- Email: [email protected]
Unbalanced thermoregulation in experimental autoimmune encephalitis induced in Lewis rats
Journal of Thermal Biology xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Thermal Biology journal homepage: http://www.elsevier.com/locate/jtherbio
Unbalanced thermoregulation in experimental autoimmune encephalitis induced in Lewis rats Sylwia Wrotek a, *, Anna Nowakowska b, Michał Caputa b, Wiesław Kozak a a
Department of Immunology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland Department of Animal Physiology and Neurobiology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Experimental autoimmune encephalitis (EAE) Multiple sclerosis Preferred ambient temperature (Ta) Body temperature (Tb) Thermal gradient Set point
Thermoregulation in patients suffering from multiple sclerosis (MS) is impaired and may result in either increases or decreases in body temperature. We have found that rat experimental autoimmune encephalitis (EAE), being a model of MS, is associated with body temperature disturbances as well. The purpose of the current study was to examine whether the altered body temperature in EAE-induced rats is due to either a deficit in thermoregulation or a controlled change in its set point. Subcutaneous injection of encephalitogenic emulsion into both pads of hind feet of the Lewis rats provoked EAE symptoms. Body temperature (Tb) of 6 rats was measured using biotelemetry system, and ambient tem perature (Ta) preferred by 6 rats of another group was analyzed using thermal gradient system. Symptoms of EAE started 11 days postinjection and progressed quickly, culminating in a complete paralysis in rats placed in the gradient, which was associated with behavioural fever (accordingly, selected Ta raised to as much as 32.8 � 0.5 � C vs 27.2 � 0.6 � C in control rats). On the other hand, EAE rats, placed at a constant Ta of 24 � C, were able to generate fever (Tb of 37.8 � 0.1 � C) at the start of the illness and then paralysis compromised fever (most likely due to an impairment of thermogenesis), which, surprisingly, resulted in recovery. We conclude that EAE onset in rats is associated with fever and its behavioural supporting leads to aggravation of the autoimmune neurotoxicity.
1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (Compston and Coles, 2008). It is the un predictable, often disabling disease that disrupts the flow of information within the brain, and between the brain and body. It has been reported that temperature regulation in MS patients is impaired and may result in both an increase of body temperature (Tb) or its drop to as low as 30.0 � C (Weiss et al., 2009; Linker et al., 2006). Experimental autoimmune encephalitis (EAE) is the most intensively exploited animal model of MS (Slaney et al., 2013; Slavin et al., 2010; Chan et al., 2008). The papers suggest that there are both similarities and differences in various disease symptoms between EAE and MS subjects. In our previous paper (Wrotek et al., 2014), we have shown that before EAE onset Tb increases and during the onset it decreases. It is well known that body temperature in mammals is regulated by
neurons located in the preoptic area of the hypothalamus. This region contains a central thermostat that keeps core body temperature within a narrow range despite broad fluctuations in ambient temperature (Ta) (Biddle, 2006). According to the current concept, Tb follows the thermostatic set point of the thermoregulatory center and in consequence adjustable increase in Tb (fever) or decrease (anapyrexia) can be observed (Cabanac and Brinnel, 1987; Caputa, 2005). Sometimes, however, Tb changes are unrelated to the set point shifts and then are considered a failure in temperature regulation, leading to hyperthermia (forced increase in Tb) or hypothermia (forced decrease in Tb) (Cabanac and Brinnel, 1987; Gordon, 1993, Wrotek et al., 2011). To date, temperature gradient chambers, in which animals have the opportunity to move along them to select a Ta adequate for their ther moregulatory needs, have been widely used as a mean of recording of the behavioral thermoregulatory responses in rodents subjected to a variety of factors (Marques et al., 1984; Spencer et al., 1990). Here, the
Abbreviations: EAE, experimental autoimmune encephalitis; MS, multiple sclerosis; Tb, body temperature; Ta, ambient temperature. * Corresponding author. Department of Immunology, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland. E-mail addresses: [email protected] (S. Wrotek), [email protected] (A. Nowakowska), [email protected] (M. Caputa), [email protected] (W. Kozak). https://doi.org/10.1016/j.jtherbio.2020.102529 Received 29 October 2019; Received in revised form 27 January 2020; Accepted 28 January 2020 Available online 30 January 2020 0306-4565/© 2020 Nicolaus Copernicus University, ToruD, Poland. Published by Elsevier Ltd.
This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: Sylwia Wrotek, Journal of Thermal Biology, https://doi.org/10.1016/j.jtherbio.2020.102529
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
thermal gradient system was used to distinguish whether altered Tb is due to a controlled resetting of the body thermostat or is the result of a deficit in the autonomic temperature regulation (Gordon, 1987). Accordingly, changes in Tb accompanied by parallel shifts in selected Ta should be regarded as an evidence of the set point shifts. The aim of the current paper was to investigate whether the altered Tb in EAE-induced rats, previously described by us (Wrotek et al., 2014), result or not from the set point changes.
Ta selected by each animal was recorded by a series of copperconstantan thermocouples (Physiotemp Instruments, Clifton, New Jer sey) connected in pairs to infrared sensors, which in turn were connected to a computer. A signal for recording the rat’s position in the apparatus was triggered when the radiation between facing transmitter (infrared light emitting diode IRED L-535F4BT) and receiver (photodiode BPW84) was blocked by the rat. Information from the corresponding thermocouple was recorded by the computer as a Ta chosen by each rat at the particular moment. All data were automatically recorded and computed at 1-min intervals using custom data acquisition computer application GRAD. Every 12 h the gradient chambers were cleaned up by the same person and the food and water were changed. Food and water were available ad libitum. A 12:12h light-dark photoperiod was maintained. The experiments were carried out in an air-conditioned room at a temperature of 24�1 � C. A group of 6 animals was placed into the gradient chambers immediately post immunization and the recording was continued according to the pro cedure described in the section 2.1. Data from the second day of the recording were collected as a control preceding EAE development. Tb was recorded for 17 days (in parallel with recording of selected Ta in rats placed in the gradient chambers).
2. Materials and methods Specific pathogen-free male Lewis rats weighing 250–300g were used throughout the experimentation. The animals were obtained from Experimental and Clinical Medical Institute Warsaw (Poland). They were housed in individual plastic cages and maintained in a temperature/humidity/light-controlled chamber set at 24 � 1 � C, 12:12 h light:dark cycle, with light on at 07:00 AM. Rodent laboratory chow and drinking water were provided ad libitum. All experimental proced ures were approved by the Local Bioethical Committee for Animal Care (permission No. 12/2011 and 1/2012). 2.1. Induction of EAE
2.4. Data analysis
EAE was induced in rats by immunization with a guinea pig spinal cord homogenate in Complete Freund’s Adjuvant (CFA) as described previously (Wrotek et al., 2014). The homogenate was emulsified in CFA to a concentration of 1 mg/ml and M. tuberculosis and was added to the emulsion (3 mg/ml of the adjuvant). The adjuvant and M. tuberculosis were purchased from Difco Laboratories Inc. (Detroit, MI). Under light isofluran anesthesia, rats were intradermally inoculated into the pads of both hind feet with 0.1 ml of the encephalitogenic emulsion. Clinical signs of EAE were graded daily at 09:00 AM (starting from the seventh day post-immunization) according to the following criteria (Constantinescu et al., 2011): 1. Floppy tail, 2. Floppy tail and one hind limb paralysis, 3. Floppy tail and complete hind limbs paral ysis, 4. Floppy tail, complete hind limbs paralysis, difficulty in breath ing. The neurological symptoms were monitored four times daily. When an animal showed signs of clinical grade 4, it was subjected to eutha nasia by means of ketamine overdose injected intraperitoneally.
