THE AUTOMATED NOCIEPTION ANALYZER (ANA)
Injection of an irritant, such as formalin into the skin will result in the persistent activation of small sensory afferents and will evoke an organized constellation of responses which includes favoring, licking and flinching of the injected paw. The incidences of these nocisponsive behaviors occur in two phases: an initial first phase (phase 1, 0-10 min) and a delayed second phase (phase 2, 10-60 min).
The formalin test is a complex model that probes the pharmacology and physiology of systems that are activated by tissue injury. The formalin test is a valid model for components of clinical pain associated with tissue injury. This places it in contrast to other models where the response is evoked by an acute, transient stimulus that does not produce tissue injury.
The formalin test is in wide use for mechanistic studies of nociception and for the evaluation of analgesic and anti-hyperalgesic agents in rats and mice.
The test involves injecting one hind paw of the rodent with a small volume (10-50µL) of formalin (1-5%). The animal is observed to favor the paw and display periodic flinches in the injected limb. These flinches are counted at periodic intervals for typically up to an hour after injection. In normal practice, the observer is required to discriminate between a response or normal movement of the animal.
Relying on human observers, the test requires considerable training to establish high “inter-observer” reliability; it is tedious, requiring uninterrupted attention on the part of the observer and finally it is labor intensive.
The ANA device was designed to address these issues. ANA automatically detect the occurrence of paw flinches. This is accomplished by measuring the movement of a small metal band (0.5 grams) that is placed on the injected paw; the animal is placed without restraint inside the observation chamber over an electromagnetic detector system. 
Paw flinches are detected by the system and counted automatically using a computer. At the end of the test, a file is written that contains the comment for each observed animal and the number of flinches per minute over time. The base system can accommodate 4 animals at a time. The upgrade package will allow the researcher to measure an additional 4 animals concurrently.
Depending upon study parameters this system can thus permit a single technician to undertake relatively large scale screening (6 runs/day x 8 rats/run). Moreover, the design of the system reduces the time required to train a technician. The time and labor to develop validating results is reduced to essentially a day.
The system as configured includes equipment and supplies to test 4 animals simultaneously and allow 4 more animals to acclimate over the period of testing. The equipment consists of, 4 detection devices, 8 clear plastic observation cylinders, signal acquisition module and BNC input interface with interconnecting cable, automated formalin testing, and data analysis software.
Included with the analysis software, is a program for searching standardized file names, and moving data from these files into an Excel spreadsheet. (template supplied) The Excel spreadsheet calculates the count, mean, standard deviation, standard error and % maximum possible effect (MPE), on the control, drug, and dose data.
The source documentation is at:
https://www.ncbi.nlm.nih.gov/pubmed/11356806
Thermal Nociception Test Device
The Thermal Nociception Test Device was developed as described by Hargreaves et al. (1988). Our PAWS device is an improvement on the Hargreaves conception. PAWS consists of a glass surface upon which the mice or rats are placed individually in Plexiglas cubicles. The glass surface temperature (either 30± 0.1°C or 25± 0.1°C) is maintained by a feedback-controlled, under-glass, forced-air heating system and is controlled by a thermocouple on the bottom of the glass plate.
The thermal nociceptive stimulus originates from a focused projection bulb mounted in a stimulus tower that is manually manipulated in a two-dimensional axis on ball bearing slides to permit the stimulus to be delivered separately to either hind paw of each test subject. This stimulus is positioned under the foot pad with the aid of an angled mirror mounted on the stimulus source, permitting an exact visual targeting of the stimulation site prior to stimulus initiation. A timer is automatically actuated with the light source, and response latency is defined as the time required for the paw to show an abrupt withdrawal.
Paw withdrawal is detected by motion sensors mounted on the stimulus tower that stops the timer and terminates the stimulus. Stimulus current from a regulated source is monitored continuously to determine the amperage delivered to the light source and, thereby, the magnitude of the radiant stimulus to which the paw is subjected. In all cases, a cut-off of 20 seconds is employed to avoid tissue injury.
Features of the device:
• CONCURRENT TESTING OF GROUPS OF ANIMALS
The large stimulus area combined with the easily adjustable stimulus probe allows for testing of up to 6 rats or mice in adjacent plastic enclosures.
• AUTOMATIC CUT OFF
Paw withdrawal leads to changes in reflectance; use of an array of 3 spatially arranged photo transistors ensure adequate sensitivity to movement and determination of latency.
• SURFACE TEMPERATURE CONTROLLER WITH DIGITAL DISPLAY
Thermocouple controlled feedback circuit controls the temperature of circulating air allowing selection of glass temperature for reliable baseline despite minor changes in room ambient temperature. A LED display updated at 10 Hz will indicate glass or stimulus temperature.
• THROUGH FLOOR STIMULUS LOCALIZATION
A mirror positioned on the moveable stimulus tower allows the investigator to position the stimulus by direct visualization of the plantar surface of the paw no matter the posture of the animal.
The full reference is given at: