WO2025064356A1

WO2025064356A1 - Apparatus and method for probing thermal or sensory pain in animals

Info

Publication number: WO2025064356A1
Application number: PCT/US2024/046947
Authority: WO
Inventors: Theanne N. GRIFFITH
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-09-21
Filing date: 2024-09-16
Publication date: 2025-03-27
Anticipated expiration: 2026-03-21

Abstract

A device (100) can include a first chamber (102) having a proximal section (108) and a distal section (110), wherein the floor of the proximal section (108) contains a thermally-insulating material, and the floor of the distal section (110) is thermally adjustable. The device (100) includes a second chamber (104) having reduced light compared to the first chamber (102) and a floor with a thermally-insulating material. The device (100) can additionally include a passageway (106) connecting the distal section (110) of the first chamber (102) and the second chamber (104).

Description

APPARATUS AND METHOD FOR PROBING THERMAL OR SENSORY PAIN IN ANIMALS

STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with Government support under Grant No. K01NS124828-01A1, awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Field

[0002] Some embodiments disclosed herein relate to thermosensory or pain assays for animals and methods for conducting such assays.

Description of the Related Art

[0003] Animals can be probed for certain behaviors, and rodents and mice are often selected for such tests due to the ubiquity of their use in laboratory environments. For example, rodents can be tested in a laboratory environment to assess cognitive or physical traits that are shared with humans or consistent across the animal kingdom.

[0004] Previously structures have been designed to test various cognitive or physical traits. These structures have ranged from mouse utopias, providing mice unlimited access to food, water, and socialization, to deprivation-based models, subjecting mice to electrical shocks or other painful stimuli.

SUMMARY

[0005] In some embodiments, the techniques described herein relate to an apparatus for probing thermosensation, pain, or avoidance and tolerance of an animal to noxious environments including: a first chamber having a proximal section and a distal section, wherein the floor of the proximal section contains a thermally -insulating material and the floor of the distal section is thermally adjustable; a second chamber having reduced light compared to the first chamber a floor with a thermally-insulating material; and a passageway connecting the distal section of the first chamber and second chamber. [0006] In some embodiments, the techniques described herein relate to an apparatus, additionally including a sensor for detecting a location, presence, or absence of the animal within the first chamber.

[0007] In some embodiments, the techniques described herein relate to an apparatus, wherein the sensor is only configured to detect the absence of a mouse in the first chamber.

[0008] In some embodiments, the techniques described herein relate to an apparatus, wherein the second chamber is enclosed on all sides by a material impermeable to light except for the passageway, and the passageway is configured for the passage of a rodent.

[0009] In some embodiments, the techniques described herein relate to an apparatus, wherein the walls of the first and second chamber are not transparent or opaque and the passageway is a hole in the wall of the first chamber.

[0010] In some embodiments, the techniques described herein relate to an apparatus, wherein the light in the first chamber is ambient light and the first chamber and second chamber are directly adjacent to each other.

[0011] In some embodiments, the techniques described herein relate to an apparatus, wherein the thermally-adjustable material in the distal section includes electronically thermally-adjustable peltier elements.

[0012] In some embodiments, the techniques described herein relate to an apparatus, wherein the proximal section includes 30% of the first chamber floor and the distal section includes 70% of the first chamber floor.

[0013] In some embodiments, the techniques described herein relate to an apparatus, wherein the distal section includes at least 80% of the first chamber floor.

[0014] In some embodiments, the techniques described herein relate to a method probing thermal or sensory pain in rodents including: providing a first chamber having a proximal section and a distal section, wherein the proximal section contains a thermally-insulating floor and the floor of the distal section is thermally adjustable; providing a second chamber having reduced visible light compared to the first chamber and a thermally-insulating floor; providing a passageway connecting the distal section of the first chamber and second chamber; adjusting the thermally-adjustable material of the distal section to a noxious temperature; and placing a rodent in the proximal section that contains the thermally-insulating floor. [0015] In some embodiments, the techniques described herein relate to a method, wherein the rodent has been subjected to brain trauma and a craniectomy.

[0016] In some embodiments, the techniques described herein relate to a method, wherein the rodent has been genetically modified to knock out one or more thermosensation ion channels.

[0017] In some embodiments, the techniques described herein relate to a method, wherein the ion channels are selected from a group consisting of Trpvl. Trpal. Trpm3, and Trpm8.

[0018] In some embodiments, the techniques described herein relate to a method, wherein placing the rodent in the proximal section includes placing the rodent without any prior habituation to the first or second chambers.

[0019] In some embodiments, the techniques described herein relate to a method, wherein the thermally-adjustable floor includes one or more peltier elements.

[0020] In some embodiments, the techniques described herein relate to a method, wherein the thermally-insulating floor is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 10 degrees Celsius.

[0021] In some embodiments, the techniques described herein relate to a method, wherein the passageway is configured for the passageway of a rodent and is vertically constrained and permanently open.

[0022] In some embodiments, the techniques described herein relate to a method, additionally including measuring the location of the rodent in the first chamber with a sensor, wherein the sensor does not measure the second chamber.

[0023] In some embodiments, the techniques described herein relate to a method, wherein the thermally-insulating material is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 20 degrees Celsius.

[0024] In some embodiments, the techniques described herein relate to a method, wherein the distal section includes at least 70% of the floor of the first chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 illustrates a non-limiting embodiment of a thermal escape apparatus according to some embodiments disclosed herein. [0026] Figure 2 illustrates a non-limiting embodiment of an additional view of a thermal escape apparatus according to some embodiments disclosed herein.

[0027] Figure 3 illustrates a non-limiting embodiment of a thermal escape apparatus with thermal plates inserted into the floor of the apparatus according to some embodiments disclosed herein.

[0028] Figure 4 illustrates a non-limiting embodiment of a side view of a thermal escape apparatus according to some embodiments disclosed herein.

[0029] Figure 5 illustrates a non-limiting embodiment of thermal escape latencies at various temperatures.

[0030] Figure 6 illustrates a non-limiting embodiment of thermal escape latencies at various temperatures.

[0031] Figure 7 illustrates a non-limiting embodiment of thermal escape latencies after habituation to a thermal escape apparatus disclosed herein.

[0032] Figure 8 illustrates a non-limiting embodiment of thermal escape latencies of rodents with various genetic variations.

[0033] Figure 9 illustrates a non-limiting embodiment of thermal escape latencies of rodents with chronic constriction injuries.

[0034] Figure 10 illustrates a non-limiting embodiment of thermal escape latencies for same day repeat testing.

[0035] Figure 11 illustrates a non-limiting embodiment of thermal escape latencies for repeat testing within the same week.

[0036] Figure 12 illustrates a non-limiting embodiment of thermal escape latencies after oxaliplatin-induced cold allodynia.

[0037] Figure 13 illustrates a non-limiting embodiment of thermal escape latencies after meloxicam-induced cold allodynia.

DETAILED DESCRIPTION

[0038] In some circumstances, it may be desirable to subject rodents to testing in order to evaluate the effectiveness of a drug, clinical procedure, or genetic variation. In many cases, rodents will be habituated to the environment prior to the introduction of stimuli. This can be time consuming and potentially interfere with the processes of a study. [0039] Rodents generally find bright light as noxious and have an aversion to it. Therefore, experiments have been structured to propose a dimly-lit environment as an incentive in a behavioral study. In some cases, the rodent may be subjected to a noxious environment at the same time as being presented with a reward. Decision making in rodents is important to understanding cognitive decision making in mammals as well as proving the effectiveness of drugs, genetic variations and gene mutations, or other clinical procedures. However, the prior art has not provided an assay to determine decision making in rodents that may be readily adopted, does not require habituation, and probes thermal pain as a barrier to an incentive.

