Integrative Physiology

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    The Effect of Nicotinic Agonist Therapy on Renal Inflammation in Mice with Systemic Lupus Erythematosus
    (2018-03-14) Pham, Grace; Uteshev, Victor; Mathis, Keisa W.; Williams, Emily
    Purpose: Systemic lupus erythematosus (SLE) is an autoimmune disease that causes chronic systemic, and specifically renal, inflammation that results in renal injury and hypertension. Under normal conditions, inflammation may be regulated by a neuroimmune reflex referred to as the cholinergic anti-inflammatory pathway; this pathway is mediated by immune cells responding to acetylcholine via the a7-subunit of nicotinic acetylcholine receptors (nAChRs) and when stimulated results in the reduction of tissue-specific inflammation. These a7nAChRs can be activated nonselectively by nicotine and we have previously shown that nicotine attenuates renal inflammation in female SLE mice. However, because nicotine is toxic and addictive, it would not be available to use as a therapy for SLE. We hypothesized that a selective a7nAChR agonist would similarly reduce renal inflammation and protect against the development of renal injury. Methods: In our pilot studies (n=2-3 mice/group), female SLE (NZBWF1) and control (NZW) mice (33 weeks of age) were surgically implanted with osmotic mini-pumps to subcutaneously administer the nicotinic agonist PNU-120596 (3.8 mg/kg/day), or vehicle (100% DMSO) at a rate of 0.25 mL/hour. At 35 weeks, mice were euthanized and tissues harvested. Results: The gene expression of renal cortical tumor necrosis factor (TNF)-a (measured by qRT-PCR using 2-DDCt method normalized to HPRT) was found to be 3-fold higher in SLE mice as compared to control mice. However, PNU-120596 treatment did not have an effect on TNF-a gene expression in SLE or control mice. Renal cortical interferon (IFN)-a gene expression was similar in SLE and control mice. By contrast, PNU-120596 treatment increased IFN-a gene expression in both SLE mice and controls (18-fold and 3-fold, respectively). Conclusions: Although inflammatory markers were increased in SLE mice, as expected, the effect of this PNU-120596 treatment on TNF-a and IFN-a is inconclusive, because statistical analysis was not performed due to the small sample size. Future studies will increase sample size and agonist dosage to further investigate the potential therapeutic effect of nicotinic agonists on SLE-induced inflammation and renal injury.
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    Immunological effects of vagus nerve stimulation in murine systemic lupus erythematosus
    (2018-03-14) Pham, Grace; Stauss, Harald; Mathis, Keisa W.; Kulp, Dennis
    Purpose: Systemic lupus erythematosus (SLE) is an autoimmune disease that principally affects women and is associated with inflammatory pathogenesis of multiple organs. Of the disseminated features of SLE, classical disease processes such as renal injury with hypertension, as well as autonomic nervous system dysregulation, are prevalent. The autonomic dysfunction in SLE is characterized by increased sympathetic activity and concomitant decreased parasympathetic nervous system (PNS) activity; however, it is unknown if impaired PNS activity promotes hypertension and renal injury in SLE. The cholinergic anti-inflammatory pathway (CAP), is an endogenous neuroimmune reflex that regulates cytokine release from immune cells; briefly, the CAP initiates with stimulation of the parasympathetic vagus nerve and culminates in the inhibition of the secretion of pro-inflammatory cytokines from macrophages and other leukocytes. Although it is known that vagal activity is suppressed in SLE, it remains unclear whether this contributes to a diminished CAP promoting inflammation in the disease. We hypothesized that chronic vagus nerve stimulation (VNS) will decrease the inflammatory cascade in SLE through enhancement of