Clinical Hemorheology and Microcirculation 60 (2015) 335–346 DOI 10.3233/CH-141854 IOS Press
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Technical report
The cooling tube: A novel small animal model of systemic hypothermia in awake Syrian Golden Hamsters (mesocricetus auratus) Jan Goedekea,∗ , Nadja Apelta and Markus Kamlerb a
Department of Pediatric Surgery, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-University, Munich, Germany b Department of Thoracic and Cardiovascular Surgery, Herzzentrum Essen-Huttrop, Essen, Germany
Abstract. Hypothermia is increasingly used as a therapeutic strategy in a diversity of clinical scenarios. Its impact on mammalian physiology, particularly on the microcirculatory changes of critical organ systems, are, however, incompletely understood. Close examination of the literature reveals a marked paucity of small animal models of rapid systemic hypothermia. All published models introduce important microvascular confounders by investigating either local cooling processes or using anaesthetised animals. Here we present the first rapid systemic hypothermia model in an awake hamster. We developed a waterstream cooled copper tube system for standardized systemic temperature control. With this novel system core body temperature (Tc ) in 14 awake animals could be precisely stabilised at temperatures of 30°C and 18°C (7 animals, respectively) within 10–20 min. Rewarming was achieved over 10–15 min. Tolerance of the procedure was excellent. Hamsters did not show any behavioural changes in the mild hypothermia group. In the deep hypothermia group 6 of 7 animals regained normal behaviour within 2–11 hs. As hypothermia was induced in dorsal skinfold chamber bearing animals this model seems suitable for investigation of microcirculatory purposes. Advantages over previously established experimental hypothermia models are significant. Amongst these, the possibility of visualization of microcirculation, the lack of microcirculation confounding factors such as anaesthetic drugs, the ability for precise Tc control and rapid induction of hypothermia are prominent. Keywords: Hypothermia, cooling tube, microcirculation, awake animal model, Syrian Golden Hamster
1. Introduction Hypothermia has long since been known to lower cellular metabolic rate and oxygen consumption [1, 8, 20]. Modern therapeutic approaches to refractory traumatic brain injury (TBI), stroke, post-haemorrhagic and cardiac shock in both infants and adults [2, 11, 13, 15, 31, 33, 36, 37, 40] as well as hypoxic ischemic encephalopathy of the newborn (CoolCap studies) all aim to use this fact to best clinical advantage. Thus, the use of systemic and local hypothermia, particularly of the central nervous system, has been expanding ∗ Corresponding author: Jan Goedeke, M.D., Department of Pediatric Surgery, Dr. von Haunersches Kinderspital, LudwigMaximilians-University, 80337 Munich, Germany. E-mail: [email protected].
1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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drastically in the intensive care (ICU) setting over recent years. While the cerebral metabolic effects of hypothermia, particularly the preservation of brain ATP supply [8], have been studied extensively, the effects of cerebral cooling on the peripheral microcirculation are quite unknown [9]. More importantly still, many systemic effects of hypothermia presently remain unstudied, not least amongst them the microcirculatory changes suffered by critical organ systems such as the heart and liver. While the need for data on the effects of hypothermia on these critical organ systems is readily apparent; the investigation of the in vivo effects of hypothermia demands accessible and cheap animal models that reliably mimic the clinical scenarios encountered in ICU. Yet, close examination of the literature reveals a marked paucity of such models. While several well-known and established experiments in large mammals such as sheep and piglets are described they unanimously carry the disadvantage of high personnel and financial expense [12, 24, 27]. This turns them inaccessible to smaller institutions. However, while generally more accessible, the numerous available small animal models in mice, hamsters and rats investigate either local cooling processes or systemic cooling processes on anaesthetised animals. This introduces important confounders in the study of hypothermia-induced microvascular changes, thus limiting their use [6, 10, 16, 19, 22, 24–26, 29, 32, 35, 38]. Here we report the novel introduction of a waterstream-cooled copper tube system that allows the study of short term systemic hypothermia and microcirculation in non-sedated small mammals, such as the Syrian Golden Hamster (mesocricetus auratus). We believe that this new animal model is apt at mimicking an array of clinical scenarios while controlling for the main confounders introduced by previous small animal models of hypothermia. Hence, this new device may help to provide significant insight into the physiopathologic and microcirculatory disturbance caused by hypothermia.
2. Materials and methods 2.1. Experimental protocol For an overview of the experimental setup and timescale please refer to Fig. 1. In brief, experiments were carried out in 21 male Syrian Golden Hamsters (two study groups, one control group, each n = 7) (age 10–14 weeks, weight 65–75 g). For the establishment of the safety and efficacy of this new experimental protocol in a non-sedated animal medico-ethical reasoning dictated use of a hibernator, as testing a wide temperature range was required to establish the temperature precision of the model and unnecessary stress on non-hibernators was feared in the lower temperature ranges. Mesocricetus auratus was chosen as the ideal laboratory animal as the dorsal skin fold chamber was established in this animal model [7]. In a first step, animals were accustomed to the copper tube system through daily training and showed no obvious discomfort. Additionally, a pipe system was installed in the cages, allowing hamsters to use the pipe system outside training hours. After successful training, dorsal skin fold chambers were implanted under general anaesthesia as previously described [7, 18]. All animals were given 3.6 ± 0.6 days (mean ± SD) for postoperative recovery before one catheter for blood pressure measuring was implanted in the carotid artery (polythene tubing 0.28 × 0.61 mm) (according to Kamler et al. [18]) Hamsters which at this stage suffered signs of inflammation, edema or bleeding spots within the dorsal skin fold chamber were suspected to suffer disturbed microcirculation before start of hypothermia and were thus excluded from further investigations. 14 animals performed systemic short term cooling, 7 of which were cooled to 30◦ C and 7 of which to 18◦ C. Temperatures were chosen to mimic the range of clinical scenarios: mild hypothermia (30◦ C), as used in cardiogenic shock syndrome and in asphyctic
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Fig. 1. Timescale of the experiments.
