A thymus tumour impairing hibernation

Background: Hibernation is a physiological and behavioural adaptation that permits survival during periods of reduced food availability and extreme environmental temperatures. This is achieved through cycles of metabolic depression and reduced body temperature (torpor) and rewarming (arousal). Rewarming from torpor is achieved through the activation of brown fat (BAT) associated with a rapid increase in ventilation frequency. Here, we studied the rate of rewarming in the European hamster by measuring both BAT temperature, core body temperature and ventilation frequency. Results: Temperature was monitored in in parallel in the BAT (IPTT tags) and peritoneal cavity (iButtons) during hibernation torpor-arousal cycling. We found that increases in brown fat temperature preceeded core body temperature rises by about 47 min, and this was accompanied by a significant increase in ventilation frequency. The rate of rewarming was slowed by the presence of a spontaneous thymus tumour in one of our animals. Core body temperature re-warming was reduced by 6.2℃*h-1 and BAT rewarming by 12℃*h-1. Ventilation frequency was increased by 77% during re-warming in the thymus tumour animal compared to a healthy animal. Inspection of the position and size of the tumour indicated that the lungs and heart were obstructed. Conclusions: We validated a minimally invasive method to monitor BAT temperature during hibernation. Using this method we showed compromised re-warming from hibernation in an animal with a thymus tumour, the likely cause of which is obstruction of the lungs and heart leading to inefficient ventilation and circulation.


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Background 33 Hibernation is a physiological and behavioural adaptation that permits survival during periods of 34 reduced food availability and extreme environmental temperatures. This is achieved through cycles of 35 metabolic depression characterised by reduced body temperature (torpor) and rewarming (arousal). 36 Entrance into torpor precisely is controlled by decreases in heart rate, ventilation frequency and 37 oxygen consumption [1,2]. Arousal occurs in a coordinated manner with increased ventilation 38 frequency and oxygen consumption subsequently followed by heart rate, blood pressure and core 39 during arousal was noted, an average BAT temperature of 15℃ was recorded at the onset of shivering 91 ( Figure 2B). 92 We plotted data from one representative individual and calculated the RWRmax in brown fat as 93 19.3℃*h -1 , a similar rate was observed in the core body temperature (18.1℃* h -1 , Figure 2C). We also 94 monitored the ventilation frequency during arousal which increased from 13 breaths per minute (bpm) 95 to a maximum of 86 bpm ( Figure 2C). Ventilation frequency increases correlated with TBAT (pearson r= 96 0.899, p-value= 0.002). 97 Brown fat re-warming is compromised by the presence of a spontaneous thymus tumour 98 We identified one individual in our study with a spontaneous thymus tumour ( Figure 3A). When 99 comparing the amount of time spent torpid or aroused the tumour bearing animal spent 63.5% 100 aroused and 14.5% torpid, compared to an average of 25.9% aroused and 54.6% torpid for the healthy 101 animals ( Figure 3B). The RWRmax of brown fat was reduced by 12℃*h -1 relative to a healthy animal 102 ( Figure 3C). The RWRmax of the core body was less compromised showing a reduction of 6.2℃*h -1 . 103 Ventilation frequency increases still correlated with TBAT (pearson r= 0.873, p-value= 0.002) but the 104 tumour animal showed a 77 % higher maximum ventilation frequency compared to a healthy animal 105 (Figure 2B compared to Figure 3C). Brown fat re-warming precedes core body temperature re-warming 106 by 53.9 minutes in the tumour animal, this is outside the confidence intervals for the healthy animals, 107 suggesting that the efficiency of BAT re-warming of the core is compromised.
increased ventilation to increase oxygen availability, followed by vasodilation, and increased heart 134 rate, to re-perfuse the circulation and re-warm the whole animal. Low oxygen availability increases 135 vasodilation [19] therefore in the tumour bearing animal lower oxygen availability may have increased 136 vasodilation leading to a compensated re-warming efficiency of the core body. Vasodilation would 137 also benefit the BAT by supplying more oxygen in the blood. In support of this the tumour bearing 138 animal in the initial stages of BAT re-warming from 10 to 15℃ appears to show a gentler slope 139 compared to 20 to 30℃ ( Figure 3C), indicating a lower efficiency in the early arousal stage which would 140 not benefit from vasodilation, this distinction is not seen in the healthy animals. Another explanation 141 for the discrepancy between BAT and core body re-warming efficiency may be the additional 142 contribution of shivering thermogenesis to core body temperature increases. 143 The thymus is a lymphoid organ involved in T-cell maturation , it is located in front of the heart 144 and found in all vertebrates (reviewed in: [20]). In general, thymus tumours are rare but are reported 145 in domestic, laboratory and wild contexts (including wild European hamsters) [21]. Characterisation of 146 European hamster thymus tumours has noted they closely match human thymic epithelial tumours 147 [22]. Human thymus tumour cases often present with tumours that obstruct the heart and lungs, 148 similar to our observations. Also in humans, an association of thymus tumour with 149 hypothermia/defective re-warming has been reported twice [23,24]. Impressively in one of the cases 150 the patient experienced a body temperature between 32.8℃ to 35℃ (Ambient room temperature: 151 22℃) [23]. However, the cause of hypothermia in these cases is unknown. 152