[0040] Some embodiments provided herein disclose thermal escape apparatus structures and methods associated therewith that overcome the drawbacks associated with thermosensory assays. For example, the thermal escape apparatus disclosed herein leverages the innate motivation of a mouse to escape from the light chamber and places this in conflict with the need to cross temperature-controlled plates to do so. Advantageously, some embodiments of the apparatus disclosed herein do not require habituation of a rodent or animal to the apparatus prior to testing. This enables streamlining of testing processes and higher fidelity in experimentation and assay. Further, the apparatus in some embodiments disclosed herein provides thermal pain as a barrier to an incentive. For example, thermally-adjustable plates may be employed to detract the animal from pursuing an incentive and act as a barrier to the animal’s pursuance of the incentive. Additionally, the animal is ideally not habituated to the apparatus prior to testing with the animal, as habituation can unexpectedly skew results of the assay. Thus, the decision making behavior of the animal can be probed as accurately as possible, which models the decision making an animal or rodent would make in the wild. For example, a rodent is enticed by the incentive but also, since the rodent has not been habituated to the apparatus, they are conflicted by the incentive, as they have not previously determined that the incentive is free of predators or other harms.

[0041] In some embodiments, a thermal escape apparatus is provided, which can be used to understand the molecular underpinnings of thermosensation and thermal pain in various animals, such as rodents or vertebrates. In some embodiments disclosed herein, the thermal escape apparatus generally comprises two chambers or compartments with a passageway between the two chambers. The passageway can be a hole in a wall between the two chambers that are directly adjacent to each other, or the passageway can be hallway that connects the two chambers spaced at a distance from each other. The first chamber is generally a well-lit chamber that receives ambient light. The second chamber has reduced light as compared to the first chamber, or is dimly lit. In some embodiments the first chamber is apportioned between two sections or zones. The first section, a proximal section, is provided with a thermally-insulating material or the floor is configured to be thermally insulating and room-temperature or substantially room temperature. The second section, a distal section, is provided with a thermally-adjustable floor or thermally - adjustable plates that can be configured to provide a noxious temperature. The wall of the distal section contains the passageway between the first chamber and the second chamber.

[0042] Figure 1 depicts an embodiment of a thermal escape apparatus 100 containing a first chamber 102 and a second chamber 104. Connecting the first chamber 102 and the second chamber 104 is a passageway 106. The first chamber 102 is provided with four walls and a floor. In some embodiments, there is no ceiling for the first chamber 102, the light in the first chamber 102 is ambient light, and the first chamber 102 and second chamber 104 are directly adjacent to each other. In some embodiments, the first chamber can be equipped with four walls and an empty bottom through which a thermally insulating or thermally-adjustable flooring sections can be inserted, such as thermally-adjustable plates. The second chamber 104 is provided with four walls, a floor, and a ceiling, which provides a confined space that has reduced light compared to the first chamber 102. In some embodiments the second chamber 104 is about 2 lux, about 5 lux, about 10 lux, about 15 lux, about 20 lux. or about 40 lux dimmer than the first chamber 102. In some embodiments, the brightness can be at least any one of the preceding values including any range defined between any two of the preceding values. In some embodiments, the second chamber 104 is enclosed on all sides by a material impermeable to light except for the passageway 106, and the passageway is configured for the passage of a rodent. In some embodiments, the walls of the first and second chamber are not transparent or opaque and the passageway 106 is a hole in the wall of the first chamber. In some embodiments, the passageway is configured for the passageway of a rodent and is vertically constrained and permanently open. As discussed, in some embodiments the first chamber 102 does not have a ceiling, causing the first chamber 102 to be well-lit. which can be noxious to animals that are provided in the thermal escape apparatus 100. However, the first chamber 102 can be provided with a ceiling and one or more lights below the ceiling to provide a noxiously lit environment. In such a scenario, a port or door can be provided in the first chamber 102 for the insertion of a rodent or animal into the thermal escape apparatus. [0043] Figure 2 illustrates a top plan view of the thermal escape apparatus 100. As shown in Figure 1, the thermal escape apparatus 100 contains a first chamber 102 and a second chamber 104. The first chamber is additionally apportioned between a proximal section 108 and a distal section 110. In the assays disclosed herein a rodent 112 is placed in the proximal section 108 prior to conductance of the study.

[0044] The proximal section 108 is provided with a thermally-insulating floor or a thermally -insulating material can be provided as the floor of the proximal section 108. Thus, as a rodent 1 12 is first provided in the thermal escape apparatus 100, the rodent 112 is only subjected the noxious environment of a well-lit environment and is not placed on a surface with a noxious temperature. The thermally-insulating material can be any thermally insulating material known in the art, such as plastics, glass, foam, cellulose, etc. In some embodiments, the thermally insulating material is selected from a group consisting of Acrylic, Polyurethane, Polystyrene, Polyisocyanurate (PIR), Polyethylene (PE), Polypropylene (PP), Polyethylene Terephthalate (PET), Polyimide (PI), Polycarbonate (PC), and Polyvinyl Chloride (PVC). In some embodiments, the thermally-insulating material is composed of 50 mm thick acrylic resin. In some embodiments, the proximal section 108 is provided such that the entire surface of the proximal section 108 is comprised of a thermally -insulating surface. In some embodiments, the floor of the proximal section 108 is comprised of one or more layers, one of which is a thermally -insulating material. In some embodiments, the first chamber 102 is provided with an insulating buffer between the proximal section 108 and the distal section 1 10 such that the temperature of the proximal section 108 does not deviate from a non-noxious temperature. In some embodiments, the material of the thermally-insulating material is configured to be maintained at room temperature. In some embodiments, the material of the thermally- insulating material is configured to be maintained near a physiological temperature of the rodent, such as 30 degrees Celsius. In some of these embodiments the thermally insulating material can be configured not to deviate from room temperature or physiological temperature by about 1 degree, about 2 degrees, about 3 degrees, or about 4 degrees Celsius.

[0045] In some embodiments, the proximal section 108 comprises at least 5% of the floor of the first chamber 102. In some embodiments, the proximal section 108 comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the floor of the first chamber 102. In some embodiments, the percentage can be at least any one of the preceding values including any range defined between any tw o of the preceding values. In some embodiments, the proximal section 108 is provided in a suitable size for the dimensions of a mouse or rate and the rest of the first chamber 102 is comprised of the distal section 110. In some embodiments, the proximal section 108 is adjustably provided in the longest dimension of the animal being probed, such as from the head to tail of a mouse or rat.

[0046] Similarly, in some embodiments the distal section 110 comprises at least 5% of the floor of the first chamber 102. In some embodiments, the distal section 110 comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the floor of the first chamber 102. In some embodiments, the percentage can be at least any one of the preceding values including any range defined between any two of the preceding values. In some embodiments, the proximal section comprises 30% of the first chamber floor and the distal section comprises 70% of the first chamber floor. In some embodiments, the distal section 110 is provided as a suitable size for the dimensions of a mouse or rate and the rest of the first chamber 102 is comprised of the proximal section 108. In some embodiments, the first chamber 102 is 465mm long x 185mm wide x 345mm high and the second chamber 104 is 100mm long x 185mm wide x 345mm high. In some embodiments, the proximal section 108 is 116 mm long and 185 mm wide. The height of the first or second chamber can be configured such that the animal is incapable of climbing or jumping out of the chamber. Further, the walls of the first and second chambers can be comprised of polished or substantially frictionless material to prevent the escape of the animal from the chambers.

[0047] The proximal section 108 and the distal section 110 can be reduced or increased in size, such as to determine the thermal tolerance of the animal and how such thermal tolerance relates to latency of the animal within the first chamber 102. For example, where the distal section 110 is larger than the proximal section 108 a rodent or other animal would have to endure a longer, more noxious path to the second chamber 104. This essentially increases the thermal pain barrier that the animal or rodent must endure. In some embodiments, the length of the distal section 110 is configured such that it is about one length, about two lengths, about three lengths, about four lengths, or at least about 5 lengths of the animal length. In some embodiments, the length can be at least any one of the preceding values including any range defined between any two of the preceding values.

[0048] The distal section 110 is comprised of athermally-adjustable material or has a thermally adjustable surface. The distal section 110 is configured such that the entirety of the surface of the distal section 110 is thermally adjustable. In this manner the animal has no escape path to the passageway 106 that does not run through a thermally-adjustable surface. The floor of the distal section 110 can be comprised of thermally-adjustable plates or be configured to be placed over thermally-adjustable plates such that the thermal profile of the plates is conducted through the surface of the distal section 110. The thermally- adjustable plates can be electrically adjustable plates, or plates that act as a heat sink for another material, such as water. Advantageously the plates are configured such that they are thermally adjustable in either direction, whether hot or cold. In some embodiments, the plates are thermally-adjustable peltier plates. In some embodiments, the thermally- adjustable material in the distal section comprises electronically thermally-adjustable peltier elements. The surface or floor of the distal section 110 can be provided with a thermally conductive material, such as metal, in order to conduct thermal energy to or from thermal plates adjacent to the distal section 110.