newborns [33, 40] and deep hypothermia (18◦ C), as the lowest temperature used during cardiovascular operations such as aortic arch replacement [39]. Hamsters usually show no signs of life-threatening impairment of the cardiovascular and respiratory system until a core body temperature lower than 10◦ C is reached [14]. Hamsters of both study groups started with an average core body temperature of 37.2◦ C (each n = 7) (Fig. 2). For introducing deep hypothermia the copper tube system was cooled to 20◦ C for 5 minutes, allowing the awake animal to adapt to the new situation. In a second step tube temperature was lowered to 10◦ C until the core body temperature of the animals reached 18.2 ± 0.9◦ C (mean ± SD). Then, tube temperature was stabilised at 18◦ C and microcirculatory exams via skinfold chamber could be performed. Cooling to 30◦ C followed the same general principles. To validate our results we used a control group of 7 animals, subjected to all procedures at 37◦ C (Fig. 3). In hamsters circadian rhythm influences both hormone levels and body temperature [28, 30]. Thus, reproducibility of results was guaranteed by performing all experiments at the same time during morning hours. This had the additional advantage of performing hypothermia on sleeping animals, thus further reducing the stress levels and adding to humane care. External influences on circadian rhythm and animal hormone levels were controlled by keeping animals in laboratory conditions with a stable circadian light source and under controlled room temperature. 2.2. Experimental setup The tube system and the further setup used is portrayed in Figs. 4–6. On the two open ends of the outside tube system short plastic tubes connected to a bypass pump were put. The pump (EDEN 104, Tauchpumpe, EDEN S.R.L, Cartigliano, Italy) was based in a 2 liter water filled beaker which was put on a heating pad with a combined magnetic stirrer (MR 3001, Heidolph Instruments GmbH, Schwabach, Germany). In this setup water of any desired temperature could be pumped through the tube system without significant loss of energy, allowing for precise regulation of the temperature of the copper tube
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Fig. 2. Sample graph of tube temperature and resulting core body temperature (Tc ) over a period of 60 minutes. The target temperature of 18◦ C core body temperature (Tc) is reached after about 20 minutes. ECC at 18◦ C core body temperature (Tc) is performed for about 30 minutes. The animal regained normal core body temperature (Tc) after 15 minutes of rewarming.
Fig. 3. Core body temperature (Tc ) of awake hamsters during the experiment. Control group (n = 7 solid bars) received normothermia. In the experimental group (n = 7 empty bars), hypothermia to 18◦ C was applied. Data are mean ± SD. The drop of core body temperature (Tc ) is statistically significant from controls as early as 10 min after onset of cooling and returns to baseline values at the end of the 60 min observation period (P < 0.05).
(Fig. 7). To guarantee uniform water temperature in the beaker a magnetic stirrer was used continuously. Room temperature was kept at 18◦ C during cooling procedures and at 25◦ C during rewarming. Water temperature and body temperature of the hamster were continuously recorded with a two channel digital thermometer (Data-Logger Thermometer, Conrad Electronic GmbH, Essen, Germany). One sensor
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Fig. 4. Original image of the copper tube system.
Fig. 5. Cross-section drawing of the tube system.
was placed in the beaker. The other was modified as a rectal thermometer measuring the hamster’s core body temperature. The temperature of the copper tube system was monitored using a probe fixed to the wall of the tube system. Data was recorded with a second data logger.
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Fig. 6. Drawing of the experimental setup.
2.3. Laboratory animal Before dorsal skinfold chamber implantation animals were kept in groups of 3–6 animals. They had to be well nourished with normal condition of coat, eyes and natural orifices. Breathing movements, posture, response to stimuli in the environment and the orientation and movement activity had to be normal. From the time of implantation, animals were kept in individual cages in order to avoid injuries. Room temperature was constant at 21 ± 2◦ C, with a relative humidity of 55 ± 5%. Drinking water and standard laboratory chow (10H10, standard diet for rat - mouse – hamster, Nohrlin GmbH, Bad Salzuflen, Germany) were always available ad libitum. Standard bedding for laboratory rodents was used. Hamsters were exposed to a 12-hour alternating light-dark cycle. Animals were observed postinterventionally for a maximum of 5 days, after which they were euthanized. High value was attributed to adequate behaviour of the animals. For cases of lack of food and fluid intake and decreasing body weight, shaggy fur, altered body orifices, high frequent breathing, changes in posture like a bended back, decreased response to environmental stimuli and lack of physical activity euthanasia was discussed following a short-term observation. All animals received humane care in compliance with the European Convention on Animal Care and the study was approved by the Institutional and Regional Committee for Animal Care. All operative procedures where performed under Narcoren (60 mg/kg body weight, Pentobarbital, Merial, Hallbergmoos, Germany) anaesthesia and aseptic conditions. Chambers and instruments were sterilized before use. 2.4. Measurement of cardiovascular parameters The arterial catheter was connected with a pressure dome (Uniflow TM , Baxter, Germany). Heart rate (fh ) [min−1 ] and mean arterial blood pressure (mABP) [mmHg] were recorded pre, during and post cooling procedure via a monitor (Servomed, Hellige, Germany) and a flatbed recorder (L250, Linseis, Selb, Germany).