The body and ambient temperature recordings were pooled into 12-h averages. Statistical significance was determined by analysis of vari ance. Two-way of ANOVA (score x night-/day-time) was applied to analyze body temperature and selected temperature at each stage of disease. As a post hoc the Tukey test was used. The threshold of statis tical significance was p < 0.05 for all tests. 3. Results 3.1. Development of neurological symptoms in EAE-induced rats The immunization of rats using the encephalitogenic emulsion resulted in a gradual development of paralytic disease (Fig. 2). It affected tail muscles (floppy tail) and then hind legs (hind limbs paral ysis) and finally progressed cranially. In rats kept at a Ta of 24 � C as well as in those kept in the thermal gradient chambers clinical signs of the disease appeared on day 11th post-immunization. Neurological assess ment demonstrated progressive development of paralysis of tail and hind limbs leading to paraplegia and loss of spinal reflexes in immunized animals. It should be stressed that rats, which were kept at a Ta of 24 � C, reached a moderate peak of the disease symptoms (the score 3) within 6 days, and gradual recovery from the illness was observed afterwards. Surprisingly, rats housed in the gradient chambers revealed stronger symptoms of EAE than the animals living at the constant Ta (Fig. 2) and after reaching signs of clinical grade 4 were subjected to the euthanasia
2.2. Body temperature recording Deep body temperature (Tb) of six EAE-induced rats was monitored in their home cages using battery-operated telemetry transmitters (model TA-F40; Data Sciences International, New Brighton, MN) implanted intra-abdominally under sterile conditions as described pre viously (Wrotek et al., 2014). Rats were briefly anaesthetized with a mixture of ketamine (Biowet, Pulawy, Poland)/xylazine (ScanVet, Poland) (87 mg/kg and 13 mg/kg, respectively) injected intramuscu larly. Then, following shaving and sterilization of the small abdomen surgical area, an incision was made in the skin and muscles of the abdomen, and a miniature temperature-sensitive telemetry device was placed into the peritoneal cavity. The abdominal muscles and the skin were separately sutures closed. All experiments started 10 days after recovery from the surgery. The effect of EAE induction on Tb of the rats was evaluated. For technical reasons, the measurement of body tem perature in thermal gradient chambers was impossible. 2.3. Analysis of ambient temperatures preferred by rats in thermal gradient Thermal behavior of six rats was examined using a battery of six custom-made, insulated aluminum chambers (1.8 m long and 0.1 m wide), cooled at one end by a cryostat, and simultaneously heated at the opposite end by a thermostat (Fig. 1). The temperatures along the chambers ranged from 5 to 35 � C.
Fig. 1. Experimental setup for recording thermal behavior of rats: A - thermal gradient chamber (temperature range from 5 to 35 � C); B - fluid chambers; C thermostat; D - cryostat; E electronic switch of thermocouples; F - scanner; G transmitters of infrared radiation; H - receivers of infrared radiation; I - ther mocouples; J - computer. 2
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
Fig. 2. Neurological symptoms of EAE in consecutive days post-immunization in rats exposed to a constant Ta of 24 � C and in those placed in the thermal gradient. Clinical score: 1. Floppy tail, 2. Floppy tail and one hind limb pa ralysis, 3. Floppy tail and complete hind limbs paralysis, 4. Floppy tail, com plete hind limbs paralysis, difficulty in breathing. Each group consisted of 6 rats.
17–19 days post-immunization. 3.2. Body temperature and ambient temperature preferences in EAEinduced rats Rats are nocturnal animals revealing low day-time and high nighttime Tb. In our previous paper (Wrotek et al., 2014) we have shown an inverted pattern of daily Tb changes during the first days after EAE manifestation. In the present research we continued Tb recording much longer, which evidenced, between 14 and 15 days post-injection, two days long increase in day- and night-time Tb comparing with their control values (Fig. 3A). Simultaneously, there was a progressive elevation in selected Ta (Fig. 3B). Two way ANOVA showed that the Tb was group-dependent (F(3.576) ¼ 186.82, p < 0.001), was highly affected by day-time (F(1.576) ¼ 506.01, p < 0.001), and interaction between those factors was also highly significant (F(3.576) ¼ 60.70, p < 0.001). The highest Tb, recorded at the stage 3 of the illness, both during day-time (37.6 � 0.1 � C) and night-time (37.8 � 0.1 � C) (Fig. 4A) correlated with the highest Ta of 31.4 � 0.5 � C and 32.8 � 0.5 � C, respectively, selected by EAE-induced rats at the same stage (Fig. 4B). It should be stressed that these ambient temperatures were as much as 7.4 and 8.8 � C higher than that available to EAE-induced rats kept in their home cages. Both the maximum Tb and Ta were highly significantly (p < 0.001) higher than those in the control period. On the other hand, the final Tbs recorded both during the day and at night tended to decrease (Fig. 4A). Day-time and night-time ambient temperatures preferred by rats at the stage 2 and 3 were highly significantly different than those recorded in control period as well as at the stage 1 of the illness (p < 0.001). Two way ANOVA was used to analyze significance of changes in Ta selected in post-injection period. Selected Ta was affected by the illness stage (F(3.40) ¼ 40.05, p < 0.001), but was unaffected by day-time (F(1.40) ¼ 1.66, p < 0.2), and there was no interaction between these factors (F(3.40) ¼ 2.79, p < 0.5). Lack of the day-time effect seems to be obvious due to a clear reversal of the pattern of daily changes in selected Ta during development of the illness (Fig. 4B). In the control period pre ceding induction of the illness rats tended to select higher Ta during the day than that at night. On the other hand, during development of the illness they tended to select higher Ta at night than that during the day and the difference tended to increase with the progression of the illness.
Fig. 3. Representative time courses of changes in body temperature of the EAE rat No. 1 housed at 24 � C (panel A) and selected ambient temperature in the EAE rat No. 3 placed in the thermal gradient chamber (panel B) in consecutive days post-immunization. The stages (1–3) of EAE are indicted by vertical lines; data from the last day and night preceding the EAE induction were used as control values. Black horizontal bars represent light-off phases of the diurnal cycle.
