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 l...

Creating a Preterm Rat Model for Early Postnatal Pain Research

<ol>
	<li>Cooper, A. H., Hanmer, J. M., Chapman, V., Hathway, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+complete+Freund's+adjuvant-induced+inflammation+does+not+induce+or+alter+hyperalgesic+priming+or+alter+adult+distributions+of+C-fibre+dorsal+horn+innervation.">Neonatal complete Freund's adjuvant-induced inflammation does not induce or alter hyperalgesic priming or alter adult distributions of C-fibre dorsal horn innervation.</a> Pain Reports. 5 (6), e872 (2020).</li><li>Davis, S. M., Rice, M., Burman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+neonatal+pain+disrupts+maternal+behavior+and+subsequent+fear+conditioning+in+a+rodent+model.">Inflammatory neonatal pain disrupts maternal behavior and subsequent fear conditioning in a rodent model.</a> Developmental Psychobiology. 62 (1), 88-98 (2020).</li><li>Mooney-Leber, S. M., Brummelte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+pain+and+reduced+maternal+care+alter+adult+behavior+and+hypothalamic-pituitary-adrenal+axis+reactivity+in+a+sex-specific+manner.">Neonatal pain and reduced maternal care alter adult behavior and hypothalamic-pituitary-adrenal axis reactivity in a sex-specific manner.</a> Developmental Psychobiology. 62 (5), 631-643 (2020).</li><li>Vilela, F. C., Vieira, J. S., Giusti-Paiva, A., Silva, M. L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiencing+early+life+maternal+separation+increases+pain+sensitivity+in+adult+offspring.">Experiencing early life maternal separation increases pain sensitivity in adult offspring.</a> International Journal of Developmental Neuroscience. 62, 8-14 (2017).</li><li>de Carvalho, R. C., 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+neonatal+needle-prick+stimulation+increases+inflammatory+mechanical+hypersensitivity+in+adult+rats.">Repeated neonatal needle-prick stimulation increases inflammatory mechanical hypersensitivity in adult rats.</a> International Journal of Developmental Neuroscience. 78, 191-197 (2019).</li><li>Carbajal, R., 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=Epidemiology+and+treatment+of+painful+procedures+in+neonates+in+intensive+care+units.">Epidemiology and treatment of painful procedures in neonates in intensive care units.</a> JAMA. 300 (1), 60-70 (2008).</li><li>Cong, X., 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=The+impact+of+cumulative+pain/stress+on+neurobehavioral+development+of+preterm+infants+in+the+NICU.">The impact of cumulative pain/stress on neurobehavioral development of preterm infants in the NICU.</a> Early Human Development. 108, 9-16 (2017).</li><li>K&#246;nig, K., Stock, E. L., Jarvis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+levels+of+neonatal+high-flow+nasal+cannula+devices+-+An+in-vitro+study.">Noise levels of neonatal high-flow nasal cannula devices - An in-vitro study.</a> Neonatology. 103 (4), 264-267 (2013).</li><li>Newnham, C. A., Inder, T. E., Milgrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+preterm+cumulative+stressors+within+the+NICU:+the+Neonatal+Infant+Stressor+Scale.">Measuring preterm cumulative stressors within the NICU: the Neonatal Infant Stressor Scale.</a> Early Human Development. 85 (9), 549-555 (2009).</li><li>De Clifford-Faug&#232;re, G., Aita, M., Arbour, C., Colson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+evaluation+and+adaptation+of+a+critical+realism+informed+theory+of+procedural+pain+management+in+preterm+infants:+The+PAIN-Neo+theory.">Development, evaluation and adaptation of a critical realism informed theory of procedural pain management in preterm infants: The PAIN-Neo theory.</a> Journal of Advanced Nursing. 79 (6), 2155-2166 (2023).</li><li>Barkhuizen, M., 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=Nitric+oxide+production+in+the+striatum+and+cerebellum+of+a+rat+model+of+preterm+global+perinatal+asphyxia.">Nitric oxide production in the striatum and cerebellum of a rat model of preterm global perinatal asphyxia.</a> Neurotoxicity Research. 31 (3), 400-409 (2017).</li><li>Remesal, A., 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=Effect+of+prenatal+steroidal+inhibition+of+sPLA2+in+a+rat+model+of+preterm+lung.">Effect of prenatal steroidal inhibition of sPLA2 in a rat model of preterm lung.</a> Pulmonary Pharmacology &amp; Therapeutics. 36, 31-36 (2016).</li><li>Corsini, I., 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=Peroxisome+proliferator-activated+receptor-&#947;+agonist+pioglitazone+reduces+the+development+of+necrotizing+enterocolitis+in+a+neonatal+preterm+rat+model.">Peroxisome proliferator-activated receptor-&#947; agonist pioglitazone reduces the development of necrotizing enterocolitis in a neonatal preterm rat model.</a> Pediatric Research. 81 (2), 364-368 (2017).</li><li>Grases-Pint&#243;, B., 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=A+preterm+rat+model+for+immunonutritional+studies.">A preterm rat model for immunonutritional studies.</a> Nutrients. 11 (5), 999 (2019).</li><li>Marcondes, F. K., Bianchi, F. J., Tanno, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+estrous+cycle+phases+of+rats:+some+helpful+considerations.">Determination of the estrous cycle phases of rats: some helpful considerations.</a> Brazilian Journal of Biology. 62 (4), 609-614 (2002).