the CAP. Methods: Female SLE (NZBWF1) mice (25 weeks of age) were implanted with electrical vagus nerve stimulators that fit the cervical vagus nerve. Only female mice of this well-characterized strain were used, reflecting the prevalence of lupus in women. The mice were then divided into two groups: VNS (n=10) and sham (n=7). Stimulators targeted the vagus nerve continuously for 2 weeks. Results: Spleen weight was slightly increased in SLE/VNS mice compared to SLE/sham mice (0.14 ± 0.03g vs. 0.11 ± 0.01; P=NS). Flow cytometry showed that SLE/VNS mice had slightly less CD3+/CD4+ bone marrow T cells when compared with SLE/sham mice (27.30 ± 9.41% vs. 40.17 ± 7.01%; P=NS). The percentage of mice with albuminuria, an index of renal injury measured by Albustix, was also decreased in SLE/VNS mice compared to SLE/sham mice (10% vs 29%). Conclusions: These results suggest the efficacy of VNS in reducing the inflammatory profile in SLE mice, and that this protection may reduce end-organ disease. Future work will investigate the role of the CAP in quelling inflammation perpetuated by neuroimmune dysregulations in SLE. Studies supported by the American Heart Association (14SDG18320033) and the National Institutes of Health (1K01HL139859)
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    Effect of Acute Bilateral Vagotomy on Tissue-Specific Inflammation in a Murine Model of Systemic Lupus Erythematosus
    (2018-03-14) Vedantam, Shyam
    Effect of Acute Bilateral Vagotomy on Tissue-Specific Inflammation in a Murine Model of Systemic Lupus Erythematosus Shyam Vedantam, Grace S Pham, Keisa W Mathis Purpose: Chronic inflammation has been implicated in the pathogenesis of hypertension. We use a model of systemic lupus erythematosus (SLE) to study this relationship since SLE is a chronic autoimmune inflammatory disorder with a high prevalence of renal injury and hypertension. SLE is also associated with diminished autonomic (vagal) tone, and this implicates impaired neuroendocrine/neuroimmune mechanisms. One example is the classic hypothalamic-pituitary-adrenal (HPA) axis, which can be activated by the afferent vagus nerve and result in cortisol production and anti-inflammatory effects. Another example is the cholinergic anti-inflammatory pathway, an endogenous vagus nerve-to-spleen pathway that reduces inflammation upon stimulation. It is thought that both of these mechanisms are dysregulated and promote chronic inflammation in SLE. To confirm the importance of the vagus nerve in regulating inflammation though these mechanisms, we performed chronic unilateral vagotomy in SLE mice and determined that this paradoxically decreased inflammatory markers in the spleen and the kidney. We hypothesized that the other vagus nerve compensated and upregulated an anti-inflammatory response and that total vagotomy would exacerbate splenic and renal inflammation. Methods: In the current study, anesthetized female SLE (NZBWF1) and control (NZW) mice (35 weeks of age; n=3 mice/group) underwent bilateral vagotomy and were euthanized 3 hours later, followed by tissue harvest. Results: Spleen tumor necrosis factor (TNF)-a (measured by Western blot and normalized to total protein) was increased in SLE mice compared to controls (4.0e6 ± 2.2e6 vs. 1.5e6 ± 3.3e5; P=NS). Acute bilateral vagotomy exacerbated this inflammation in SLE mice (5.1e6 ± 2.5e6) and controls (3.2e6 ± 6.8e5). Renal cortical TNF-a was not different in SLE and control mice (7.4e5 ± 1.6e5 vs. 6.9e5 ± 4.8e4); however, acute bilateral vagotomy exacerbated TNF-a in SLE mice (1.3e6 ± 7.2e4 vs) and controls (1.2e6 ±6.5e5). Conclusions: These findings suggest that the vagus nerve and vagally-mediated anti-inflammatory mechanisms like the HPA axis and the cholinergic anti-inflammatory pathway are critical in controlling inflammation in SLE. Future studies involving chronic bilateral vagotomy in SLE mice are necessary to confirm our hypotheses.