4. Discussion Although criticism has been levied against EAE as an accurate rep resentation of MS, having such an animal model enables completion of experiments that are not allowed in humans, and if the studies are designed appropriately, the relevance of the results to MS is unques tionable. Therefore, the precise analysis concerning all aspects of this model is needed. In our previous paper (Wrotek et al., 2014) we showed that EAE model reflects Tb disturbances which likewise were observed in MS patients (Weiss et al., 2009. Linker et al., 2006; White et al., 1996). We have found that EAE-induced rats react with increased Tb before EAE onset, whereas just after EAE manifestation, a gradual decrease of Tb was recorded (Wrotek et al., 2014). However, we have been unable to determine whether these changes were due to a failure in thermoregu lation or were the set point-dependent. To resolve this problem in the present investigation we used the temperature gradient system. It should be stressed that control injection of complete Freund’s adjuvant without the spinal cord antigens in the above mentioned paper did not affect body temperature of rats over a period of the first three days of EAE manifestation. The increase in Tb together with the raising Ta preferences recorded 3
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
therefore, they must have been able to progressively enhance their fever response, with the development of the illness. Such a failure of ther mogenesis seems to be obvious in EAE rats due to paralysis started with tail muscles and hind limbs and then progressing cranially. However, nonshivering thermogenesis, which is well developed in rodents (Gor don, 1993) must have not been sufficient to fully compensate for the failure. Since rats counteracted the reduction in Tb by selecting pro gressively higher Ta, we think that the thermoregulatory center of EAE-induced rats works properly, but its autonomic effector mecha nisms are insufficient. This is why EAE rats rely more and more on behavioral mechanisms supporting fever. Unfortunately, for technical reasons, we were unable to use our telemetric system of Tb recording in the thermal gradient chambers. Therefore, in this group of rats we could only apply a rough extrapolation of a trend in Tb shifts from changes in selected Ta. Rats are nocturnal animals revealing low day-time and high night-time Tb (Gordon, 1987, 1993, 1994). In our control EAE rats the diurnal rhythm of Tb was maintained throughout the recording period (preceding devel opment of the paralytic disease) (see Figs. 3A and 4A). On the other hand, the relationship of the rhythm to daily shifts in thermal preference of EAE rats changed with the occurrence of the paralytic symptoms. A couple of authors have found that the diurnal rhythm of selected Ta in rodents is opposite to the rhythm of Tb (Gordon, 1993). Accordingly, rats select significantly higher Ta during their day-time rest than during their night-time activity (Gordon, 1987, 1993, 1994). This suggests that they compensate the metabolic heat excess, resulting from their nocturnal motor activity, with a reduction in selected Ta, comparing with that recorded during their day-time rest. In the period preceding development of the paralysis our EAE-induced rats presented the above mentioned pattern of diurnal changes in selected Ta (see Figs. 3B and 4B), although, due to the extremely small number of the experimental animals, the changes did not reach the threshold of statistical significance. With the development of paralytic symptoms the pattern of diurnal changes in selected Ta inverted, i.e. the changes became parallel to diurnal Tb shifts. This finding is compatible with a strong inhibition of the night-time motor activity in rats suffering from EAE, recorded in our previous paper (Wrotek et al., 2014). An explanation for the reversal in the pattern is the necessity of behavioral supporting of the normal diurnal rhythm of Tb. In our pre vious paper (Wrotek et al., 2014) we have shown that (without such a support) the Tb rhythm was inverted on the first and second day of EAE manifestation. At first glance, the most unexpected result of the present investigation was a clear-cut aggravation of the illness in rats given the opportunity to select Ta (see Fig. 2), and so to compensate for the failure of the muscular thermogenesis, resulting from the progressive paralysis. However, sustained fever must have led to increased neurotoxicity. Such a relationship has been repeatedly shown in patients suffering from spontaneous subarachnoid hemorrhage (Takagi et al., 2003), acute cerebral infraction (Fukuda et al., 1999) and acute ischemic stroke (Wrotek et al., 2011, Reith et al., 1996). Accordingly, fever (which generally exerts strong cytotoxic effects) exacerbates the neuronal injury while the reduction in Tb provides a natural neuroprotection. Therefore, autoimmune diseases belong to exceptions to the rule of the beneficial role of fever. Our EAE rats placed in the thermal gradient, besides being exposed to the aggravation of the autoimmune neurotoxicity, must have also been suffering from cerebral hypoxia due to difficulty in breathing at stage 4 of the illness, which must have exacerbated the injury further. Our main finding is also consistent with an enhancement of the multiple sclerosis symptoms in patients due to higher Tb or Ta, which is a well- known phenomenon. The worsening has been reported due to exercise (van Diemen et al., 1992), hot shower (Waxman and Geschwind, 1983), and sunbathing (Avis and Pryse-Phillips, 1995). Even fluctuations in the circadian Tb from the morning to the afternoon can influence the symp toms (Romani et al., 2000).
Fig. 4. Twelve hours averages of day-time and night-time body temperatures of EAE-induced rats housed at a constant Ta of 24 � C (n ¼ 6) (panel A) and the averages of preferred ambient temperatures (n ¼ 6) in EAE rats placed in the thermal gradient chambers (panel B) recorded at the stages 1–3 of the illness. Data from the last day and night preceding the EAE induction were used as control values. Asterisks indicate significant differences (***p < 0.001).
