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Introduction: Functional MRI in rats under anesthesia can largely minimize motion artifacts and attenuate the stress of the animal. However, two issues remain to be clarified and improved. First, fMRI results obtained with different types of anesthesia during surgical preparation and imaging show a large variability, which could be caused by the variable stress level of the rodents. Second, the most common surgical procedure used for anesthesia, blood gas analysis and mean arterial blood-pressure (MABP) monitoring is the femoral vein and artery catheterization that makes longitudinal studies difficult., Methods: In order to examine the variability of the stress level with three different anesthesia protocols using isoflurane (Iso), medetomidine-ketamine (MK) or propofol-remifentanil (PR), we measured the plasma corticosterone (CORT) concentration with (125)I-radioimmunoassay in blood samples collected prior to, immediately after and 60min after surgery. Tail-artery and vein catheterization was adapted for long-term monitoring of MABP with periodic blood sampling and is proposed as a less invasive and technically simple alternative to femoral vessel catheterization in fMRI preparation protocols., Results: We show that the CORT concentration depends on the anesthesia protocol with both alternatives providing more efficient stress reduction than the protocol using Iso. However, only the protocol using PR achieved a significant hormone reduction during surgery. Stress was not reliably manifested in changes in heart-rate and breathing-rate. Anesthesia and strain related changes in these two physiological parameters may be assigned to the pharmacological effects of the premedication and anesthetic agents. The results indicate also that MABP can be monitored over a long period of time (e.g. functional imaging session) through an arterial access point in the rat tail after cannulation with the proposed procedure., Discussion and Conclusion: Animals can experience stress during fMRI preparation protocols without obvious signs in commonly monitored physiological parameters. Our results challenge the efficiency of surgical protocols using Iso as mono-anesthetic agent, even when extended with topical analgesia. It was demonstrated that the CORT-based stress-level measurement through tail-artery cannulation can be used for developing anesthesia protocols (i.e. the presented PR protocol) when setting up future fMRI studies. The proposed surgical method for the tail is expected to facilitate longitudinal fMRI studies with permanent arterial access., (Copyright © 2014 Elsevier Inc. All rights reserved.)