Integrative Physiology
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30815
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Browsing Integrative Physiology by Author "Cunningham, Joseph"
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Item Norepinephrine innervation of the supraoptic nucleus contributes to dilutional hyponatremia in male BDL rats(2022) Aikins, Ato; Little, Joel; Rybalchenko, Nataliya; Cunningham, JosephPurpose: Dilutional hyponatremia is a common complication associated with liver cirrhosis that is linked to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention and hypoosmolality. In the cirrhotic liver, there is increase in resistance to blood flow resulting in portal hypertension, ascites formation, mesenteric vasodilation due to release of vasodilators and increased pooling of blood in the splanchnic circulation. The fluid redistribution decreases central vascular blood volume which is sensed by peripheral baroreceptors and volume receptors located in the aortic arch and heart. This could be relayed to A1 neurons in the caudal ventrolateral medulla (CVLM) and the A2 neurons in the nucleus tractus solitarius (NTS). The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON). We propose that the A1 and A2 norepinephrine neurons in the hindbrain contribute to the activation of AVP-secreting neurons in the supraoptic nucleus (SON) leading to inappropriate AVP release and dilutional hyponatremia. Method: Anti-DBH saporin [IT-03] (Advanced Targeting Systems), a cytotoxin conjugated to an antibody against DBH was injected to the SON to lesion the norepinephrine innervation of SON including A1/A2 neurons. After two weeks, adult male rats received bile duct ligation surgery (BDL) which was used to model liver cirrhosis. In this model, the common duct that drains bile from the liver to the intestine is cauterized between two ligatures leading to obstructive cholestasis and liver cirrhosis. Four weeks after BDL surgery, rats were anesthetized with inactin (thiobutabarbital sodium salt hydrate; 100 mg/kg, i.p.). Blood samples were taken for plasma copeptin, osmolality, and hematocrit measurements. The rats were then perfused transcardially with 1M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (4% PFA) in 1M PBS. Plasma copeptin concentration was measured as a surrogate marker for AVP using commercially available copeptin ELISA kits. The brains were removed and processed for delta FosB (a marker of chronic activation), dopamine β-hydroxylase (DBH) and AVP immunohistochemistry. Results: Anti-DBH saporin (ADS) injection in the SON of BDL rats significantly decreased the number of cells positive for both delta FosB and DBH in the A1 and A2 cell groups as compared to vehicle injection (A1 neurons, ADS/BDL vs Vehicle/BDL P< 0.001; A2 neurons, ADS/BDL vs Vehicle/BDL P< 0.05). ADS treated BDL rats had fewer SON cells positive for both AVP and delta FosB as compared to vehicle injection (ADS/BDL vs Vehicle/BDL P< 0.05). Reduced colocalization of delta FosB and DBH in A1/A2 neurons was associated with a significantly lower plasma copeptin concentration in BDL rats. (ADS/BDL vs Vehicle/BDL P< 0.05). Similar effects were seen for plasma osmolality and hematocrit (ADS/BDL vs Vehicle/BDL P< 0.05). Conclusion: The result suggests that an increase in cells positive for both delta FosB and DBH in A1/A2 neurons is associated with an increase in plasma AVP and hypoosmolality in male BDL rats. Anti-DBH saporin lesions of SON prevented increases in plasma copeptin and neural activation of A1/A2 and AVP SON neurons associated with BDL.Item Spatial Transcriptomics Reveal Potential Sex Differences in Supraoptic Nucleus Gene Expression of Adult Rats(2022) Nguyen, Dianna H.; Phillips, Nicole; Cunningham, JosephPurpose: The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well-known sexually dimorphic regions of the hypothalamus, little is known about possible sex differences in gene expression in the SON. Our study aims to address this knowledge gap by leveraging spatially-resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Methods: Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially-resolved gene expression data for the SON of adult male (n=4) and female (n=4) Sprague-Dawley rats. Briefly, each brain was sectioned at 10µm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the following workflow: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics' Space Ranger and Loupe Browser applications and other bioinformatic tools. Results: Gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. From the list of significant genes after performing differential expression analysis on the SON region via Loupe Browser, 22 genes (e.g., Avp and Oxt) were common to both sexes, 24 genes were unique to the females, and no genes were unique to the males. Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females and 2) endomembrane system and glycerophospholipid metabolism pathway for the significant genes unique to females, as some examples. Further interrogation of the significant genes with Ingenuity Pathway Analysis showed some overlapping networks and common upstream regulators; however, differences between the male and female groups were also identified in upstream regulators, such as Creb, Pka, and LEPR unique to the males and insulin, Cdkal1, and HIF1A unique to the females. Conclusions: These spatially-resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with basic endomembrane structure and function and phospholipid metabolism/signaling. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.