A new formalin test allowing simultaneous evaluation of cardiovascular and nociceptive responses

The formalin test for nociception, predominantly used with rodents, is characterized by continuous pain due to tissue injury induced by formalin. In addition to the pain generated by formalin itself, the classical formalin test for nociception comprises a number of additional strong stressful events such as restraint and injury caused by needle insertion. These events have hampered its use as a model of stress employing continuous pain as a stress stimulus. We describe here a new, simple method of a subcutaneous application of formalin in conscious rats without restraint and needle insertion. Formalin or physiological saline (controls) was injected subcutaneously through a chronically implanted catheter in the region of the lower leg. In most animals, the cardiovascular and behavioural responses to subcutaneously injected formalin were biphasic. The early phase was quantitatively characterized. Formalin (2.5%, 50 mu L) induced a marked increase in mean arterial pressure and heart rate. Maximal increases occurred during the first 3 min after formalin injection and were followed by a gradual decline of both cardiovascular parameters to levels higher than the preinjection baseline. Subcutaneous saline injection did not induce any changes in mean arterial pressure and heart rate. The behavioural response to formalin featured intensive licking and biting of the injection site. The behavioural response to subcutaneous saline did not differ from the spontaneous behavioural activity recorded in noninjected rats. Subcutaneously injected formalin induced an immediate increase in plasma corticotrophin (ACTH), corticosterone, noradrenaline, and adrenaline levels, and all hormones remained increased during the whole observation period (60 min). Adrenaline and noradrenaline levels in plasma were slightly, but significantly, elevated during the initial 5 min after subcutaneous saline application but returned to basal values after 15 min. The magnitude of the hormonal responses characterizes the described formalin test as a moderate stress stimulus. The complex response pattern to formalin injected through the subcutaneous catheter is brought about exclusively by pain generated by formalin-induced tissue injury. The described technique of subcutaneous formalin injection represents a new tool to study mechanisms activating brain neuronal circuits that generate the cardiovascular, endocrine, and behavioural responses of the reaction to pain.