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

1. Obtain adult male and nulliparous Wistar female rats (approximately eight weeks of age) from the Central Animal Facility of the Federal University of Alfenas.
2. House the rats under controlled temperature and humidity conditions in a 12:12 h light: dark cycle and feed them with chow and water ad libitum in the Faculty of Physiotherapy animal facilities (Federal University of Alfenas).

2. Checking for pregnancy

1. For 1 month, every morning between 8:00 and 9:00 a.m., transport each animal cage to the experimental room. To perform a vaginal wash, carefully introduce a plastic pipette containing 10 µL of 0.9% NaCl solution into the rat's vagina. Ensure a shallow insertion to avoid deep penetration, and then gently withdraw to gather vaginal secretions.
2. Place the collected vaginal fluid on individual glass slides, assigning a separate slide to each cage of animals.
   1. Obtain a single drop of unstained material from each rat using a clean pipette tip. Examine the material under a light microscope using 10x and 40x objective lenses, without using the condenser lens.
   2. Identify three distinct cell types based on their characteristics: round and nucleated epithelial cells, irregular cornified cells without a nucleus, and small and round leukocytes¹⁵.
3. Expose female Wistar rats with intense keratinization and vaginal desquamation, characteristic of the estrous cycle and indicating increased male receptivity, to mating. Mate the rats by placing 2 females and 1 male per cage. Determine gestational day 0 by checking for the presence of sperm and estrous phase cells in vaginal smears.

3. Classification and management of pregnant dams and their offspring

1. Dispose of three types of pregnant dams (2 dams each) based on their gestational stage: Preterm, Term, and Surrogate dams. Allow the Term pregnant dams to deliver naturally on day 22 of gestation, and use their litters for the term group.
2. On day 19 of gestation, perform a cesarean section on the preterm pregnant dams, which is 3 days prior to their expected delivery date. Utilize these litters for the preterm group. Given that this procedure is not conducive to the mother's survival, entrust the preterm pups to surrogate dams, which had given birth naturally 2 days prior to the cesarean section of the preterm dams.
3. Keep the surrogate dam's pups with her for a few hours, allowing their scents to intermingle with the Preterm litters. After this period, introduce the Preterm litters and remove the surrogate's original pups, subsequently sacrificing them with high doses inhalation of isoflurane.
   NOTE: This step is essential to ensure that the surrogate dams have already stimulated milk production in their mammary glands, positioning them to effectively nurse the preterm pups¹⁶.

4. Cesarean surgery

1. Partially anesthetize the Preterm pregnant dams with 2% isoflurane and euthanize them 3 days before the expected delivery date using cervical dislocation. Following euthanasia, extract the offspring one by one through hysterectomy.
2. Make a 3 cm cut in the midline of the lower abdomen, followed by a 2 cm longitudinal cut. Make an incision along the antimesenteric border in the middle portion of each uterine tube from these cuts.
3. Gently extract rat pups and placenta via hysterectomy^17,18. Extract the offspring one by one immediately.

5. Post-operative care and preparation of pups for adoption

1. Use the adoption procedure to prevent the operated mother from exhibiting inappropriate behavior due to the surgical procedure and potential pain, which could affect the adult behavior of the puppies.
2. Clear the airways of the rat pups using paper towels after birth. Clean the rat pups by giving them a bath to prevent cannibalism by surrogate dams. Wash the rat pups in water at 28 °C and dry them.
3. Remove any traces of blood and place the pups in Petri dishes under heated infrared lighting, maintaining a temperature of approximately 28 °C until their breathing becomes regular.
4. Cut their umbilical cords just below the placenta and use cotton soaked in H₂O₂ to stop any bleeding from the umbilical cord. Finally, offer them to the foster mothers for adoption.

6. Foster mother interaction and adoption of preterm offspring

1. House each foster mother with their litter in separate plastic cages. Before transferring, mark each term puppy from the foster hand. This marking is crucial for subsequent maternal behavior analyses. Maintain the integrity of the foster mothers' cages throughout the adoption process.
2. Initially, place the Preterm pups outside the nest. This strategy allows the surrogate mother to recognize and become familiar with the new pups' scent in a neutral territory. Also, select one or two of the foster mother's own pups and place them outside the nest alongside the preterm pups. This mix prompts the surrogate mother to collect both her own and the preterm pups, thus promoting acceptance and integration of the new pups into her nest.
3. Mix the adoptive offspring with the biological offspring initially. Confirm effective adoption. Remove the foster mother's pups from the nest and sacrifice them¹⁹.
   NOTE: In this study, the viability of preterm rats was 100% and they were not rejected by the surrogate dam.

7. Litters standardization

1. Maintain a litter size of 8 pups per litter in all groups of surrogate-reared pups, with 4 males and 4 females. Sacrifice the remaining pup rats after 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.

1. Utilize a total of 20 pregnant dams for the procedures. Divide them into two experimental groups, each consisting of 10 dams.
   1. Stimulate the pups in the first group, the PP group, with pinprick stimuli from PND 2 to PND 15.
   2. Designate the second group as the CC group, which serves as the control, with the pups in this group not receiving pinprick stimulation.
   3. Monitor the maternal behavior of the dams and the weight of the litters diligently throughout this period.