[0049] Figure 3 is another depiction of a thermal escape apparatus 100 according to some embodiments herein. This embodiment shows the first chamber 102 and second chamber 104, as shown in Figures 1 and 2. The figure additionally shows the passageway 106 directly connecting first chamber 102 and distal section 110. In some embodiments, distal section 110 is provided with two independently thermally-adjustable plates, first plate 110a and second plate 110b. In some embodiments, the distal section 110 is not provided with a floor and the thermally-adjustable plates, 110a and 110b, are provided as the floor. In this depiction the thermally-adjustable plates 1 10a and 1 10b comprise approximately 70% of the area of the first chamber 102, with each thermally- adjustable plate comprising approximately 35% of the first chamber 102 surface area.

[0050] The first plate 110a or second plate 110b can be thermally adjusted to a temperature that is approximately room temperature (approximately 21° Celsius) or near physiological temperature (30° Celsius or temperature-dependent on the type of animal) for control experiments, or they can be configured to be hotter or cooler than these temperatures by at least about 2° Celsius, at least about 5° Celsius, at least about 10° Celsius, at least about 15° Celsius, at least about 20° Celsius, at least about 25° Celsius, or at least about 30° Celsius. In some embodiments, the temperature can be at least any one of the preceding values including any range defined between any two of the preceding values. In some embodiments, the thermally-insulating floor is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 10 degrees Celsius. In some embodiments, the thermally-insulating material is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 20 degrees Celsius. The first plate 110a or second plate 110b can also be controlled to differ from the temperature of the other plate (1 10a or 110b) by at least about 1° Celsius, at least about 2° Celsius, at least about 5° Celsius, at least about 10° Celsius, at least about 15° Celsius, at least about 20° Celsius, or at least about 25° Celsius. In some embodiments, the temperature can be at least any one of the preceding values including any range defined between any two of the preceding values. Advantageously this can enable the use of disparate noxious temperatures, hot and cold temperatures or hot/ cold and neutral temperatures, in order to validate genetic variations, such as genetic knockouts of Trpvl, Trpal, Trpm3, and Trpm8 receptors or other temperature regulation pathways in various animals. This can also be advantageous in older to probe temperature tolerance, such as temperature tolerance of both 5° and 18° Celsius, in the same thermal escape experiment.

[0051] In Figure 3 second chamber 104 contains a lid that prevents ambient light from entering the second chamber 104 from the top of the chamber. In this way, the second chamber 104 has reduced visible light compared to first chamber 102. The first chamber 102 can also be configured with one or more lights directly above the first chamber (not shown) in order to provide increased light compared to ambient light and increased light compared to the second chamber 104.

[0052] Figure 4 illustrates a side view of a thermal escape apparatus 300. In some embodiments, a sensor 1 14 is provided above the first chamber in order to detect the location, presence, heat signature, or absence of the animal in the first chamber 102. The sensor 114 can be a camera, such as depicted in the figure, or there can be one or more sensors provided to detect an animal in the first chamber 102. Other sensors can be used such as one or more Passive Infrared (PIR) Sensors, Ultrasonic Sensors, Acoustic Sensors, Thermal Imaging Cameras, proximity sensors, motion detectors, or the floor of the first chamber can be equipped with one or more pressure sensitive plates. The pressure sensitive plates can be placed in the proximal section 108 or the distal section 110. Where the pressure sensitive plates are provided in the distal section they can be configured to work in tandem with thermally-adjustable plates or conductors. Advantageously, a thermal imaging camera can be used to detect the presence of one or more animals in the thermal escape apparatus 300, as animals give off heat signatures that can be detected in the infrared spectrum. Additionally, the thermal imaging camera can provide temperature validation or calibration for the thermally-adjustable surfaces. It should also be noted that sensor can be provided in the second chamber 104, but providing a sensor only in the first chamber 102 can advantageously enable fewer sensors, less complexity, and a lower cost for the apparatus. In some embodiments, the sensor 114 is only configured to detect the absence of a mouse in the first chamber 102. In some embodiments, the sensor 114 is configured for measuring the location of the rodent in the first chamber, wherein the sensor 114 does not measure the second chamber. The sensor or sensors provided in the second chamber 104 can be the same as those provided in the first chamber 102. or they can be different from those provided in the first chamber 102. The sensor 114 advantageously allows humans or physical observers to not be in view of the animal during testing, which can skew results of the study. To this end, the walls of the first chamber 102 and second chamber 104 are advantageously non-transparent, to prevent interference with testing.

[0053] In Figure 4 the thermal escape apparatus 300 is provided on a thermal regulation apparatus 116. The thermal regulation apparatus 116 can be integrated with the first chamber 102 and second chamber 104, or the thermal regulation apparatus 116 can be detachable and separable from the first chamber 102 and second chamber 104. The thermal regulation apparatus 116 can be configured to provide regulation to all or some of the first chamber 102 and the second chamber 104. For example, the thermal regulation apparatus 116 can be configured to provide the proximal section 108 or the distal section 110 at room temperature, substantially physiological temperature of the animal, or any of the temperatures discussed above. The thermal regulation apparatus 116 can also provide the distal section 1 10 with temperature regulation, for example by providing the distal section with temperature regulation at substantially room temperature or substantially physiological temperature. Advantageously the distal section 110 cannot be provided with a temperature regulation plate and be comprised of thermally -insulating materials, such as the thermally insulating materials of the distal section 110, thus allowing the apparatus to be easier to control, more efficient, and lower cost. In some embodiments, the thermal regulation apparatus 116 comprises the tw o metal temperature-controlled plates (sourced from Bioseb) whose temperature is controlled by a processor or microcontroller running control software (BIO-T2CT v2).

[0054] In each plot discussed below, the escape latency, or the time required for the mouse to move from the proximal section 108 of the first chamber 102 to the second chamber 104, is measured from the time the mouse is placed in the proximal section 108 to the time that all four paws of the mouse entered to the second chamber 104. The time trials were capped at 180 seconds and the trial ended at 180 seconds where the mouse did not enter the second chamber 104. The escape latencies were measured with a sensor, such as a camera, with no humans visibly in view, and the recordings were analyzed post hoc. Only one trial was performed per temperature for each mouse. After each trial, the mouse is returned to their home cage and allowed to reacclimate for at least fifteen minutes before beginning the next trial. The apparatus and metal plates were cleaned with a 10% bleach solution in between trials, and all trials were performed on the same day.

[0055] As an example, first chamber 102 is exposed to ambient light and the floor of the proximal section 108 is acclimated to room temperature. The second chamber 104 is covered and has reduced light compared to the first chamber 102 by at least 5 lux and has a floor surface that is acclimated to room temperature. The floor of the distal section 110 is temperature controlled to a temperature at least 10 degrees warmer or colder than the room temperature. A mouse is lowered into proximal section 108 by a human or machine, and the human or machine is retracted from the chamber. As the mouse is unconstrained in the proximal section 108 of the first chamber 102, the time of the trial begins. No human is in direct view during the duration of each timed trial. A sensor measures the signal or location of the mouse within the first chamber, and the time for each trial is measured until the first moment that all four of the mouse’s paws touch the second chamber 104 or the first moment that all four of the mouse’s paw s are absent from the first chamber 102. The mouse is removed from the apparatus after advancing to the second chamber 104 or after 180 seconds expire, whichever occurs first. Another mouse is probed at the same temperature. After all the mice have been probed at the designated temperature a new' temperature profile is set for the distal section 110. In some embodiments, the method of assaying a mouse in the thermal escape apparatus may be performed by following steps, either sequentially or non-sequentially: providing a first chamber 102 having a proximal section 108 and a distal section 110, wherein the proximal section contains a thermally- insulating floor and the floor of the distal section is thermally adjustable; providing a second chamber 104 having reduced visible light compared to the first chamber and a thermally -insulating floor; providing a passageway 106 connecting the distal section of the first chamber and second chamber: adjusting the thermally-adjustable material of the distal section 110 to a noxious temperature; and placing a rodent in the proximal section 108 that contains the thermally-insulating floor. In some embodiments, the thermally-insulating floor of the proximal section 108 can be a thermally neutral floor or acclimated to room temperature and the thermally-adjustable floor comprises one or more peltier elements. [0056] As discussed below, prior to assaying the rodent the rodent may be subjected to one or more physical or molecular modifications. In some embodiments, the rodent is subjected to brain trauma and a craniectomy. In some embodiments, the rodent is genetically modified to knockout one or more thermosensation ion channels, and the ion channels are selected from a group consisting of Trpvl, Trpal, Trpm3, and Trpm8.