</li><li>Zhu, M. -. R., Liu, H. -. Y., Liu, P. -. P., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+patent+ductus+arteriosus+model+in+preterm+rats.">Establishment of the patent ductus arteriosus model in preterm rats.</a> Chinese Journal of Contemporary Pediatrics. 18, 372-375 (2016).</li><li>Bowers, D., McKenzie, D., Dutta, D., Wheeless, C. R., Cohen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone+treatment+after+cesarean+delivery+in+rats+increases+the+strength+of+the+uterine+scar.">Growth hormone treatment after cesarean delivery in rats increases the strength of the uterine scar.</a> American Journal of Obstetrics and Gynecology. 185 (3), 614-617 (2001).</li><li>Wang, 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=Whey+peptides+improve+wound+healing+following+caesarean+section+in+rats.">Whey peptides improve wound healing following caesarean section in rats.</a> The British Journal of Nutrition. 104 (11), 1621-1627 (2010).</li><li>Appleby, C. J., Towner, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+pulmonary+damage+in+the+term+and+premature+rat+neonate+exposed+to+hyperoxia.">Magnetic resonance imaging of pulmonary damage in the term and premature rat neonate exposed to hyperoxia.</a> Pediatric Research. 50 (4), 502-507 (2001).</li><li>Anand, K. J., Coskun, V., Thrivikraman, K. V., Nemeroff, C. B., Plotsky, P. M. <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+behavioral+effects+of+repetitive+pain+in+neonatal+rat+pups.">Long-term behavioral effects of repetitive pain in neonatal rat pups.</a> Physiology &amp; Behavior. 66 (4), 627-637 (1999).</li><li>Tal, M., Bennett, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extra-territorial+pain+in+rats+with+a+peripheral+mononeuropathy:+mechano-hyperalgesia+and+mechano-allodynia+in+the+territory+of+an+uninjured+nerve.">Extra-territorial pain in rats with a peripheral mononeuropathy: mechano-hyperalgesia and mechano-allodynia in the territory of an uninjured nerve.</a> Pain. 57 (3), 375-382 (1994).</li><li>Chen, M., 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=Neonatal+repetitive+pain+in+rats+leads+to+impaired+spatial+learning+and+dysregulated+hypothalamic-pituitary-adrenal+axis+function+in+later+life.">Neonatal repetitive pain in rats leads to impaired spatial learning and dysregulated hypothalamic-pituitary-adrenal axis function in later life.</a> Scientific Reports. 6 (1), 39159 (2016).</li><li>Costa, H. H., Vilela, F. C., Giusti-Paiva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+central+infusion+of+cannabinoid+receptor+agonist+WIN+55,212-2+decreases+maternal+care+in+lactating+rats:+consequences+for+fear+conditioning+in+adulthood+males.">Continuous central infusion of cannabinoid receptor agonist WIN 55,212-2 decreases maternal care in lactating rats: consequences for fear conditioning in adulthood males.</a> Behavioural Brain Research. 257, 31-38 (2013).</li><li>Vilela, F. C., Ruginsk, S. G., de Melo, C. M., Giusti-Paiva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CB1+cannabinoid+receptor+mediates+glucocorticoid-induced+effects+on+behavioural+and+neuronal+responses+during+lactation.">The CB1 cannabinoid receptor mediates glucocorticoid-induced effects on behavioural and neuronal responses during lactation.</a> Pflugers Archiv: European Journal of Physiology. 465 (8), 1197-1207 (2013).</li><li>Fehrenbacher, J. C., Vasko, M. R., Duarte, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+inflammation:+Carrageenan-+or+complete+Freund's+Adjuvant+(CFA)-induced+edema+and+hypersensitivity+in+the+rat.">Models of inflammation: Carrageenan- or complete Freund's Adjuvant (CFA)-induced edema and hypersensitivity in the rat.</a> Current Protocols in Pharmacology. , (2012).</li><li>de Carvalho, R. C., 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=Effects+of+repetitive+pinprick+stimulation+on+preterm+offspring:+Alterations+in+nociceptive+responses+and+inflammatory+hypersensitivity+in+adulthood.">Effects of repetitive pinprick stimulation on preterm offspring: Alterations in nociceptive responses and inflammatory hypersensitivity in adulthood.</a> Behavioural Brain Research. 454, 114633 (2023).</li><li>Gier&#233;, C., 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=Towards+a+central+origin+of+nociceptive+hypersensitivity+in+adult+rats+after+a+neonatal+maternal+separation.">Towards a central origin of nociceptive hypersensitivity in adult rats after a neonatal maternal separation.</a> The European Journal of Neuroscience. 58 (10), 4155-4165 (2023).</li><li>Chang, P., Fabrizi, L., Fitzgerald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+life+pain+experience+changes+adult+functional+pain+connectivity+in+the+rat+somatosensory+and+the+medial+prefrontal+cortex.">Early life pain experience changes adult functional pain connectivity in the rat somatosensory and the medial prefrontal cortex.</a> The Journal of Neuroscience. 42 (44), 8284-8296 (2022).</li><li>vanden Hoogen, N. 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>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% NaCl solution</td>
			<td>Concare, Brazil</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic cages (42 cm &#215; 24 cm &#215; 15 cm) with wire grid floors</td>
			<td>Insight Equipamentos, Brazil</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s Adjuvant (CFA)&#160;</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>H2O2 (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&#225;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...