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Fig. 7. Sample graph of water temperature and resulting tube temperature over a period of 10 minutes. The experimental set-up allowed a precisely regulation of the copper tube temperature via regulation of the water temperature.
2.5. Microscopic observation Microscopic observations in the skinfold chamber could be performed prior, during and after the cooling procedure using an intravital microscope (Leica DMLM, Wetzlar, Germany) with a 20xSW 0.40 BD NA objective (Leica Fluotar, Wetzlar, Germany) (Fig. 8). A 100-W Hg light source was used for epiillumination. Contrast enhancement for transillumination was accomplished with a blue filter (420 nm), which selectively passes light in the maximum absorption band of hemoglobin, causing red blood cells to appear as dark objects in an otherwise gray background. A heat filter was placed in the light path prior to the condenser. Microscopic images were viewed by a closed circuit video system consisting of a CCD camera (Kappa CF 8/4 NIR, Gleichen, Germany) and a RGB-monitor (Sony, Japan). Leukocytes were stained with rhodamine 6 (Sigma, St Louis, MO) and classified by fluorescence microscopy according to their interaction with the endothelial lining as adherent, rolling or free flowing cells. Functional capillary density (FCD) was assessed in nine successive microscopic fields by transillumination in a region of approximately 1.3 mm2 ; the initial field was chosen where microvessels were in focus and there were usually between two and five RBC-perfused capillaries in the field of view. FCD was evaluated by measuring the length of capillaries that had red blood cell flow. A capillary was defined to be functional if passing RBCs were noted within the entire 20-s observation period. Microvascular permeability was analyzed by quantifying the extravasation of the macromolecular fluorescent marker FITC-Dextran (Mw 150,000) with the computer-assisted microcirculation analysis system (CAPIMAGE) [21]. Measurements were performed 15 min after intravenous injection of the fluorescent marker at the end of each study. In each vessel, gray-levels over the tissue directly adjacent to the vessel wall (E1) and over the marginal cell-free plasma layer within the vessel (E2) were determined over a length of at least 100 m. Extravasation (E) was then calculated as EZE1/E2.
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Fig. 8. The capillaries (arrows) are visible in their strictly parallel course. They show cross-links between each other. The distance is about 30–50 microns. In the background one can see a postcapillary venule. (Intravital fluorescence microscopy, contrast enhancement with FITC-dextran in blue-light epi-illumination, scale: 50 microns).
3. Results 3.1. Experimental tolerance Overall, hamsters showed good tolerance of the described procedure. After rewarming, 6 hamsters cooled to 18◦ C regained normal behaviour within 2 to 11 hours, five of which did not show behavioural sequelae over the next 5 days. One hamster cooled to 18◦ C died 28 hours after the experiment because of an aortic clot probably caused by the arterial catheter. The seventh hamster cooled to 18◦ C was euthanized 18 hours after the experiment because of abnormal feeding habits and behaviour. In the mild hypothermia group and in the 37◦ C control group no detrimental effects of the procedure were observed until sacrifice. 3.2. Cardiovascular parameters At the beginning and at the end of experiments mean arterial blood pressure amounted to 80–90 mmHg in all groups. During the 18◦ C cooling process, blood pressure decreased significantly by up to 50 % as compared to baseline. In the other groups it only showed non-significant physiological changes during the whole trial period (Table 1). No significant differences in heart rate fh [min-1] were observed either amongst or within groups during cooling. Mean fh ranged between 260 – 310 beats [min-1] and was within physiological limits. In the group of animals cooled to 18◦ C a significant decrease of heart rate to 25 % of baseline-value could be observed during cooling (Table 1) while animals of other groups showed only non-significant physiological changes in heart rate. These results are consistent with published literature and are within physiologic limits for species and body temperature [14].
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Table 1 Mean arterial blood pressure and heart rate during normothermia and hypothermia Parameter
Experiment
Baseline
Goal temperature
End
37◦ C 30◦ C 18◦ C
81.8 ± 8.9 83.4 ± 5.9 82.9 ± 7.3
84.2 ± 10.3 80.3 ± 6.2 45.8 ± 6.4*
83.1 ± 7.9 80.4 ± 9.1 86.2 ± 10.7
37◦ C 30◦ C 18◦ C
278.2 ± 18.4 273.7 ± 14.3 286.4 ± 20.9
286.4 ± 16.9 276.1 ± 12.1 76.3 ± 14.4*
289.8 ± 20.2 280.3 ± 18.2 290.9 ± 17.3
mABP [mmHg]
fh [min−1 ]
Data are mean ± SD, each group n = 7; ∗ P < 0.05 hypothermia [18◦ C] vs. normothermia [37◦ C].
