Fawn-Hooded Research Articles

A new inbred strain of Fawn-Hooded rats demonstrates mania-like behavioural and monoaminergic abnormalities

The Fawn-Hooded (FH) rat carries a gene mutation that results in a dysfunctional serotoninergic system. However, previous studies have reported differing features between the FH/Wjd and FH/Har strains. We aimed to compare the behavioural and neurobiological features of FH/HamSlc rats with those of Fischer 344 rats. We performed the open field, elevated minus-maze, Y-maze spontaneous alternation, and forced swim tests to investigate behavioural alterations. We also assessed neurobiological characteristics by quantifying monoamines and their related compounds in the prefrontal cortex, hippocampus, and striatum using high-performance liquid chromatography with an electrochemical detection system. FH/HamSlc rats showed hyperactivity and a high impulsivity tendency in the open field and the elevated minus maze test, but no cognitive dysfunction. In addition, the hyperactivity was suppressed immediately after the forced swim test. FH/HamSlc rats showed low dopamine levels, but high dopamine turnover in the striatum. Serotonin and noradrenaline levels were low in the prefrontal cortex and the hippocampus of FH/HamSlc rats, but high serotonin turnover was observed in the prefrontal cortex, hippocampus, and striatum. FH/HamSlc rats show (1) mania-like behavioural characteristics that are different from those of other strains of FH rats; (2) stimulus dependent suppression of hyperactivity similar to the clinical findings that exercise alleviates the symptoms of bipolar disorder; and (3) monoaminergic dysregulation such as monoamine imbalance and hyperturnover that may be associated with mania-related behavioural characteristics. Thus, the FH/HamSlc rat is a new animal model for mania including bipolar disorder.

Sex Differences in the Progression of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of chronic renal disease. To explore the age‐related progression and sexual dimorphisms in the development of DN, we used a rat model of type 2 diabetic nephropathy (T2DN). The T2DN rat was previously created by introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive rat into the background of the Goto‐Kakizaki (GK; type 2 diabetic) rat. Type 2 diabetes in T2DN rats is accompanied by renal histologic abnormalities that are characteristic of DN, similar to clinical observations in human patients. The experimental groups were Wistar (non‐diabetic controls), GK, or T2DN rats at the early stages of DN (12 weeks old) or the late stages of the disease (>40 weeks of age). At the early stage of DN, male T2DN and GK rats showed significant increases in blood glucose (374 ± 96 and 329 ± 45 vs. 280 ± 24 mg/dL in Wistar, respectively) and kidneys to body weight ratio (10.05 ± 1.19 and 9.39 ± 0.78 vs. 7.31 ± 1.13 mg/g, respectively). In the late stage of the disease progression, T2DN rats had a significant increase in renal damage compared with GK and Wistar rats, indicated by progressive albuminuria, diuresis, a high prevalence of medullary protein cats, and glomerular lesions. Besides a striking DN phenotype in the T2DN model, we found significant sex differences in the development and progression of the disease. The male weight was double that of females (414 ± 27 vs. 264 ± 19 g, respectively), which correlates with a significant difference in postprandial blood glucose levels (435 ± 50 vs. 353 ± 46 mg/dL, respectively). To further evaluate changes in type 2 diabetes between the sexes, we performed the glucose tolerance test (GTT) in fasting animals. The male GTT indicates significant impairment in the body's response to sugar load compared to the female (155 ± 18 vs. 118 ± 5 at control and 378 ± 19 vs. 183 ± 48 mg/dL after oral glucose gavage; male vs female respectively). Blood tests also show elevated levels of serum alkaline phosphatase (158 ± 32 U/L) and cholesterol in males, similar to the clinical observations in patients with diabetes mellitus. Analysis of the renal damage between the sexes reveals significant increases in protein cast formation and glomerular abnormalities indicated by a high percentage of damaged glomeruli in males (28% vs. 4% in females). Aged male T2DN rats also had increases in nephrin shedding in their urine, which correlates with the dramatic differences in albuminuria. In T2DN males, there was an inverse correlation in serum levels (2.5 ± 0.2 vs. 3.3 ± 0.1 g/dL) and urinary excretion of albumin (11.8 ± 5.7 vs. 0.98 ± 0.64 Alb/Cre ratio) in comparison to females of similar age. Here we report that T2DN rat develops renal and physiological lesions to those seen in patients with type 2 diabetes, and there is a dramatic sex difference in the progression of renal damage.Support or Funding InformationNHLBI R35 HL135749 and P01 HL116264AHA 17SDG33660149 and 18PRE34030127ADA 1‐15‐BS‐172This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative effects of stressors on behavioral and neuroimmune responses of fawn-hooded (FH/Wjd) and Wistar rats: Implications for models of depression

