received epidural infusion (Genie syringe pump, World Precision Instruments, WPI, Berlin, Germany) of 15 μL/h isotonic saline solution 0.9%. Epidural anesthesia (EPI, n = 8) was performed by epidural infusion of 15 μl/h bupivacaine 0.5%. In each animal, two sets of measurements were completed: The first measurement was done 24 h after surgery (d1), the second set was performed on the third postoperative day (d3). The stability of epidural drug effects during a prolonged use of the epidural catheter was analyzed by comparison between d1 (n = 8) and d3 (n = 8) within the EPI group. Consequently, 10 and 16 sets of measurement were evaluated in both groups. Variables were recorded at baseline and every 30 min during 120 min of epidural infusion.
Mean arterial blood pressure (MAP) was continuously recorded using a standard transducer (PMSET 1DT, Becton Dickinson, Heidelberg, Germany) and a monitor (Siemens Sirecust 404, Siemens, Muenster, Germany). Heart rate (HR) was recorded using the arterial pressure curve.
Respiratory rate was recorded every 30 min. In addition, arterial blood gas analysis was performed to measure partial pressure of CO2 after 30 and 120 min.
At baseline and every 30 min during epidural infusion, muscular tone and function of front and hind limbs was assessed by an experienced observer who was blinded concerning group assignment. An established and reliable motor score was used. It was derived from Bromage score and adapted to rats (17). Categories of this score are as follows: 0 = no motor deficit, normal tone, free movement of the limb; 1 = mild reduction in muscular tone, able to stand but reduced power and control when moving; 2 = moderately reduced power, difficulties standing; 3 = complete loss of power, inability to stand, flat body posture.
Changes in sympathetic tone were indirectly assessed by changes in skin temperature measured by a fast response thermometer (BAT 10; World Precision Instruments) and a solid surface probe (BT 1, World Precision Instruments). The equilibration time of this system is 0.15 s with accuracy of 0.1°C in the range of −20°C to 50°C. Sites of measurement were the front paws, high-thoracic, mid-thoracic, and low-thoracic regions, the hind paws, and the proximal and distal tail. The thoracic measurement sites were marked at the sides of the body. High thoracic mark was set with permanent color immediately caudal of the shoulder; low thoracic measurement was done at the lowest ribs. The mid-thoracic mark was set at the midpoint between the high-thoracic and low-thoracic marks.
All temperature measurements were done in the awake and freely moving rat. The probe was placed gently but firmly on the marked site on the shaved skin. The animals tolerated this procedure well with minimal aversive reactions or efforts to evade. To exclude the impact of movement and increased activity 2 sets of sham measurement 30 and 15 min before baseline measurement were performed. Afterwards, the skin temperature was recorded at baseline and every 30 min during infusion. For direct comparison of the temperature changes between the CON and EPI groups, the temperature difference ΔT = T − Tbase was calculated for each time and measurement site. Infrared thermography (Agema 600; FLIR, Frankfurt, Germany) was used to visualize temperature changes in awake rats. The camera registers infrared wavelength between 3.6 and 5 μm and has a sensitivity of 0.1 degree. The spatial resolution with a 20° lens is 1 mm. Because continuous movement and changes in posture of the unrestricted rats prevented a retrieval of the predefined anatomical landmark, quantitative evaluation of thermography was not performed.
For statistical analysis Sigma Stat 2.03 software (SPSS, Chicago, IL) was used. Effects of time and treatment infusion were evaluated appropriately for a two-way repeated-measures design. Student-Newman-Keuls test was used for pairwise comparisons. For assessment of the nonlinear motor score a repeated measurement analysis of variance on ranks and Tukey’s test for pairwise comparison was performed. Variance was analyzed by F-test. For all statistical tests significance was assumed at P < 0.05. Data are presented as the mean ± sem.
All animals survived the surgical procedure and recovered quickly. They started to drink, eat, and groom on the day of surgery. One animal had to be excluded from the study as a result of a postoperative motor deficit in the right hind limb.
Autopsy showed a median position of the epidural catheter on the level of Th6 (25%–75% percentile, Th5–Th7). No catheter was placed intradurally.
Hemodynamics and Respiration
MAP remained constant in the CON group. At baseline, MAP was 128 ± 16 mm Hg and 127 ± 12 mm Hg after 2 h. In the EPI group, no changes in MAP occurred, with 124 ± 17 mm Hg at baseline and 115 ± 9 mm Hg after 120 min. Differences in MAP between the EPI and CON groups did not occur at any time. HR varied within physiological values and did not change within or between groups throughout the observational period.
Respiratory rate ranged from 115 ± 20/min to 138 ± 26/min and was unchanged in both groups. Arterial Pco2 was comparable at baseline and did not change over time in the two groups.
