A Preterm Rat Model for Pain Studies

Summary

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

Abstract

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund’s adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Introduction

During the neonatal period, nociceptive pathways undergo significant structural and functional maturation, and the presence of tissue damage and associated pain has profound implications for the development of somatosensory processing¹.

Utilizing animal models allows for the controlled experimental manipulation of nonhuman animals, enabling a deeper comprehension of the consequences of neonatal pain on behavior later in life while mitigating potential confounding variables^2,3. A commonly observed outcome is the influence of neonatal pain on heightened pain sensitivity in adulthood^2,4,5. In the neonatal intensive care unit (NICU), neonatal pain is a highly prevalent source of stress, with preterm infants typically undergoing a median of 10 invasive procedures per day⁶. Premature neonates in the NICU encounter a range of stressors, encompassing pain, limited maternal contact, auditory stimuli, and excessive lighting^7,8,9.

The utilization of animal models is essential for advancing our understanding of the underlying mechanisms involved in these processes and facilitating new advances in this area. In particular, employing preterm animal models in studies can greatly contribute to expanding the body of knowledge on premature infants and provide valuable insights into pain management interventions for preterm neonates¹⁰.

Currently, there are a limited number of rodent models that specifically address prematurity, with the majority of these studies primarily investigating the effects of prematurity on the brain¹¹, lung development¹², necrotizing enterocolitis¹³, or immune nutritional studies¹⁴. However, none of these models examine the maturation of the pain system, which is particularly vulnerable in cases of prematurity.

Premature birth and its consequences for early postnatal pain management remain crucial areas of study. Therefore, the present work aimed to contribute to the literature by establishing a preterm rat model. This model provides insights into the impact of neonatal pinprick stimuli on pain thresholds during later stages of life, enhancing our understanding of prematurity-related pain.

Protocol

All experimental procedures followed the Guide for the Care and Use of Laboratory Animals adopted by the Ethical Committee for Animal Experimentation of the Federal University of Alfenas (protocol 32/2016). 1. Animals Obtain adult male and nulliparous Wistar female rats (approximately eight weeks of age) from the Central Animal Facility of the Federal University of Alfenas. House the rats under controlled temperature and humidity conditions in a 12:12 h l…

Representative Results

In this study, there were no differences in maternal or nonmaternal behavior between mothers, irrespective of whether their offspring underwent pinprick experimentation during the neonatal period or were preterm or term (Figure 1). Regarding the maternal behavior of adoptive mothers of preterm offspring, two-way ANOVA showed that there was an effect of PND (postnatal day) but no effect of pinprick stimuli or any interaction between the two factors on evaluating the observed maternal behavior…

Discussion

In this investigation, we observed that maternal and nonmaternal behaviors of mothers remained unaffected by neonatal pinprick experimentation. This trend extended to nonmaternal behavior as well. Furthermore, the weight gain of preterm litters during the pinprick stimulus period was not significantly different between the control and pinprick groups. Paw withdrawal threshold analyses revealed a noteworthy reduction in both male and female pups from the pinprick and CFA groups compared to those from the control groups. P…

Materials

0.9% NaCl solution	Concare, Brazil
Acrylic cages (42 cm × 24 cm × 15 cm) with wire grid floors	Insight Equipamentos, Brazil
Complete Freund's Adjuvant (CFA)	Sigma Aldrich, Brazil
Electronic von Frey,	Insight Equipamentos, Brazil
H2O2 (hydrogen peroxide)	ACS Cientifica, Brazil
Infrared lighting	Carci, Brazil
Isoflurane (2%)	Cristália, Brazil
Upright microscope	Nikon, Brazil	ECLIPSE Ei	Microscope with 10x and 40x objective lenses