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    High Salt Loading Increases Brain Derived Neurotrophic Factor in Supraoptic Vasopressin Neurons
    (2018-03-14) Little, Joel; Cunningham, J. Thomas; Balapattabi, Kirthikaa
    Purpose: Salt loading (SL) upregulates Brain Derived Neurotrophic Factor (BDNF) and causes increased arginine vasopressin (AVP) release from supraoptic nucleus of the hypothalamus (SON). BDNF diminishes or reverses the GABAA inhibition in the SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrKB) phosphorylation. This creates a feed forward loop that drives AVP release. However, the source of BDNF is not known. Hypothesis: We hypothesize that SON is the source of BDNF contributing to increased AVP release in SL rats. Methods: Adult male Sprague Dawley rats were anesthetized with isoflurane (2-3%) and bilaterally injected in the SON (300 nl/side) with an AAV2 vector with a U6 promoter containing either shRNA against BDNF or a control construct with an mCherry reporter. The vectors were injected at a titer of 1.0 X 1013 GC/ml (Vector Biolabs). Two weeks after the stereotaxic injections, the rats were provided with either water or 2%NaCl to drink for 7 days. At the end of the protocol, rats were anesthetized with inactin (100 mg/kg IP) and brains were collected and flash frozen. Fresh frozen brains were prepared for Laser Capture Microdissection (LCM) by cutting 10μm thick coronal sections through the hypothalamus at the level of the SON. Using LCM, we verified the accuracy of the injections by visualizing the mCherry reporter and collected the SON to measure changes in the BDNF mRNA and AVP hnRNA expression using quantitative Real Time PCR. Subset of brains from each group were used for Western blot analysis of punch samples containing the SON. Rats that did not have successful virus injections in the SON were separately analyzed. Plasma osmolality, hematocrit, and AVP concentration were measured. Data were analyzed by one-way ANOVA with Bonferroni comparisons. Results:SL was associated with significant increases in BDNF mRNA and AVP hnRNA in SON (P Conclusion: The results indicate that BDNF produced in the SON contributes to increased AVP secretion during SL. Supported by R01 HL119458
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    Hypertensive young adult female obese zucker rats (OZR) do not have the blunted baroreflexes and poor glycemic control observed in age-matched hypertensive male OZR
    (2018-03-14) Schreihofer, Ann; Chaudhary, Parul
    Purpose: Obese Zucker rats (OZR) have hyperphagia owing to dysfunctional leptin receptors and gain excess weight compared to lean Zucker rats (LZR) with functional leptin receptors. Young adult male OZR (12-14 wks) develop hypertension and impaired baroreflexes coincident with blunted activation of the nucleus tractus solitarius (NTS), the brain stem site that receives baroreceptor afferent inputs. Male OZR at this age have hyperinsulinemia and chronically elevated fed glucose levels (despite normal fasting glucose) with significant glucose intolerance as measured by telemetry (DSI). Treatment with metformin (300mg/day, 4 wks) restores glycemic control in OZR without eliminating hypertension or hyperinsulinemia, with no effect on these measures in LZR. After treatment with metformin male OZR have improved baroreflexes and activation of the NTS, suggesting hyperglycemia contributes to these deficits in adult male OZR. In contrast to males, at this age female OZR are hypertensive but do not develop impaired baroreflex-mediated changes in heart rate (HR; Tenorio et al., 2013). The present study examined whether preserved baroreflex-mediated changes in HR in female OZR extend to sympathetic baroreflexes and diminished activation of the NTS and whether female OZR maintain glycemic control at this age (13-14 wks). Method: Female zucker rats were instrumented with indwelling femoral catheters run through a tether to record MAP and HR and infuse drugs while rats were conscious and undisturbed. After 24hours of rest, baseline parameters were recorded, and phenylephrine (PE) was infused through the venous line to raise MAP by 40 mmHg to evoke bradycardia response. PE infusion was continued for 90 min to evoke PE-induced c-Fos expression in the brainstem. A different set of rats was anesthetized with isoflurane for surgical preparation to measure MAP (femoral artery) and splanchnic sympathetic nerve activity (SNA) and to infuse drugs (femoral vein). Following this, the rat was anesthetized with urethane (1.5g/kg iv LZR body weight), ventilated, and paralyzed. Additionally, a set of female rats was instrumented with telemetry to monitor continuous glucose recording in undisturbed female zucker rats. Results: Female OZR had excess weight gain (215±8 g vs. 483±14 g in 9 LZR and 10 OZR, P Conclusion: These data suggest better maintenance of glycemic control in hypertensive, hyperinsulinemic young adult female OZR may preserve activation of the NTS and baroreflexes. In addition, these data highlight differing mechanisms for the development of hypertension and impaired control of SNA and HR by baroreflexes in the setting of metabolic syndrome.
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    DREADD-induced inhibition of the MnPO affects drinking behavior and neuroendocrine function in adult male rats
    (2018-03-14) Farmer, George; Wang, Lei; Cunningham, J. Thomas; Marciante, Alexandria B.
