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Abstract

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Protocol

Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

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

Published: November 14th, 2018

DOI:

10.3791/58341

1Department of Psychiatry, Columbia University, 2Department of Pathology & Cell Biology, Columbia University Medical Center, 3Department of Psychiatry, Division of Molecular Imaging and Neuropathology, New York State Psychiatric Institute, 4EB Sciences

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 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-κB (NF-kB) and inhibitor κB (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.

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 region....

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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

  1. Order timed pregnant rats from vendor.
  2. 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.
  3. Remove pups with a gloved hand by their tail.......

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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 .......

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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.......

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

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Name Company Catalog Number Comments
Bradford solution Bio Rad
Protein lysis kit Protein simple CBS403 Bicine/CHAPS
WES kits Protein simple 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)
anti-mouse IgG HRP conjugate Protein simple
Rabbit anti-phospho-eIF2a Cell Signaling technology SER51, 9721
mouse mAb anti-PKR Cell Signaling technology 2103
Rabbit anti-phospho-PKR Millipore Thr451, 07-886
Rabbit mAb anti-PKR Cell Signaling technology 12297
rabbit mAb anti-GAPDH Cell Signaling technology 2118
mouse mAb anti-phospho-IKB Cell Signaling technology 9246
mouse mAb anti-IKB Cell Signaling technology 4814
rabbit anti-BiP Cell Signaling technology 3183
Rabbit anti GCN2 Cell Signaling technology 3302
Rabbit mAb anti-phospho-GCN2 BIORBYT T899
pregnant Sprague-Dawley rats Charles River Laboratories
Punch device WellTech Rapid Core or Harris Uni-Core 0.35, 0.50, 0.75, 1.0, 1.20, 1.50

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