In the CON group no motor deficit occurred. In the EPI group, weak to moderate motor deficits of the hind limbs were observed in four of 16 experiments. Motor score (MS) was 0 (0/0) at 30 min, 0 (0/0.5) at 60 min, 0 (0/1.5) at 90 min, and 0 (0/1.0) at 120 min (not significant versus baseline after 30, 60, 90, and 120 min). The front paws were not affected.
Baseline temperatures did not differ between groups (Table 1). TEA induced an increase in thoracic skin temperature (Fig. 1). ΔT were higher in the EPI group compared with the CON group in high-thoracic, mid-thoracic, and lower-thoracic segments throughout the entire observational period of 120 min (P < 0.001 versus CON after 30, 60, 90, and 120 min) (Fig. 2). In the distal tail temperature decreased to −0.84 ± 0.24°C during TEA in the EPI group (P < 0.05 versus CON after 60, 90, and 120 min) (Fig. 3).
In contrast to the consistent warming of the thoracic region and cooling of the distal tail, ΔT in the front paws, hind paws, and proximal tail showed a different pattern. There was either marked decrease or a strong increase in skin temperature with ΔT in both the front and the hind paws of the EPI group ranging between −4.1°C and 10.2°C, resulting in an increased scatter of ΔT (F-test P < 0.001 versus CON in front and hind paws after 30, 60, 90, and 120 min).
In the EPI group, baseline values did not differ between the first and third postoperative days. TEA-induced ΔT on d3 were the same as on d1 with the one exception of an increased ΔT at the mid-thoracic level on d3 (P < 0.05) (Fig. 4). There were no differences in ΔT for the front paws, the hind paws, and the tail when compared between d1 and d3.
In awake rats, we demonstrated that during continuous TEA using 15 μl/hour bupivacaine 0.5% for 120 min skin temperature in the thoracic segments increased, whereas it decreased in the distal tail, thus indicating a segmental sympathetic block with compensatory increase in sympathetic activity outside the anesthetized segments. Cardiorespiratory side effects or extensive motor blockade were not observed.
The surgical technique of thoracic epidural catheterization has been described by our group (16). We used a lumbar insertion to avoid surgical trauma of the thoracic region. The remote site of catheter insertion is expected to reduce the risk of drug leakage from the epidural space, which is a problem in experimental epidural anesthesia in rats (18,19).
Alteration of epidural drug effects has also been addressed as a problem in chronic rat epidural catheters. Nishiyama and Haraoka (20) noted altered pattern of dye spread 4 days after implantation in a lumbar catheterization model and warned against more than 48 hours’ use. Durant and Yaksh (18) reported severe fibrosis with effective hindrance of drug effect after 10 days. In contrast, Grouls et al. (17) found no fibrinous reactions after 4 days. In our model, the tip of the epidural catheter was placed remote from the site of insertion, where the surgical trauma was thought to increase inflammatory responses. There seemed to be no change in drug spread and efficacy after 3 days, even though the catheter was not used continuously. Many experimental studies of critical illness, such as sepsis or necrotizing pancreatitis, are performed with an observation period of up to 48 hours. Some models allow a recovery period after instrumentation that is typically 24 hours. Therefore, TEA should be effective for up to 72 hours. Consequently, the described technique of continuous TEA may be reliably used in these experimental models.
Evaluation of Sympathetic Activity
Both direct and indirect techniques are available for the measurement of sympathetic nerve activity.
Microneurography allows direct measurement of sympathetic tone and discrimination between muscle and skin sympathetic activity (13,21,22). Indirect techniques for assessment of sympathetic activity include the measurement of skin conductance response (21,23) and HR variability (23,24). Most investigators, however, have focused on measurements of skin perfusion. Heat washout techniques determine cutaneous blood flow by the rate of temperature equilibration (25). Doppler flow probes have also been used to quantify skin perfusion in regional anesthesia (26) but it has been argued that Doppler measurements may underestimate or even fail to detect periods of increased skin perfusion during TEA (25). All of these techniques require an anesthetized rat.
To evaluate changes in skin sympathetic activity ΔT has often been used as an indirect marker (27–29). This approach is based on the physiological fact that skin perfusion is regulated mainly by sympathetic tone in the presence of constant ambient temperature, constant internal heat production, and a constant emotional state (25,27,30). Under such circumstances, any change of sympathetic tone can be expected to induce changes in skin perfusion followed by subsequent changes in skin temperature.
In the current study, we used repeated measurement of skin temperature. The procedure was noninvasive and did not require restriction or anesthesia. Two sets of sham measurement procedures were performed before baseline to reduce interference between the effects of TEA and a potential change in skin sympathetic activity owing to the measurements. This approach appeared to be the most feasible method to assess changes in sympathetic activity in awake rats.
