1Department of Pharmacology and Toxicology, Wright State University, 2Human Movement Laboratory, Universidade São Judas Tadeu
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Alshahrani, S., Fernandez-Conti, F., Araujo, A., DiFulvio, M. Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats. J. Vis. Exp. (63), e3785, doi:10.3791/3785 (2012).
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 temperatures3,5. 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.
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 temperatures.
1. Equipment Set-up
2. Testing Normal Animals
Either young or adult Wistar rats [post-natal (p) age 21-25 days], or mice (p21-25) of any genetic background or gender can be used. The mice used in our experiments are of the strain C57BL/6J.
3. Testing Diabetic Animals
The streptozotocin (STZ)-induced diabetic rat is the most commonly used animal model to study mechanisms of PDN and to assess analgesic drugs and therapies11,12. Rats made diabetic by a single intraperitoneal dose of STZ (60 mg/kg) can be tested for thermal hyperalgesia during the 3rd week after onset of diabetes11. It is important to note that not all diabetic rats develop PDN. However, animals that do develop hyperalgesia (~50%) can be easily identified. Indeed, their TNTs are lower than normal*.
*To determine TNTs of STZ-diabetic rats, a starting plate temperature of 15 °C instead of 28 °C is used. The reason for these change is that most diabetic animals with PDN are expected to be hyperalgesic2,11. Therefore, they will have lower than normal noxious heat thresholds (e.g., <45 °C).
4. Testing Analgesia
The anti-nociceptive properties of drugs (e.g., analgesics) as well as the determination of pharmacologic parameters such as the minimum effective dose can be easily obtained by using the steps explained above. It is predicted that proper doses of an analgesic administered to rats/mice would significantly increase their TNTs. This is particularly sensitive in animals with thermal hyperalgesia3. However, to avoid bias in the interpretation of the results, the investigators involved in the behavioral observation of an animal group must not know or be aware if the animals had been treated or not. Further, drugs to be injected by the observer may be prepared by another investigator and labeled A, B, C, etc. Dosage is provided in a printout, listing individual doses per identified animal.
Depending on the drug being tested, different doses of a potential analgesic may be administered to normal or diabetic animals 5-15 min prior behavioral tests. Proper controls must be included (i.e., age-matched animals injected with vehicle alone and/or diabetic rats without PDN) and tested using identical conditions (e.g., same starting temperature of the plate). Chronic effects of drugs can be tested as well.
5. Representative Results
The lowest plate temperature evoking nocifensive reactions in either hind-paw of young adult normal non-treated rats or mice were determined following steps 2-1 to 2-6. As shown in Figure 1, TNT of normal young adult rats and mice were 47.2 ± 0.2 °C and 47.5 ± 0.5 °C, respectively.
We have observed neither significant daily variation of individual's TNTs (Figure 2A) nor significant changes of TNTs among individuals of the same age (Figure 2B). TNT differences due to gender were not observed (not shown).
TNTs of young adult STZ-diabetic rats were determined in a similar fashion as for normal rats (Steps 3.1 to 3.3). However, the starting plate's temperature was set to 15 °C instead of 28 °C. The nocifensive behavior of STZ-diabetic rats was assessed at least three times in an individual and in a daily fashion starting eleven days post-STZ injection. As shown in Figure 3, a significant drop (p<0.01) in the TNT of STZ-diabetic rats becomes evident two weeks post-STZ injection. The mean TNT of STZ-diabetic rats was 45.6 ± 0.1 °C (n=16, pooled values from days 14-23).
Diabetic rats exhibiting significantly lower TNT than normal were considered to have PDN. These rats were used to test the acute anti-nociceptive properties of an analgesic (compound A). TNTs were determined in normal and diabetic rats injected with either a single dose of the compound or vehicle alone five minutes before the test. As shown in Figure 4, a single intra-peritoneal dose of the compound significantly increased TNTs of diabetic rats when compared to rats treated with vehicle alone (45.6 °C vs 47.6 °C, respectively). The anti-nocifensive effect of compound A was also observed in normal rats; although significant (p<0.01), it was less pronounced (47.4 °C vs 48.0 °C, respectively).
Figure 1. Determination of TNTs of normal young adult rats or mice: The TNTs of young adult rats (p21-24) or mice (p20-21) were tested in the iHPAM following the steps outlined above. A total of 12 rats and 25 mice were tested. Measurements were performed during several days and the results were pooled. Results are expressed as mean ± SEM. The minimum and maximum TNT values recorded in these rats were 46.2 °C and 48.6 °C respectively. In the case of mice, the maximum and minimum TNT values recorded were 48.8 °C and 44.9 °C, respectively.
Figure 2. Age-dependency of TNTs in rats and mice: A) Plotted are the TNTs of four normal rats (n=4) of 21 days of age determined at least three times daily during 5 days. B) TNTs of mice littermates determined once at indicated ages. The numbers on top of each determination point represents the number of littermates used.
Figure 3. Diabetic rats become hyperalgesic 3 weeks after the onset of diabetes: TNTs of six (n=6) age matched young adult STZ-diabetic rats were determined every day starting 11 days post-induction of diabetes. Shown are daily TNT averages (mean ± SEM). Diabetic TNTs determined between 11-13 days post-STZ injection were within the normal range (red line). Two weeks after STZ injection TNTs decreased significantly to 45.1 ± 0.4 °C, (p<0.05).
Figure 4. Acute anti-nocifensive effects of compound B in STZ-diabetic rats. Young adult normal rats (blue group n=12) and age matched STZ-diabetic hyperalgesic rats (green group n=6) were tested in the iHPAM after intraperitoneal injection of vehicle (dark bars) or compound A (light bars). The mean TNT ± SEM for each treatment (vehicle control or compound A-treated) are shown. Asterisks denote statistical differences (p<0.05).
Similarly to the classic hot plate test to quantify thermal hyperalgesia4,13, 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).
Under the conditions described, normal young adult rats and mice showed nocifensive behavior at temperatures surrounding 47 °C. Although these thresholds are slightly higher than those reported by other investigators3,5,13, our results are in agreement with the thermal nociceptive threshold of 46-48 °C observed in humans14, monkeys15 and other animal models10. The differences in TNTs from our studies and those published3,5,13 may be related to the age of the animals used. Indeed, thermal nociceptive thresholds are higher in young adult rats than those of rats that are 4 wk of age or older16.
In conclusion, following the steps outlined above, the incremental hot-plate analgesia meter allows determination of the thermo-nociceptive thresholds of normal/diabetic rats or mice in a reproducible manner. Further, upon administration of analgesics, particularly to hyperalgesic rats, an increase in the TNT can be statistically detected. This method can be useful to screen potential analgesics in mice and rats under conditions of minimal stress.
We have decided not to disclose the identity of the analgesic used in these experiments. Part of the results shown here has been presented in the 71st Scientific Sessions of the American Diabetes Association (San Diego CA) and the 47th Annual Meeting of the European Association for the Study of Diabetes (Lisbon, Portugal).
This work was funded by the American Diabetes Association (ADA), Grant JF1-10-14 (MDiF). We would like to thank the personnel of the Laboratory of Animal Resources at WSU. Authors gratefully acknowledge assistance with the statistical analysis of data from Neil Paton, Ph.D.
|Incremental Hot-Plate Analgesia Meter||IITC Inc. Life Science||Part #PE34|
|Soft Series 8||IITC Inc. Life Science||Part # Series8|