in the present investigation suggests appearance of fever but by no means hyperthermia in EAE-induced rats. This finding is consistent with observation by Bolton and coworkers (1984) who detected in the spinal cord of EAE-induced rats prostaglandin E, which is a key factor trig gering fever (Kozak et al., 2006). The authors have found that the earliest change recorded in EAE-induced rats was an elevation of PGE 5–7 days post-inoculation and prior to the appearance of clinical symptoms. PGE content continued to rise until days 12–14 post-injection when the animals displayed paralytic EAE (Bolton et al., 1984). PGE reached its maximum at the onset of neurological symptoms, 11–12 days after inoculation (Bolton et al., 1984). In accordance, Schiffmann et al., 2014 demonstrated that PGE2 synthesized by monocytes in the early preclinical phase promoted the development of EAE in an EP4 receptor dependent manner. Compatible with the above mentioned findings are those described in recently published papers, which indicate that anti pyretic agents such as aspirin inhibit EAE (Mondal et al., 2018; Pahan and Pahan, 2019). At the stage 2 of the illness our rats placed in the thermal gradient chamber started to show warmth-seeking behavior (see Figs. 3B and 4B). At that time, in our previous research (Wrotek et al., 2014) we noticed a night-time Tb decrease in comparison with that in control rats. Hansen and Pender (1989) recorded a deep hypothermia of 30 � C in EAE rats. This observation, taken together with that of a tendency to reduce final Tb in the present investigation, indicates that the illness is associated with a failure of shivering thermogenesis. The rats placed in the gradient chambers compensated for the failure by warmth-seeking behavior, and 4
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
5. Conclusions
Gordon, C.J., 1993. Temperature Regulation in Laboratory Rodents. Cambridge University Press. Gordon, C.J., 1994. 24-hour control of body temperature in rats. I. Integration of behavioral and autonomic effectors. Am. J. Physiol. 36, R71–R77. Hansen, L.A., Pender, M.P., 1989. Hypothermia due to an ascending impairment of shivering in hyperacute experimental allergic encephalomyelitis in the Lewis rat. J. Neurol. Sci. 94, 231–240. Kozak, W., Wrotek, S., Kozak, A., 2006. Pyrogenicity of CpG-DNA in mice: role of interleukin-6, cyclooxygenases, and nuclear factor-kappaB. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R871–R880. Linker, R.A., Mohr, A., Cepek, L., et al., 2006. Core hypothermia in multiple sclerosis: case report with magnetic resonance imaging localization of a thalamic lesion. Mult. Scler. 12, 112–115. Marques, P.R., Spencer, R.L., Burks, T.F., McDougal, J.N., 1984. Behavioral thermoregulation, core temperature, and motor activity: simultaneous quantitative assessment in rats after dopamine and prostaglandin E1. Behav. Neurosci. 98, 858–867. Mondal, S., Jana, M., Dasarathi, S., Roy, A., Pahan, K., 2018. Aspirin ameliorates experimental autoimmune encephalomyelitis through interleukin-11-mediated protection of regulatory T cells. Sci. Signal. 11 (558) https://doi.org/10.1126/ scisignal.aar8278 pii: eaar8278. Pahan, S., Pahan, K., 2019. Mode of action of aspirin in experimental autoimmune encephalomyelitis. DNA Cell Biol. 38 (7), 593–596. Reith, J., Jørgensen, H.S., Pedersen, P.M., Nakayama, H., Raaschou, H.O., Jeppesen, L.L., Olsen, T.S., 1996. Pedersen PM et al. Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome. Lancet 347, 422–425. Romani, A., Bergamaschi, R., Versino, M., Zilioli, A., Callieco, R., Cosi, V., 2000. Circadian and hypothermia-induced effects on visual and auditory evoked potentials in multiple sclerosis. Clin. Neurophysiol. 111, 1602–1606. Schiffmann, S., Weigert, A., M€ annich, J., Eberle, M., Birod, K., H€ aussler, A., et al., 2014. PGE2/EP4 signaling in peripheral immune cells promotes development of experimental autoimmune encephalomyelitis. Biochem. Pharmacol. 87, 625–635. Slaney, C.Y., Toker, A., Fraser, J.D., et al., 2013. A modified superantigen rescues Ly6GCD11bþblood monocyte suppressor function and suppresses antigen-specific inflammation in EAE. Autoimmunity 46, 269–278. Slavin, A., Kelly-Modis, L., Labadia, M., et al., 2010. Pathogenic mechanisms and experimental models of multiple sclerosis. Autoimmunity 43, 504–513. Spencer, R.L., Hruby, V.J., Burks, T.F., 1990. Alteration of thermoregulatory set point with opioid agonists. J. Pharmacol. Exp. Therapeut. 252, 696–705. Takagi, K., Tsuchiya, Y., Okinaga, K., Hirata, M., Nakagomi, Y., Tamura, A., 2003. Natural hypothermia immediately after transient global cerebral ischemia induced by spontaneous subarachnoid hemorrhage. J. Neurosurg. 98, 50–56. van Diemen, H.A., van Dongen, M.M., Dammers, J.W., Polman, C.H., 1992. Increased visual impairment after exercise (Uhthoff’s phenomenon) in multiple sclerosis: therapeutic possibilities. Eur. Neurol. 32, 231–234. Waxman, G.S., Geschwind, N., 1983. Major morbidity related to hyperthermia in multiple sclerosis. Ann. Neurol. 13, 348. Weiss, N., Hasboun, D., Demeret, S., et al., 2009. Paroxysmal hypothermia as a clinical feature of multiple sclerosis. Neurology 13, 193–195. White, K.D., Scoones, D.J., Newman, P.K., 1996. Hypothermia in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 61, 369–375. Wrotek, S.E., Kozak, W.E., Hess, D.C., Fagan, S.C., 2011. Treatment of fever after stroke: conflicting evidence. Pharmacotherapy 31, 1085–1091. Wrotek, S., Rosochowicz, T., Nowakowska, A., Kozak, W., 2014. Thermal and motor behavior in experimental autoimmune encephalitis in Lewis rats. Autoimmunity 47, 334–340.
The results of the present investigation show that: I. There is an upward resetting of the temperature regulation in EAE rats. II. Acute symptoms of EAE are exacerbated by fever, which means that autoimmune diseases belong to exceptions to the rule of the beneficial role of fever. III. Both the fever and normal pattern of the diurnal Tb shifts in EAE rats need a behavioral support to compensate for a strong deficit in muscular thermogenesis. IV. Accordingly, thermoregulation appears to be a highly redundant system. CRediT authorship contribution statement Sylwia Wrotek: Conceptualization, Investigation, Formal analysis, Visualization, Writing - original draft. Anna Nowakowska: Formal analysis, Investigation, Visualization, Writing - original draft. Michał Caputa: Conceptualization, Formal analysis, Supervision, Visualization, Writing - review & editing. Wiesław Kozak: Conceptualization, Supervision. References Avis, S.P., Pryse-Phillips, W.E., 1995. Sudden death in multiple sclerosis associated with sun exposure: a report of two cases. Can. J. Neurol. Sci. 22, 305–307. Biddle, C., 2006. The neurobiology of the human febrile response. AANA J. (Am. Assoc. Nurse Anesth.) 74, 145–150. Bolton, C., Gordon, D., Turk, J.L., 1984. Prostaglandin and thromboxane levels in central nervous system tissues from rats during the induction and development of experimental allergic encephalomyelitis (EAE). Immunopharmacology 7, 101–107. Cabanac, M., Brinnel, H., 1987. The pathology of human temperature regulation: thermiatrics. Experientia 43, 19–27. Caputa, M., 2005. Comments on "Do fever and anapyrexia exist? Analysis of set pointbased definitions. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R281 author reply R281-2. Chan, J., Ban, E.J., Chun, K.H., et al., 2008. Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis. Autoimmunity 41, 405–413. Compston, A., Coles, A., 2008. Multiple sclerosis. Lancet 25, 1502–1517. Constantinescu, C.S., Farooqi, N., O’Brien, K., Gran, B., 2011. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br. J. Pharmacol. 164, 1079–1106. Fukuda, H., Kitani, M., Takahashi, K., 1999. Body temperature correlates with functional outcome and the lesion size of cerebral infarction. Acta Neurol. Scand. 100, 385–390. Gordon, C.J., 1987. Relationship between preferred ambient temperature and autonomic thermoregulatory function in rat. Am. J. Physiol. 252, R1130–R1137.