Patients with depression and rodent models of depression show increased cytokines and activated microglia. Fawn Hooded (FH/Wjd) rats have long been used as a model of depression based on their depressive-like behaviors, high basal corticosterone levels and altered serotonergic levels, but little is known about the neuroimmune function in this model. To test whether depressive-like behaviors relate to dysfunction of the neuroimmune system, depressive-like behaviors in the forced swim test (FST) and corticosterone (CORT) response to the swim test were compared in male Fawn Hooded versus Wistar rats, and cytokine levels in plasma and brain and plasma CORT in response to lipopolysaccharide (LPS, an endotoxin that activates the neuroimmune system) or 1 h restraint were measured. Fawn Hooded rats had more depressive-like behaviors in the FST (decreased swim time and increased immobility) and increased overall plasma CORT compared with Wistar rats. Additionally, Fawn Hooded rats exhibited blunted brain and plasma cytokine response to LPS compared with Wistar rats, an effect that might be related to the blunted plasma CORT response to LPS. No strain differences were found on these measures in response to restraint stress. These results suggest that Fawn Hooded rats have a depressive-like phenotype potentially more closely associated with serotonin dysregulation and a dysregulated HPA axis and remain a relevant model for further defining the role of these systems in depressive conditions.

Abstract P124: Role of Add3 Dysfunction in Renal Epithelial Cells in the Development of Renal Injury in Fhh Rats

The Fawn Hooded Hypertensive Rat (FHH) is a genetic model of hypertension, in which Add3 has been identified as a candidate gene for renal injury but the mechanism is unknown. The present study examined the effects of knockdown of Add3 on rat renal epithelial (NRK) cells which are the model system for podocytes and proximal tubular cells. Knockdown of the expression of Add3 using a 27-mer Dicer-substrate RNAi (DsiRNA) decreased cell viability using an MTS assay by 50% in comparison to cells treated with vehicle or scrambled DSiRNA (Add3DsiRNA, OD 0.5±0.1; untreated cells 0.9±0.1; NC-1 0.9±0.1; n=8, P<0.001). F-actin immunostaining revealed that Add3 DSiRNA markedly disrupted the cytoskeleton, and the F-actin staining intensity/cell ratio was significantly reduced (untreated 543±47, n=552 cells; NC-1 592±39, n=566 cells; Add3DsiRNA 430±58, n=385 cells, P<0.05). The maximum mitochondrial respiratory rate was reduced in Add3 DsiRNA treated cells (untreated 444±42; NC-1 414±32; Add3 DsiRNA 275±16 pmole/min/μg, P<0.01). Proton leak was higher (untreated 42±3; NC-1 40±4; Add3 DsiRNA 63±5 pmole/min/μg, P<0.002) and oxygen consumption coupled to ATP production was decreased in Add3 DsiRNA treated cells (untreated 229±12, n=9; NC-1 200±12, n=8; Add3 DsiRNA 146±11 pmole/min/μg, n=9, P<0.001). Spare respiratory capacity, an index of cell fitness, was reduced by knockdown of Add3 (untreated 154±42, n=9: NC-1 143±32, n=8; Add3DsiRNA 33±16 pmole/min/μg, n=9, P<0.001). Non-mitochondrial oxygen consumption rate (OCR/μg protein) increased in cells treated with Add3 DsiRNA (untreated 57±7; NC-1 80±11; Add3DsiRNA 116±15 pmole/min/μg, P<0.01) suggesting a possible metabolic switch to glycolytic oxygen consumption. In conclusion, knockdown of Add3 in NRK cells disrupts the actin cytoskeleton, promotes mitochondrial dysfunction and decreases cell viability. Loss of Add3 function may contribute to proteinuria in FHH rats by altering the cytoskeleton and promoting effacement and loss of podocytes and the reuptake of filtered protein in the proximal tubule.

Dynamic Bayesian Graphical Modeling to Predict Regulatory Networks in Hypertensive Rats

