Name: assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding
Uploaded: 2018-11-14T14:15:00
Description: Watch this Scientific Journal Video about Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding at JoVE.co

The authors thank Manon Ranger and Alexandra Schulz for their assistance in preparing this protocol.

This study was approved by the Institutional Animal Care and Use Committees at Columbia University and the New York State Psychiatric Institute.
1. Tissue Preparation
<ol>
	<li>Order timed pregnant rats from vendor.</li>
	<li>Follow timed pregnant rats by observing their growing abdomens in the weeks after their arrival and subsequently looking for pups on the expected delivery date by inspecting the cage every 2 h until delivery begins.</li>
	<li>Remove pups with a gloved hand by their tail...

<ol>
	<li>Hietaniemi, 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=Gene+expression+profiles+in+fetal+and+neonatal+rat+offspring+of+energy-restricted+dams.">Gene expression profiles in fetal and neonatal rat offspring of energy-restricted dams.</a> Journal of Nutrigenetics and Nutrigenomics. 2 (4-5), 173-183 (2009).</li><li>Okabe, 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=Homogenous+glycine+receptor+expression+in+cortical+plate+neurons+and+Cajal-Retzius+cells+of+neonatal+rat+cerebral+cortex.">Homogenous glycine receptor expression in cortical plate neurons and Cajal-Retzius cells of neonatal rat cerebral cortex.</a> Neuroscience. 123 (3), 715-724 (2004).</li><li>Mailleux, P., Takazawa, K., Erneux, C., Vanderhaeghen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+the+neurons+containing+inositol+1,4,5-trisphosphate+3-kinase+and+its+messenger+RNA+in+the+developing+rat+brain.">Distribution of the neurons containing inositol 1,4,5-trisphosphate 3-kinase and its messenger RNA in the developing rat brain.</a> Journal of Comparative Neurology. 327 (4), 618-629 (1993).</li><li>Carter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+and+Human+Evolution.">Oxytocin and Human Evolution.</a> Current Topics in Behavioral Neuroscience. , (2017).</li><li>Sippel, L. 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=Oxytocin+and+Stress-related+Disorders:+Neurobiological+Mechanisms+and+Treatment+Opportunities.">Oxytocin and Stress-related Disorders: Neurobiological Mechanisms and Treatment Opportunities.</a> Chronic Stress (Thousand Oaks). 1, (2017).</li><li>Agnati, L. F., 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=Aspects+on+the+integrative+actions+of+the+brain+from+neural+networks+to+"brain-body+medicine".">Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".</a> Journal of Receptors and Signal Transduction Research. 32 (4), 163-180 (2012).</li><li>Welch, M. G., Margolis, K. G., Li, Z., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+regulates+gastrointestinal+motility,+inflammation,+macromolecular+permeability,+and+mucosal+maintenance+in+mice.">Oxytocin regulates gastrointestinal motility, inflammation, macromolecular permeability, and mucosal maintenance in mice.</a> American Journal Physiology: Gastrointestinal and Liver Physiology. 307 (8), G848-G862 (2014).</li><li>Welch, M. G., 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=Expression+and+developmental+regulation+of+oxytocin+(OT)+and+oxytocin+receptors+(OTR)+in+the+enteric+nervous+system+(ENS)+and+intestinal+epithelium.">Expression and developmental regulation of oxytocin (OT) and oxytocin receptors (OTR) in the enteric nervous system (ENS) and intestinal epithelium.</a> Journal of Comparative Neurology. 512 (2), 256-270 (2009).</li><li>Prakash, B. S., Paul, V., Kliem, H., Kulozik, U., Meyer, H. H. <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+oxytocin+in+milk+of+cows+administered+oxytocin.">Determination of oxytocin in milk of cows administered oxytocin.</a> Analytica Chimica Acta. 636 (1), 111-115 (2009).</li><li>Solangi, A. R., Memon, S. Q., Mallah, A., Khuhawar, M. Y., Bhanger, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+separation+of+oxytocin,+norfloxacin+and+diclofenac+sodium+in+milk+samples+using+capillary+electrophoresis.">Quantitative separation of oxytocin, norfloxacin and diclofenac sodium in milk samples using capillary electrophoresis.</a> Biomedical Chromatography. 23 (9), 1007-1013 (2009).</li><li>Klein, B. Y., 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=Oxytocin+modulates+markers+of+the+unfolded+protein+response+in+Caco2BB+gut+cells.">Oxytocin modulates markers of the unfolded protein response in Caco2BB gut cells.</a> Cell Stress and Chaperones. 19 (4), 465-477 (2014).