[0057] Figure 5 is an experimental plot of escape latencies of non-habituated 8-12-week-old male and female C57B1/6 mice at various temperatures, 5° Celsius, 18° Celsius, 30° Celsius, and 52° Celsius using the thermal escape apparatus 100/300. The experiment in Figure 5 was conducted to test the effect of mouse behavior in the thermal escape apparatus 100 with a starting temperature of 30°C. The temperature trials were presented in the following order: 30°C, 5°C, 18°C, and 52°C, with 30°C operating as the control temperature. Figure 5 A shows the escape latencies of 21 male mice whereas Figure 5B shows the escape latencies of 25 female mice. Individual dots represent biological replicates.

[0058] Figure 5A and 5B Mean show that the escape latencies for males were: 5°C: 112.3 s ± 13.96 s; 18°C: 98.43 s ± 14.02 s; 30°C: 43.33 s ± 6.216 s, 52°C: 129.0 s ± 15.73 s. Mean escape latencies for females were: 5°C: 73.52 s ± 13.75 s; 18°C: 79.68 s ± 12.56 s; 30°C: 27.72 s ± 4.363 s, 52°C: 98.2 s ± 13.65 s. Statistical analyses found that escape latencies were significantly longer for 5°C, 18°C, and 52°C as compared to 30°C (5°C vs. 30°C. p=0.0192; 18°C vs. 30°C, p=0.0066; and 30°C vs. 52°C. mean ± SEM, p=0.0002). This demonstrates the thermal escape apparatus serves as a cost-benefit valuation assay that can be used to examine physiological thermosensing. Significance was determined using a One-way ANOVA with Dunnetfs multiple comparisons: *p<0.05, **p< 0.01, ***p<0.001, ****p< 0.0001.

[0059] Figure 6 is an experimental plot of escape latencies of non-habituated 8-12-week-old male and female C57B1/6 mice at various temperatures, 5° Celsius, 18° Celsius, 30° Celsius, and 52° Celsius using the thermal escape apparatus 100. In contrast with Figure 6, the temperature trials were presented in the following order: 5°C, 18°C, 30°C and 52°C, with 30°C operating as the control temperature, and the experiment of Figure 6 was conducted to probe the behavior of mice in the thermal escape apparatus with a starting temperature of 5°C. Thus, the results in Figure 6 show whether beginning the assay with a noxious temperature would affect escape latencies in subsequent trials. Figure 6A shows the escape latencies of 17 male mice whereas Figure 6B shows the escape latencies of 20 female mice. Individual dots represent biological replicates. _o Mean escape latencies for males were: 5°C: 112.6 s ± 16.2 s; 18°C: 77.82 s ± 15.44 s; 30°C: 86.18 s ± 15.66 s, 52°C: 136.9 s ± 15.51 s. Mean escape latencies for females were: 5°C: 124.3 s ± 14.93 s; 18°C: 89.2 s ± 14.05 s; 30°C: 77.85 s ± 13.76 s, 52°C: 134.5 s ± 15.9 s. Male and female escape latencies compared to baseline (30°C) were not significantly different between temperatures, except in the case of female mice when comparing escape latencies at 30°C and 52°C (p= 0.0215). Thus, beginning the assay with a trial at 5°C led to longer escape latencies in subsequent trials (compare Figure 5). Significance was determined using a Oneway ANOVA with Dunnett’s multiple comparisons. Thus, in some circumstances, starting a trial at a temperature other than the control temperature can be undesirable.

[0060] Figure 7 shows experimental testing of the behavior of mice in the thermal escape apparatus 100 after habituation. Figure 7 is an experimental plot of escape latencies of habituated (30 minutes of habituation the day before behavioral testing), showing that habitation to the thermal escape apparatus for 30 minutes the day before behavioral testing unexpectedly had a negative effect on performance in the assay. As shown, the latency of the mice in the chamber is greatly reduced after habituation. This is highly advantageous compared to other thermosensation apparatuses. 30 minutes of habituation prior to the testing requires a significant amount of time when 50 animals are involved in the testing and each animal can be individually habituated. Figure 7 demonstrates that the habituation is has a negative influence on the results of the study (comparing Figure 7 and Figure 5. which were conducted with the same temperature order of 30°C, 5°C, 18°C, and 52°C). The average escape latencies for each temperature tested were 5°C: 81.56 s ± 30.99 s; 18°C: 17.22 s ± 9.973 s; 30°C: 5.778 s ± 1.331 s, 52°C: 22.56 s ± 13.34 s, which were much shorter than when mice were not habituated to the thermal escape apparatus 100. In the testing for Figure 7. male and female mice were combined, and the assay length was increased to 300 seconds. No significant differences in escape latencies were observed between temperatures, except for 5°C vs. 30°C (n=9 mice, p=0.0127). As shown, the latency, or the amount of decision-making, such as cost-benefit decision making, that the mice had to undertake was very low when compared to the tests shown in Figures 5 and 6. Thus, habituation was shown as a cognitive shortcut for the mice, and decision-making behavior was skewed. Therefore, the thermal escape apparatus 100 disclosed herein is superior and unexpected as athermosensory assay at least in this regard. In some embodiments, the non-habituation period can be a period less than about 1 hour, less than about 45 minutes, less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, less than about 3 minutes, less than about 1 minute, less than about 30 seconds, less than about 10 seconds, or less than about 5 seconds. In some embodiments, placing the rodent in the proximal section comprises placing the rodent without any prior habituation to the first or second chambers.

[0061] Figure 8 shows the results of an experiment to test the behavior of mice in the thermal escape apparatus 100 where the mice have genetic modifications in Trpm8 and Trpvl ion channels. Figure 8 is an experimental plot of escape latencies of these genetically modified mice at various temperatures. Figure 8A and 8B began each trial at 30°C, followed by trials at 5°C, 18°C and 52°C. Figure 8A shows Trpm8-/- males (13 mice) and 8B shows Trpm8-/- female mice (14 mice). Trpm8-/- refers to a double knockout of genes for the thermotransduction channel Trpm8, which is activated by cold and provides a sensation of cold temperatures. Mean escape latencies for Trpm8-/- males were: 5°C: 29.15 s ± 10.12 s: 18°C: 34.33 s ± 8.281 s; 30°C: 33.62 s ± 4.464 s, 52°C: 63.77 s ± 14.82 s. Mean escape latencies for Trpm8-/- females were: 5°C: 26.5 s ± 8.9 s; 18°C: 13.43 s ± 1.68 s; 30°C: 25.43 s ± 3.587 s, 52°C: 28.5 s ± 8.338 s. Statistical analyses found no significant differences in escape latencies compared to 30°C and was determined using a One-way ANOVA with Dunnetf s multiple comparisons. Thus, this provides validation that the thermal escape apparatus 100 does provide a difference for mice lacking cold sensation, when compared to the latencies of mice that do not have this genetic modification.

