Browsing by Subject "Rats, Sprague-Dawley"
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Item A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells(PLOS, 2017-09-22) Wang, Junming; Ma, Hai-Ying; Krishnamoorthy, Raghu R.; Yorio, Thomas; He, Shaoqingc-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein beta (C/EBPbeta) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPbeta in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPbeta as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPbeta augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPbeta was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPbeta and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPbeta appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.Item Angiotensin II type 1 receptor agonistic autoantibody blockade improves postpartum hypertension and cardiac mitochondrial function in rat model of preeclampsia(BioMed Central Ltd., 2021-11-02) Booz, George W.; Kennedy, Daniel; Bowling, Michael; Robinson, Taprieka; Azubuike, Daniel; Fisher, Brandon; Brooks, Karen; Chinthakuntla, Pooja; Hoang, Ngoc H.; Hosler, Jonathan P.; Cunningham, Mark W., Jr.Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence ('n7AAc') improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + 'n7AAc' (n = 16). Gestational day 14, RUPP surgery was performed and 'n7AAc' (144 mug/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 +/- 4 vs. 116 +/- 3 mmHg, p < 0.05), but was normalized in in RUPP + 'n7AAc' (109 +/- 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + 'n7AAc' vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with 'n7AAc'. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.Item [beta-Glu(2)]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain(MDPI, 2021-06-09) Prokai-Tatrai, Katalin; Nguyen, Vien; Prokai, LaszloSelective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu(2)]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [beta-Glu(2)]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [beta-Glu(2)]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [beta-Glu(2)]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.Item In vitro and in vivo neuroprotective effects of cJun N-terminal kinase inhibitors on retinal ganglion cells(BioMed Central Ltd., 2016-04-21) Kim, Byung-Jin; Silverman, Sean M.; Liu, Yang; Wordinger, Robert J.; Pang, Iok-Hou; Clark, Abbot F.BACKGROUND: The c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied. RESULTS: JNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p < 0.05) protected against glutamate excitotoxicity and trophic factor withdrawal induced RGC death in culture. In the I/R model, phosphorylation of JNK (pJNK) in the retina was significantly (p < 0.05) increased after injury. I/R injury significantly (p < 0.05) decreased the thickness of retinal layers, including the whole retina, inner plexiform layer, and inner nuclear layer and cell numbers in the GCL. Administration of SP600125 for 28 days protected against all these degenerative morphological changes (p < 0.05). In addition, SP600125 significantly (p < 0.05) protected against I/R-induced reduction in scotopic ERG b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. SP600125 also protected against the I/R-induced losses in volume and levels of synaptic markers in the SC. Moreover, the protective effects of SP600125 in the retina and SC were also detected even with only 7 days (Days 1-7 after I/R) of SP600125 treatment. CONCLUSIONS: Our results demonstrate the important role the JNK pathway plays in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection of RGCs in the retina.Item Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion(Ivyspring International Publisher, 2020-01-01) Zhang, Lin-Yuan; Pan, Jiaji; Mamtilahun, Muyassar; Zhu, Yuan; Wang, Liping; Venkatesh, Ashwin; Shi, Rubing; Tu, Xuanqiang; Jin, Kunlin; Wang, Yongting; Zhang, Zhijun; Yang, Guo-YuanMicroglial activation participates in white matter injury after cerebral hypoperfusion. However, the underlying mechanism is unclear. Here, we explore whether activated microglia aggravate white matter injury via complement C3-C3aR pathway after chronic cerebral hypoperfusion. Methods: Adult male Sprague-Dawley rats (n = 80) underwent bilateral common carotid artery occlusion for 7, 14, and 28 days. Cerebral vessel density and blood flow were examined by synchrotron radiation angiography and three-dimensional arterial spin labeling. Neurobehavioral assessments, CLARITY imaging, and immunohistochemistry were performed to evaluate activation of microglia and C3-C3aR pathway. Furthermore, C3aR knockout mice were used to establish