En enkel og billig metode til bestemmelse Kolde Følsomhed og tilpasning i mus
Summary
The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.
Abstract
Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.
Introduction
Måling kold lydhørhed hos gnavere er vigtig for at forbedre forståelsen af de potentielle mekanismer i kulde følsomhed hos mennesker under både normale og patologiske tilstande. Den kolde Plantar Assay (CPA), der oprindeligt blev udviklet for flere år siden 1, er designet til at generere reproducerbare, utvetydige murine adfærdsmæssige reaktioner på en kold stimulus leveret ved stuetemperatur. Nyere forbedringer af dette assay har tilladt reproducerbar måling af koldt følsomhed ved en lang række temperaturer 2. Begge versioner er også designet til at være relativt højt gennemløb, og billig at anvende.
En stor del af der er gjort fremskridt i forståelsen af mekanismerne i koldt følsomhed ved hjælp af andre adfærdsmæssige metoder. En fremgangsmåde er acetone fordampning test, som indebærer duppe eller sprøjtning acetone på musen pote og måling af mængden af tid, som musen bruger på at stille pote 3,4. Desværresvarene til acetone fordampning er forvirret af våd fornemmelse og lugten af acetone. Ligeledes kan den kolde stimulus, der anvendes i acetone fordampning test variere baseret på mængden af acetone anvendes, og er vanskelig at kvantificere. Endelig uskadte mus har minimale reaktioner på acetone ved baseline, hvilket gør det umuligt at måle analgesi i fravær af overfølsomhed med denne metode.
En anden klassisk assay til kolde svar er halen svirp assay, hvor latenstiden til tilbagetrækningen er målt efter halen er nedsænket i koldt vand 5,6. Mens de adfærdsmæssige reaktioner i denne analyse er entydige og analysen måler svar på en bestemt temperatur, skal dyrene fastholdes under test, som kan ændre kold lydhørhed gennem velbeskrevne stressinducerede smertestillende mekanismer 7.
En anden almindeligt anvendt værktøj er den kolde plade test, som måler de adfærdsmæssigereaktioner af mus efter de er placeret på et Peltier-kølet plade 8-10. Mens dette værktøj indeholder oplysninger om dyr reaktioner på bestemte temperaturer, er det også blevet inkonsekvent anvendt; forskellige grupper har målt forskellige reaktioner, herunder antal hopper 8,11, latenstiden til første reaktion 8,11- 13, og antallet af pote løfter 11,13,14 med meget forskellige resultater. Den kolde plade assay er også relativt lav gennemløb som kun et dyr kan testes på et tidspunkt, og det kræver et dyrt og skrøbelige Peltier indretning.
2-plade præference temperatur test er en almindeligt anvendt derivat af den kolde plade test, der måler den relative mængde af tid, som dyrene tilbringer på 2 forbundne plader af forskellige temperaturer 9,15- 17. En anden lignende almindeligt anvendt assay er den termiske gradient assay, hvor mængden af tid, at mus tilbringer i forskellige temperaturzonermellem 5 ° C og 45 ° C på en lang metalplade måles 16. Mens disse analyser kunne sammenligne temperaturer, er det uklart, om den adfærd repræsenterer temperaturen aversion eller præference temperatur.
Endelig har den dynamiske kold plade assay blevet anvendt til at måle, hvordan mus reagere på skiftende temperaturer 18. Denne metode indebærer at placere mus på en RT Peltier enhed og rampe ned til 1 ° C under måling, hvor meget musene hoppe eller slikker deres poter ved forskellige plade temperaturer. Mens dette tester hvordan mus tilpasse sig et kølende miljø, betyder det ikke en måde at teste, hvordan mus reagere på en kold stimulus i fastsættelsen af en køligere omgivende temperatur. Derudover kræver dyrt udstyr til at udføre og giver ikke en måde at akklimatisere mus til det udstyr, før måling af deres kolde følsomhed.
For at supplere disse analyser, CPA tester acclimated svar på et veldefineret koldt stimulus på en række temperaturområder, eller i løbet af processen med tilpasning til kolde temperaturer. Det kan teste op til 14 mus ad gangen med vores aktuelle apparat med potentialet til at blive billigt opskaleres for high-throughput test.
Protocol
Representative Results
Discussion
The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.
Materials
| Name | Company | Catalog Number | Comments |
| T-type thermocouple probe | Physitemp | IT-24p | Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/) |
| Glass plate | Local glass company (in St. Louis, Stemmerich Inc) | N/A | We use pyrex glass (borosilicate float). Our lab generally uses 1/4'', but 3/16'' and 1/8'' are also useful |
| Thermal Data logger | Extech | EA15 | Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&prodid=408) |
| 3mL Syringe | BD | 309657 | The top is cut off, and dry ice is compressed in the syringe to generate a cold probe |
| Computer | If using Extech logger, any Pcwill work | N/A | |
| Aluminum boxes | Washington University in St. Louis machine shop | N/A | boxes are 3' long, 4.5'' wide, and 3'' tall with a sealed lid. There is a 1/2'' hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water. |
| Heated water circulator | VWR | Any water circulator model with a pump will work | |
| 21 gauge needle | BD | 305165 | The exact needle size is not important |
| Hand timer | N/A | Any hand timer will work | |
| Mirror | N/A | Any flat mirror will work |
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