Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Summary

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Abstract

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli¹. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus². However, as stated by Bölcskei et al, determination of "pain" in animal models is challenging due to its subjective nature³. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity³. For instance, by using the hot-plate method of Ankier⁴, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 °C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures^3,5. As the recently described method of Bölcskei et al.⁶, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Almási et al ⁵ and Bölcskei et al ³. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Protocol

TNTs of mice and rats are determined by using the incremental hot-plate analgesia meter [iHPAM, IITC Inc. Life Science (Woodland Hills, CA)]. The equipment consists of several components: an aluminum plate (10 x 20 cm) with the heating system underneath and a Plexiglas observation chamber above; a heat controlling unit, software for data acquisition (IITC Part # Soft Series 8), a PC (personal computer) and a footswitch for remote start, stop or reset control of the unit. The heating system also allows starting/stopping/resetting of the heating process manually through a keypad in front of the equipment. The iHPAM can be set to various heating rates, standby/cutoff tem....

Discussion

Similarly to the classic hot plate test to quantify thermal hyperalgesia^4,13, the assay of nociception described here permits a fast and reliable way to quantify nocifensive behavior in rats and mice. However, contrary to classic test, the incremental hot-plate method is non-invasive and virtually stress-free. Although some restrain is necessary to perform the test (i.e., the animal must be in the observation chamber), rats or mice are accustomed to similar areas (e.g., housing cages).

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Incremental Hot-Plate Analgesia Meter	IITC Inc. Life Science	Part #PE34
Soft Series 8	IITC Inc. Life Science	Part # Series8
Streptozotocin	Calbiochem	572201