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 ...

To begin, obtain a partially anesthetized pre-term pregnant dam. After euthanizing, make a three centimeter cut in the midline of the lower abdomen, followed by a two centimeter longitudinal cut. Then, make an incision along the antimesenteric border in the midline portion of each uterine tube from these cuts.Then perform a hysterectomy and gently extract the rat pups and placenta one by one. Bathe the pup to prevent cannibalism by surrogate dams. After drying the pup, remove any traces of blood.Using paper towels, clear the airways of the pup thoroughly. Next, cut the umbilical cord just below the placenta, and using cotton soaked in hydrogen peroxide, stop the bleeding from the cord area. Place the pup in a Petri dish under heated infrared lighting at 28 degrees Celsius until breathing regularizes.For the adoption procedure, house each foster mother with the litter separately, and maintain the integrity of the cages. Mark each term puppy from the foster hand. Then, place the pre-term pup along with the foster mother's own pups outside the nest for familiarization with the new pups'scent.Remove the foster mother's pups from the nest and mix the adoptive offspring with the biological offspring. Confirm effective adoption when the surrogate mother collects both her own and the pre-term pups into her nest.

Research

JoVE Journal

Neuroscience

180 ビュー.  UNIFAL-MG. まず、部分的に麻酔をかけられた早産の母体を入手します。安楽死させた後、下腹部の正中線に3センチの切り込みを入れ、続いて縦に2センチの切り込みを入れます。次に、これらの切り傷から各子宮管の正中線部分の無腸境界に沿って切開を行います。その後、子宮摘出術を行い、ラットの子犬と胎盤を1匹ずつ優しく摘出します。代理母...

ビデオ: 疼痛研究のための早産ラットモデル

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Preterm EEG: A Multimodal Neurophysiological Protocol

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. 
In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked.
In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...