Supplementary Material for: Neonatal anoxia impacts rats' maternal behavior and offspring development due to ultrasonic vocalization's impairment

Hypoxic-ischemic encephalopathy is a severe clinical condition, among others, affecting the brain after offspring exposure to neonatal anoxia, which causes persistent sensorimotor and cognitive deficits. During peripartum, maternal behaviors are crucial for the healthy development of the offspring. In rats, the vocalization of newborns, around 40 kHz, corresponds to separation calls that encourage their mothers to retrieve them. Alterations in this pattern affect the maternal behavior addressed to the offspring. This study aimed to evaluate the maternal behavior of primiparous rats whose offspring were exposed to neonatal Anoxia in P2 (postpartum day) during the lactation period, to assess mother-pup interactions through the pups' vocalization from P3 to P18. It also intends to quantify eventual neuronal alterations in the mothers' medial preoptic area (MPOA) after the last weaning (P21) through FOS protein expression. Anoxia offspring were found to reduce maternal behaviors towards them, increased frequency of separation calls in the male anoxia group, and reduced vocalization rate in the female anoxia group compared to their respective controls. Body weight gain reduction of males' and females' anoxia was observed. We concluded that anoxia exerts deleterious effects on the vocalization patterns of the pups, with sex differences that alter maternal behavior towards them. Impaired USV makes an additional negative impact on the already noxious effects of neonatal anoxia. Understanding those phenomena apply/contributes to guiding procedures and strategies to mitigate the deleterious outcomes and orient research concerning the complexity of neonatal anoxia events and the influence of maternal care quality concerning the pups, which should also be considered sex differences.

缺氧缺血性脑病（hypoxic-ischemic encephalopathy）是一类严重的临床病症，当子代暴露于新生儿缺氧环境后可引发脑部损伤，进而导致持续的感觉运动与认知功能缺损。围产期阶段，母性行为对子代的健康发育至关重要。在大鼠模型中，新生幼崽约40kHz的发声对应诱导母鼠衔回幼崽的分离叫声，该发声模式的改变会影响母鼠对子代的抚育行为。本研究旨在评估泌乳期内，幼崽于产后第2日（P2）暴露于新生儿缺氧环境的初产雌鼠的母性行为，并通过产后第3日（P3）至第18日（P18）幼崽的发声情况评估母鼠与幼崽的互动关系；同时还拟在末次断乳（P21）后，通过检测母鼠内侧视前区（medial preoptic area, MPOA）的FOS蛋白（FOS protein）表达，量化该脑区的神经元改变情况。研究结果显示：相较于各自的对照组，缺氧组幼崽所获得的母性行为照料水平降低；雄性缺氧组幼崽的分离叫声频率升高，而雌性缺氧组幼崽的发声速率降低；同时雌雄缺氧组幼崽的体重增长均出现下降。本研究结论为：缺氧会对幼崽的发声模式产生有害影响，且该影响存在性别差异，进而改变母鼠对子代的抚育行为。受损的超声波发声（Ultrasonic Vocalization, USV）会进一步加重新生儿缺氧已造成的不良影响。明晰此类现象，有助于指导缓解新生儿缺氧有害结局的相关操作与策略，并为围绕新生儿缺氧事件的复杂性、母鼠照料质量对幼崽的影响开展研究提供方向，而性别差异也应被纳入此类研究的考量范畴。