3.3. Microvascular parameters 3.3.1. Leukocyte/endothelial cell interaction in arterioles and postcapillary venules The majority of fluorescently labeled leukocytes did not interact with endothelial cells in arterioles and postcapillary venules. There were no significant differences between the 18◦ C cooling and the 37◦ C control group prior and after the procedure up to 8hs post intervention observation time. 3.3.2. Functional capillary density FCD was significantly less in animals at 18◦ C body temperature compared with that seen in the 37◦ C and 30◦ C group (84 ± 15 vs. 151 ± 14 vs. 142 ± 16 cm/cm2 , P < 0.05), but it restored after rewarming. 3.3.3. Microvascular permeability Deep hypothermia significantly increased the edema formation in all microvascular compartments. In the perivascular tissue around the arterioles it was about 20% higher after the 18◦ C cooling process compared to the 30◦ C group and the 37◦ C control group (microvascular permeability compared in % of BL; P < 0.05 18◦ C vs. 37◦ C). 4. Discussion Hypothermia is increasingly used as a therapeutic strategy in a diversity of clinical scenarios from perinatal asphyxia to trauma, shock and cardiac surgery. Its impact on mammalian physiology, particularly on the microcirculatory changes of critical organ systems, are, however, incompletely understood. A better understanding of the pathophysiologic effects of systemic hypothermia in mammals is thus not only urgent but paramount to the safe clinical use of this promising therapeutic approach. Cheap, efficient and accessible animal models are a crucial step in answering the most pressing scientific questions surrounding the clinical use of hypothermia. Hypothermia in humans is carried out both in the presence and absence of anaesthesia and ECC. Thus, a good animal model should allow its effects to be studied in all of these potential settings. However, none of the previously described small animal models of hypothermia offered these possibilities [6, 22, 26, 29]. Moreover, they either required the animal to be anaesthetised or
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achieved only inaccurate core body temperature control, thus introducing important confounders, such as the proven detrimental effect of general anaesthesia on microvascular perfusion [3, 5, 23]. The experimental setup herein presented thus offers a vast improvement over previously available small animal models, allowing for systemic hypothermia effects to be studied both in the presence and absence of anaesthesia and ECC, as required. In the presents study a hibernator was chosen for medico-ethical reasoning to establish the tolerance to our experimental setup in non-sedated mammals. Our data confirms excellent tolerance of the procedure with low stress levels for the animals, making an experimental model with a tube setup a humane and animal-friendly option providing high body surface contact for any tube-dwelling mammal. We expect these results to be applicable to other non-hibernating tube dwelling animals. Copper was chosen in the production of the tube as it is an extremely efficient thermal conductor, easy to purchase and inexpensive compared to other metals with the same characteristics. Our results regarding the microcirculatory effects of deep hypothermia strongly support the need for further investigation, as we found not only a markedly reduced microvascular perfusion, but also increased vascular permeability and extravasation. Furthermore we observed a significantly reduced L/E cell interaction in the early post-ECC period. Despite a reduced number of adherent leukocytes we saw no protection of endothelial barrier function after ECC in deep hypothermia compared to ECC under normothermia [17]. In summary, the experimental setup herein described is cheap, animal friendly and can be potentially modified for any small mammal, including the possibilities for ECC, dorsal skinfold chamber and anaesthesia but not requiring any of these to work. It presents significant improvements on other established small animal hypothermia models, not least because of the precision of Tc achieved. Furthermore, the present setup may serve as an improvement for available perinatal asphyxia and hypothermia models in rats [4, 34]. Thus, we believe that this novel hypothermia small animal model provides invaluable new possibilities for hypothermia research across all specialties. Acknowledgments None. References [1] P.N. Amess, J. Penrice, E.B. Cady, A. Lorek, M. Wylezinska, C.E. Cooper, P. D’Souza, L. Tyszczuk, M. Thoresen, A.D. Edwards, J.S. Wyatt and E.O. Reynolds, Mild hypothermia after severe transient hypoxia-ischemia reduces the delayed rise in cerebral lactate in the newborn piglet, Pediatr Res 41(6) (1997), 803–808. [2] J.F. Ashruf, H.A. Bruining and C. Ince, New insights into the pathophysiology of cardiogenic shock: The role of the microcirculation, Curr Opin Crit Care 19(5) (2013), 381–386. [3] Z. Brookes, C. Reillyand and N. Brown, Differential effects of propofol, ketamine, and thiopental anaesthesia on the skeletal muscle microcirculation of normotensive and hypertensive rats in vivo, Br J Anaesth 93 (2004), 249–256. [4] M.L. Dalen, X. Liu, M. Elstad, E.M. Løberg, O.D. Saugstad, T. Rootwelt and M. Thoresen, Resuscitation with 100% oxygen increases injury and counteracts the neuroprotective effect of therapeutic hypothermia in the neonatal rat, Pediatr Res 71(3) (2012), 247–252. [5] R.A. De Blasi, S. Palmisani, M. Boezi, R. Arcioni, S. Collini, F. Troisi and G. Pinto, Effects of remifentanil-based general anaesthesia with propofol or sevoflurane on muscle microcirculation as assessed by near-infrared spectroscopy, Br J Anaesth 101(2) (2008), 171–177. [6] D. Deveci and S. Egginton, Effects of acute and chronic cooling on cardiorespiratory depression in rodents, J Physiol Sci 57(1) (2007), 73–79.