5
Contents lists available at ScienceDirect
Journal of Thermal Biology journal homepage: http://www.elsevier.com/locate/jtherbio
Unbalanced thermoregulation in experimental autoimmune encephalitis induced in Lewis rats Sylwia Wrotek a, *, Anna Nowakowska b, Michał Caputa b, Wiesław Kozak a a
Department of Immunology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland Department of Animal Physiology and Neurobiology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Experimental autoimmune encephalitis (EAE) Multiple sclerosis Preferred ambient temperature (Ta) Body temperature (Tb) Thermal gradient Set point
Thermoregulation in patients suffering from multiple sclerosis (MS) is impaired and may result in either increases or decreases in body temperature. We have found that rat experimental autoimmune encephalitis (EAE), being a model of MS, is associated with body temperature disturbances as well. The purpose of the current study was to examine whether the altered body temperature in EAE-induced rats is due to either a deficit in thermoregulation or a controlled change in its set point. Subcutaneous injection of encephalitogenic emulsion into both pads of hind feet of the Lewis rats provoked EAE symptoms. Body temperature (Tb) of 6 rats was measured using biotelemetry system, and ambient tem perature (Ta) preferred by 6 rats of another group was analyzed using thermal gradient system. Symptoms of EAE started 11 days postinjection and progressed quickly, culminating in a complete paralysis in rats placed in the gradient, which was associated with behavioural fever (accordingly, selected Ta raised to as much as 32.8 � 0.5 � C vs 27.2 � 0.6 � C in control rats). On the other hand, EAE rats, placed at a constant Ta of 24 � C, were able to generate fever (Tb of 37.8 � 0.1 � C) at the start of the illness and then paralysis compromised fever (most likely due to an impairment of thermogenesis), which, surprisingly, resulted in recovery. We conclude that EAE onset in rats is associated with fever and its behavioural supporting leads to aggravation of the autoimmune neurotoxicity.
1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (Compston and Coles, 2008). It is the un predictable, often disabling disease that disrupts the flow of information within the brain, and between the brain and body. It has been reported that temperature regulation in MS patients is impaired and may result in both an increase of body temperature (Tb) or its drop to as low as 30.0 � C (Weiss et al., 2009; Linker et al., 2006). Experimental autoimmune encephalitis (EAE) is the most intensively exploited animal model of MS (Slaney et al., 2013; Slavin et al., 2010; Chan et al., 2008). The papers suggest that there are both similarities and differences in various disease symptoms between EAE and MS subjects. In our previous paper (Wrotek et al., 2014), we have shown that before EAE onset Tb increases and during the onset it decreases. It is well known that body temperature in mammals is regulated by
neurons located in the preoptic area of the hypothalamus. This region contains a central thermostat that keeps core body temperature within a narrow range despite broad fluctuations in ambient temperature (Ta) (Biddle, 2006). According to the current concept, Tb follows the thermostatic set point of the thermoregulatory center and in consequence adjustable increase in Tb (fever) or decrease (anapyrexia) can be observed (Cabanac and Brinnel, 1987; Caputa, 2005). Sometimes, however, Tb changes are unrelated to the set point shifts and then are considered a failure in temperature regulation, leading to hyperthermia (forced increase in Tb) or hypothermia (forced decrease in Tb) (Cabanac and Brinnel, 1987; Gordon, 1993, Wrotek et al., 2011). To date, temperature gradient chambers, in which animals have the opportunity to move along them to select a Ta adequate for their ther moregulatory needs, have been widely used as a mean of recording of the behavioral thermoregulatory responses in rodents subjected to a variety of factors (Marques et al., 1984; Spencer et al., 1990). Here, the
Abbreviations: EAE, experimental autoimmune encephalitis; MS, multiple sclerosis; Tb, body temperature; Ta, ambient temperature. * Corresponding author. Department of Immunology, Nicolaus Copernicus University, 1 Lwowska Str., 87-100, Torun, Poland. E-mail addresses: [email protected] (S. Wrotek), [email protected] (A. Nowakowska), [email protected] (M. Caputa), [email protected] (W. Kozak). https://doi.org/10.1016/j.jtherbio.2020.102529 Received 29 October 2019; Received in revised form 27 January 2020; Accepted 28 January 2020 Available online 30 January 2020 0306-4565/© 2020 Nicolaus Copernicus University, ToruD, Poland. Published by Elsevier Ltd.
This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: Sylwia Wrotek, Journal of Thermal Biology, https://doi.org/10.1016/j.jtherbio.2020.102529
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
thermal gradient system was used to distinguish whether altered Tb is due to a controlled resetting of the body thermostat or is the result of a deficit in the autonomic temperature regulation (Gordon, 1987). Accordingly, changes in Tb accompanied by parallel shifts in selected Ta should be regarded as an evidence of the set point shifts. The aim of the current paper was to investigate whether the altered Tb in EAE-induced rats, previously described by us (Wrotek et al., 2014), result or not from the set point changes.
Ta selected by each animal was recorded by a series of copperconstantan thermocouples (Physiotemp Instruments, Clifton, New Jer sey) connected in pairs to infrared sensors, which in turn were connected to a computer. A signal for recording the rat’s position in the apparatus was triggered when the radiation between facing transmitter (infrared light emitting diode IRED L-535F4BT) and receiver (photodiode BPW84) was blocked by the rat. Information from the corresponding thermocouple was recorded by the computer as a Ta chosen by each rat at the particular moment. All data were automatically recorded and computed at 1-min intervals using custom data acquisition computer application GRAD. Every 12 h the gradient chambers were cleaned up by the same person and the food and water were changed. Food and water were available ad libitum. A 12:12h light-dark photoperiod was maintained. The experiments were carried out in an air-conditioned room at a temperature of 24�1 � C. A group of 6 animals was placed into the gradient chambers immediately post immunization and the recording was continued according to the pro cedure described in the section 2.1. Data from the second day of the recording were collected as a control preceding EAE development. Tb was recorded for 17 days (in parallel with recording of selected Ta in rats placed in the gradient chambers).
2. Materials and methods Specific pathogen-free male Lewis rats weighing 250–300g were used throughout the experimentation. The animals were obtained from Experimental and Clinical Medical Institute Warsaw (Poland). They were housed in individual plastic cages and maintained in a temperature/humidity/light-controlled chamber set at 24 � 1 � C, 12:12 h light:dark cycle, with light on at 07:00 AM. Rodent laboratory chow and drinking water were provided ad libitum. All experimental proced ures were approved by the Local Bioethical Committee for Animal Care (permission No. 12/2011 and 1/2012). 2.1. Induction of EAE
2.4. Data analysis
EAE was induced in rats by immunization with a guinea pig spinal cord homogenate in Complete Freund’s Adjuvant (CFA) as described previously (Wrotek et al., 2014). The homogenate was emulsified in CFA to a concentration of 1 mg/ml and M. tuberculosis and was added to the emulsion (3 mg/ml of the adjuvant). The adjuvant and M. tuberculosis were purchased from Difco Laboratories Inc. (Detroit, MI). Under light isofluran anesthesia, rats were intradermally inoculated into the pads of both hind feet with 0.1 ml of the encephalitogenic emulsion. Clinical signs of EAE were graded daily at 09:00 AM (starting from the seventh day post-immunization) according to the following criteria (Constantinescu et al., 2011): 1. Floppy tail, 2. Floppy tail and one hind limb paralysis, 3. Floppy tail and complete hind limbs paral ysis, 4. Floppy tail, complete hind limbs paralysis, difficulty in breath ing. The neurological symptoms were monitored four times daily. When an animal showed signs of clinical grade 4, it was subjected to eutha nasia by means of ketamine overdose injected intraperitoneally.