We have previously used Bayesian graphical methods to detect networks of interacting genes relevant to the development of salt sensitive hypertension. However, these previous approaches are “static” or time‐insensitive methods that cannot infer causality to gene interactions. Here, we present a dynamic Bayesian graphical modeling approach which is used to connect potential target genes to other genes and networks.Dynamic Bayesian graphical networking approaches require large amounts of time‐series data, in addition to being computationally complex. Data was generated by isolating medullary thick ascending limb (mTAL) cells from 3 rat strains: the Dahl Salt‐sensitive rat (SS), the Fawn Hooded Hypertensive rat (FHH) and the Spontaneously Hypertensive Rat (SHR). After isolation, the freshly isolated mTAL cells were exposed to one of two stimuli: H2O2 (500 nM) or TNF‐α (50 ng/mL). RNA was then collected at 30 minute intervals for 8 hours, resulting in a 16‐point time‐series for each strain and stimulus. Each time‐series was duplicated, and duplicates were pooled to reduce overall measurement noise. As there are 6 combinations of strains (3) and stimuli (2) this procedure resulted in 96 individual time‐points. RNA was then isolated and sequenced on an Illumina Hi‐Seq 2500.The Dynamic Bayesian graphical model attempts to find connections between genes by looking at the sequence of activation/inactivation of genes across the entire 8 hour time‐series. For example, in a time‐series generated by treatment of SS mTAL cells with 500 nM H2O2, we find that the gene Dusp5 undergoes a 4‐fold increase in RNA expression one hour before a similar 4‐fold increase in the gene Rasgrp3, a GWAS gene for hypertension that has no known function in the mTAL. The Bayesian graphical modeling algorithm looks at such changes across each time‐series for the different rat strains and stimuli, and detects the most probable connections between the analyzed genes.This analysis represents the first attempt to create a dynamic Bayesian graph in non‐cancer mammalian cells. The use of such a graphical modeling approach in mTAL, a cell type of particular relevance to the pathophysiology of hypertension, will give us insight into the regulation of critical genes in hypertension and allow us to better understand, and potentially treat, this important disease.Support or Funding InformationA. Dayton supported by NIH Fellowship: 1F30HL127979‐01A1.Work funded by NIH grant: 4P01HL116264.

Elevated K+ channel activity opposes vasoconstrictor response to serotonin in cerebral arteries of the Fawn Hooded Hypertensive rat.

Previous studies suggest that middle cerebral arteries (MCAs) of Fawn Hooded Hypertensive (FHH) rats exhibit impaired myogenic response and introgression of a small region of Brown Norway chromosome 1 containing 15 genes restored the response in FHH.1BN congenic rat. The impaired myogenic response in FHH rats is associated with an increase in the activity of the large conductance potassium (BK) channel in vascular smooth muscle cells (VSMCs). The present study examined whether the increased BK channel function in FHH rat alters vasoconstrictor response to serotonin (5-HT). Basal myogenic tone and spontaneous myogenic response of the MCA was attenuated by about twofold and about fivefold, respectively in FHH compared with FHH.1BN rats. 5-HT (0.1 μM)-mediated vasoconstriction was about twofold lower, and inhibition of the BK channel increased the vasoconstrictor response by about threefold in FHH compared with FHH.1BN rats. 5-HT (3 μM) decreased BK channel and spontaneous transient outward currents in VSMCs isolated from FHH.1BN but had no effect in FHH rats. 5-HT significantly depolarized the membrane potential in MCAs of FHH.1BN than FHH rats. Blockade of the BK channel normalized 5-HT-induced depolarization in MCAs of FHH rats. The 5-HT-mediated increase in cytosolic calcium concentration was significantly reduced in plateau phase in the VSMCs of FHH relative to FHH.1BN rats. These findings suggest that sequence variants in the genes located in the small region of FHH rat chromosome 1 impairs 5-HT-mediated vasoconstriction by decreasing its ability to inhibit BK channel activity, depolarize the membrane and blunt the rise in cytosolic calcium concentration.

Gene-environment interactions in a rat model of depression. Maternal separation affects neurotensin in selected brain regions

Although the etiology of major psychiatric disorders has not been elucidated, accumulating evidence indicates that both genetic and early environmental factors play a role.We have previously demonstrated behavioral and neurochemical changes both in non-manipulated genetic rat models of depression, such as Flinders Sensitive Line (FSL) and Fawn Hooded (FH), and in normal rats following maternal separation (MS). The aim of the present study was to extend this work by exploring whether neurotensin (NT), a peptide implicated in several psychiatric disorders, is altered in a new animal model based on gene – environment interactions.More specifically, we used the FSL rats as a genetic model of depression and the Flinders Resistant Line (FRL) as controls and subjected them to MS. Pups randomly assigned to the MS procedure were separated from the dam as a litter for 180min daily between postnatal day 2 to 14. On postnatal day 90, rats were weighed and sacrificed by a two second high energy focused microwave irradiation and several brain regions were obtained by micropuncture. Neurotensin-like immunoreactivity (NT-LI) was measured by radioimmunoassay (RIA).The results showed that the FSL rats compared to the FRL rats have higher baseline NT-LI concentrations in the temporal cortex and periaqueductal gray and a markedly different response to maternal separation. The only observed change following maternal separation in the FRL rats was an NT-LI increase in the periaqueductal gray. In contrast, in the FSL significant increases were found in the nucleus accumbens, hippocampus, and entorhinal cortex and a decrease was seen in the temporal cortex after MS.The present study revealed baseline regional differences in NT-LI concentrations between the FSL and FRL strains and demonstrated that early MD differentially affects the two strains. The relevance of these alterations for depression as well as possible mechanisms underlying this gene-environment interaction are discussed.

Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food

Ghrelin, a peptide hormone produced by the stomach, is the endogenous ligand for the Growth Hormone Secretagogue Receptor (GHSR). Ghrelin acts on the GHSR to increase food intake, appetitive behaviors, and adiposity. Recently, a rat model with a null mutation to the GHSR gene (FHH-GHSRm1/Mcwi) was generated and used in behavioral studies, but the basic metabolic phenotype of this strain as well as that of the background strain (Fawn Hooded Hypertensive, FHH) has not been characterized in detail. Here we compared male FHH-GHSRm1/Mcwi rats with their wild-type littermates (FHH-WT) in a number of metabolic parameters. In the 24h of recovery following an acute overnight fast, FHH-GHSRm1/Mcwi rats consumed less food than FHH-WT animals, and relative to their body weights, adult FHH-GHSRm1/Mcwi rats consumed fewer calories when placed on a high-fat diet. Despite this, FHH-GHSRm1/Mcwi rats did not show a difference in diet-induced obesity or weight gain. Fasted FHH-GHSRm1/Mcwi rats exhibited increased Agouti-Related Peptide (AgRP) and Neuropeptide Y (NPY) expression in the Arcuate Nucleus (ARC), indicative of altered central regulation of feeding and energy balance. FHH-GHSRm1/Mcwi rats exhibited lower levels of home cage locomotor behavior over the entire light/dark cycle, and reduced levels of food anticipatory activity when placed on a restricted feeding schedule. Finally, FHH-GHSRm1/Mcwi rats consumed less of a palatable dessert (cookie dough) given after the completion of the scheduled meal. Altogether, our data show that rats lacking a functional GHSR tend to eat less than their wild-type counterparts in the face of acute fasts, chronic high-fat diet exposure, and exposure to a palatable dessert, despite not showing differences in body weight and glucose homeostasis that are characteristic of GHSR null mice. These data indicate that many, but not all responses to GHSR ablation are conserved between rats and mice. The FHH-GHSRm1/Mcwi rat thus represents a novel and useful model for studying GHSR function in rats.

Detection of TRPV4 channel current‐like activity in Fawn Hooded Hypertensive (FHH) rat cerebral arterial muscle

The FHH rat strain is associated impaired pressure‐dependent myogenic tone and exaggerated cerebral arterial muscle KCa channel current opening. In the present study we examined if the FHH rat cerebral arterial muscle cells exhibit TRPV4 channel current‐like activity. Western blot analysis and RT‐PCR revealed expression of protein and transcript for TRPV4 channel in cerebral arteries of FHH rat strain. Immunostaining using the TRPV4 channel antibody detected presence of TRPV4 channel protein in freshly isolated FHH rat cerebral arterial myocytes. Patch clamp recording of single‐channel current activity in FHH rat cerebral arterial myocytes identified presence of a non‐selective cation channel current with unitary conductances of ~96 pS and ~84.6 pS at depolarizing and hyperpolarizing potentials, respectively, that were sensitive to inhibition by the TRPV4 channel inhibitor RT‐1734 (5 μM) or HC 067074 (0.2 μM) and activated by the potent TRPV4 channel agonist GSK‐101690A (100 nM). These findings indicate that the FHH rat cerebral arterial myocytes appear to express TRPV4 channel current‐like activity that could have relevance for the regulation of calcium movement and calcium‐dependent mechanisms in these cells.Support or Funding InformationThis work is supported by grants from NIH (R01 NIH/NHLBI, HL033833 and HL105997)

Disruption In PKC‐mediated K Channel Regulation Contributes To Enhanced Serotonin Mediated Carotid Vessel Constriction from the Fawn Hooded Hypertensive Rat

Previous studies indicate Fawn Hooded Hypertensive (FHH) rats exhibit cerebral damage due to diminished cerebrovascular myogenic function associated with elevated BK channel function. Introgression of the 2.4 Mbp region of BN chromosome 1 into the FHH rat restored myogenic response and BK channel function in a FHH.1BN congenic rat. However, results obtained from resistance vessels cannot be extrapolated to conduit vessels due to structural and functional differences. Thus, the present study addresses the hypothesis that carotid vessels of FHH rats exhibit altered serotonin (5‐HT)‐mediated contraction compared to FHH.1BN rats. Using tension myography to measure conduit vascular function, we found that carotid vessels of FHH rats exhibited greater 5‐HT‐mediated contraction than FHH.1BN rats. Activation of BK channels (NS1619: 5 to 30μM) relaxed carotid vessels to a lesser extent (1.17 times less) in FHH rats. On the other hand, inhibition of BK channels (Paxilline or Iberiotoxin: 100nM) had no effect in either strain. Inhibition of voltage gated calcium channels (VGCC, Diltiazem: 10μM) or store operated calcium (SOC) channels (2‐APB: 75μM) relaxed carotid vessels similarly in both strains. Furthermore, PKC activation (PMA: 100nM) or inhibition (GF109203X: 3μM) increased and decreased 5‐HT‐mediated constriction respectively in FHH.1BN rats but had minimal effect in FHH rats. These results suggest that a mutation in the genes located in 2.4Mbp region in FHH rats contribute to a disruption in PKC and its regulation of K channels, causing enhanced 5‐HT‐mediated contraction.Support or Funding InformationThis research has received funding support from the American Heart Association, Scientist Development Grant (13SDG14000006) and the IOSP Undergraduate Summer Research Fellowship supported by the American Physiological Society and a grant from the National Science Foundation Integrative Organismal Systems Award No. IOS‐ 1238831.