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Technical report
The cooling tube: A novel small animal model of systemic hypothermia in awake Syrian Golden Hamsters (mesocricetus auratus) Jan Goedekea,∗ , Nadja Apelta and Markus Kamlerb a
Department of Pediatric Surgery, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-University, Munich, Germany b Department of Thoracic and Cardiovascular Surgery, Herzzentrum Essen-Huttrop, Essen, Germany
Abstract. Hypothermia is increasingly used as a therapeutic strategy in a diversity of clinical scenarios. Its impact on mammalian physiology, particularly on the microcirculatory changes of critical organ systems, are, however, incompletely understood. Close examination of the literature reveals a marked paucity of small animal models of rapid systemic hypothermia. All published models introduce important microvascular confounders by investigating either local cooling processes or using anaesthetised animals. Here we present the first rapid systemic hypothermia model in an awake hamster. We developed a waterstream cooled copper tube system for standardized systemic temperature control. With this novel system core body temperature (Tc ) in 14 awake animals could be precisely stabilised at temperatures of 30°C and 18°C (7 animals, respectively) within 10–20 min. Rewarming was achieved over 10–15 min. Tolerance of the procedure was excellent. Hamsters did not show any behavioural changes in the mild hypothermia group. In the deep hypothermia group 6 of 7 animals regained normal behaviour within 2–11 hs. As hypothermia was induced in dorsal skinfold chamber bearing animals this model seems suitable for investigation of microcirculatory purposes. Advantages over previously established experimental hypothermia models are significant. Amongst these, the possibility of visualization of microcirculation, the lack of microcirculation confounding factors such as anaesthetic drugs, the ability for precise Tc control and rapid induction of hypothermia are prominent. Keywords: Hypothermia, cooling tube, microcirculation, awake animal model, Syrian Golden Hamster
1. Introduction Hypothermia has long since been known to lower cellular metabolic rate and oxygen consumption [1, 8, 20]. Modern therapeutic approaches to refractory traumatic brain injury (TBI), stroke, post-haemorrhagic and cardiac shock in both infants and adults [2, 11, 13, 15, 31, 33, 36, 37, 40] as well as hypoxic ischemic encephalopathy of the newborn (CoolCap studies) all aim to use this fact to best clinical advantage. Thus, the use of systemic and local hypothermia, particularly of the central nervous system, has been expanding ∗ Corresponding author: Jan Goedeke, M.D., Department of Pediatric Surgery, Dr. von Haunersches Kinderspital, LudwigMaximilians-University, 80337 Munich, Germany. E-mail: [email protected].
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drastically in the intensive care (ICU) setting over recent years. While the cerebral metabolic effects of hypothermia, particularly the preservation of brain ATP supply [8], have been studied extensively, the effects of cerebral cooling on the peripheral microcirculation are quite unknown [9]. More importantly still, many systemic effects of hypothermia presently remain unstudied, not least amongst them the microcirculatory changes suffered by critical organ systems such as the heart and liver. While the need for data on the effects of hypothermia on these critical organ systems is readily apparent; the investigation of the in vivo effects of hypothermia demands accessible and cheap animal models that reliably mimic the clinical scenarios encountered in ICU. Yet, close examination of the literature reveals a marked paucity of such models. While several well-known and established experiments in large mammals such as sheep and piglets are described they unanimously carry the disadvantage of high personnel and financial expense [12, 24, 27]. This turns them inaccessible to smaller institutions. However, while generally more accessible, the numerous available small animal models in mice, hamsters and rats investigate either local cooling processes or systemic cooling processes on anaesthetised animals. This introduces important confounders in the study of hypothermia-induced microvascular changes, thus limiting their use [6, 10, 16, 19, 22, 24–26, 29, 32, 35, 38]. Here we report the novel introduction of a waterstream-cooled copper tube system that allows the study of short term systemic hypothermia and microcirculation in non-sedated small mammals, such as the Syrian Golden Hamster (mesocricetus auratus). We believe that this new animal model is apt at mimicking an array of clinical scenarios while controlling for the main confounders introduced by previous small animal models of hypothermia. Hence, this new device may help to provide significant insight into the physiopathologic and microcirculatory disturbance caused by hypothermia.
2. Materials and methods 2.1. Experimental protocol For an overview of the experimental setup and timescale please refer to Fig. 1. In brief, experiments were carried out in 21 male Syrian Golden Hamsters (two study groups, one control group, each n = 7) (age 10–14 weeks, weight 65–75 g). For the establishment of the safety and efficacy of this new experimental protocol in a non-sedated animal medico-ethical reasoning dictated use of a hibernator, as testing a wide temperature range was required to establish the temperature precision of the model and unnecessary stress on non-hibernators was feared in the lower temperature ranges. Mesocricetus auratus was chosen as the ideal laboratory animal as the dorsal skin fold chamber was established in this animal model [7]. In a first step, animals were accustomed to the copper tube system through daily training and showed no obvious discomfort. Additionally, a pipe system was installed in the cages, allowing hamsters to use the pipe system outside training hours. After successful training, dorsal skin fold chambers were implanted under general anaesthesia as previously described [7, 18]. All animals were given 3.6 ± 0.6 days (mean ± SD) for postoperative recovery before one catheter for blood pressure measuring was implanted in the carotid artery (polythene tubing 0.28 × 0.61 mm) (according to Kamler et al. [18]) Hamsters which at this stage suffered signs of inflammation, edema or bleeding spots within the dorsal skin fold chamber were suspected to suffer disturbed microcirculation before start of hypothermia and were thus excluded from further investigations. 14 animals performed systemic short term cooling, 7 of which were cooled to 30◦ C and 7 of which to 18◦ C. Temperatures were chosen to mimic the range of clinical scenarios: mild hypothermia (30◦ C), as used in cardiogenic shock syndrome and in asphyctic
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Fig. 1. Timescale of the experiments.