</li><li>Klein, B. Y., Tamir, H., Hirschberg, D. L., Glickstein, S. B., Welch, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colostrum+oxytocin+modulates+cellular+stress+response,+inflammation,+and+autophagy+markers+in+newborn+rat+gut+villi.">Colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi.</a> Biochemical an Biophysical Research Communications. 487 (1), 47-53 (2017).</li><li>Donnet-Hughes, 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=Potential+role+of+the+intestinal+microbiota+of+the+mother+in+neonatal+immune+education.">Potential role of the intestinal microbiota of the mother in neonatal immune education.</a> Proceedings of the Nutritional Society. 69 (3), 407-415 (2010).</li><li>Perez, P. 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R., Salinas, A., Buffenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+Postnatal+Brain+Development+in+the+Longest-Lived+Rodent:+Prolonged+Maintenance+of+Neotenous+Traits+in+the+Naked+Mole-Rat+Brain.">Extended Postnatal Brain Development in the Longest-Lived Rodent: Prolonged Maintenance of Neotenous Traits in the Naked Mole-Rat Brain.</a> Frontiers in Neuroscience. 10, 504 (2016).</li><li>Hansson, 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=Time-resolved+quantitative+proteome+analysis+of+in+vivo+intestinal+development.">Time-resolved quantitative proteome analysis of in vivo intestinal development.</a> Molecular and Cellular Proteomics. 10 (3), (2011).</li><li>Mochizuki, K., Yorita, S., Goda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+the+jejunum+of+rats+during+the+transient+suckling-weaning+period.">Gene expression changes in the jejunum of rats during the transient suckling-weaning period.</a> Journal of Nutritional Science and Vitaminology (Tokyo). 55 (2), 139-148 (2009).</li><li>Rinaman, L., Banihashemi, L., Koehnle, T. J. <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+experience+shapes+the+functional+organization+of+stress-responsive+visceral+circuits.">Early life experience shapes the functional organization of stress-responsive visceral circuits.</a> Physiology and Behavior. 104 (4), 632-640 (2011).</li><li>Johannes, G., Sarnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+polysomal+association+of+natural+mRNAs+encoding+c-myc,+BiP,+and+eIF4G+conferred+by+internal+ribosome+entry+sites.">Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.</a> RNA. 4 (12), 1500-1513 (1998).</li><li>Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindbrain+noradrenergic+A2+neurons:+diverse+roles+in+autonomic,+endocrine,+cognitive,+and+behavioral+functions.">Hindbrain noradrenergic A2 neurons: diverse roles in autonomic, endocrine, cognitive, and behavioral functions.</a> American Journal of Physiology: Regulatory, Integrative and Comparative Physiology. 300 (2), R222-R235 (2011).</li><li>Walker, C. D., Toufexis, D. J., Burlet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+and+limbic+expression+of+CRF+and+vasopressin+during+lactation:+implications+for+the+control+of+ACTH+secretion+and+stress+hyporesponsiveness.">Hypothalamic and limbic expression of CRF and vasopressin during lactation: implications for the control of ACTH secretion and stress hyporesponsiveness.</a> Progress in Brain Research. 133, 99-110 (2001).</li><li>Montiel-Castro, A. J., Gonzalez-Cervantes, R. M., Bravo-Ruiseco, G., Pacheco-Lopez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiota-gut-brain+axis:+neurobehavioral+correlates,+health+and+sociality.">The microbiota-gut-brain axis: neurobehavioral correlates, health and sociality.</a> Frontiers in Integrative Neuroscience. 7, 70 (2013).</li><li>Blevins, J. E., Ho, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+oxytocin+signaling+in+the+regulation+of+body+weight.">Role of oxytocin signaling in the regulation of body weight.</a> Reviews in Endocrine and Metabolic Disorders. 14 (4), 311-329 (2013).</li><li>Welch, M. G., 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=Combined+administration+of+secretin+and+oxytocin+inhibits+chronic+colitis+and+associated+activation+of+forebrain+neurons.">Combined administration of secretin and oxytocin inhibits chronic colitis and associated activation of forebrain neurons.</a> Neurogastroenterology Motility. 22 (6), 654 (2010).</li><li>Berthoud, H. R., Neuhuber, W. 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A technique for microdissection of discrete, OTR-rich brain nuclei in the neonatal rat brain is presented in this paper. It is well recognized that neurons are highly specialized, even within well-characterized nuclei in the brain. This highly reproducible approach to isolate specific OTR-rich nuclei enables robust hypothesis testing. Using automated Western blotting, the consistency and reproducibility of the results were further improved. While a limitation of this technique remains modest brain punch variability; this...

