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.
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.
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 variables2,3. A commonly observed outcome is the influence of neonatal pain on heightened pain sensitivity in adulthood2,4,5. 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 lighting7,8,9.
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.
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
2. Checking for pregnancy
3. Classification and management of pregnant dams and their offspring
4. Cesarean surgery
5. Post-operative care and preparation of pups for adoption
6. Foster mother interaction and adoption of preterm offspring
7. Litters standardization
8. Experimental design and protocol implementation
NOTE: The aim of this protocol was to obtain viable preterm pups for the development of following experimental procedures.
9. Post-weaning assessments and behavioral testing
10. Repeated neonatal pain induction
11. Evaluation of maternal behaviors
12. Recording of maternal and non-maternal behaviors
13. Litter weight assessment
14. Mechanical threshold test
15. Data analysis
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 at 8 a.m. [PND factor: F(13, 140) = 6.31, p < 0.001; pinprick stimulus factor: F(1, 140) = 1.04, p = 0.30; pinprick stimulus x PND interaction: F(13, 140) = 0.55, p = 0.88; Figure 1A]; or at 3 p.m. [PND factor: F(13, 140) = 16.97, p < 0.001; pinprick stimulus factor: F(1, 140) = 3.27, p = 0.07; pinprick stimulus x PND interaction: F(13, 140) = 1.82, p = 0.04; Figure 1C]. In terms of nonmaternal behavior, there was a noticeable effect due to PND, but there was no significant influence from the pinprick stimuli or interaction between these two factors at 8 a.m. [PND factor: F(13, 140) = 6.31, p < 0.001; pinprick stimulus factor: F(1, 140) = 1.04, p = 0.30; interaction between the pinprick stimulus and PND: F(13, 140) = 0.55, p = 0.88; see Figure 1B]. Similarly, while the PND effect persisted, the influence of the pinprick stimuli and its interaction with PND were not statistically significant at 3 p.m. [PND factor: F(13, 140) = 16.97, p < 0.001; pinprick stimulus factor: F(1, 140) = 3.27, p = 0.07. Two-way ANOVA revealed a noteworthy effect of PND, and, importantly, a significant interaction between pinprick stimuli and PND at 3:00 p.m. (PND factor: F(13, 182) = 13.82, p < 0.001; pinprick stimuli factor: F(1, 182) = 3.78, p = 0.05; PND x pinprick stimuli interaction: F(13, 182) = 1.82, p = 0.04; refer to Figure 1D]. This interaction underscores a distinct impact of pinprick stimuli on non-maternal behavior, particularly evident in the afternoon evaluation.
Figure 1: Effects of pinpricking during the neonatal period (PND 2-15) on the maternal behavior of adoptive mothers of preterm offspring. (A) Number of recorded maternal behaviors assessed at 8:00 a.m. (B) Number of recorded nonmaternal behaviors assessed at 8:00 a.m. (C) Number of recorded maternal behaviors assessed at 3:00 p.m. (D) Number of recorded nonmaternal behaviors assessed at 3:00 p.m. Each point represents the mean ± SEM. Please click here to view a larger version of this figure.
Figure 2 displays the weight gain of the preterm litter during the period in which the pinprick stimulus was applied (PND 2-15). No changes in litter weight were observed between the CC (control) group and the PP (pinprick) group. Two-way ANOVA revealed a significant effect of PND but no significant effects of pinprick stimulus or interaction between the two factors on litter weight [PND factor: F(13, 140) = 247.5, p < 0.001; pinprick stimulus factor: F(1, 140) = 0.89, p = 0.34; pinprick stimulus × PND interaction: F(13, 140) = 0.05, p = 1.00].
Figure 2 – Effects of pinpricking during the neonatal period (PND 2-15) on the weight of the preterm litter in grams. Each point represents the mean ± SEM of 8 animals. Please click here to view a larger version of this figure.