Conclusions 153
In conclusion we have used temperature logging in BAT and intraperitoneal cavity to study progression 154 of arousal in hibernating hamsters. We show compromised re-warming from hibernation in an animal 155 with a thymus tumour, the likely cause of which is obstruction of the lungs and heart leading to 156 inefficient ventilation and circulation.

Animals and ethics statement 159
European hamsters were bred from stock animals at the Chronobiotron, an animal facility dedicated 160 to the study of biological rhythms. Animals were housed in an environment controlled room in 161 separate cages according to legislations provided in European Commission directive 2010/63/EU. 162 They were provided with ad libitum access to food (Safe®) and water throughout the study 163 period. The animals were kept under a long photoperiod (14L:10D) and at 22℃ (LP22) until the start 164 of the experiment. To initiate the preparation for hibernation (pre-hibernal period) the animals were 165 transferred to a short photoperiod (10L:14D) at 22℃ (SP22). After 8 weeks, the temperature was Surgeries were performed on each animal to implant iButtons (thermochron DS1922L, Maxim 183 integrated) and IPTT tags. Each animal was anesthetized by 3 % isoflurane, surgery was performed 184 under 3% isoflurane and 95 % oxygen. The IPTT-tag was implanted subcutaneously into the classical 185 brown adipose tissue (BAT) depot, using a standard IPTT-tag injector (supplied by the manufacturer). 186 Post-mortem verification ensured the tag was in contact with the BAT. The iButton was implanted in 187 the abdominal cavity with the use of laparotomy. Local anaesthetic agents (lidocaine and bupivacaine) 188 was injected intraperitoneally (2.5 mg/kg) before the incision was made. Analgesic agent (Metacam®, 189 injectable, 2mg/kg) was dorsally injected subcutaneously. In addition, analgesic agent was dissolved in 190 drinking water and provided for 3 days post-surgery (Metacam® drinkable 1.5 mg/ml, 1 mg/kg). The 191 animals were extensively monitored after surgery and allowed to recover for two weeks. 192

Behaviour and temperature monitoring 193
To keep track of individual torpor-arousal patterns, behavioural recordings were made twice per day; 194 1 to 2 hours after lights on and 9-10 hours after lights on. We defined torpid as a ventilation frequency 195 (VF) < 10 per minute, curled up position in hibernacula, immobile, unresponsive and an IPTT reading 196 <13.5℃. Signs of arousal were; increased IPTT tag temperature and increased ventilation frequency. 197 We identified animals beginning to arouse and monitored IPTT tag temperatures every two minutes, 198 ventilation frequency was counted in regular intervals for one minute. IButton core temperature 199 recordings were collected post-mortem and time matched with the calibrated IPTT measurements.