Essential Involvement of Purinergic Signaling in Calcium Handling in the Podocytes of Type 2 Diabetic Rats

Purinergic signaling (initiated via interaction of ATP with P2 receptors) is one of the major triggers of intracellular calcium influx in the epithelial cells of the glomeruli ‐ podocytes. Excessive elevation of intracellular calcium concentration in these cells results in their loss, subsequent damage of the glomeruli filtration barrier and, eventually, proteinuria. Growing evidence suggests that the activation of extracellular ATP signaling may play a crucial role in the development of diabetic nephropathy. The goal of this study was to characterize purinergic signaling in the podocytes of Type 2 Diabetic (T2D) rats. Here we used 12 week old and aged (40–60 weeks old) Wistar (control), Goto‐Kakizaki (GK) and Type 2 Diabetic Nephropathy (T2DN) rats. The GK rat is a non‐obese Wistar substrain which develops T2D early in life. T2DN rat (created by an introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive (FHH) rat into the background of GK rat) is a model of T2D accompanied with renal histologic abnormalities characteristic for diabetic nephropathy (DN). Using real‐time confocal microscopy we measured acute effects of ATP on intracellular calcium dynamics in the podocytes of glomeruli freshly isolated from these rat strains. We have shown that basal intracellular calcium concentration was within normal range in Wistar and GK podocytes (150.1 ± 10.8 and 174.7 ± 29.7 nM, respectively), whereas in T2DN rats it was pathologically elevated up to 232.9 ± 20.1 nM. Effect of ATP in Wistar rat podocytes (in the range of 1 to 200 μM) was dose dependent and similar to what we previously reported for Sprague Dawley rats. However, ATP‐triggered calcium influx in GK rats was substantially enhanced with dose‐response curve shifted towards stronger activation of calcium influx with lower concentrations of ATP. Importantly, the response to ATP was further increased in T2DN compared to GK rat podocytes, and was found to be dose‐independent within the same ATP concentration range. The same phenomenon was observed in aged (approximately 12 month old) GK and T2DN animals. Furthermore, experiments in calcium‐free solutions and studies using alpha, beta‐methylene‐ATP revealed a major role of extracellular calcium influx as distinct from intracellular depot depletion, as well as a likely involvement of the ionotropic P2X receptors, or calcium channels activated by P2Y‐evoked metabotropic GPCR cascades. Western blotting revealed a significantly higher expression of P2X7 receptors in the cortex of T2DN and GK strains compared to Wistar rats, and a decrease in P2Y1 expression. Thus, our data indicate that in T2DN podocytes ATP‐triggered calcium influx is enhanced compared to control animals with even low concentrations of ATP causing activation of calcium flux and augmented intracellular calcium concentration, correspondingly. This mechanism might be an important determinant of podocyte injury resulting from pathological remodeling from metabotropic P2Y to ionotropic P2X receptors occurring in DN.Support or Funding InformationSupported by ASN Ben J. Lipps fellowship, ADA 1‐15‐BS‐172, NIH DK105160 and HL122662.

Abstract 616: Diminished K Channel Inhibition Impairs Serotonin-Mediated Vasoconstriction in the Middle Cerebral Arteries From the Fawn Hooded Hypertensive Rat