In contrast to our understanding of early brain development occurring over the course of days-to-weeks postpartum, relatively little is known about the myriad of dynamic changes occurring in the first hours of life in rats. A key challenge has been the small size of the neonatal rat brain and a requirement for high-tech tools to isolate discrete brain regions or single cells. Studies often assess gene transcription and not translation1,2, which does not give a firm understanding of functional levels of activated signaling molecules. Others examine expression using immunohistochemistry to reference brain regions, which does not allow for the quantification of expression levels3. No study to date has examined the activation of signaling pathways associated with rats&#39; first colostrum feed in discrete brain regions, which requires rapid isolation and sacrifice and measurement of protein expression and protein phosphorylation using Western blotting. While brain microdissection is performed on older and larger brains, we have not identified a reference performing a non-single-cell brain punch in a P0 brain. This paper presents a protocol for isolating restricted regions of the neonatal brain using a relatively low-tech punch technique and a Western blotting procedure to measure protein expression in relatively small samples. This protocol may be suitable for research questions that require the assessment of protein expression and post-translational modifications (e.g., phosphorylation) in relatively restricted regions of small brains of any species, provided that the user can visually identify the brain region of interest with an atlas and identifiable landmarks.
This technique was developed to understand changes occurring in the brain as a result of the neonatal rats&#39; first colostrum feed, which is rich in oxytocin (OT). OT has been long known for its ability to stimulate milk let-down and uterine contraction. However, OT is now known to play a wide range of roles in the regulation of many bodily functions and behaviors4. For example, OT opposes stress and inflammation in conjunction with adaptive affiliative behaviors5, delays gastric emptying, and slows intestinal transit. OT receptors (OTR) have been identified in enteric neurons and intestinal epithelium6,7,8. The gastrointestinal effects of OT are particularly important to the infant during the early postnatal period. For instance, breastfeeding is associated with the delivery of significant quantities of OT to the neonatal gut9,10, and data show that the OTR is heavily overexpressed in duodenal villi during the milk suckling period8.
In vitro experiments using a gut cell line have demonstrated at the cellular level that oxytocin modulates important molecules in the stress signaling pathway11,12 and plays a regulatory role in translation of proteins12. These studies suggest that components of milk, including exogenous oxytocin from the mother, are important in the unfolded protein response in neonates to reduce cellular stress13.
In vivo and ex vivo studies have shown that colostrum OT modulates the cellular stress response, inflammation, and autophagy markers in newborn rat gut villi. Newborn enterocytes suffer substantial cellular stress on their luminal side when the gut is simultaneously exposed to microbiota from the mother in colostrum14,15 and numerous proteins, including hormones such as OT9,10,16.
The effects of OT on the brain have been studied17. However, the OT signaling mechanisms demonstrated in the gut during the early postnatal period have not been studied in the brain. In this paper, a method for isolating discrete brain nuclei in the neonatal rat brainstem and hypothalamus using electrophoresis is used to profile isolated brain regions. The overall goal of this method is to capture the state of cell signaling in brain areas as close as possible to birth, before and after the first milk suckling, in brain tissue with the lowest glial/neuronal index. The rationale for the development of this technique is that it allows for the rapid isolation of restricted, microscopic brain regions in neonatal pups with a more homogenous collection of neurons for ex vivo studies using an automated Western blotting methodology, offering highly consistent results on relatively small dissected samples. A shortcoming of prior work includes more gross dissection (brain slices or whole brain) and older animals18,19. The brains of young pups are incredibly dynamic, featuring waves of glial differentiation after birth. In order to study brain changes influenced by the pups&#39; first feeding, studying restricted neuronal nuclei with reproducible dissection is necessary.
Milk feed is usually analyzed for its immunological and nutritional impact on health or gene expression (for example, in enterocytes20,21), whereas its effect on brain areas during brain development is rarely studied. The effect of milk transit in the gut on brain function was analyzed in reference to gut cholecystokinin receptors vagal relay to brain stem nuclei, but not to intracellular signaling pathways22. There is a vast literature on vulnerability of the developing neonate brain to malnutrition of mothers during pregnancy23, but the stress and inflammation signals are not addressed. Importantly, the current method takes advantage of a phenomenon in day-zero rat newborns that isolates the blood-born colostrum stimuli from vagal relay of visceral stimuli. This is the so-called stress hypo-responsiveness period characterized by immature nucleus tractus solitarius (NTS)-hypothalamic circuit immediately after birth24,25 that restricts NTS, paraventricular nucleus (PVN), and supraoptic nucleus (SON) signals to blood-born stimuli.
This method is useful for analysis of multiple signaling pathways and relatively restricted to neuronal cells, provided that brain tissue is harvested at postnatal day-0 in rats, in addition to whether mothers have been challenged or not by any kind of treatment during pregnancy. Litters can be analyzed for the effects of colostrum feed versus pre-feeding signaling. When comparing signals between brain areas with poor versus rich protein yield, this method enables in-capillary determination of total protein of the polypeptide bands in capillaries run parallel to immune-quantitation of protein antigens. This method enables the quantitative comparison, using arbitrary units, of results obtained by the same antibody without standard quantitative curves and by reference to total protein per capillary. Comparing results obtained by different antibodies is possible only using quantitative standard curves.
This method allowed for the assessment of bidirectional signaling occurring between the gut and the brain and that can impact function in both organs26. The association between oxytocin and food intake, which has been extensively studied in recent years27, supports a link between increased oxytocin signaling and nutrient availability. These studies also support the converse concept that energy deficits are coupled with reductions in hypothalamic oxytocin signaling.
Earlier studies of the effect of OT on brain activity demonstrated that induced gut inflammation elicited cFos transcription in hypothalamic PVN, amygdala, and piriform cortex which was refractory to vagotomy28. However, systemic infusion of OT with secretin decreased the brain cFos response to the provoked inflammatory reaction in the gut28. This suggested that the effect of exogenous OT was carried out by routes other than vagal relays, possibly via blood-borne signaling molecules carried through the area postrema6,29.
In this study, the cellular stress signaling pathways that have previously observed in the gut were assessed in the brain. The hypothesis was that milk components may protect or defer the effect of inflammation on gut permeability to microbial and other metabolites, and in turn, the effects on brain function. The clear antagonistic differences in IkB versus BiP signaling found in villi, before and after priming by colostrum13, suggested that the brains of neonates, still in the process of developing, may sense these colostrum-induced gut signals.
Signaling protein markers used in previous gut experiments that are associated with endoplasmic reticulum stress were measured. They include the ER chaperone BiP, translation initiation factor eIF2a (which serves as a stress response integrator30), eIF2a kinase p-PKR, and two inflammation-signaling proteins (NF-kB and its inhibitor, IkB).
Six brain regions based on their ability in adults to secrete or respond to OT were chosen. The NTS, located at the upper medulla, is the first relay of the visceral input and receives direct signaling from vagal sensory neurons in the gut31 and possibly blood-born cytokines, toxins, and hormones via the adjacent area-postrema32. The PVN, supraoptic nucleus (SON), striatum nuclei (STR), cerebral cortex (CX), and medial preoptic nucleus (MPO) receive signaling from the gut via the NTS.
Results showed that the cellular stress response during the immediate postnatal period prior to colostrum priming and immediately after first feeding is different in NTS compared to PVN and SON. Signaling in CX, STR, and MPO differed from that of PVN and SON, as well. The distinct protective functions of OT shown previously to modulate cell stress and inflammation in the gut are likely sensed by some areas of the brain. Collectively, the data indicate that at the cellular level, during the first hours after birth, the brain responds to the metabolic stress associated with nutrient insufficiency. The data also show that the extent and direction of the modulating effects of the colostrum feed are region-dependent and that in some regions, they mirror OT effects shown previously in the gut.