Significant main effects of pinprick stimuli and CFA on paw withdrawal threshold were observed, with a substantial decrease (p < 0.001) evident in male pups from the CC/CFA and PP/CFA groups at all time points compared to those in the CC/Sal and PP/Sal groups (Figure 3A). This underscores the robust impact of both pinprick stimuli and CFA on nociceptive responses in male pups. Notably, 4 h following the CFA injection, a significant reduction in the PWT (p < 0.001) was observed in the PP/CFA group compared to the CC/CFA group [CFA factor: F(4,112) = 13.12, p < 0.001; pinprick stimulus factor: F(3,112) = 14.45, p < 0.05; CFA x pinprick stimulus interaction: F(12,112) = 5.14, p < 0.05]. Concerning female pups (Figure 3B), a decrease in the withdrawal threshold (p < 0.001) was noted in the CC/CFA and PP/CFA groups at all time points compared to those in the CC/Sal and PP/Sal groups. Specifically, 4 h after the CFA injection, a significant reduction in the withdrawal threshold (p < 0.05) was observed in the PP/CFA group compared to the CC/CFA group [CFA factor: F(4,112) = 31.16, p < 0.001; pinprick stimulus factor: F(3,112) = 18.22, p < 0.01; CFA x pinprick stimulus interaction: F(12,112) = 58.13, p < 0.01]. Both male and female adults showcased a reduced paw withdrawal threshold between the PP/CFA group and the CC/CFA group at all time points starting from the 4 h mark.
Figure 3 – Effects of pinpricking during the neonatal period (PND 2-15) in preterm litters on nociception according to the von Frey test before and after injection of intraplantar CFA or saline. Paw withdrawal threshold, in grams, in (A) male rats or (B) female rats. Each point represents the mean ± SEM of 8 animals. * p < 0.05 and *** p < 0.001 compared to the Control and PP/Saline groups against the Control and PP/CFA groups; # p < 0.01 comparing the Control CFA group to the PP/CFA group. BASAL represents the nociceptive threshold measured prior to intraplantar injection of CFA or saline. The arrow indicates the time of intraplantar injection of CFA or saline. Please click here to view a larger version of this figure.
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. Particularly striking was the observation of a further reduction in the paw withdrawal threshold 4 h post-CFA injection in the pinprick/CFA group compared to the control/CFA group. These nuanced results underscore the multifaceted effects of neonatal pinprick stimulation on maternal behavior, litter weight gain, and nociceptive responses in offspring, emphasizing the importance of considering both preterm and term conditions when interpreting outcomes.
Our exploration of nociceptive responses aligns with and extends the findings of de Carvalho et al.26, who reported alterations in nociceptive responses and inflammatory hypersensitivity in adulthood resulting from repetitive pinprick stimulation in preterm offspring. This convergence of results underscores the enduring impact of neonatal experiences on nociceptive pathways, emphasizing the robustness of these outcomes across studies. The observed increased sensitivity to noxious stimuli in both male and female pups subjected to neonatal pinprick stimulation suggested a consistent trend in the modulation of nociceptive responses, further contributing to our understanding of the long-term consequences of early-life stressors.
The findings of this study also align with the work of Gieré et al.27, who explored nociceptive hypersensitivity in adult rats following neonatal maternal separation. Their study suggested a central origin of nociceptive hypersensitivity, reinforcing the notion that early-life stressors can induce enduring changes in pain processing mechanisms. The convergence of the results emphasizes the complex interplay between early-life events and nociceptive responses, further underscoring the need for a comprehensive understanding of the central mechanisms contributing to long-term alterations in pain sensitivity.
The impact of early-life experiences on nociceptive pathways is further supported by the findings of Chang et al.28, who investigated alterations in functional pain connectivity in the rat somatosensory and medial prefrontal cortex following early-life pain experiences. Their work highlighted long-term changes in pain processing mechanisms induced by early-life stressors, emphasizing the importance of understanding the neural correlates of nociceptive responses. Integrating these results with the observations of heightened sensitivity to noxious stimuli in preterm offspring subjected to neonatal pinprick stimulation contributes to a more comprehensive understanding of the enduring consequences of early-life pain experiences on adult pain circuitry.