newborns [33, 40] and deep hypothermia (18◦ C), as the lowest temperature used during cardiovascular operations such as aortic arch replacement [39]. Hamsters usually show no signs of life-threatening impairment of the cardiovascular and respiratory system until a core body temperature lower than 10◦ C is reached [14]. Hamsters of both study groups started with an average core body temperature of 37.2◦ C (each n = 7) (Fig. 2). For introducing deep hypothermia the copper tube system was cooled to 20◦ C for 5 minutes, allowing the awake animal to adapt to the new situation. In a second step tube temperature was lowered to 10◦ C until the core body temperature of the animals reached 18.2 ± 0.9◦ C (mean ± SD). Then, tube temperature was stabilised at 18◦ C and microcirculatory exams via skinfold chamber could be performed. Cooling to 30◦ C followed the same general principles. To validate our results we used a control group of 7 animals, subjected to all procedures at 37◦ C (Fig. 3). In hamsters circadian rhythm influences both hormone levels and body temperature [28, 30]. Thus, reproducibility of results was guaranteed by performing all experiments at the same time during morning hours. This had the additional advantage of performing hypothermia on sleeping animals, thus further reducing the stress levels and adding to humane care. External influences on circadian rhythm and animal hormone levels were controlled by keeping animals in laboratory conditions with a stable circadian light source and under controlled room temperature. 2.2. Experimental setup The tube system and the further setup used is portrayed in Figs. 4–6. On the two open ends of the outside tube system short plastic tubes connected to a bypass pump were put. The pump (EDEN 104, Tauchpumpe, EDEN S.R.L, Cartigliano, Italy) was based in a 2 liter water filled beaker which was put on a heating pad with a combined magnetic stirrer (MR 3001, Heidolph Instruments GmbH, Schwabach, Germany). In this setup water of any desired temperature could be pumped through the tube system without significant loss of energy, allowing for precise regulation of the temperature of the copper tube
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Fig. 2. Sample graph of tube temperature and resulting core body temperature (Tc ) over a period of 60 minutes. The target temperature of 18◦ C core body temperature (Tc) is reached after about 20 minutes. ECC at 18◦ C core body temperature (Tc) is performed for about 30 minutes. The animal regained normal core body temperature (Tc) after 15 minutes of rewarming.
Fig. 3. Core body temperature (Tc ) of awake hamsters during the experiment. Control group (n = 7 solid bars) received normothermia. In the experimental group (n = 7 empty bars), hypothermia to 18◦ C was applied. Data are mean ± SD. The drop of core body temperature (Tc ) is statistically significant from controls as early as 10 min after onset of cooling and returns to baseline values at the end of the 60 min observation period (P < 0.05).
(Fig. 7). To guarantee uniform water temperature in the beaker a magnetic stirrer was used continuously. Room temperature was kept at 18◦ C during cooling procedures and at 25◦ C during rewarming. Water temperature and body temperature of the hamster were continuously recorded with a two channel digital thermometer (Data-Logger Thermometer, Conrad Electronic GmbH, Essen, Germany). One sensor
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Fig. 4. Original image of the copper tube system.
Fig. 5. Cross-section drawing of the tube system.
was placed in the beaker. The other was modified as a rectal thermometer measuring the hamster’s core body temperature. The temperature of the copper tube system was monitored using a probe fixed to the wall of the tube system. Data was recorded with a second data logger.
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Fig. 6. Drawing of the experimental setup.
2.3. Laboratory animal Before dorsal skinfold chamber implantation animals were kept in groups of 3–6 animals. They had to be well nourished with normal condition of coat, eyes and natural orifices. Breathing movements, posture, response to stimuli in the environment and the orientation and movement activity had to be normal. From the time of implantation, animals were kept in individual cages in order to avoid injuries. Room temperature was constant at 21 ± 2◦ C, with a relative humidity of 55 ± 5%. Drinking water and standard laboratory chow (10H10, standard diet for rat - mouse – hamster, Nohrlin GmbH, Bad Salzuflen, Germany) were always available ad libitum. Standard bedding for laboratory rodents was used. Hamsters were exposed to a 12-hour alternating light-dark cycle. Animals were observed postinterventionally for a maximum of 5 days, after which they were euthanized. High value was attributed to adequate behaviour of the animals. For cases of lack of food and fluid intake and decreasing body weight, shaggy fur, altered body orifices, high frequent breathing, changes in posture like a bended back, decreased response to environmental stimuli and lack of physical activity euthanasia was discussed following a short-term observation. All animals received humane care in compliance with the European Convention on Animal Care and the study was approved by the Institutional and Regional Committee for Animal Care. All operative procedures where performed under Narcoren (60 mg/kg body weight, Pentobarbital, Merial, Hallbergmoos, Germany) anaesthesia and aseptic conditions. Chambers and instruments were sterilized before use. 2.4. Measurement of cardiovascular parameters The arterial catheter was connected with a pressure dome (Uniflow TM , Baxter, Germany). Heart rate (fh ) [min−1 ] and mean arterial blood pressure (mABP) [mmHg] were recorded pre, during and post cooling procedure via a monitor (Servomed, Hellige, Germany) and a flatbed recorder (L250, Linseis, Selb, Germany).