the causal relationship of C3-C3aR signaling on microglia activation and white matter injury after hypoperfusion. Results: Cerebral vessel density and blood flow were reduced after hypoperfusion (p<0.05). Spatial learning and memory deficits and white matter injury were shown (p<0.05). These impairments were correlated with aberrant microglia activation and an increase in the number of reactive microglia adhering to and phagocytosed myelin in the hypoperfusion group (p<0.05), which were accompanied by the up-regulation of complement C3 and its receptors C3aR (p<0.05). Genetic deletion of C3ar1 significantly inhibited aberrant microglial activation and reversed white matter injury after hypoperfusion (p<0.05). Furthermore, the C3aR antagonist SB290157 decreased the number of microglia adhering to myelin (p<0.05), attenuated white matter injury and cognitive deficits in chronic hypoperfusion rats (p<0.05). Conclusions: Our results demonstrated that aberrant activated microglia aggravate white matter injury via C3-C3aR pathway during chronic hypoperfusion. These findings indicate C3aR plays a critical role in mediating neuroinflammation and white matter injury through aberrant microglia activation, which provides a novel therapeutic target for the small vessel disease and vascular dementia.Item Oxazolone as a Model to Induce Edema in the Lower Limb of Rats(2018-05) Pastrana, Christopher; Hodge, Lisa M.; Simecka, Jerry W.; Mathis, Keisa W.; Sumien, NathaliePurpose. Peripheral edema is a condition characterized by the accumulation of excess interstitial fluid in distal tissues and commonly manifests in the arms or legs. Untreated complex peripheral edema can progress into chronic lymphedema as impaired fluid drainage and chronic inflammation cause irreversible damage to the surrounding tissue and local lymphatics. The overall goal of our research is to study the effectiveness of osteopathic manipulative medicine techniques (OMT) for the treatment of edema, infection and inflammation. The aim of this study was to evaluate oxazolone (OXA) as a model to induce acute inflammation and lower limb edema in the rat. Specifically, we hypothesized that a single application of OXA would induce a local inflammatory response and induce edema in the lower limb. Methods. Female Sprague Dawley rats, weighing 200-250 g, were used for this study. On day 0 the right lower limb was shaved and 750 µl of 5% OXA-acetone solution or 750 µl of a 5% phosphate buffered saline (PBS)-acetone solution vehicle (VEH) was applied to the exposed skin. Lower limb measurements were made at days 0 and 6 using a Vernier caliper. At day 6, the rats were euthanized, and the bilateral hind paws were removed above the calcaneus and weighed. The spleen and bilateral inguinal lymph nodes were removed, homogenized, centrifuged and cells were stained with phycoerythrin (PE) mouse anti-rat granulocytes, fluorescein (FITC) anti-rat CD3, and allophycocyanin (APC) anti-rat CD161 antibodies. The percentage of granulocytes, T cells and dendritic cells were measured by flow cytometry. Data were analyzed by analysis of variance (ANOVA) followed by Tukey-Kramer post-test or by Student's t-test. Comparisons were made between OXA and VEH groups at day 6 post-induction. Results. OXA did not induce significant (p[greater than]0.05) changes in either hind paw thickness or hind paw volume. OXA significantly (P[less than]0.05) increased the concentration of macrophages, neutrophils, and dendritic cells, and T cells within inguinal lymph nodes. Conclusions. OXA induced an acute local inflammatory response in the draining inguinal lymph nodes. However, as used in this approach, OXA did not induce peripheral edema. In future studies we will investigate alternate strategies to induce lower limb edema in the rat.Item Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications(MDPI, 2021-02-08) Prokai, Laszlo; Rahlouni, Fatima; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, KatalinThe widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17beta-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.Item Selectively Inhibiting the Median Preoptic Nucleus Attenuates Angiotensin II and Hyperosmotic-Induced Drinking Behavior and Vasopressin Release in Adult Male Rats(Society for Neuroscience, 2019-03-26) Marciante, Alexandria B.; Wang, Lei A.; Farmer, George E.; Cunningham, J. ThomasThe median preoptic nucleus (MnPO) is a putative integrative region that contributes to body fluid balance. Activation of the MnPO can influence thirst, but it is not clear how these responses are linked to body fluid homeostasis. We used designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of