    Purpose: Angiotensin II (Ang II) is a peptide hormone that contributes to body fluid balance and hypertension. Forebrain circumventricular organs (CVOs) are sensitive to circulating Ang II and project to the median preoptic nucleus (MnPO). The MnPO projects to the paraventricular nucleus (PVN) and contributes to elevated sympathetic tone and thirst. Methods: We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the MnPO in thirst and neuroendocrine responses to Ang II in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory (Gi) DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After 2 weeks of recovery, each rat was administered 10 mg/kg of exogenous Clozapine-N-Oxide (CNO) ip to inhibit DREADD expressing cells or vehicle ip followed by 2 mg/kg Ang II sc twice per week for 4 weeks. Rats were anesthetized with inactin (10 mg/kg ip) and transcardially perfused 90 minutes after CNO and Ang II treatments. Brains were processed for cFos and mCherry immunohistochemistry. Results: DREADD-injected rats treated with CNO during Ang II exposure had a significantly attenuated drinking response compared to vehicle treatments or to control virus injected rats treated with CNO and Ang II (pIn vitro loose-cell voltage clamp recordings from DREADD-transfected MnPO slices indicated focal CNO (10 uM) application significantly reduces firing rates of these neurons. In situ hybridization experiments of DREADD-transfected MnPO neurons and vesicular glutamate transporter 2 indicated neurons transfected with the DREADD virus containing the CaMKIIa promotor are largely glutamatergic (89.17+1.32%). Conclusion: The results indicate CNO-induced inhibition of excitatory, CaMKIIa-expressing MnPO neurons influences drinking behavior and neuroendocrine function.
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    Oxidative Stress During Simulated Hemorrhage Elicited by Lower Body Negative Pressure
    (2018-03-14) Kay, Victoria; Anderson, Garen; Sprick, Justin; Rickards, Caroline; Park, Flora
    Purpose: Hemorrhage is a leading cause of potentially preventable death in both civilian and military trauma settings. Hemorrhage also elicits an oxidative stress response as a direct result of losing blood volume, or as an indirect response to ischemia-reperfusion injury. Lower body negative pressure (LBNP) is a well validated, non-invasive, and reproducible approach to simulate hemorrhage by inducing central hypovolemia in healthy conscious humans. The oxidative stress response to simulated hemorrhage via LBNP has not been quantified. We hypothesized that systemic markers of oxidative stress would increase with application of LBNP. Methods: 15 healthy human subjects (11M, 4F; 27 ± 1 y) were recruited for a step-wise LBNP exposure to presyncope (systolic blood pressuresymptoms). After baseline, LBNP pressure progressively decreased every 5 minutes to -15, -30, -45, -60, -70, -80, -90, and -100 mmHg. Arterial pressure and stroke volume were measured continuously via finger photoplethysmography, and venous blood samples were collected at baseline and during the LBNP profile. Plasma samples were analyzed for F2-isoprostanes, a global marker of oxidative stress, via gas chromatography/mass spectrometry. Results: The magnitude of central hypovolemia, indexed by the % change in stroke volume, ranged from a 27% to 74%. LBNP induced a -12.6 ± 2.6 % decrease in MAP (%Δ MAP) from baseline (P Conclusion: Simulated hemorrhage elicited by step-wise LBNP to presyncope elicited an increase in a global marker of oxidative stress. These findings have important implications in the study of hemorrhage and potential application of targeted interventions. Funding Source: US Army Medical Research and Materiel Command (W81XWH-11-2-0137) & Owens Foundation Grant. Analysis of eicosanoids (F2-isoprostanes) were performed in the Vanderbilt University Eicosanoid Core Laboratory.
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    Sex-related Effects on Treatment Success in Obstructive Sleep Apnea
    (2018-03-14) Elias, Christopher; Burgess, Lauren; Burk, John; Smith, Michael; Davis, Tyler
    Abstract Purpose: Obstructive Sleep Apnea (OSA) has a high prevalence in adults in the US (over 20 million estimated diagnoses) for which the current gold standard treatment for OSA is Positive Airway Pressure (PAP). Current literature suggests a much higher occurrence in men than women and there appear to be sex-related disparities for treatment compliance as well. Current standards for treatment adequacy only require 4 hours/night and 5 days/week. We have developed a more rigorous Treatment Success Index (TSI) that combines key measures of PAP patient compliance and treatment efficacy. The purpose of this study was to determine whether there is a gender difference in TSI and, if so, what factors appear to contribute to these differences. Methods: Retrospective analyses of sleep data from 150 patients were performed (#2018-019) to assess sex-related differences in TSI and determine how several treatment parameters and demographic factors affect the TSI. Results: The findings revealed three main points. First, length of treatment versus TSI was significant for the males (P = 0.0307) but not the females (P = 0.0760). Second, age versus TSI was not significant for either gender (P [greater than] 0.05). Third, BMI versus TSI was significant for females (P = 0.0031) but not males (P = 0.1182). This suggests a longer treatment time in males and a higher baseline BMI in females are significant predictors of treatment success. Conclusions: These data further define sex-related distinctions in the treatment efficacy of OSA and should be considered when physicians consider the management of their patients with OSA. Further studies will focus on the effects these factors might have on clinical outcomes.