[0062] Figure 8C and 8D likewise show escape latencies of genetically modified (Trpvl -/-) mice with trials beginning at 30°C, followed by trials at 5°C and 52°C. Trpvl-/- refers to a double knocked of genes for the thermotransduction channel Trpvl, which is activated by heat and provides a sensation of hot temperatures. Mean escape latencies for Trpvl-/- males (12 mice) were: 5°C: 53.83 s ± 16.0 s: 30°C: 31.83 s ± 5.352 s; 52°C: 47.0 s ± 13.37 s. Mean escape latencies for Trpvl-/- females (22 mice) were: 5°C: 79.18 s ± 14.42 s; 30°C: 24.09 s ± 2.632 s, 52°C: 78.23 a ± 12.85 s. Figure 8C shows no significant differences in mean escape latency for male Trpvl-/- mice compared to either 5°C or 52°C. Conversely, Female Trpvl-/- mice did have significantly slower escape latencies at 5°C and 52°C compared to 30°C, as shown in Figure 8D (n=22, 5°C vs. 30°C. p=0.0019; 30°C vs. 52°C, p=0.0023). Significance was determined using a One-way ANOVA with Dunnetf s multiple comparisons. Nevertheless, the data in Figures 8C and 8D associated with Trpvl-/- mice highlights the utility of the thermal escape apparatus 100 for the detection of deficits in thermosensation. In particular, Trpvl activity is regulated by sex hormones which could explain the sex differences between Trpvl-/- mice in the thermal escape apparatus 100. Further, Trpvl is activated by heat, though its role in temperature sensing is more complex than that of Trpm8. Data from figures 8C and 8D confirms that triple knockout of Trpvl, Trpal, and Trpm3 can be required to completely abolish noxious heat sensation. Thus, these results again validate the use of the thermal escape apparatus 100 as a means of probing thermsensation or lack thereof.

[0063] Figure 9 shows the results of an experiment to test the behavior of mice in the thermal escape apparatus 100 where the mice have undergone chronic constriction injury in order to induce thermal hypersensitivity. Figure 9 depicts escape latencies of mice in accordance with the CCI neuropathic pain model. Prior to this experiment a chronic constriction injury (CCI) was produced by ligation of the left common sciatic nerve in male and female 10-12-week-old C57B1/6 mice. Control (sham) mice also had the same surgery without the ligation of the left common sciatic nerve. 12-days post injury the animals were assayed in the thermal escape apparatus. Filled bars represent CCI-treated mice and clear bars represent sham controls. Individual dots represent biological replicates.

[0064] CCI mimics peripheral nerve injury and is one of the most important models to study neuropathic pain, as one of the most common pain behaviors observed following CCI is thermal hyperalgesia (thermal hypersensitivity). The mice in Figure 9 were first tested mice at the innocuous temperature of 30°C, followed by trials at 18°C and 10°C. Mean escape latencies were combined (30 mice) for male and female mice: 10°C: 73.07 s ± 10.29 s; 18°C: 20.90 s ± 2.541 s; 30°C: 19.33 s ± 1.304 s. Mean escape latencies for sham mice (8 mice) were: 10°C: 26.38 s ± 6.138 s; 18°C: 16.13 s ± 2.573 s; 30°C: 19.63 mean ± 4.855 s. Statistical analyses found escape latencies were significantly longer for CCI mice at 10°C compared to sham mice (CCI n= 30 mice and sham n=8 mice, p=0.0004), demonstrating the thermal escape apparatus 100 can be used to quantify injury- induced thermal hyperalgesia. Significance was determined using a Two-way ANOVA with Sidak’s multiple comparisons. Interestingly, the experiment did not observe significant differences in escape latencies for CCI animals at 18°C, though naive animals (animals that had not undergone any surgical procedure) had significantly longer escape latencies at 18°C compared to 30°C. It is possible that the thermal hypersensitivity induced by CCI injury motivates escape behavior up to a certain temperature threshold. Nonetheless, the results support the use the thermal escape box in preclinical pain studies to analyze the motivation and affective aspects of thermal pain.

[0065] Figure 10 is an experimental plot of escape latencies of same day repeat testing. 8-10-week-old C57B1/6 mice of both sexes were assayed in the thermal escape box assay at two different temperatures three times in the same day, with 90 minutes between trials. The temperature order was as follows: 30°C, 5°C. (A) For same day testing, mice were assayed three times on the same day with 90 minutes between each trial. Significant differences were observed between the trials at 5° C (n = 10 mice, Trial 1 vs. Trial 2, p = 0.0171; Trial 1 vs. Trial 3, p = 0.0380) and at 30° C (n = 10 mice, Trial 1 vs. Trial 3, p = 0.0013; Trial 2 vs. Trial 3, p = 0.03469). (B) For repeat testing during a one-week period, mice were assayed three times, and each trial was separated by one day. Significant differences were observed between the temperatures in the first trial (n = 10 mice, 5° C vs. 30° C, p = 0.0442) and the second trial (n = 10 mice, 5° C vs. 30° C, p = 0.0005). No significant differences in escape latencies were observed in the third trial. Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 27) = 2.774, P = 0.0802; Row Factor: F (1, 27) = 15.09, P = 0.0006; Column Factor: F (2, 27) = 7.724, P = 0.0022) with Tukey’s multiple comparisons. Individual dots represent biological replicates. Thus, same day repeat testing in the thermal escape box lengthens escape latencies.

[0066] Figure 11 is an experimental plot of repeat testing with the thermal escape box 100 within the same week. 8-10-week-old C57B1/6 mice of both sexes were assayed in the thermal escape box 100 at four different temperatures three times in the same week, with subsequent runs on every other day. The temperature order was as follows: 30 °C, 5 °C, 18 °C, and 52 °C. Significant differences were observed at 5 °C between Trial 1 and Trial 2 (A, n = 27 mice, p = 0.0016) and Trial 1 and Trial 3 (A, n = 27 mice, p = 0.0003), F-value = 14.15. Significant differences were observed at 18 °C between Trial 1 and Trial 3 (B, n = 27 mice, p = 0.0062). No significant differences in escape latencies w ere observed at 30 °C and 52 °C (C, D). Significance was determined using a Friedman’s One-way ANOVA (A-P = 0.0001; B-P = 0.0001; C-P = 0.3914; D-P = 0.3311) with Dunn’s multiple comparisons. Individual dots represent biological replicates. The average escape latencies for each temperature were: Trial 1 — 5 °C: 83.64 s ± 14.56 s, 18 °C: 97.14 s ± 13.2 s; 30 °C: 77.74 s ± 10.70 s, 52 °C: 138.1 s ± 11.05 s; Trial 2— 5 °C: 142.8 s ± 10.64 s, 18 °C: 126.3 s ± 12.37 s; 30 °C: 77.37 s ± 13.12 s. 52 °C: 145.9 s ± 1 1.63 s; Tnal 3—5 °C: 147.4 s ± 10.55 s, 18 °C: 133.7 s ± 11.62 s; 30 °C: 82.75 s ± 13.12 s, 52 °C: 133.4 s ± 12.18 s. Thus, repeat testing with the thermal escape box within the same w eek can be carried out and can impact performance.

[0067] Figure 12 is an experimental plot of the effect of thermal pain on effortbased decision making in chemotherapeutic induced cold allodynia. 10-12-week-old C57B1/6 female mice were assayed in the thermal escape box 100 following induced cold allodynia from chronic oxaliplatin injection. Mice were assayed at 5 days post final injection. The temperature order was as follows: 30 C, 18 C, and 5° C. Significant differences in escape latencies were observed between oxaliplatin and vehicle groups at 5°C (oxaliplatin n = 30 mice and vehicle n = 10 mice, p = 0.02). Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 76) = 1.479, P = 0.2343; Row Factor: F (1.085, 42.23) = 1 1.53, P = 0.0012: Column Factor: F (1, 38) = 3.008, P = 0.0910) with Tukey’s multiple comparisons. Filled bars represent oxaliplatin- treated mice and clear bars represent vehicle controls. Individual dots represent biological replicates. Thus, the thermal escape box detects the effect of thermal pain on effort-based decision making in chemotherapeutic induced cold allodynia.

[0068] Figure 13 is an experimental plot of the effect of thermal pain on effortbased decision making in chemotherapeutic induced cold allodynia. 10-12-week-old C57B1/6 female mice were assayed in the thermal escape box 100 following induced cold allodynia from chronic oxaliplatin injection. Mice were assayed at 5 days post final injection. Four hours prior to being assayed, mice were injected with the analgesic meloxicam (5 mg/kg), or a saline vehicle control. The temperature order was as follows: 30° C, 18° C, and 5° C. Significant differences in escape latencies were observed between meloxicam and vehicle groups at 5° C (meloxicam n = 10 mice and vehicle n = 10 mice, p = 0.0193). Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 36) = 2.280, P = 0.1169; Row Factor: F (2, 36) = 1 1.53, P < 0.0001 ; Column Factor: F (1, 18) = 1.455, P = 0.2434) with Tukey’s multiple comparisons. Filled bars represent meloxicam-treated mice and clear bars represent vehicle controls. Individual dots represent biological replicates. Thus, analgesic efficacy can be determined using the thermal escape box assay.