The body and ambient temperature recordings were pooled into 12-h averages. Statistical significance was determined by analysis of vari ance. Two-way of ANOVA (score x night-/day-time) was applied to analyze body temperature and selected temperature at each stage of disease. As a post hoc the Tukey test was used. The threshold of statis tical significance was p < 0.05 for all tests. 3. Results 3.1. Development of neurological symptoms in EAE-induced rats The immunization of rats using the encephalitogenic emulsion resulted in a gradual development of paralytic disease (Fig. 2). It affected tail muscles (floppy tail) and then hind legs (hind limbs paral ysis) and finally progressed cranially. In rats kept at a Ta of 24 � C as well as in those kept in the thermal gradient chambers clinical signs of the disease appeared on day 11th post-immunization. Neurological assess ment demonstrated progressive development of paralysis of tail and hind limbs leading to paraplegia and loss of spinal reflexes in immunized animals. It should be stressed that rats, which were kept at a Ta of 24 � C, reached a moderate peak of the disease symptoms (the score 3) within 6 days, and gradual recovery from the illness was observed afterwards. Surprisingly, rats housed in the gradient chambers revealed stronger symptoms of EAE than the animals living at the constant Ta (Fig. 2) and after reaching signs of clinical grade 4 were subjected to the euthanasia
2.2. Body temperature recording Deep body temperature (Tb) of six EAE-induced rats was monitored in their home cages using battery-operated telemetry transmitters (model TA-F40; Data Sciences International, New Brighton, MN) implanted intra-abdominally under sterile conditions as described pre viously (Wrotek et al., 2014). Rats were briefly anaesthetized with a mixture of ketamine (Biowet, Pulawy, Poland)/xylazine (ScanVet, Poland) (87 mg/kg and 13 mg/kg, respectively) injected intramuscu larly. Then, following shaving and sterilization of the small abdomen surgical area, an incision was made in the skin and muscles of the abdomen, and a miniature temperature-sensitive telemetry device was placed into the peritoneal cavity. The abdominal muscles and the skin were separately sutures closed. All experiments started 10 days after recovery from the surgery. The effect of EAE induction on Tb of the rats was evaluated. For technical reasons, the measurement of body tem perature in thermal gradient chambers was impossible. 2.3. Analysis of ambient temperatures preferred by rats in thermal gradient Thermal behavior of six rats was examined using a battery of six custom-made, insulated aluminum chambers (1.8 m long and 0.1 m wide), cooled at one end by a cryostat, and simultaneously heated at the opposite end by a thermostat (Fig. 1). The temperatures along the chambers ranged from 5 to 35 � C.
Fig. 1. Experimental setup for recording thermal behavior of rats: A - thermal gradient chamber (temperature range from 5 to 35 � C); B - fluid chambers; C thermostat; D - cryostat; E electronic switch of thermocouples; F - scanner; G transmitters of infrared radiation; H - receivers of infrared radiation; I - ther mocouples; J - computer. 2
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
Fig. 2. Neurological symptoms of EAE in consecutive days post-immunization in rats exposed to a constant Ta of 24 � C and in those placed in the thermal gradient. Clinical score: 1. Floppy tail, 2. Floppy tail and one hind limb pa ralysis, 3. Floppy tail and complete hind limbs paralysis, 4. Floppy tail, com plete hind limbs paralysis, difficulty in breathing. Each group consisted of 6 rats.
17–19 days post-immunization. 3.2. Body temperature and ambient temperature preferences in EAEinduced rats Rats are nocturnal animals revealing low day-time and high nighttime Tb. In our previous paper (Wrotek et al., 2014) we have shown an inverted pattern of daily Tb changes during the first days after EAE manifestation. In the present research we continued Tb recording much longer, which evidenced, between 14 and 15 days post-injection, two days long increase in day- and night-time Tb comparing with their control values (Fig. 3A). Simultaneously, there was a progressive elevation in selected Ta (Fig. 3B). Two way ANOVA showed that the Tb was group-dependent (F(3.576) ¼ 186.82, p < 0.001), was highly affected by day-time (F(1.576) ¼ 506.01, p < 0.001), and interaction between those factors was also highly significant (F(3.576) ¼ 60.70, p < 0.001). The highest Tb, recorded at the stage 3 of the illness, both during day-time (37.6 � 0.1 � C) and night-time (37.8 � 0.1 � C) (Fig. 4A) correlated with the highest Ta of 31.4 � 0.5 � C and 32.8 � 0.5 � C, respectively, selected by EAE-induced rats at the same stage (Fig. 4B). It should be stressed that these ambient temperatures were as much as 7.4 and 8.8 � C higher than that available to EAE-induced rats kept in their home cages. Both the maximum Tb and Ta were highly significantly (p < 0.001) higher than those in the control period. On the other hand, the final Tbs recorded both during the day and at night tended to decrease (Fig. 4A). Day-time and night-time ambient temperatures preferred by rats at the stage 2 and 3 were highly significantly different than those recorded in control period as well as at the stage 1 of the illness (p < 0.001). Two way ANOVA was used to analyze significance of changes in Ta selected in post-injection period. Selected Ta was affected by the illness stage (F(3.40) ¼ 40.05, p < 0.001), but was unaffected by day-time (F(1.40) ¼ 1.66, p < 0.2), and there was no interaction between these factors (F(3.40) ¼ 2.79, p < 0.5). Lack of the day-time effect seems to be obvious due to a clear reversal of the pattern of daily changes in selected Ta during development of the illness (Fig. 4B). In the control period pre ceding induction of the illness rats tended to select higher Ta during the day than that at night. On the other hand, during development of the illness they tended to select higher Ta at night than that during the day and the difference tended to increase with the progression of the illness.
Fig. 3. Representative time courses of changes in body temperature of the EAE rat No. 1 housed at 24 � C (panel A) and selected ambient temperature in the EAE rat No. 3 placed in the thermal gradient chamber (panel B) in consecutive days post-immunization. The stages (1–3) of EAE are indicted by vertical lines; data from the last day and night preceding the EAE induction were used as control values. Black horizontal bars represent light-off phases of the diurnal cycle.