the MnPO in drinking behavior and vasopressin release in response to peripheral angiotensin II (ANG II) or 3% hypertonic saline (3% HTN) in adult male Sprague Dawley rats (250-300 g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After two weeks' recovery, a subset of rats was used for extracellular recordings to verify functional effects of ANG II or hyperosmotic challenges in MnPO slice preparations. Remaining rats were used in drinking behavior studies. Each rat was administered either 10 mg/kg of exogenous clozapine-N-oxide (CNO) to inhibit DREADD-expressing cells or vehicle intraperitoneal followed by a test treatment with either 2-mg/kg ANG II or 3% HTN (1 ml/100-g bw, s.c.), twice per week for two separate treatment weeks. CNO-induced inhibition during either test treatment significantly attenuated drinking responses compared to vehicle treatments and controls. Brain tissue processed for cFos immunohistochemistry showed decreased expression with CNO-induced inhibition during either test treatment in the MnPO and downstream nuclei compared to controls. CNO-mediated inhibition significantly attenuated treatment-induced increases in plasma vasopressin compared to controls. The results indicate inhibition of CaMKIIa-expressing MnPO neurons significantly reduces drinking and vasopressin release in response to ANG II or hyperosmotic challenge.Item Small Immunomodulatory Molecules as Potential Therapeutics in Experimental Murine Models of Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS)(MDPI, 2021-03-04) Shah, Dilip; Das, Pragnya; Acharya, Suchismita; Agarwal, Beamon; Christensen, Dale J.; Robertson, Stella M.; Bhandari, VineetBACKGROUND: Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary process triggered by a variety of insults including sepsis, viral or bacterial pneumonia, and mechanical ventilator-induced trauma. Currently, there are no effective therapies available for ARDS. We have recently reported that a novel small molecule AVR-25 derived from chitin molecule (a long-chain polymer of N-acetylglucosamine) showed anti-inflammatory effects in the lungs. The goal of this study was to determine the efficacy of two chitin-derived compounds, AVR-25 and AVR-48, in multiple mouse models of ALI/ARDS. We further determined the safety and pharmacokinetic (PK) profile of the lead compound AVR-48 in rats. METHODS: ALI in mice was induced by intratracheal instillation of a single dose of lipopolysaccharide (LPS; 100 µg) for 24 h or exposed to hyperoxia (100% oxygen) for 48 h or undergoing cecal ligation and puncture (CLP) procedure and observation for 10 days. RESULTS: Both chitin derivatives, AVR-25 and AVR-48, showed decreased neutrophil recruitment and reduced inflammation in the lungs of ALI mice. Further, AVR-25 and AVR-48 mediated diminished lung inflammation was associated with reduced expression of lung adhesion molecules with improvement in pulmonary endothelial barrier function, pulmonary edema, and lung injury. Consistent with these results, CLP-induced sepsis mice treated with AVR-48 showed a significant increase in survival of the mice (80%) and improved lung histopathology in the treated CLP group. AVR-48, the lead chitin derivative compound, demonstrated a good safety profile. CONCLUSION: Both AVR-25 and AVR-48 demonstrate the potential to be developed as therapeutic agents to treat ALI/ARDS.Item Sniffer cells for the detection of neural Angiotensin II in vitro(Springer Nature, 2019-06-19) Farmer, George E.; Amune, Anna; Bachelor, Martha E.; Duong, Phong; Yuan, Joseph P.; Cunningham, J. ThomasNeuropeptide release in the brain has traditionally been difficult to observe. Existing methods lack temporal and spatial resolution that is consistent with the function and size of neurons. We use cultured "sniffer cells" to improve the temporal and spatial resolution of observing neuropeptide release. Sniffer cells were created by stably transfecting Chinese Hamster Ovary (CHO) cells with plasmids encoding the rat angiotensin type 1a receptor and a genetically encoded Ca2+ sensor. Isolated, cultured sniffer cells showed dose-dependent increases in fluorescence in response to exogenously applied angiotensin II and III, but not other common neurotransmitters. Sniffer cells placed on the median preoptic nucleus (a presumptive site of angiotensin release) displayed spontaneous activity and evoked responses to either electrical or optogenetic stimulation of the subfornical organ. Stable sniffer cell lines could be a viable method for detecting neuropeptide release in vitro, while still being able to distinguish differences in neuropeptide concentration.