[0069] The results shown herein demonstrate that the thermal escape apparatus 100 is advantageously configured to study mechanisms of physiological thermosensation and thermal pain. The thermal escape apparatus 100 assay relies on unlearned, naturalistic escape behaviors to evaluate how temperature effects cost-benefit decision making. The thermal escape apparatus 100 forces mice to choose between staying in an aversive, brightly lit chamber, or traversing temperature-controlled plates to escape to a covered dark chamber. Experiments shown herein demonstrate that wild-type mice readily crossed plates set to 30°C, a preferred ambient temperature for mice. Conversely, deviation from this preferred temperature resulted in significantly longer escape latencies, suggesting mice took more time to evaluate the cost-benefit relationship of experiencing non-preferred temperatures to avoid an aversive environment. Performance in this assay does not require training, and habituation to the thermal escape apparatus decreased escape latencies to nonpreferred plate temperatures. On the other hand, the order of temperature presentation is an important experimental design consideration, as beginning the assay with a noxious temperature can increase escape latencies during subsequent trials, even at preferred temperatures. The thermal escape apparatus 100 has been validated (as shown in the plots in Figures 5-9) for detecting deficits in thermosensation using genetically modified mice and CCI modified mice, whereas other assays have not been validated with regard to genetically modified and CCI modified mice. Thus, the thermal escape apparatus is a novel decision-based behavioral paradigm for the study of thermosensation and thermal pain.

[0070] In this disclosure the term “non-habituated” should be given its ordinary meaning as would be understood by a person having ordinary skill in the art, but can also be interpreted as an animal that has not been habituated to a measuring apparatus. For example, the animal has not been exposed to the testing portion (such as the inside of the apparatus) of the apparatus for longer than 180 seconds prior to initiation of the study.

[0071] In this disclosure the term ‘'thermosensation” generally refers to the ability to ability to detect temperature and distinguish between temperatures that are innocuous or noxious. Thermal pain, which includes thermal allodynia and thermal hyperalgesia, can be an indication of many pathological conditions, and can negatively affect daily activities. Thermosensation is important to survival.

[0072] In this disclosure the terms “not transparent” or “not opaque” should be given their ordinary meanings as would be understood by a person having ordinary skill in the art, but can also be interpreted as blocking visible light.

[0073] In this disclosure the term “reduced light” should be given its ordinary meaning as would be understood by a person having ordinary skill in the art, but can also be interpreted as light that is at least 10 lux lower than a reference luminosity.

[0074] In this disclosure the term “noxious temperature” should be given its ordinary meaning as would be understood by a person having ordinary skill in the art. but can also be interpreted as a temperature that is at least 10 degrees Celsius higher or lower than a desirable temperature (such as 30 degrees Celsius). Additional Embodiments

[0075] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than restrictive sense.

[0076] Indeed, although this invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. In addition, while several variations of the embodiments of the invention have been shown and described in detail, other modifications, which are within the scope of this invention, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the invention. It should be understood that vanous features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes of the embodiments of the disclosed invention. Any methods disclosed herein need not be performed in the order recited. Thus, it is intended that the scope of the invention herein disclosed should not be limited by the particular embodiments described above.

[0077] It will be appreciated that the systems and methods of the disclosure each have several innovative aspects, no single one of which is solely responsible or required for the desirable attributes disclosed herein. The various features and processes described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure.

[0078] Certain features that are described in this specification in the context of separate embodiments also may be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment also may be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination may in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination. No single feature or group of features is necessary or indispensable to each and every embodiment.

[0079] It will also be appreciated that conditional language used herein, such as, among others, ‘'can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment. The terms “comprising,” “including,” “having,” and the like are synonymous and are used inclusively, in an open- ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. In addition, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list. In addition, the articles “a.” “an.” and “the” as used in this application and the appended claims are to be construed to mean “one or more” or “at least one” unless specified otherwise.

[0080] Further, while the methods and devices described herein may be susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but. to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various implementations described and the appended claims. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an implementation or embodiment can be used in all other implementations or embodiments set forth herein. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein may include certain actions taken by a practitioner; however, the methods can also include any third-party instruction of those actions, either expressly or by implication. The ranges disclosed herein also encompass any and all overlap, subranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between.” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers and should be interpreted based on the circumstances (e.g., as accurate as reasonably possible under the circumstances, for example ±1%, ±5%, ±10%, ±15%. etc.). For example, "about 3.5 mm” includes ‘'3.5 mm.” Phrases preceded by a term such as '‘substantially” include the recited phrase and should be interpreted based on the circumstances (e.g., as much as reasonably possible under the circumstances). For example, “substantially constant” includes “constant.” Unless stated otherwise, all measurements are at standard conditions including temperature and pressure.

[0081] As used herein, a phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of: A, B, or C” is intended to cover: A, B, C, A and B. A and C, B and C, and A, B. and C. Conjunctive language such as the phrase “at least one of X, Y and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be at least one of X, Y or Z. Thus, such conjunctive language is not generally intended to imply that certain embodiments require at least one of X, at least one of Y, and at least one of Z to each be present. The headings provided herein, if any. are for convenience only and do not necessarily affect the scope or meaning of the devices and methods disclosed herein.

[0082] Accordingly, the claims are not intended to be limited to the embodiments shown herein, but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein.

Examples

Example 1

[0083] The present non-limiting example was performed to test the escape latencies of 8-12-week-old male and female C57B1/6 mice beginning with a thermally- adjustable floor temperature of 30°C. C57B1/6 (stock no. 000664), Trpvl-/- ( stock no.003770) and Trpm8-/- (stock no. 008198) were obtained from The Jackson Laboratory. Mice were maintained in pathogen free conditions and studies Animal use was conducted according to guidelines from the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of UC Davis (#22438). Mice were maintained on a 12 hour light/dark cycle, and food and water was provided ad libitum. Genotyping was outsourced to Transnetyx. [0084] Behavioral testing was conducted in a quiet, isolated room maintained at ambient temperature and humidity. No other behavioral assay was conducted during testing sessions. Mice underwent room acclimation for 60 minutes prior to the start of behavioral testing on each experiment day. Mice were handled and transported to the testing room on 4-5 separate days before the start of data collection to habituate them to human contact and cart transport, respectively. The Thermal Escape Box was always positioned in the exact same location of the testing room during test sessions to maintain consistent ambient tight conditions.

[0085] The Thermal Escape Box consisted of conjoined light (465mm L x 185mm W x 345mm H) and dark (lOOmm L x 185mm W x 345mm H) chambers. The light and dark chambers were constructed from acrylic resin (50mm thick) and were solid white and black, respectively. The light chamber is open air with no lid and the dark chamber has a black lid and a narrowed entry way (40mm x 40mm) for access from the light chamber. The apparatus was placed on top of two metal temperature-controlled plates (Bioseb) whose temperature is controlled by external software (BIO-T2CT v2). The metal plates fit into an opening in a portion of the bottom of the light chamber. When assembled, the plates were flush with the bottom of the light and dark chambers. A video₀ camera was placed above the apparatus to record each trial, keeping investigator interference to a minimum.

[0086] Mice were placed individually in the center of the acrylic platform of the light chamber. Latency to escape to the dark chamber was video recorded and analyzed post hoc. Trial timing began when all four paws touched down on the white acrylic platform. Trial time ended when all four paws entered the dark chamber or after 180 seconds, which ever came first. Escape latency was recorded at each temperature, with one trial per temperature for each mouse. After each trial, the₀ mouse is returned to their home cage and allowed to reacclimate for at least 15 minutes before beginning the next trial. Repeating trials at the same temperature resulted in animal acclimation and learning (data not shown). Therefore, each mouse only performed one trial at each temperature. The apparatus and metal plates were cleaned with a 10% bleach solution in between trials. All trials were performed on the same day.

