Iptt 300

Male and female 13-lined ground squirrels, I. tridecemlineatus ranging from 150 to 300 g (at the time of capture) were caught from the wild by USDA licensed trappers (TLS Research, Bartlett, IL, USA) annually in August. Animal housing and experimental procedures followed the guidelines set by the NINDS animal care and use committee (ACUC). Once caught, the squirrels were brought to the NIH and kept in quarantine to be sure they contained no infectious agents. As a prophylactic measure, all incoming squirrels were treated with 0.05 ml/squirrel of Profender to kill any parasites (3 mg/kg emodepside + 12 mg/kg praziquantel). Each animal was briefly anesthetized with 5% isofluorane and injected subcutaneously in the intrascapular area with a sterile programmable temperature transponder (IPTT-300, Bio medic Data Systems) to allow daily monitoring of Tb without touching or disturbing the squirrel. While this system does not provide an exact measure of core Tb, it does provide a close estimate (±2–3°C) (data not shown); therefore, indicating the stage of hibernation in which the squirrels belonged. Ground squirrels were then housed in individual cages and fed water and standard rodent chow ad libitum. Animals were housed in the holding colony at 21°C under a 12 h light:12 h dark cycle until used for experimental purposes. To enable a natural transition into torpor, some squirrels were transferred to constant darkness in an environmental chamber held at 4–5°C. A red safe light (3–5 lux) was used when entering the chamber so as not to disturb torpid squirrels. Also, a heavy dark curtain was used to shield the shelves containing the cages and block the light and sound resulting from opening and closing the door to the environmental chamber. Once a squirrel was observed to descent into torpor, a small amount of saw dust was dropped on the back of the animal to be sure it did not move (arouse) in between monitoring periods. Tissues were collected from animals at six different stages of the hibernation cycle based on Tb, time, and respiration rate. ACR = active in the cold room for at least 3 days (Tb = 34–37°C). ACR animals were capable of entering torpor, but had not done so in the past 72 h and were chosen as the reference group to rule out environmental light and temperature effects. EN = entrance phase of torpor (18°C ≤ Tb ≤ 31°C) (animals were only harvested for an EN time point after at least two successive temperatures showed a declining Tb), ET = early torpor phase (Tb = 5–8°C for 1 day), LT = late torpor phase (Tb = 5–8°C for at least 5 days), E-AR = early arousal, spontaneously arousing from torpor with an increasing respiration rate ≥60/min and an increasing yet still reduced Tb (Tb = 9–12°C). The final phase IA = interbout arousal. These animals had previously been in deep torpor, but had since returned to normal Tb (Tb = 34–37°C).
To collect tissues at the desired time points, the squirrels were first anesthetized with 5% isofluorane and the rectal temperature was measured to verify the accuracy of the temperature transponder. The animal was then decapitated with a guillotine. The desired tissues were removed quickly, rinsed with ice-cold phosphate-buffered saline (PBS) and frozen instantly in 2-methylbutane chilled with dry ice (−50°C) and then stored in a −70°C freezer until use.

For cytokine measurements, mice were treated intraperitoneally with OI (50 mg kg⁻¹) in 40% cyclodextrin in PBS or vehicle control for 2 h before stimulation with LPS (Sigma; 2.5 mg kg⁻¹) intraperitoneally for 2 h. Mice were euthanized in a CO₂ chamber, blood samples were collected and serum was isolated. Cytokines were measured using R&D ELISA kits according to manufacturer’s protocol. For temperature recording, mice (n = 10 per group) were treated intraperitoneally with OI (50 mg kg⁻¹) in 40% cyclodextrin in PBS or vehicle control for 2 h before stimulation with LPS (5 mg kg⁻¹) and monitored for temperature at 1, 2, 3, 4, 6, 12, 18 and 24 h after LPS treatment. Temperature was monitored using subcutaneously implanted temperature transponder chips (Bio Medic Data Systems; IPTT 300) which were injected between the shoulder blades 48 h before experiment. At defined times, body temperature was measured by scanning the transponder with a corresponding BMDS Smart Probe. Animals were additionally monitored for clinical signs of endotoxic shock, based on temperature change, body condition, physical condition and unprovoked behaviour, with a combined score of 9 indicating the humane end point for the experiment.

Mice were treated intragastrically with either sterile PBS or OVA (Sigma-Aldrich) (250μg) together with 10μg SEB (Toxin Technology) in PBS (Treg) once weekly for 8 weeks. In some experiments, mice were gavaged with SEB (10 μg) alone. On week 9, mice were challenged intragastrically with 150 mg of OVA. Anaphylaxis was assessed by measuring changes in total body core temperature with transponders placed subcutaneously 2 days before challenge (IPTT-300; Bio Medic Data Systems) and a DAS-6001 console (Bio Medic Data Systems).