Long-term in vivo imaging in adult zebrafish (i.e., 1-24 hours) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults, with respiratory arrest occurring within ~10-50 mins using MS-222 or MS-222 cocktails [1]. Here, we report the potential for dynamic administration of benzocaine to enable long-term anesthesia of up to 24 hours in adult zebrafish. We developed a computer-controlled perfusion system comprised of a 3D printed imaging chamber coupled to programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 hours and 8-hour survival of 14.3%. We postulated that an intermittent regimen consisting of ~100% of the average time to sedation and ~50% of the average time to recovery would prolong time to respiratory arrest compared to continuous administration. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 hours and 8-hour survival of 71.4%. To examine the potential for this regimen to extend anesthesia beyond an 8-hour period, we performed a single 24-hour trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire period, with respiratory rates comparable to those observed in fish that survived to 8 hours in intermittent benzocaine trials. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to significantly extend anesthetic periods in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 hours.
References
1. Huang WC, et al.: Combined use of MS-222 (tricaine) and isoflurane extends anesthesia time and minimizes cardiac rhythm side effects in adult zebrafish. Zebrafish. 2010;7:297-304.