[0087] 8 -12-week-old male and female C57B1/6 mice (21 male mice and 25 female mice) were assayed in the thermal escape box and quantified escape latencies for four different temperatures. Temperatures were presented in the following order: 30°C, 5°C, 18°C, and 52°C. Mean escape latencies for males were: 5°C: 112.3 s ± 13.96 s; 18°C: 98.43 s ± 14.02 s: 30°C: 43.33 s ± 6.216 s, 52°C: 129.0 s ± 15.73 s. Mean escape latencies for females were: 5°C: 73.52 s ± 13.75 s; 18°C: 79.68 s ± 12.56 s; 30°C: 27.72 s ± 4.363 s, 52°C: 98.2 s ± 13.65 s. Experiment 1 results are shown in Figure 5. Statistical analyses found that escape latencies were significantly longer for 5°C, 18°C, and 52°C as compared to 30°C. Significance was determined using a One-way ANOVA with Dunnett’s multiple comparisons: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Individual dots in the figures represent biological replicates. Therefore, this example demonstrates the thermal escape box serves as a cost-benefit valuation assay that can be used to examine physiological thermosensing.

Example 2

[0088] The present non-limiting example was performed to test whether the escape latencies of 8-12-week-old male and female C57B1/6 mice were longer if the assay begins with a thermally-adjustable floor temperature of 5 °C. The escape latencies of 17 male mice and 20 female mice were probed. The animals were obtained, maintained, and monitored as in Example 1 and assayed using the thermal escape box of Example 1.

[0089] The mice were assayed using the following temperature order: 5°C. 18°C, 30°C and 52°C. Mean escape latencies for males were: 5°C: 112.6 s ± 16.2 s; 18°C: 77.82 s ± 15.44 s; 30°C: 86.18 s ± 15.66 s, 52°C: 136.9 s ± 15.51 s. Mean escape latencies for females were: 5°C: 124.3 s ± 14.93 s; 18°C: 89.2 s ± 14.05 s; 30°C: 77.85 s ± 13.76 s, 52°C: 134.5 s ± 15.9 s. Experiment 2 results are shown in Figure 6. Significance was determined using a One-way ANOVA with Dunnetf s multiple comparisons. Individual dots in the figures represent biological replicates. Male and female escape latencies compared to baseline (30°C) were not significantly different between temperatures, except in the case of female mice when comparing escape latencies at 30°C and 52°C. Thus, this example demonstrates that beginning the assay with a trial at 5°C led to longer escape latencies.

Example 3

[0090] The present non-limiting example was performed to test whether habituation to the thermal escape box affected the escape latencies of nine 8-12-week-old male and female C57B1/6 mice. The animals were obtained, maintained, and monitored as in Example 1 and assayed using the thermal escape box of Example 1. The mice were habituated to the first and second chambers of the thermal escape box apparatus for 30 minutes the day prior to behavioral testing. [0091] The mice were assayed using the following temperature order: 30°C, 5°C, 18°C, and 52°C. The average escape latencies for each temperature tested were 5°C: 81.56 s ± 30.99 s: 18°C: 17.22 s ± 9.973 s; 30°C: 5.778 s ± 1.331 s, 52°C: 22.56 s ± 13.34 s, which were much shorter than when mice were not habituated to the apparatus. Experiment 3 results are shown in Figure 7. Significance was determined using a One-way ANOVA with Dunnetf s multiple comparisons. Individual dots in the figures represent biological replicates. We only found a significant difference in escape latencies when comparing 30°C and 5°C (p=0.0127). Thus, this experiment shows that habituation to the thermal escape box negatively affects behavioral performance and decreases latency.

Example 4

[0092] The present non-limiting example was performed to test the escape latencies of Trpm8-/- male and female mice to see if a reduction in cold sensing affected latency. The animals were obtained, maintained, and monitored as in Example 1 and assayed with the thermal escape box of Example 1.

[0093] We tested Trpm8-/- male and female mice at 30°C, followed by trials at 5°C, 18°C and 52°C. Mean escape latencies for Trpm8-/- males (13 mice) were: 5°C: 29.15 s ± 10.12 s; 18°C: 34.33 s ± 8.281 s; 30°C: 33.62 s ± 4.464 s, 52°C: 63.77 s ± 14.82 s. Mean escape latencies for Trpm8-/- females (14 mice) were: 5°C: 26.5 s ± 8.9 s; 18°C: 13.43 s ± 1.68 s; 30°C: 25.43 s ± 3.587 s, 52°C: 28.5 s ± 8.338 s. Experiment 4 results are shown in Figure 8 A and 8B. Significance was determined using a One-way ANOVA with Dunnetf s multiple comparisons. Individual dots in the figures represent biological replicates. Statistical analyses found no significant differences in escape latencies compared to 30°C. Thus, this demonstrates that demonstrates that the thermal escape box is effective at detecting deficits in cold sensing via decreased latency.

Example 5

[0094] The present non-limiting example was performed to test the escape latencies of Trpvl-/- male and female mice to see if a reduction in heat sensing affected latency. The animals were obtained, maintained, and monitored as in Example 1 and assayed with the thermal escape box of Example 1.

[0095] Male and female Trpvl-/- mice were assayed at 30°C, followed by trials at_o 5°C and 52°C. This test excluded 18°C when testing Trpvl-/- mice, as the test did not anticipate any effect of loss of Trpvl on temperature sensing at 18°C. Mean escape latencies for Trpvl-/- males (12 mice) were: 5°C: 53.83 s ± 16.0 s; 30°C: 31.83 s ± 5.352 s: 52°C: 47.0 s ± 13.37 s. Mean escape latencies for Trpvl-/- females (22 mice) were: 5°C: 79.18 s ± 14.42 s: 30°C: 24.09 s ± 2.632 s, 52°C: 78.23 a ± 12.85 s. Experiment 5 results are shown in Figure 8C and 8D. The data shows no significant differences in mean escape latency for male Trpvl-/- mice compared to either 5°C or 52°C. Conversely, Female Trpvl - /- mice did have significantly slower escape latencies at 5°C and 52°C compared to 30°C, but differences between male and female mice can be explained by the differences in the sexes, as Trpvl activity is regulated by sex hormones. Thus, this example demonstrates that a reduction in heat sensing affected latency in male mice and the thermal escape box may be used to detect this deficit.

Example 6

[0096] The present non-limiting example was performed to test whether the escape latencies differed in mice with chronic constriction injury and the thermal escape box can be used to quantify injury -induced thermal hyperalgesia. The animals were obtained, maintained, and monitored as in Example 1.

[0097] A chronic constriction injury (CCI) was produced by ligation of the left common sciatic nerve in male and female 10-12-week-old C57B1/6 mice. Following standard aseptic techniques for survival surgery in rodents, mice were anesthetized in an induction chamber using 5% isoflurane in 02, then anesthesia was maintained during surgery via nose cone delivering isoflurane ant 1 -5% in 02. Lubricating ophthalmic ointment was applied to the animal’s eyes to prevent drying and post-surgical discomfort. The animal w as placed onto a thermo-regulated heating mat at 37°C.

[0098] The left hind leg of the animal was shaved and sterilized with three alternating applications of 70% isopropyl alcohol and iodine solution. An incision in the skin was made 3-4 mm below the femur and a cut w as made through the connective tissue between the gluteus superficialis and the biceps femoris muscles. A retractor was used to widen the gap between the two muscles, allowing clear visualization of the sciatic nerve. Approximately 10 mm of the sciatic nerve (proximal to the sciatic trifurcation) was freed from the surrounding connective tissue. Four ligatures (chromic gut 4.0) were tied with a double knot, 1 mm apart, proximal to the trifurcation of the sciatic nerve. A second loop was placed on top of the first to complete the knot. The loose ends of the ligature w ere cut to around 1mm. Constriction of the nerve in this manner is minimal and immediately stopped if a brief twitch is observed to prevent arresting of the epineural blood flow. Chromic gut sutures were used to close the muscle layer, and non-absorbable sutures (prolene 5.0) were used to close the skin. Mice were assayed in the thermal escape box 12 days post operation to allow for post-surgical inflammation to subside and for mice to reacclimate to_Q ambient cage conditions following injury. Sham animals underwent the same surgical procedures but in the absence of ligature placement.