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    Optimizing Treatment Success in Sleep Apnea
    (2018-03-14) Davis, Tyler; Burgess, Lauren; Burk, John; Smith, Michael; Elias, Christopher
    Abstract Purpose: Obstructive Sleep Apnea (OSA) is an exceedingly common disorder in America with estimates of disease prevalence ranging from 3-7% of the entire population, and it is likely that this is a gross underestimation. OSA is considered a multifactorial disorder with a diverse range of influences including developmental, environmental, and genetic factors. The current gold standard of treatment is Positive Airway Pressure (PAP). PAP treatment, while proven to be effective, is heavily influenced by many patient features. As a result, treatment success of OSA is difficult to predict and current standards do not take all the critical factors into account. The purpose of this study was to determine which patient traits can be used to predict treatment success. Methods: We performed a retrospective analysis (IRB #2018-019) of de-identified patient data from sleep studies in 150 patients over time to calculate a newly developed Treatment Success Index (TSI). TSI is a novel measure that comprehensively measures patient treatment success by combining Apnea Hypopnea Index derived from the sleep data (AHI, a clinical measurement of disease severity) and specific measures of patient PAP compliance. We performed predictive statistical analyses to determine how several different parameters affected the calculated TSI. Results: A linear regression was performed between BMI and TSI, which revealed a significant increase in treatment success secondary to increasing patient BMI (p=0.00002). In addition, patients were divided into three groups based on their length of treatment (LoTx), and a linear regression between the group average LoTx’s and their respective TSI’s revealed significant results (p=0.003). Conclusions: These findings present new insights into factors that best predict treatment efficacy for OSA and may assist in optimizing patient treatment. Future studies will expand the scope of the utility of TSI as a new measure of treatment efficacy for OSA.
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    Cytokine Responses to Cyclical Blood Flow Restriction Exercise
    (2018-03-14) Romero, Steven; Rickards, Caroline; Sprick, Justin
    Introduction: We developed a novel adaptation of blood flow restriction exercise, cyclical blood flow restriction exercise (C-BFRE), as a method to potentially augment the cardio-protective effects associated with conventional exercise (CE) and remote ischemic preconditioning (RIPC). As the cytokines IL-6 and IL-10 have been implicated in mediating the acute cardio-protection associated with both exercise training and RIPC when performed independently, we hypothesized that C-BFRE would augment the systemic release of these factors compared to an acute bout of either CE or RIPC alone. Methods: 13 young healthy subjects (6M/7F, age 28±2 y) completed 3 experimental sessions separated by at least 1 month each. Aerobic exercise was performed via 40-min of treadmill walking at 65-70% HRmax with (C-BFRE) and without (CE) application of bilateral thigh cuffs inflated to 220 mmHg (4 x 5-min inflation periods followed by 5-min reperfusion periods). RIPC consisted of 4 x 5-min thigh cuff inflation periods (220 mmHg) followed by 5-min reperfusion periods in the absence of exercise. Venous blood samples (via an antecubital vein) were collected at baseline and immediately following completion of each protocol for analysis of IL-6 and IL-10 via multiplex ELISA. Results: Plasma IL-6 increased from baseline following CE (Pre, 0.26 ± 0.03 vs. Post, 0.40 ± 0.07 pg/ml; P=0.03) but not with C-BFRE (Pre, 0.28 ± 0.05 vs. Post, 0.34 ± 0.07 pg/ml; P=0.75) or RIPC (Pre, 0.31 ± 0.04 vs. Post, 0.32 ± 0.03 pg/ml; P=1.0). There were no changes in plasma IL-10 from pre- to post-intervention for any of the three conditions (P=0.42 for main effect of time). Discussion: Contrary to our hypothesis, we observed no change in plasma IL-10 or IL-6 with application of acute C-BFRE. These findings suggest that the model of C-BFRE utilized in this study does not augment the release of these cytokines in young healthy individuals. This may be due to the timing of the blood samples, the use of a standard occlusive pressure across all subjects, and the young, healthy subjects tested in this study. Future work should delineate the inflammatory response induced by C-BFRE at more extended recovery time points, with use of individualized occlusive pressures, and in more clinically relevant populations in both acute and long term settings.