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Fig. 7. Sample graph of water temperature and resulting tube temperature over a period of 10 minutes. The experimental set-up allowed a precisely regulation of the copper tube temperature via regulation of the water temperature.
2.5. Microscopic observation Microscopic observations in the skinfold chamber could be performed prior, during and after the cooling procedure using an intravital microscope (Leica DMLM, Wetzlar, Germany) with a 20xSW 0.40 BD NA objective (Leica Fluotar, Wetzlar, Germany) (Fig. 8). A 100-W Hg light source was used for epiillumination. Contrast enhancement for transillumination was accomplished with a blue filter (420 nm), which selectively passes light in the maximum absorption band of hemoglobin, causing red blood cells to appear as dark objects in an otherwise gray background. A heat filter was placed in the light path prior to the condenser. Microscopic images were viewed by a closed circuit video system consisting of a CCD camera (Kappa CF 8/4 NIR, Gleichen, Germany) and a RGB-monitor (Sony, Japan). Leukocytes were stained with rhodamine 6 (Sigma, St Louis, MO) and classified by fluorescence microscopy according to their interaction with the endothelial lining as adherent, rolling or free flowing cells. Functional capillary density (FCD) was assessed in nine successive microscopic fields by transillumination in a region of approximately 1.3 mm2 ; the initial field was chosen where microvessels were in focus and there were usually between two and five RBC-perfused capillaries in the field of view. FCD was evaluated by measuring the length of capillaries that had red blood cell flow. A capillary was defined to be functional if passing RBCs were noted within the entire 20-s observation period. Microvascular permeability was analyzed by quantifying the extravasation of the macromolecular fluorescent marker FITC-Dextran (Mw 150,000) with the computer-assisted microcirculation analysis system (CAPIMAGE) [21]. Measurements were performed 15 min after intravenous injection of the fluorescent marker at the end of each study. In each vessel, gray-levels over the tissue directly adjacent to the vessel wall (E1) and over the marginal cell-free plasma layer within the vessel (E2) were determined over a length of at least 100 m. Extravasation (E) was then calculated as EZE1/E2.
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Fig. 8. The capillaries (arrows) are visible in their strictly parallel course. They show cross-links between each other. The distance is about 30–50 microns. In the background one can see a postcapillary venule. (Intravital fluorescence microscopy, contrast enhancement with FITC-dextran in blue-light epi-illumination, scale: 50 microns).
3. Results 3.1. Experimental tolerance Overall, hamsters showed good tolerance of the described procedure. After rewarming, 6 hamsters cooled to 18◦ C regained normal behaviour within 2 to 11 hours, five of which did not show behavioural sequelae over the next 5 days. One hamster cooled to 18◦ C died 28 hours after the experiment because of an aortic clot probably caused by the arterial catheter. The seventh hamster cooled to 18◦ C was euthanized 18 hours after the experiment because of abnormal feeding habits and behaviour. In the mild hypothermia group and in the 37◦ C control group no detrimental effects of the procedure were observed until sacrifice. 3.2. Cardiovascular parameters At the beginning and at the end of experiments mean arterial blood pressure amounted to 80–90 mmHg in all groups. During the 18◦ C cooling process, blood pressure decreased significantly by up to 50 % as compared to baseline. In the other groups it only showed non-significant physiological changes during the whole trial period (Table 1). No significant differences in heart rate fh [min-1] were observed either amongst or within groups during cooling. Mean fh ranged between 260 – 310 beats [min-1] and was within physiological limits. In the group of animals cooled to 18◦ C a significant decrease of heart rate to 25 % of baseline-value could be observed during cooling (Table 1) while animals of other groups showed only non-significant physiological changes in heart rate. These results are consistent with published literature and are within physiologic limits for species and body temperature [14].
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Table 1 Mean arterial blood pressure and heart rate during normothermia and hypothermia Parameter
Experiment
Baseline
Goal temperature
End
37◦ C 30◦ C 18◦ C
81.8 ± 8.9 83.4 ± 5.9 82.9 ± 7.3
84.2 ± 10.3 80.3 ± 6.2 45.8 ± 6.4*
83.1 ± 7.9 80.4 ± 9.1 86.2 ± 10.7
37◦ C 30◦ C 18◦ C
278.2 ± 18.4 273.7 ± 14.3 286.4 ± 20.9
286.4 ± 16.9 276.1 ± 12.1 76.3 ± 14.4*
289.8 ± 20.2 280.3 ± 18.2 290.9 ± 17.3
mABP [mmHg]
fh [min−1 ]
Data are mean ± SD, each group n = 7; ∗ P < 0.05 hypothermia [18◦ C] vs. normothermia [37◦ C].