9. Post-weaning assessments and behavioral testing

1. Wean the offspring on PND 22. Sort them by gender and house them in cages with a maximum capacity of 4 animals each until they reach approximately 8 weeks of age.
2. Afterward, subject these animals to evaluations for sensitivity to painful stimuli using the electronic von Frey test. Focus specifically on inflammation-induced pain from CFA.
3. To mitigate any potential litter-related influences, select 1 male and 1 female rat from each litter for each experimental group to undergo behavioral testing during adulthood.
4. Use each animal in a single experiment to avoid hormonal factor interference in the nociceptive responses of female offspring rats. Specifically, ensure that tests are conducted on females during the diestrus phase of their estrous cycle.

10. Repeated neonatal pain induction

1. Induce repeated neonatal pain using a pinprick technique similar to the one described in a previous study²⁰. Initiate daily pinprick stimuli for the rat pups starting from postnatal day 2 (PND 2) and continue this practice until PND 15.
2. Carefully insert a 22 G needle to a shallow depth into the mid-plantar area of the right hind paw.
   1. Ensure that the penetration is just enough to stimulate without causing undue injury. Calibrate the gauge to prevent deeper penetration, considering the risk of it passing entirely through the paw at this age.
   2. If bleeding occurs, promptly stop it using a cotton-tipped swab; typically, this intervention lasts only a few seconds. Administer stimuli 4 times, maintaining a 2 min gap between each, totaling 8 pricks a day.
3. To minimize potential confounding factors related to maternal separation and neonatal handling, separate the rat pups from their dams for a maximum of 5 min. Apply the same separation duration to the control group. Following each set of stimuli, promptly return the rat pups to their dams^5,21,22.

11. Evaluation of maternal behaviors

1. To evaluate maternal behavior, assess the behavior of dams in both experimental groups (n = 10 per group) from PND 2 to PND 15. Conduct the assessment in two sessions: one in the morning, before the pinprick stimuli on the rat pups (between 08:00 and 09:30), and one in the afternoon, after the pinprick stimuli on the rat pups (between 15:00 and 16:30).
2. During these sessions, diligently observe, record, and score each mother's behavior every 3 min during these sessions, leading to 30 observations per period per day. This accumulates to a total of 60 observations per mother per day.

12. Recording of maternal and non-maternal behaviors

1. Record maternal behavior parameters that encompass actions such as grooming or licking (on the body or anogenital region), nursing, maintaining an arched-back in a "blanket" like posture by lying over the pups, lying passively on its back or side while nursing, engaging in building nests, and maternal self-grooming (including stimulation of breasts through self-cleaning).
2. Document non-maternal behavior parameters, including actions such as feeding, exploring the cage housing, not exploring, and the absence of maternal self-grooming.
3. Present the data as the percentage of total maternal behavior and non-maternal behavior. Divide the number of target behavior recorded observations by the total number of observations and multiply the result by 100^5,23,24.

13. Litter weight assessment

1. Throughout the pinprick stimulation phase (PND 2-15), monitor the weight of the litters in both the PP and CC groups, each comprising 8 litters.
2. During the pinprick stimulation phase (PND 2-15), keep a continuous check on the weight of the litters in both the PP and CC groups, each consisting of 8 litters.

14. Mechanical threshold test

1. In this experiment, administer injections of either saline or CFA, each in a volume of 100 µL, to rats (8 weeks old) from the PP and CC groups. Afterward, individually place them in acrylic cages (42 cm × 24 cm × 15 cm) with wire grid floors 15-30 min before the test to assess mechanical hyperalgesia.
2. In the test, induce a hind paw flexion reflex using a hand-held force transducer equipped with a 0.5 mm² polypropylene tip (Electronic von Frey).
   1. Gradually apply the tip between the five distal foot-pads of the right hind paw, increasing pressure until a response is observed.
      NOTE: When paw retraction occurs, the stimulus automatically ceases, and its strength is documented. The test concludes with a clear flinch response followed by paw withdrawal. Subcutaneous administration of CFA induces prolonged inflammation, peaking at 24 h and persisting for at least 7 days²⁵.
3. Conduct tests on the animals before and at 4 h, 7 h, 10 h, and 24 h after the administration of saline or CFA⁴. Present the results in terms of withdrawal threshold, measured in grams (g), and calculate it by averaging three measurements.
4. To prevent the interference of potential hormonal factors in nociceptive responses among female offspring, ensure the tests are performed exclusively during the diestrus phase of their estrous cycle.

15. Data analysis

1. Process the data using statistical analysis software and present it as mean ± Standard Error of the Mean (SEM). To identify statistically significant differences between groups, apply a two-way analysis of variance (ANOVA) with repeated measures, considering factors such as the assessment of maternal versus non-maternal parameters and litter weight evaluation.
2. Specifically, analyze PND and pinprick stimuli for maternal parameters and von Frey: CFA and pinprick stimuli for litter weight assessment. Conduct post hoc analysis using the Bonferroni test when required.

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

List of materials used in this article

Name	Company	Catalog Number	Comments
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