[0099] Because the effect on escape latency at 5°C and 52°C was so strong in Example 1. these temperatures were excluded from analysis in CCI and shame mice. Mice were first tested mice at the innocuous temperature of 30°C, followed by trials at 18°C and 10°C. Mean escape latencies were combined (30 mice) for male and female CCI mice: 10°C: 73.07 s ± 10.29 s; 18°C: 20.90 s ± 2.541 s; 30°C: 19.33 s ± 1.304 s. Mean escape latencies for sham mice (8 mice) were: 10°C: 26.38 s ± 6.138 s; 18°C: 16.13 s ± 2.573 s; 30°C: 19.63 mean ± 4.855 s. Experiment 6 results are shown in Figure 9. Statistical analyses found escape latencies were significantly longer for CCI mice at 10°C compared to sham mice (CCI n= 30 mice and sham n=8 mice, p=0.0004). This is shown in Figure 9. Significance was determined using a Two-way ANOVA with Sidak's multiple comparisons. Thus, this example demonstrates that the thermal escape box can be used to quantify injury-induced thermal hyperalgesia.

Example 7

[0100] The present non-limiting example was performed to examine the effect of same-day repeat testing and repeat testing during a one week period on escape latencies. The animals were obtained, maintained, and monitored as in Example 1.

[0101] For same day testing, mice were assayed three times on the same day, with 90 minutes in between each trial. To limit total testing time, two temperatures were chosen, 30°C and 5°C. The average escape latencies for each temperature were: Trial 1: 30°C: 53.60 s ± 15.11 s, 5°C: 97.30 s ± 20.30 s; Trial 2: 30°C: 86.10 s ± 22.72 s, 5°C: 168.3 s ± 7.817 s; Trial 3: 30°C: 147.0 s ± 18.71 s, 5°C: 160.4 s ± 17.49 s. Within each temperature, escape latencies significantly increased with subsequent trials (Fig. 10A). Additionally, statistical significance was lost by the third trial when companng escape latencies at 30°C to 5 °C (Fig. 10B).

[0102] For repeat testing during a one-week period, mice were assayed three times, and each trial was separated by one day. The following temperature order was used during each trial: 30 °C, 5°C. 18°C. and 52 C. Escape latencies for 30°C and 52°C remained stable over the three trials; however, there were significant increases in escape latencies at 5°C and 18°C following repeat testing during the same week (Fig. 11, mean ± SEM values can be found in the figure legend). Collectively, these data indicate that repeat testing on the same day or on different days can be carried out, though the potential for increased escape latencies should be considered during experimental planification.

Example 8

[0103] The present non-limiting example was performed to examine whether the thermal escape box detects the effect of thermal pain on effort-based decision making in chemotherapeutic induced cold allodynia. The animals were obtained, maintained, and monitored as in Example 1.

[0104] We next examined the utility of the thermal escape box in detecting cold pain using the oxaliplatin-induced cold allodynia model. Female mice were injected intraperitoneally for five consecutive days with oxaliplatin (3 mg/kg) or vehicle (5% glucose), followed by five consecutive days of rest and a final five-day course of oxaliplatin, for a cumulative dose of 30 mg/kg (Braden et al., 2022). Mice were assayed five days after the final dose of oxaliplatin and escape latencies were quantified for the following temperature order: 30°C, 18°C and 5°C. Mean escape latencies were: 5°C: 80.38 s ± 13.79 s; 18’C: 19.70 s ± 1.856 s; 30°C: 24.36 s ± 2.929 s for oxaliplatin-treated mice. Mean escape latencies for vehicle- injected mice were 5°C: 44.21 s ± 16.54 s; 18°C: 18.67 s ± 1.258 s; 30°C: 12.48 mean ± 1.593 s. Statistical analyses show that escape latencies were significantly longer for oxaliplatin-injected mice at 5°C compared to vehicle-injected controls (Fig. 12).

[0105] Collectively, these results demonstrate the thermal escape box can be used to quantify chemotherapy-induced cold pain.

[0106] Finally, we asked if quantification of escape latencies in the thermal escape box were sensitive to analgesic drug affects. To accomplish this, we treated female mice with oxaliplatin as described above in Figure 12; however, four hours prior to testing, half of the cohort received a subcutaneous injection of the analgesic mel oxicam (5 mg/kg), whereas the other half received vehicle (saline). Mice were assayed using the following temperature order: 30°C, 18°C and 5 C. Mean escape latencies for meloxicam-treated mice were 5 C: 60.37 s ± 26.12 s; 18 C: 26.58 s ± 7.175 s; 30 C: 26.50 s ± 6.674 s. Mean escape latencies for vehicle-injected mice were: 5°C: 109.1 s ± 19.64 s; 18°C: 26.71 s ± 7.091 s; 30°C: 24.17 mean ± 2.656 s. Meloxicam treatment significantly reduced escape latencies at 5 C compared to vehicle treatment (Fig. 13).

[0107] Thus, analgesic efficacy can be determined using the thermal escape box assay.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for probing thermosensation, pain, or avoidance and tolerance of an animal to noxious environments comprising: a first chamber having a proximal section and a distal section, wherein the floor of the proximal section contains a thermally -insulating material and the floor of the distal section is thermally adjustable; a second chamber having reduced light compared to the first chamber a floor with a thermally-insulating material; and a passageway connecting the distal section of the first chamber and second chamber.

2. The apparatus of Claim 1, additionally comprising a sensor for detecting a location, presence, heat signature, or absence of the animal within the first chamber.

3. The apparatus of Claim 2, wherein the sensor is only configured to detect the absence of a mouse in the first chamber.

4. The apparatus of Claim 1. wherein the second chamber is enclosed on all sides by a material impermeable to light except for the passageway, and the passageway is configured for the passage of a rodent.

5. The apparatus of Claim 1, wherein the walls of the first and second chamber are not transparent or opaque and the passageway is a hole in the wall of the first chamber.

6. The apparatus of Claim 1, wherein the light in the first chamber is ambient light and the first chamber and second chamber are directly adjacent to each other.

7. The apparatus of Claim 1, wherein the thermally-adjustable material in the distal section comprises electronically thermally-adjustable peltier elements.

8. The apparatus of Claim 1, wherein the proximal section comprises 30% of the first chamber floor and the distal section comprises 70% of the first chamber floor.

9. The apparatus of Claim 1, wherein the distal section comprises at least 80% of the first chamber floor.

10. A method probing thermal or sensory pain in rodents comprising: providing a first chamber having a proximal section and a distal section, wherein the proximal section contains a thermally-insulating floor and the floor of the distal section is thermally adjustable; providing a second chamber having reduced visible light compared to the first chamber and a thermally-insulating floor; providing a passageway connecting the distal section of the first chamber and second chamber; adjusting the thermally-adjustable material of the distal section to a noxious temperature; and placing a rodent in the proximal section that contains the thermally- insulating floor.

11. The method of Claim 10, wherein the rodent has been subjected to brain trauma and a craniectomy.

12. The method of Claim 10, wherein the rodent has been genetically modified to knock out one or more thermosensation ion channels.

13. The method of Claim 12, wherein the ion channels are selected from a group consisting of Trpvl, Trpal, Trpm3, and Trpm8.

14. The method of Claim 10, wherein the thermal pain is induced by a chemotherapeutic agent.

15. The method of Claim 10, wherein the rodent is treated with an analgesic prior to being placed in the proximal section.

16. The method of Claim 10, wherein placing the rodent in the proximal section comprises placing the rodent without any prior habituation to the first or second chambers.

17. The method of Claim 10, wherein the rodent is placed in the proximal section more than one time in a 24-hour period.

18. The method of Claim 10, wherein the rodent is placed in the proximal section more than one time in a 7-day period with at least 24 hours between each placement.

19. The method of Claim 10, the thermally-adjustable floor comprises one or more peltier elements.

20. The method of Claim 10, wherein the thermally-insulating floor is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 10 degrees Celsius.

21. The method of Claim 10, wherein the passageway is configured for the passageway of a rodent and is vertically constrained and permanently open.

22. The method of Claim 10, additionally comprising measuring the location of the rodent in the first chamber with a sensor, wherein the sensor does not measure the second chamber.

23. The method of Claim 10, wherein the thermally-insulating material is substantially room temperature and the noxious temperature is hotter or colder than room temperature by at least 20 degrees Celsius.

24. The method of Claim 10, wherein the distal section comprises at least 70% of the floor of the first chamber.