3.3. Microvascular parameters 3.3.1. Leukocyte/endothelial cell interaction in arterioles and postcapillary venules The majority of fluorescently labeled leukocytes did not interact with endothelial cells in arterioles and postcapillary venules. There were no significant differences between the 18◦ C cooling and the 37◦ C control group prior and after the procedure up to 8hs post intervention observation time. 3.3.2. Functional capillary density FCD was significantly less in animals at 18◦ C body temperature compared with that seen in the 37◦ C and 30◦ C group (84 ± 15 vs. 151 ± 14 vs. 142 ± 16 cm/cm2 , P < 0.05), but it restored after rewarming. 3.3.3. Microvascular permeability Deep hypothermia significantly increased the edema formation in all microvascular compartments. In the perivascular tissue around the arterioles it was about 20% higher after the 18◦ C cooling process compared to the 30◦ C group and the 37◦ C control group (microvascular permeability compared in % of BL; P < 0.05 18◦ C vs. 37◦ C). 4. Discussion Hypothermia is increasingly used as a therapeutic strategy in a diversity of clinical scenarios from perinatal asphyxia to trauma, shock and cardiac surgery. Its impact on mammalian physiology, particularly on the microcirculatory changes of critical organ systems, are, however, incompletely understood. A better understanding of the pathophysiologic effects of systemic hypothermia in mammals is thus not only urgent but paramount to the safe clinical use of this promising therapeutic approach. Cheap, efficient and accessible animal models are a crucial step in answering the most pressing scientific questions surrounding the clinical use of hypothermia. Hypothermia in humans is carried out both in the presence and absence of anaesthesia and ECC. Thus, a good animal model should allow its effects to be studied in all of these potential settings. However, none of the previously described small animal models of hypothermia offered these possibilities [6, 22, 26, 29]. Moreover, they either required the animal to be anaesthetised or
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achieved only inaccurate core body temperature control, thus introducing important confounders, such as the proven detrimental effect of general anaesthesia on microvascular perfusion [3, 5, 23]. The experimental setup herein presented thus offers a vast improvement over previously available small animal models, allowing for systemic hypothermia effects to be studied both in the presence and absence of anaesthesia and ECC, as required. In the presents study a hibernator was chosen for medico-ethical reasoning to establish the tolerance to our experimental setup in non-sedated mammals. Our data confirms excellent tolerance of the procedure with low stress levels for the animals, making an experimental model with a tube setup a humane and animal-friendly option providing high body surface contact for any tube-dwelling mammal. We expect these results to be applicable to other non-hibernating tube dwelling animals. Copper was chosen in the production of the tube as it is an extremely efficient thermal conductor, easy to purchase and inexpensive compared to other metals with the same characteristics. Our results regarding the microcirculatory effects of deep hypothermia strongly support the need for further investigation, as we found not only a markedly reduced microvascular perfusion, but also increased vascular permeability and extravasation. Furthermore we observed a significantly reduced L/E cell interaction in the early post-ECC period. Despite a reduced number of adherent leukocytes we saw no protection of endothelial barrier function after ECC in deep hypothermia compared to ECC under normothermia [17]. In summary, the experimental setup herein described is cheap, animal friendly and can be potentially modified for any small mammal, including the possibilities for ECC, dorsal skinfold chamber and anaesthesia but not requiring any of these to work. It presents significant improvements on other established small animal hypothermia models, not least because of the precision of Tc achieved. Furthermore, the present setup may serve as an improvement for available perinatal asphyxia and hypothermia models in rats [4, 34]. Thus, we believe that this novel hypothermia small animal model provides invaluable new possibilities for hypothermia research across all specialties. Acknowledgments None. References [1] P.N. Amess, J. Penrice, E.B. Cady, A. Lorek, M. Wylezinska, C.E. Cooper, P. D’Souza, L. Tyszczuk, M. Thoresen, A.D. Edwards, J.S. Wyatt and E.O. Reynolds, Mild hypothermia after severe transient hypoxia-ischemia reduces the delayed rise in cerebral lactate in the newborn piglet, Pediatr Res 41(6) (1997), 803–808. [2] J.F. Ashruf, H.A. Bruining and C. Ince, New insights into the pathophysiology of cardiogenic shock: The role of the microcirculation, Curr Opin Crit Care 19(5) (2013), 381–386. [3] Z. Brookes, C. Reillyand and N. Brown, Differential effects of propofol, ketamine, and thiopental anaesthesia on the skeletal muscle microcirculation of normotensive and hypertensive rats in vivo, Br J Anaesth 93 (2004), 249–256. [4] M.L. Dalen, X. Liu, M. Elstad, E.M. Løberg, O.D. Saugstad, T. Rootwelt and M. Thoresen, Resuscitation with 100% oxygen increases injury and counteracts the neuroprotective effect of therapeutic hypothermia in the neonatal rat, Pediatr Res 71(3) (2012), 247–252. [5] R.A. De Blasi, S. Palmisani, M. Boezi, R. Arcioni, S. Collini, F. Troisi and G. Pinto, Effects of remifentanil-based general anaesthesia with propofol or sevoflurane on muscle microcirculation as assessed by near-infrared spectroscopy, Br J Anaesth 101(2) (2008), 171–177. [6] D. Deveci and S. Egginton, Effects of acute and chronic cooling on cardiorespiratory depression in rodents, J Physiol Sci 57(1) (2007), 73–79.
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