4. Discussion Although criticism has been levied against EAE as an accurate rep resentation of MS, having such an animal model enables completion of experiments that are not allowed in humans, and if the studies are designed appropriately, the relevance of the results to MS is unques tionable. Therefore, the precise analysis concerning all aspects of this model is needed. In our previous paper (Wrotek et al., 2014) we showed that EAE model reflects Tb disturbances which likewise were observed in MS patients (Weiss et al., 2009. Linker et al., 2006; White et al., 1996). We have found that EAE-induced rats react with increased Tb before EAE onset, whereas just after EAE manifestation, a gradual decrease of Tb was recorded (Wrotek et al., 2014). However, we have been unable to determine whether these changes were due to a failure in thermoregu lation or were the set point-dependent. To resolve this problem in the present investigation we used the temperature gradient system. It should be stressed that control injection of complete Freund’s adjuvant without the spinal cord antigens in the above mentioned paper did not affect body temperature of rats over a period of the first three days of EAE manifestation. The increase in Tb together with the raising Ta preferences recorded 3
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
therefore, they must have been able to progressively enhance their fever response, with the development of the illness. Such a failure of ther mogenesis seems to be obvious in EAE rats due to paralysis started with tail muscles and hind limbs and then progressing cranially. However, nonshivering thermogenesis, which is well developed in rodents (Gor don, 1993) must have not been sufficient to fully compensate for the failure. Since rats counteracted the reduction in Tb by selecting pro gressively higher Ta, we think that the thermoregulatory center of EAE-induced rats works properly, but its autonomic effector mecha nisms are insufficient. This is why EAE rats rely more and more on behavioral mechanisms supporting fever. Unfortunately, for technical reasons, we were unable to use our telemetric system of Tb recording in the thermal gradient chambers. Therefore, in this group of rats we could only apply a rough extrapolation of a trend in Tb shifts from changes in selected Ta. Rats are nocturnal animals revealing low day-time and high night-time Tb (Gordon, 1987, 1993, 1994). In our control EAE rats the diurnal rhythm of Tb was maintained throughout the recording period (preceding devel opment of the paralytic disease) (see Figs. 3A and 4A). On the other hand, the relationship of the rhythm to daily shifts in thermal preference of EAE rats changed with the occurrence of the paralytic symptoms. A couple of authors have found that the diurnal rhythm of selected Ta in rodents is opposite to the rhythm of Tb (Gordon, 1993). Accordingly, rats select significantly higher Ta during their day-time rest than during their night-time activity (Gordon, 1987, 1993, 1994). This suggests that they compensate the metabolic heat excess, resulting from their nocturnal motor activity, with a reduction in selected Ta, comparing with that recorded during their day-time rest. In the period preceding development of the paralysis our EAE-induced rats presented the above mentioned pattern of diurnal changes in selected Ta (see Figs. 3B and 4B), although, due to the extremely small number of the experimental animals, the changes did not reach the threshold of statistical significance. With the development of paralytic symptoms the pattern of diurnal changes in selected Ta inverted, i.e. the changes became parallel to diurnal Tb shifts. This finding is compatible with a strong inhibition of the night-time motor activity in rats suffering from EAE, recorded in our previous paper (Wrotek et al., 2014). An explanation for the reversal in the pattern is the necessity of behavioral supporting of the normal diurnal rhythm of Tb. In our pre vious paper (Wrotek et al., 2014) we have shown that (without such a support) the Tb rhythm was inverted on the first and second day of EAE manifestation. At first glance, the most unexpected result of the present investigation was a clear-cut aggravation of the illness in rats given the opportunity to select Ta (see Fig. 2), and so to compensate for the failure of the muscular thermogenesis, resulting from the progressive paralysis. However, sustained fever must have led to increased neurotoxicity. Such a relationship has been repeatedly shown in patients suffering from spontaneous subarachnoid hemorrhage (Takagi et al., 2003), acute cerebral infraction (Fukuda et al., 1999) and acute ischemic stroke (Wrotek et al., 2011, Reith et al., 1996). Accordingly, fever (which generally exerts strong cytotoxic effects) exacerbates the neuronal injury while the reduction in Tb provides a natural neuroprotection. Therefore, autoimmune diseases belong to exceptions to the rule of the beneficial role of fever. Our EAE rats placed in the thermal gradient, besides being exposed to the aggravation of the autoimmune neurotoxicity, must have also been suffering from cerebral hypoxia due to difficulty in breathing at stage 4 of the illness, which must have exacerbated the injury further. Our main finding is also consistent with an enhancement of the multiple sclerosis symptoms in patients due to higher Tb or Ta, which is a well- known phenomenon. The worsening has been reported due to exercise (van Diemen et al., 1992), hot shower (Waxman and Geschwind, 1983), and sunbathing (Avis and Pryse-Phillips, 1995). Even fluctuations in the circadian Tb from the morning to the afternoon can influence the symp toms (Romani et al., 2000).
Fig. 4. Twelve hours averages of day-time and night-time body temperatures of EAE-induced rats housed at a constant Ta of 24 � C (n ¼ 6) (panel A) and the averages of preferred ambient temperatures (n ¼ 6) in EAE rats placed in the thermal gradient chambers (panel B) recorded at the stages 1–3 of the illness. Data from the last day and night preceding the EAE induction were used as control values. Asterisks indicate significant differences (***p < 0.001).
in the present investigation suggests appearance of fever but by no means hyperthermia in EAE-induced rats. This finding is consistent with observation by Bolton and coworkers (1984) who detected in the spinal cord of EAE-induced rats prostaglandin E, which is a key factor trig gering fever (Kozak et al., 2006). The authors have found that the earliest change recorded in EAE-induced rats was an elevation of PGE 5–7 days post-inoculation and prior to the appearance of clinical symptoms. PGE content continued to rise until days 12–14 post-injection when the animals displayed paralytic EAE (Bolton et al., 1984). PGE reached its maximum at the onset of neurological symptoms, 11–12 days after inoculation (Bolton et al., 1984). In accordance, Schiffmann et al., 2014 demonstrated that PGE2 synthesized by monocytes in the early preclinical phase promoted the development of EAE in an EP4 receptor dependent manner. Compatible with the above mentioned findings are those described in recently published papers, which indicate that anti pyretic agents such as aspirin inhibit EAE (Mondal et al., 2018; Pahan and Pahan, 2019). At the stage 2 of the illness our rats placed in the thermal gradient chamber started to show warmth-seeking behavior (see Figs. 3B and 4B). At that time, in our previous research (Wrotek et al., 2014) we noticed a night-time Tb decrease in comparison with that in control rats. Hansen and Pender (1989) recorded a deep hypothermia of 30 � C in EAE rats. This observation, taken together with that of a tendency to reduce final Tb in the present investigation, indicates that the illness is associated with a failure of shivering thermogenesis. The rats placed in the gradient chambers compensated for the failure by warmth-seeking behavior, and 4