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    Oxazolone as a Model to Induce Edema in the Lower Limb of Rats
    (2018-03-14) Castillo, Rudy; Hodge, Lisa M.; Pastrana, Christopher
    Purpose: 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 treatments on edema, infection and inflammation. The aim of this research was to evaluate to the oxazolone (OXA)-induced acute skin inflammation model to induce lower limb edema in the rat. Specifically, we hypothesized that the application of OXA would induce create a local inflammatory response, induce edema in the lower limbs decrease lymphatic vessel function. Methods: Female Sprague Dawley rats, weighing 200-250 g, were used for this study. To induce edema, on day zero the right lower limb was shaved and 750 µl of 5% OXA-acetone solution or 5% PBS-acetone solution vehicle (VEH) was applied to the exposed skin. Lower limb measurements were made at days zero and six using a Vernier caliper. Measurements were taken at the midpoints of the hind paw, ankle, tibia and femur of their right lower limbs to establish paw thickness; distance between paw-to-ankle and ankle-to-tibia were taken to calculate lower limb volume by truncated cone formula. At day six, 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 washed. Cell pellets were stained with PE mouse anti-rat granulocytes, FITC anti-rat CD3, and APC anti-rat CD161 antibodies. The percentage of granulocytes, T cells and dendritic cells were measured by flow cytometry. Data were analyzed by ANOVA followed a Tukey post-test. Comparisons were made between OXA and VEH groups at day six post-induction. Results: OXA did not induce significant (p [greater than] 0.05) changes in either hind paw thickness or lower limb volume. OXA significantly (P Conclusions: OXA induced an acute inflammatory response in the draining inguinal lymph nodes. However, as used in this approach, OXA did not induce peripheral edema. In future studies we investigate alternate strategies to induce lower limb edema in the rat.
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    Free access to lard, sucrose, and chow results in expansion of rat periuterine adipose tissue.
    (2018-03-14) Goulopoulou, Styliani; Ahmed, Hijab
    Purpose: Adipose tissue expansion, a common feature of obesity, is associated with metabolic dysfunction, endocrine dysregulation, and adipokine imbalance. Adipocyte hypertrophy and hyperplasia contribute to adipose tissue expansion in a depot-specific manner. Our objective was to determine whether a high-fat, high-carbohydrate diet induces expansion of periuterine adipose tissue (PUT: adipose tissue surrounding the uterus). We hypothesized that free access to lard, sucrose, and chow results in hypertrophy and hyperplasia of adipocytes from rat PUT. Methods: Sixteen Sprague-Dawley female rats were divided into 2 weight-matched groups (n=8 rats/group) after 5 days of baseline measurements of food intake and body weight. One group was offered free access to chow, 30% sucrose solution, and lard (choice group) and the other group remained on chow (chow group) for 3 weeks. Energy intake and body weight were recorded daily. After euthanization, PUT was collected, weighed, and fixed in 4% paraformaldehyde. Samples were embedded in paraffin, sliced into 5 μm sections, and stained with hematoxylin and eosin. To determine hypertrophy, hyperplasia, and cell morphology, we used NIS Elements software to measure cross-sectional area/cell, number of cells/unit area, and adipocyte size distribution, respectively. Results: Total energy intake was greater in choice rats than chow rats (1590 ± 40.60 kcal/21 days vs. 1036 ± 19.00 kcal/21 days, p2/cell vs. 492.0 ± 27.76 μm2/cell, p=0.0001). The number of cells/unit area was smaller in PUT from choice rats compared to chow rats (13.9 x 10-5 ± 0.880 x 10-5 cells/μm2 vs. 20.4 x 10-5 ±0.910 x 10-5 cells/μm2, p=0.0001). PUT from choice rats had large adipocytes in greater frequency compared to PUT from chow rats. Conclusion: Obesity induced by free access to chow, lard, and sucrose resulted in hyperplasia and hypertrophy of adipocytes, and in cell size distribution changes in PUT of female rats of breeding age. Future studies will investigate if obesity-induced PUT expansion influences reproductive capacity and gestational outcomes.