Repeated infrared thermography also allows determination of changes in sympathetic nerve activity and data acquisition with a high resolution in time and space (27). Although an animal’s continuous movement impairs identification of predefined measurement sites and thus limits the use of thermography in unrestricted rats, it is well suited for visualization of skin temperature changes.
Effects of TEA on Sympathetic Activity
In the present study, continuous TEA in awake rats induced a segmental increase in thoracic skin temperature. A transition zone that either showed marked increase or decrease of temperature in front paws, hind paws, and the proximal tail was located both cranial and caudal of the thoracic region. The skin temperature of the distal tail decreased in all animals in the EPI group. The results suggest a sympatholysis in the thoracic segments and a compensatory increase in sympathetic nerve activity in the tail and partially in the front and hind paws.
In this model, TEA consistently blocked the sympathetic preganglionic neurons in the thoracic spinal cord from Th1 down to segment Th13. Consequently, the block in all animals included the preganglionic neurons of the splanchnic nerve that supplies most of the abdominal organs and the intestinal tract up to the colon transversum (31). The hind paw and the proximal tail are innervated by sympathetic neurons located on the level of L1 and L2 (32). Depending on the individual spread of TEA, they are either included in the sympathetic blockade or contribute to reflectory vasoconstriction. This transition zone includes the intermesenteric nerve and the lumbar colonic nerves innervating the pelvic organs (31,33). The distal tail that is innervated by sympathetic preganglionic neurons of L3 or below was consistently colder, indicating an increase in sympathetic activity.
The temperature changes in paws and tail are more pronounced, probably as a result of the increased range of regulation necessary in the hairless skin. The variation in the spread of sympatholysis and the occasional occurrence of motor block represent the differences in animal size, catheter position, and fluid spread in the epidural space.
Our results support the concept of segmental, limited spread of sympathetic block during TEA, which is thought to be a key mechanism of the protective cardiovascular and intestinal effects of perioperative TEA. However, clinical and experimental data concerning the presence of segmental sympatholysis are in conflict. Two clinical studies reported a blockade of the sympathetic nerve activity distal to the sensory blockade with a significant increase in skin temperature of the foot during TEA (27,34). They used high thoracic TEA (C7–Th3) with 4–8 mL of 0.75% bupivacaine. In contrast, Magnusdottir et al. (21) reported a constant sympathetic tone of the lower extremities during TEA (T3–T5) with 4 mL 0.5% bupivacaine.
In an experimental dog model, TEA induced a temperature increase of the hind limb during high-thoracic and mid-thoracic TEA with limited sensory block (28). In contrast, two experimental studies using direct assessment of sympathetic activity reported a reflectory increase of sympathetic activity. In cats, TEA increased sympathetic activity caudal to the blocked segments in the kidney. Baroreceptor-dependant sympathetic reflexes contributed to hemodynamic stability during segmental sympatholysis in this study (22). In rabbits, a decreased sympathetic activity was demonstrated in mesenteric vessels during TEA with concomitant hypotension (13). In lumbar epidural anesthesia, an increased intestinal sympathetic activity and mesenteric venoconstriction were demonstrated and hypotension was markedly attenuated.
Recently, another model of continuous TEA in anesthetized rats has been described. In this study an epidural bolus infusion of 30 μL lidocaine 2% was given (35). The initial bolus infusion was followed by a continuous infusion of 100 μL/hour lidocaine 2%. Because the animals were anesthetized and ventilated, no data about motor blockade were obtained. Therefore, it remains unclear whether such an increased dose of local anesthetics interferes with respiratory function in awake animals. In our own pilot experiments a motor blockade of the front paws occurred during infusion of only 30 μL/hour bupivacaine 0.5%.
This is the first experimental investigation of continuous TEA in awake rats. The new technique induces segmental thoracic sympathetic block without cardiorespiratory depression or motor blockade. Continuous TEA is applicable in awake and unrestricted rats and can be reliably used for at least 72 h.
To study TEA in experimental models of critical illness, e.g., CLP-sepsis and pancreatitis-induced systemic inflammatory response syndrome (SIRS), a prolonged period of observation, exceeding 24 hours, is required. The animals need to be awake and extensive motor blockade, respiratory depression, or prolonged hypotension must be avoided. In addition, TEA needs to be reliably effective for the observational period. The results of the presented investigation may demonstrate the suitability of our continuous TEA technique for such purposes. In contrast to single-shot techniques, this method provides the possibility of valid experimental studies of the impact of epidural anesthesia on the microcirculation, regional perfusion, and immunologic responses under conditions of health and in prolonged experimental models of critical illness.
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