S. Wrotek et al.
Journal of Thermal Biology xxx (xxxx) xxx
5. Conclusions
Gordon, C.J., 1993. Temperature Regulation in Laboratory Rodents. Cambridge University Press. Gordon, C.J., 1994. 24-hour control of body temperature in rats. I. Integration of behavioral and autonomic effectors. Am. J. Physiol. 36, R71–R77. Hansen, L.A., Pender, M.P., 1989. Hypothermia due to an ascending impairment of shivering in hyperacute experimental allergic encephalomyelitis in the Lewis rat. J. Neurol. Sci. 94, 231–240. Kozak, W., Wrotek, S., Kozak, A., 2006. Pyrogenicity of CpG-DNA in mice: role of interleukin-6, cyclooxygenases, and nuclear factor-kappaB. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R871–R880. Linker, R.A., Mohr, A., Cepek, L., et al., 2006. Core hypothermia in multiple sclerosis: case report with magnetic resonance imaging localization of a thalamic lesion. Mult. Scler. 12, 112–115. Marques, P.R., Spencer, R.L., Burks, T.F., McDougal, J.N., 1984. Behavioral thermoregulation, core temperature, and motor activity: simultaneous quantitative assessment in rats after dopamine and prostaglandin E1. Behav. Neurosci. 98, 858–867. Mondal, S., Jana, M., Dasarathi, S., Roy, A., Pahan, K., 2018. Aspirin ameliorates experimental autoimmune encephalomyelitis through interleukin-11-mediated protection of regulatory T cells. Sci. Signal. 11 (558) https://doi.org/10.1126/ scisignal.aar8278 pii: eaar8278. Pahan, S., Pahan, K., 2019. Mode of action of aspirin in experimental autoimmune encephalomyelitis. DNA Cell Biol. 38 (7), 593–596. Reith, J., Jørgensen, H.S., Pedersen, P.M., Nakayama, H., Raaschou, H.O., Jeppesen, L.L., Olsen, T.S., 1996. Pedersen PM et al. Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome. Lancet 347, 422–425. Romani, A., Bergamaschi, R., Versino, M., Zilioli, A., Callieco, R., Cosi, V., 2000. Circadian and hypothermia-induced effects on visual and auditory evoked potentials in multiple sclerosis. Clin. Neurophysiol. 111, 1602–1606. Schiffmann, S., Weigert, A., M€ annich, J., Eberle, M., Birod, K., H€ aussler, A., et al., 2014. PGE2/EP4 signaling in peripheral immune cells promotes development of experimental autoimmune encephalomyelitis. Biochem. Pharmacol. 87, 625–635. Slaney, C.Y., Toker, A., Fraser, J.D., et al., 2013. A modified superantigen rescues Ly6GCD11bþblood monocyte suppressor function and suppresses antigen-specific inflammation in EAE. Autoimmunity 46, 269–278. Slavin, A., Kelly-Modis, L., Labadia, M., et al., 2010. Pathogenic mechanisms and experimental models of multiple sclerosis. Autoimmunity 43, 504–513. Spencer, R.L., Hruby, V.J., Burks, T.F., 1990. Alteration of thermoregulatory set point with opioid agonists. J. Pharmacol. Exp. Therapeut. 252, 696–705. Takagi, K., Tsuchiya, Y., Okinaga, K., Hirata, M., Nakagomi, Y., Tamura, A., 2003. Natural hypothermia immediately after transient global cerebral ischemia induced by spontaneous subarachnoid hemorrhage. J. Neurosurg. 98, 50–56. van Diemen, H.A., van Dongen, M.M., Dammers, J.W., Polman, C.H., 1992. Increased visual impairment after exercise (Uhthoff’s phenomenon) in multiple sclerosis: therapeutic possibilities. Eur. Neurol. 32, 231–234. Waxman, G.S., Geschwind, N., 1983. Major morbidity related to hyperthermia in multiple sclerosis. Ann. Neurol. 13, 348. Weiss, N., Hasboun, D., Demeret, S., et al., 2009. Paroxysmal hypothermia as a clinical feature of multiple sclerosis. Neurology 13, 193–195. White, K.D., Scoones, D.J., Newman, P.K., 1996. Hypothermia in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 61, 369–375. Wrotek, S.E., Kozak, W.E., Hess, D.C., Fagan, S.C., 2011. Treatment of fever after stroke: conflicting evidence. Pharmacotherapy 31, 1085–1091. Wrotek, S., Rosochowicz, T., Nowakowska, A., Kozak, W., 2014. Thermal and motor behavior in experimental autoimmune encephalitis in Lewis rats. Autoimmunity 47, 334–340.
The results of the present investigation show that: I. There is an upward resetting of the temperature regulation in EAE rats. II. Acute symptoms of EAE are exacerbated by fever, which means that autoimmune diseases belong to exceptions to the rule of the beneficial role of fever. III. Both the fever and normal pattern of the diurnal Tb shifts in EAE rats need a behavioral support to compensate for a strong deficit in muscular thermogenesis. IV. Accordingly, thermoregulation appears to be a highly redundant system. CRediT authorship contribution statement Sylwia Wrotek: Conceptualization, Investigation, Formal analysis, Visualization, Writing - original draft. Anna Nowakowska: Formal analysis, Investigation, Visualization, Writing - original draft. Michał Caputa: Conceptualization, Formal analysis, Supervision, Visualization, Writing - review & editing. Wiesław Kozak: Conceptualization, Supervision. References Avis, S.P., Pryse-Phillips, W.E., 1995. Sudden death in multiple sclerosis associated with sun exposure: a report of two cases. Can. J. Neurol. Sci. 22, 305–307. Biddle, C., 2006. The neurobiology of the human febrile response. AANA J. (Am. Assoc. Nurse Anesth.) 74, 145–150. Bolton, C., Gordon, D., Turk, J.L., 1984. Prostaglandin and thromboxane levels in central nervous system tissues from rats during the induction and development of experimental allergic encephalomyelitis (EAE). Immunopharmacology 7, 101–107. Cabanac, M., Brinnel, H., 1987. The pathology of human temperature regulation: thermiatrics. Experientia 43, 19–27. Caputa, M., 2005. Comments on "Do fever and anapyrexia exist? Analysis of set pointbased definitions. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R281 author reply R281-2. Chan, J., Ban, E.J., Chun, K.H., et al., 2008. Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis. Autoimmunity 41, 405–413. Compston, A., Coles, A., 2008. Multiple sclerosis. Lancet 25, 1502–1517. Constantinescu, C.S., Farooqi, N., O’Brien, K., Gran, B., 2011. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br. J. Pharmacol. 164, 1079–1106. Fukuda, H., Kitani, M., Takahashi, K., 1999. Body temperature correlates with functional outcome and the lesion size of cerebral infarction. Acta Neurol. Scand. 100, 385–390. Gordon, C.J., 1987. Relationship between preferred ambient temperature and autonomic thermoregulatory function in rat. Am. J. Physiol. 252, R1130–R1137.
5