Fawn Hooded Hypertensive (FHH) rat exhibit impaired pressure-mediated myogenic response (MR) in the middle cerebral arteries (MCAs) that is associated with an increase in large conductance potassium (BK) channel function. Introgression of 2.4 Mbp region of BN (Brown Norway) chromosome 1 into FHH rats (FHH.1BN) restored MR and BK channel function. The present study assessed the hypothesis that FHH rats also exhibit impaired serotonin (5-HT)-mediated vasoconstriction due to diminished BK channel inhibition compared to FHH.1BN rats. We used pressure myography and patch-clamp to measure vessel response and K channel function respectively and Fluo 4 method to measure cytosolic calcium. Basal myogenic tone of MCAs as measured by change in diameter from 22oC to 37oC temperature was ~2.6 fold lower in FHH rats compared to FHH.1BN rats (FHH, 8.1 ± 2% and FHH.1BN, 21.7 ± 3%, n = 4; p<0.05). Vascular smooth muscle cells (VSMCs) of FHH rats have significantly more negative membrane potentials (Em) (–32 ± 1 mV; n=9) compared to FHH.1BN rats (-16 ± 2mV; n=10). 5-HT-mediated vasoconstriction was lower in MCAs isolated from FHH rats compared to FHH.1BN rats (5-HT: 1μM; FHH, 36 ± 5%; FHH.1BN rats, 58 ± 9%; n = 4; p<0.05). 5-HT-mediated BK channel inhibition was less in FHH rats (3μM 5-HT: FHH, 8 ± 3%; FHH.1BN, 39 ± 4%; n = 4; p<0.05). 5-HT did not affect VSMC membrane potential in FHH rats compared to FHH.1BN rats (delta change in Em: FHH, -2 ± 2mV; n = 4; FHH.1BN, -9 ± 1 mV; n = 5; p<0.05). 5-HT-mediated increase in calcium fluorescence (F/Fo) during plateau phase was blunted in the VSMCs isolated from FHH rats compared to FHH.1BN rats (FHH: 1 + 0.01% Vs 1.22 + 0.02% in FHH.1BN (3 rats)). Finally, inhibition of BK channel restored 5-HT-mediated vasoconstriction in MCAs of FHH rats but did not affect FHH.1BN vessels (5-HT + Paxilline; FHH, 2.6 ± 0.6 fold; FHH.1BN, 1.4 ± 0.4 fold; n = 5; p<0.05). In conclusion, mutation in the genes located in 2.4 Mbp region of FHH rats disrupts 5-HT-mediated BK channel inhibition that prevents the raise in [Ca2+]i and this may contribute to impaired 5HT-mediated vasoconstriction.

Bleeding Diathesis in Fawn Hooded Rats-Possible Implications for Invasive Procedures and Refinement Strategies.

Simple SummaryThe Fawn Hooded (FH) rat population is commonly used in biomedical research. It is widely acknowledged that the FH rat has a bleeding disorder, which may lead to abundant bleeding. However, the clinical consequences of this bleeding disorder have not been described in current literature. During our study, surgical procedures on FH rats led to an unanticipated loss of animals due to abundant bleeding. Adjustments made to minimize the impact of this disorder in animals undergoing invasive procedures are described. It is strongly recommended to take the bleeding diathesis into account when performing invasive procedures in FH rats and to apply the described refinements.The Fawn hooded (FH) rat is commonly used in biomedical research. It is widely acknowledged that the FH rat has a bleeding disorder; leading to abundant bleedings. Although this bleeding disorder is investigated to model the storage pool defect; its impact on commonly performed invasive laboratory procedures has not yet been described. Our research group experienced clinically significant consequences of this bleeding disorder following invasive procedures (including intraperitoneal injections and neurocranial surgery) in the Rjlbm: FH stock. The clinical consequences of the surgical and anesthetic protocols applied; are described including the subsequent procedural refinements applied to minimize the impact of this disorder. It is strongly recommended to take the bleeding diathesis into account when performing invasive procedures in FH rats and to apply the suggested refinement of procedures.

The Role of Nitric Oxide System in The Regulation of Renal Function During Prehypertensive and Hypertensive Period in Fawn‐Hooded Rat

Objective: Fawn-Hooded (FH) rat strain represents unique model of hypertension with renal failure. Normotensive FHL rats serve as a control whereas FHH develop early hypertension and exhibit increased susceptibility to renal disease due to impaired autoregulatory efficiency of the kidney. Aim of this study was to examine the influence of nitric oxide (NO) system in FHL and FHH rats in prehypertensive and hypertensive period. Design and method: 3 and 9 months old FHH and FHL rats were anesthetized, NO synthase inhibitor (L-NAME) were infused via jugular vein in dose 50 μg/kg of body weight and mean arterial pressure (MAP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UF) and sodium excretion responses were measured. Results: FHH rats exhibited significantly higher basal MAP compare to FHL in 3 month (123 ± 2,9 vs. 101 ± 2,5 mmHg) and also in 9 month (141 ± 2,1 vs. 102 ± 2,4 mmHg). Young FHH had higher basal RBF than FHL (6,5 ± 0,2 vs. 5,4 ± 0,5 ml/min/g) although older FHH rats had slightly lower RBF compare to FHL (3,7 ± 0,5 vs. 4,9 ± 0,5 ml/min/g) and also GFR (0,47 ± 0,07 vs. 0,66 ± 0,09 ml/min/g). L-NAME administration led to similar responses in both ages in MAP, RBF and GFR but hypertensive FHH had significantly lower increase in UF (from 3,7 ± 0,4 to 13,6 ± 2,5 µl/min/g) compare to FHL (from 4,4 ± 0,8 to 29,7 ± 2,6 µl/min/g) and also in sodium excretion (from 0,19 ± 0,07 to 1,57 ± 0,54 µmol/min/g) than in FHL (from 0,14 ± 0,08 to 3,77 ± 0,57 µmol/min/g). Conclusions: This study indicates that FHH rats with developed hypertension exhibit impaired renal hemodynamics and tubular function that may be caused by relative NO deficiency in the kidney.

