This work was supported by the Federal University of Alfenas - UNIFAL-MG and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior - Brazil (CAPES Fellowship, Laura Pereira Generoso; Natalie Lange Candido and Maria Gabriela Maziero Capello) - Finance Code 001.

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
<ol>
	<li>Obtain adult male and nulliparous Wistar female rats (approximately eight weeks of age) from the Central Animal Facility of the Federal University of Alfenas.</li>
	<li>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).</li>
</ol>
2. Checking for pregnancy
<ol>
	<li>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 &#181;L of 0.9% NaCl solution into the rat&#39;s vagina. Ensure a shallow insertion to avoid deep penetration, and then gently withdraw to gather vaginal secretions.</li>
	<li>Place the collected vaginal fluid on individual glass slides, assigning a separate slide to each cage of animals.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Classification and management of pregnant dams and their offspring
<ol>
	<li>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.</li>
	<li>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&#39;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.</li>
	<li>Keep the surrogate dam&#39;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&#39;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.</li>
</ol>
4. Cesarean surgery
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Gently extract rat pups and placenta via hysterectomy17,18. Extract the offspring one by one immediately.</li>
</ol>
5. Post-operative care and preparation of pups for adoption
<ol>
	<li>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.</li>
	<li>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 &#176;C and dry them.</li>
	<li>Remove any traces of blood and place the pups in Petri dishes under heated infrared lighting, maintaining a temperature of approximately 28 &#176;C until their breathing becomes regular.</li>
	<li>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.</li>
</ol>
6. Foster mother interaction and adoption of preterm offspring
<ol>
	<li>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&#39; cages throughout the adoption process.</li>
	<li>Initially, place the Preterm pups outside the nest. This strategy allows the surrogate mother to recognize and become familiar with the new pups&#39; scent in a neutral territory. Also, select one or two of the foster mother&#39;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.</li>
	<li>Mix the adoptive offspring with the biological offspring initially. Confirm effective adoption. Remove the foster mother&#39;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.</li>
</ol>
7. Litters standardization
<ol>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Utilize a total of 20 pregnant dams for the procedures. Divide them into two experimental groups, each consisting of 10 dams.
	<ol>
		<li>Stimulate the pups in the first group, the PP group, with pinprick stimuli from PND 2 to PND 15.</li>
		<li>Designate the second group as the CC group, which serves as the control, with the pups in this group not receiving pinprick stimulation.</li>
		<li>Monitor the maternal behavior of the dams and the weight of the litters diligently throughout this period.</li>
	</ol>
	</li>
</ol>
9. Post-weaning assessments and behavioral testing
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
10. Repeated neonatal pain induction
<ol>
	<li>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.</li>
	<li>Carefully insert a 22 G needle to a shallow depth into the mid-plantar area of the right hind paw.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 dams5,21,22.</li>
</ol>
11. Evaluation of maternal behaviors
<ol>
	<li>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).</li>
	<li>During these sessions, diligently observe, record, and score each mother&#39;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.</li>
</ol>
12. Recording of maternal and non-maternal behaviors
<ol>
	<li>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 &#34;blanket&#34; 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).</li>
	<li>Document non-maternal behavior parameters, including actions such as feeding, exploring the cage housing, not exploring, and the absence of maternal self-grooming.</li>
	<li>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 1005,23,24.</li>
</ol>
13. Litter weight assessment
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
14. Mechanical threshold test
<ol>
	<li>In this experiment, administer injections of either saline or CFA, each in a volume of 100 &#181;L, to rats (8 weeks old) from the PP and CC groups. Afterward, individually place them in acrylic cages (42 cm &#215; 24 cm &#215; 15 cm) with wire grid floors 15-30 min before the test to assess mechanical hyperalgesia.</li>
	<li>In the test, induce a hind paw flexion reflex using a hand-held force transducer equipped with a 0.5 mm2&#160;polypropylene tip (Electronic von Frey).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
</ol>
15. Data analysis
<ol>
	<li>Process the data using statistical analysis software and present it as mean &#177; 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.</li>
	<li>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.</li>
</ol>

Creating a Preterm Rat Model for Early Postnatal Pain Research

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+touch+and+needle-prick+stimulation+in+the+neonatal+period+increases+the+baseline+mechanical+sensitivity+and+postinjury+hypersensitivity+of+adult+spinal+sensory+neurons.">Repeated touch and needle-prick stimulation in the neonatal period increases the baseline mechanical sensitivity and postinjury hypersensitivity of adult spinal sensory neurons.</a> Pain. 159 (6), 1166-1175 (2018).</li><li>Butkevich, I. P., Mikhailenko, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+neonatal+pain+and+stress+on+reactivity+of+the+nociceptive+system.">Long-term effects of neonatal pain and stress on reactivity of the nociceptive system.</a> Bulletin of Experimental Biology and Medicine. 161 (6), 755-758 (2016).</li></ol>

We have nothing to disclose.