<table>
	<tbody>
		<tr>
			<td>Bradford solution</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein lysis kit</td>
			<td>Protein simple</td>
			<td>CBS403</td>
			<td>Bicine/CHAPS</td>
		</tr>
		<tr>
			<td>WES kits</td>
			<td>Protein simple</td>
			<td>WES-Mouse 12-230 master kit (PS-MK15), WES-Rabbit 12-230 master kit (PS-MK14), WES 12-230 kDa total Protein master kit (PS-TP07)</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG HRP conjugate</td>
			<td>Protein simple</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phospho-eIF2a</td>
			<td>Cell Signaling technology</td>
			<td>SER51, 9721</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-PKR</td>
			<td>Cell Signaling technology</td>
			<td>2103</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phospho-PKR</td>
			<td>Millipore</td>
			<td>Thr451, 07-886</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit mAb anti-PKR</td>
			<td>Cell Signaling technology</td>
			<td>12297</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit mAb anti-GAPDH</td>
			<td>Cell Signaling technology</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-phospho-IKB</td>
			<td>Cell Signaling technology</td>
			<td>9246</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-IKB</td>
			<td>Cell Signaling technology</td>
			<td>4814</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-BiP</td>
			<td>Cell Signaling technology</td>
			<td>3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti GCN2</td>
			<td>Cell Signaling technology</td>
			<td>3302</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit mAb anti-phospho-GCN2</td>
			<td>BIORBYT</td>
			<td>T899</td>
			<td></td>
		</tr>
		<tr>
			<td>pregnant Sprague-Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch device</td>
			<td>WellTech Rapid Core or Harris Uni-Core</td>
			<td>0.35, 0.50, 0.75, 1.0, 1.20, 1.50</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

The representative bands of immunoreactivity relative to total protein show that there are brain nuclei with very low harvested protein. This requires the use of the automated Western blot technique, which is highly sensitive compared to the canonical Western blot. This approach can be run with fortyfold less protein per capillary compared to the per-lane in Western blots.
Differential effects of colostrum priming on BiP levels in ...

Watch this Scientific Journal Video about Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding at JoVE.co

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

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