Additionally, van den Hoogen et al.29 demonstrated that repeated touch and needle-prick stimulation during the neonatal period increased baseline mechanical sensitivity and postinjury hypersensitivity in adult spinal sensory neurons. The current findings, aligning with previous research, underscore the enduring consequences of neonatal pain experiences on nociceptive pathways. Together, these studies emphasize the importance of recognizing the long-term impact of early-life experiences on adult pain sensitivity, contributing to a comprehensive understanding of the complex interplay between neonatal stimuli and nociceptive responses.
By combining preterm birth with exposure to painful stimuli during the neonatal period, we developed a model that closely mimics the early life experiences of human preterm infants, accounting for the imperative need for intensive care necessitated by prematurity. Nevertheless, the translational relevance of this model, particularly in relation to the NICU experiences of preterm infants, requires further elucidation. Notably, no studies employing a similar prematurity model to the one utilized in the present study were identified. However, when considering the initial days of life (1-2) as a representation of prematurity, prior research has demonstrated that males exhibit greater vulnerability to nociceptive stimuli than females during this critical period. This vulnerability was confirmed through nociceptive tests applied in adulthood, providing partial justification for the observed results of this study30.
The present study pioneered the use of preterm animals born by cesarean section at 19 days of gestation to evaluate the nociceptive threshold in adulthood. This novel model for studying pain in preterm neonates provides a unique perspective on this population. This model raises new questions regarding nociceptive tests, such as the von Frey test, in adult animals of both sexes, as well as all aspects involved in the nociceptive thresholds of these animals, whether during the neonatal period or in adulthood.
While the current study primarily focused on the impact of neonatal pinprick stimuli on pain thresholds in later stages of life, there is a promising avenue for extending this research to interventions and postnatal analgesic strategies. Future studies could assess the efficacy of various pain management interventions in a preterm rat model, exploring potential avenues for mitigating the long-term effects of neonatal pain. This may include investigating novel analgesic approaches, assessing the duration and intensity of interventions needed, and exploring the underlying mechanisms influencing the effectiveness of these interventions.
In conclusion, the comprehensive investigation carried out in this study aimed to dissect the intricate interplay of neonatal pinprick stimulation, maternal behavior, and preterm birth conditions on nociceptive responses in offspring. The meticulous analysis of maternal behavior, coupled with the exclusion of potential confounding factors such as premature birth and adoptive caregiving, reaffirmed the resilience of maternal behavior to the administered nociceptive stimulus. The weight gain of the preterm litters remained unaffected, indicating that the observed alterations in nociceptive responses during adulthood were more likely attributed to early-life pinprick stimulation than maternal care or offspring development. The findings of this study align with the literature on the enduring consequences of neonatal pain experiences, emphasizing heightened sensitivity to noxious stimuli in adulthood. Moreover, the exploration of potential mechanistic theories, including alterations in neural processing and glucocorticoid receptor function, provides valuable insights into the underlying pathways contributing to nociceptive alterations. Together, the results presented here and those of previous studies underscore the complexity of early-life experiences in nociceptive pathways, shedding light on the enduring consequences of neonatal stimuli on adult pain circuitry. While further research is warranted to elucidate the nuanced underlying mechanisms involved, this study contributes to the growing body of knowledge aimed at revealing the long-term impact of early-life events on nociceptive responses in adult offspring.
The authors have nothing to disclose.
This work was supported by the Federal University of Alfenas – UNIFAL-MG and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES Fellowship, Laura Pereira Generoso; Natalie Lange Candido and Maria Gabriela Maziero Capello) – Finance Code 001.
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 |
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