Enhanced Serotonin (5‐HT)‐Mediated Carotid Vessel Constriction From The Fawn Hooded Hypertensive (FHH) Rat

Impaired myogenic response associated with an increase in BK (large conductance potassium) channel function was suggested in cerebral resistance vessels of FHH rats. Introgression of BN (Brown Norway) 2.4 Mbp region of chromosome 1 into FHH rats restored the function of these vessels by normalizing BK channel function in an FHH.1BN congenic rat. However, conclusions derived from resistance vessels cannot be extrapolated to conduit vessels such as the carotid or aorta since they differ in structure, function, and expression of contractile proteins. Here we tested agonist‐mediated contraction and the regulatory role of PKC in mediating K channel function using isometric measurement of tension in carotid vessels isolated from FHH and FHH.1BN rats. Serotonin (5‐HT)‐mediated tension is enhanced in carotid rings isolated from FHH rats when compared to FHH.1BN rats. 5‐HT‐mediated constriction is enhanced further more by inhibition of Kv channel (4AP; 1mM) but not BK channel (Paxilline or Iberiotoxin; 100nM) in FHH rats. 5‐HT‐mediated constriction is not affected by PKC activation (PMA; 100nM) in FHH rats but significantly enhanced in FHH.1BN rats. While inhibition of PKC (GF 109203X, 3µM) diminished 5‐HT‐mediated contraction in both strains it has significantly less effect in FHH rats compared to FHH.1BN rats. Together, these results suggest that mutation in the genes located in 2.4 Mbp region of FHH rat may enhance PKC function that inhibit K channels contributing to augmented 5‐HT‐mediated carotid vessel constriction. This research has received partial funding support from the American Heart Association, Scientist Development Grant (13SDG14000006).

Cardiac mRNA Expression in Inbred Rat Strains Divergent in Survival Time after Hemorrhage

We previously identified inbred rat strains differing in survival time after severe controlled hemorrhage (StaH). To identify early biomarkers of hemorrhagic shock and genes that might explain differences in STaH, rats from three strains [Brown Norway (BN); Dark Agouti (DA), and Fawn Hooded Hypertensive (FHH)] with different StaH (DA = FHH > BN; 4-5 rats/group) were assigned to one of three treatment groups: non-operated controls (CNTL), surgically catheterized rats (CATH), or rats catheterized and bled (~46% hemorrhage) 24 h post-surgery (BLD). Rats were euthanized 30 min after handling or 30 min after start of a 26 min bleed. After euthanasia, hearts were removed and frozen in liquid nitrogen. Total RNA was isolated, fluorescently labeled, hybridized to Agilent rat whole genome microarrays, and statistically analyzed with BRB Array Tools. After controlling for multiple comparisons via the False Discovery Rate (<10%), mRNA expression for 65 known genes differed (P<.01). There were no inbred rat strain-related differences in mRNA expression (P>.05). Expression of 26 genes changed after BLD (20 decreased, 6 increased). These changes ranged from 5 to 556-fold. Genes with hemorrhage-associated changes included: Hif1α (22-fold decrease; oxygen homeostasis); Map4 (45-fold decrease; mitochondrial permeabilization); Mmp21 (24-fold decrease; Ca++ binding); Tubb5 (556-fold decrease; phagocytosis and protein complex binding); and Rnase10 (70-fold increase; nucleic acid binding). In the absence of inbred rat strain differences in mRNA expression, our data do not identify cardiac genes associated with differences in STaH. However, the data do provide new information concerning cardiac mRNA expression that rapidly changes after hemorrhage. Funding: US Army MRMC.

Tacrolimus-induced hypertension and nephrotoxicity in Fawn-Hooded rats are attenuated by dual inhibition of renin–angiotensin system

