Comparison of Novel and Traditional Bleeding Techniques in Neonatal and Juvenile Mice.

Blood collection is frequently used for neonatal and juvenile mice in toxicology, developmental, and immunology studies and is often a terminal procedure. However, the use of nonterminal blood collection techniques, including the submandibular and the submental collection techniques described for adult mice, may offer opportunities to reduce animal numbers and refine current methods. The use of the submental technique has not been described for neonatal or juvenile mice. In this study, we compared the submental and submandibular blood collection techniques to determine their suitability for use in neonatal and juvenile mice. Male and female CD1 mice, ages 7, 14, 21, and 28 d, were randomized by sex into submental (n = 16), submandibular (n = 16), or control (n = 8) groups. Each mouse was weighed, bled per its assigned group (or only restrained in the case of control mice), and then decapitated without anesthesia for terminal blood collection. Blood collection volume and corticosterone concentrations were measured. The 2 methods showed significant differences in the volume of blood collected at ages 14 and 28, with the submandibular technique yielding significantly higher volumes. No significant differences were detected in corticosterone levels between the 2 techniques based on age or sex. A subset of mice (n = 8, 2 per age group) were bled via submental or submandibular technique and were evaluated 48 h later for gross and histopathologic evidence of trauma. Seven of the 8 mice showed expected inflammation and healing at the collection sites, with 4 mice having embedded strands of fur in the tissue. These data indicate that the submental blood collection is a viable method for nonterminal blood collection method in neonatal and juvenile mice, especially when smaller amounts of blood are needed.

Acute Effects of Hypothermia and Inhalant Anesthesia on Ultrasonic Vocalizations and Neuroendocrine Markers in Neonatal Rats.

Neonatal rodents undergo anesthesia for numerous procedures and for euthanasia by anesthetic overdose. However, data regarding whether neonatal anesthesia is humane are limited. Hypothermia (cryoanesthesia) is the most commonly used anesthetic protocol for neonatal rats 10 d of age or younger. However, hypothermia has recently been restricted in several countries due to perceived painful effects, including pain on rewarming. Minimizing the potential pain and distress of neonates in research is imperative, although very challenging. Traditional validated and nonvalidated behavioral and physiologic outcome measures used for adult rats undergoing anesthesia are unsuitable for evaluating neonates. Therefore, we investigated the effects of several anesthetic methods on neonatal rats by using the innovative objective approaches of noninvasive ultrasonic vocalizations and more invasive neuroendocrine responses (i. e., serum corticosterone, norepinephrine, glucose). Our results show that hypothermia leads to heightened acute distress in neonatal rats as indicated by prolonged recovery times, increased duration of vocalizations, and elevated corticosterone levels, as compared with neonates undergoing inhalational anesthesia. We demonstrate that inhalational anesthesia is preferable to cryoanesthesia for neonatal rats, and researchers using hypothermia anesthesia should consider using inhalational anesthesia as an alternative method.