Een eenvoudige en goedkope methode voor het bepalen Koude Gevoeligheid en aanpassing in Muizen

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

Meten koude responsiviteit bij knaagdieren is van belang voor een beter begrip van de mogelijke mechanismen van koude gevoeligheid bij mensen zowel onder normale en pathologische omstandigheden. De Koude Plantaire Assay (CPA), oorspronkelijk ontwikkeld enkele jaren geleden ^1, is ontworpen om reproduceerbare, eenduidige muizen gedragsreacties op een koude stimulus afgeleverd bij RT genereren. Meer recente verbeteringen van deze assay kon de reproduceerbare meting koude gevoeligheid uiteenlopende temperaturen ^2. Beide versies zijn ook ontworpen om relatief hoge doorvoer, en goedkoop te gebruiken.

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Protocol

Alle muis protocollen waren in overeenstemming met de National Institutes of Health richtlijnen en werden goedgekeurd door de Animal Studies Committee van de Washington University School of Medicine (St. Louis, MO).

1. Voorbereiding van de testplaat en behuizingen

1. Reinig het glasoppervlak.
2. Zet het T-type filament thermokoppel aan de oppervlakte in het midden van de glasplaat met laboratorium tape.
3. Plaats de dierenverblijven op de glasplaat in één lijn langs het midden van de plaat.
4. Rijg de thermokoppelsonde door het centrum dierenverblijf en steek de stekker in de datalogger. Draai de datalogger op....

Results

De gedragsreacties ontlokt muizen beginnend bij 30 ° C, 23 ° C, 17 ° C en 12 ° C zijn zeer reproduceerbaar (figuur 4A) ^20. Om de koude stimulus onder de achterpoot wordt gegenereerd meten, werden muizen geanesthetiseerd met ketamine / xylazine / acepromazine cocktail en de poten zijn bevestigd aan het glas op een T-type thermokoppel filament (figuur 4B) ^20. Het glas werd gekoeld of verwarmd tot de gewenste testbereik. Hoewel de plaat gelijkmatig gekoeld langs de le.......

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.......

Materials

List of materials used in this article

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)		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)
3 ml 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
Aluminum boxes	Washington University in St. Louis machine shop		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 G needle	BD	305165	The exact needle size is not important
Hand timer			Any hand timer will work
Mirror			Any flat mirror will work