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 Gier&#233; 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.

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.

<table><tbody><tr><td>0.9% NaCl solution</td><td>Concare, Brazil</td><td></td><td></td></tr><tr><td>Acrylic cages (42 cm &amp;times; 24 cm &amp;times; 15 cm) with wire grid floors</td><td>Insight Equipamentos, Brazil</td><td></td><td></td></tr><tr><td>Complete Freund&#039;s Adjuvant (CFA)&amp;nbsp;</td><td>Sigma Aldrich, Brazil</td><td></td><td></td></tr><tr><td>Electronic von Frey,</td><td>Insight Equipamentos, Brazil</td><td></td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; (hydrogen peroxide)</td><td>ACS Cientifica, Brazil</td><td></td><td></td></tr><tr><td>Infrared lighting</td><td>Carci, Brazil</td><td></td><td></td></tr><tr><td>Isoflurane (2%)</td><td>Crist&amp;aacute;lia, Brazil</td><td></td><td></td></tr><tr><td>Upright microscope</td><td>Nikon, Brazil</td><td>ECLIPSE Ei</td><td>Microscope with 10x and 40x objective lenses</td></tr></tbody></table>

a preterm rat model for pain studies

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.

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 &#60; 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 &#60; 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 &#60; 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 &#60; 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 &#60; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65800/65800fig01.jpg" /> 
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 &#177; SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65800/65800fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 &#60; 0.001; pinprick stimulus factor: F(1, 140) = 0.89, p = 0.34; pinprick stimulus &#215; PND interaction: F(13, 140) = 0.05, p = 1.00].
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65800/65800fig02.jpg" /> 
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 &#177; SEM of 8 animals. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65800/65800fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Significant main effects of pinprick stimuli and CFA on paw withdrawal threshold were observed, with a substantial decrease (p &#60; 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 &#60; 0.001) was observed in the PP/CFA group compared to the CC/CFA group [CFA factor: F(4,112) = 13.12, p &#60; 0.001; pinprick stimulus factor: F(3,112) = 14.45, p &#60; 0.05; CFA x pinprick stimulus interaction: F(12,112) = 5.14, p &#60; 0.05]. Concerning female pups (Figure 3B), a decrease in the withdrawal threshold (p &#60; 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 &#60; 0.05) was observed in the PP/CFA group compared to the CC/CFA group&#160;[CFA factor: F(4,112) = 31.16, p &#60; 0.001; pinprick stimulus factor: F(3,112) = 18.22, p &#60; 0.01; CFA x pinprick stimulus interaction: F(12,112) = 58.13, p &#60; 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.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65800/65800fig3v3.jpg" /> 
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 &#177; SEM of 8 animals. * p &#60; 0.05 and *** p &#60; 0.001 compared to the Control and PP/Saline groups against the Control and PP/CFA groups; # p &#60; 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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65800/65800fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about A Preterm Rat Model for Pain Studies at JoVE.com

A Preterm Rat Model for Pain Studies

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 ...

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

312 Views.  UNIFAL-MG. 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. 

Video: A Preterm Rat Model for Pain Studies

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Behavior

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

A Neuronal Apoptosis Model induced by Spinal Cord Compression in Rat

As a severe progressive degenerative disease, cervical spondylotic myelopathy (CSM) has a poor prognosis and is associated with physical pain, stiffness, motor or sensory dysfunction, and a high risk of spinal cord injury and acroparalysis. Thus, therapeutic strategies that promote efficient spinal cord regeneration in this chronic and progressive disease are urgently needed. Effective and reproducible animal spinal cord compression models are required to understand the complex biological mechanism underlying CSM. Most spinal cord injury models reflect acute and structural destructive conditions, whereas animal models of CSM present a chronic compression in the spinal cord. This paper presents a protocol to generate a rat spinal cord compression model, which was further evaluated by assessing the behavioral score and observing the compressed spinal cord region. The behavioral assessments showed decreased monitor motor disability, including joint movements, stepping ability, coordination, trunk stability, and limb muscle strength. Hematoxylin and eosin (H&#38;E) staining and immunostaining revealed considerable neuronal apoptosis in the compressed region of the spinal cord.

Here, we present a protocol to generate a rat spinal cord compression model, assess its behavioral score, and observe the compressed spinal cord region. The behavioral assessments showed decreased monitor motor disability. Hematoxylin and eosin staining and immunostaining revealed considerable neuronal apoptosis in the compressed region of the spinal cord.

As a severe progressive degenerative disease, cervical spondylotic myelopathy (CSM) has a poor prognosis and is associated with physical pain, stiffness, ...

Assessing Tuina Therapy's Pain Relief in Chronic Constriction Rats

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...