Chronic immunosuppressive therapy is often complicated by the development of both arterial hypertension and renal dysfunction. The principal aim of this study was to assess the effects of dual inhibition of renin-angiotensin system (RAS) and other antihypertensive treatment on blood pressure and renal function in normotensive and hypertensive Fawn-Hooded (FH) strains during chronic calcineurin inhibitor (CNI) administration. Combinations of perindopril (5 mg kg(-1) per day) and losartan (50 mg kg(-1) per day) or amlodipine (6 mg kg(-1) per day) and metoprolol (80 mg kg(-1) per day) were administered to normotensive (FHL) and hypertensive (FHH) rats, fed with diet containing tacrolimus (Tac; 12 mg kg(-1) per day). Tac-induced arterial hypertension in both animal strains (FHL: 151±4; FHH: 198±6 mm Hg) was prevented by dual RAS inhibition (FHL: 132±3 mm Hg, P<0.05; FHH: 153±3 mm Hg, P<0.05) as well as by a combination of amlodipine and metoprolol (FHL: 136±3 mm Hg, P<0.05; FHH: 166±4 mm Hg, P<0.05). However, significant nephroprotection was observed only in animals on dual RAS inhibition where albuminuria was reduced in both FHL (51.1±3.9 vs. 68.3±4.5 μg per day; P<0.05) and FHH rats (13.1±0.3 vs. 18.8±0.7 mg per day; P<0.05). We also found Tac-induced enhancement in renal angiotensin II activity that was significantly reduced by dual RAS inhibition in both FHL (63.5±3.2 vs. 23.1±3.0 fmol g(-1)) and FHH (79.8±8.5 vs. 32.2±5.8 fmol g(-1)). In addition, histological analysis revealed that RAS inhibition noticeably diminished glomerulosclerosis and tubulo-interstitial injury. This study indicates that dual blockade of RAS significantly attenuates Tac-induced arterial hypertension and nephrotoxicity in FH rats and further supports the notion that RAS inhibitors display efficient renoprotective properties during CNI treatment.

Integration of background genome variation to dissect complex cardiovascular phenotypes (697.5)

The genetic underpinnings of cardiovascular (CV) disease are far more complex than originally imagined. Human genome wide association studies (GWAS) have identified candidate genes, but to date have predicted only a small fraction of the genetic basis. Some of the “missing heritability” is likely due to the interaction of multiple genes and alleles contributing to the disease. Phenotypic and genotypic characterization of two complete comsomic rat panels provides the foundation to analyze the influence of genetic background on CV traits. Phenotyping of PhysGen consomic strains, developed by introgressing Brown Norway (BN) chromosomes onto the Salt‐Sensitive (SS) or Fawn Hooded Hypertensive (FHH) backgrounds, identified consomic strains in introgressed BN chromosomes alter phenotypes on only one genomic background or under only one environmental condition. Introgression of BN chromosome 1 differentially regulates left ventricle infarct size following ischemia on SS and FHH backgrounds. HIF pathway genes on chromosome 1 are nearly identical between SS and FHH, although 12 genes on background chromosomes contain non‐synonymous SNPs (2 in SS only, 6 in FHH only, and 4 in both compared to BN), potentially altering the HIF pathway and CV phenotypes in response to chronic hypoxia. Additional in silico analysis, using gene, QTL and phenotype annotations, in conjunction with strain‐specific sequence variation, enables virtual variant profiles for complex phenotypes or disease pathways to be created for each consomic strain.Grant Funding Source: Supported by R01HL094271 and R01HL064541

Enhanced large conductance K+channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1(BN) congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1(BN) congenic strain. Inside-out patches indicated that the BK channel open probability (NPo) is 10-fold higher and IBTX reduced NPo to a greater extent in VSMCs isolated from FHH than in FHH.1(BN) rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1(BN) rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1(BN) rats. However, the expression of the BK-α and -β-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity.

Nociception and conditioned fear in rats: strains matter.

When using rats in pain research, strain-related differences in outcomes of tests for pain and nociception are acknowledged. However, very little is known about the specific characteristics of these strain differences. In this study four phylogenetically distant inbred rat strains, i.e. Wistar Kyoto (WKY), Fawn Hooded (FH), Brown Norway (BN) and Lewis (LE), were investigated in different tests related to pain and nociception. During Pavlovian fear conditioning, the LE and WKY showed a significantly longer duration of freezing behaviour than the FH and BN. Additionally, differences in c-Fos expression in subregions of the prefrontal cortex and amygdala between rat strains during retrieval and expression of conditioned fear were found. For example, the BN did not show recruitment of the basolateral amygdala, whereas the WKY, FH and LE did. During the hot plate test, the WKY and LE showed a lower thermal threshold compared to the BN and FH. In a follow-up experiment, the two most contrasting strains regarding behaviour during the hot plate test and Pavlovian fear conditioning (i.e. FH and WKY) were selected and the hot plate test, Von Frey test and somatosensory-evoked potential (SEP) were investigated. During the Von Frey test, the WKY showed a lower mechanical threshold compared to the FH. When measuring the SEP, the FH appeared to be less reactive to increasing stimulus intensities when considering both peak amplitudes and latencies. Altogether, the combined results indicate various differences between rat strains in Pavlovian fear conditioning, nociception related behaviours and nociceptive processing. These findings demonstrate the necessity of using multiple rat strains when using tests including noxious stimuli and suggest that the choice of rat strains should be considered. When selecting a strain for a particular study it should be considered how this strain behaves during the tests used in that study.