Fawn-Hooded Research Articles

Derivation and Genetic Modification of Embryonic Stem Cells from Disease-Model Inbred Rat Strains

The lack of rat embryonic stem cells (ESCs) and approaches for manipulation of their genomes have restricted the ability to create new genetic models and to explore the function of a single gene in complex diseases in the laboratory rat. The recent breakthrough in isolating germline-competent ESCs from rat and subsequent demonstration of gene knockout has propelled the field forward, but such tools do not yet exist for many disease-model rat strains. Here we derive new ESCs from several commonly used rat models including the Dahl Salt Sensitive (SS), the sequenced Brown Norway (BN), and Fischer (F344) rat and establish the first germline-competent ESCs from a hypertension disease model strain, the Fawn Hooded Hypertensive (FHH) rat. Genetic manipulations including transgenesis mediated by lentivirus, routine homologous recombination, and homologous recombination mediated by zinc-finger nucleases (ZFNs) were performed effectively in FHH rat ESCs. Our results showed these rat ESC lines, isolated from inner cell masses using mechanical splitting, had germline competency; the Pparg gene locus and homologous genomic region to the mouse Rosa26 locus can be targeted effectively in these rat ESCs. Furthermore, our results also demonstrated that ZFNs increased the efficiency of proper homologous recombination in FHH rat ESCs using targeting vectors with short homology arms. These rat ESC lines and advancements in genetic manipulation pave the way to novel genetic approaches in this valuable biomedical model species and for exploration of complex disease in these strains.

Cardiac CARD9 mRNA expression in inbred rat strains divergent in survival time after severe hemorrhage (STaH)

We identified 3‐fold differences among inbred rat strains in STaH (Physiol Genomics 43: 758, 2011). We hypothesized that a protein of the caspase recruitment domain (CARD9) might be a specific cellular mediator associated with these differences. CARD9 is a known activator of nuclear factor κβ (JBC 275: 41082, 2000), a transcription factor that mediates both protective and detrimental effects on myocardial function (Clin Sci 118: 593, 2010). To test this hypothesis, we measured CARD9 mRNA levels using real‐time PCR in cardiac tissue in three inbred rat strains [Dark Agouti (DA), Fawn Hooded Hypertensive (FHH), and Brown Norway (BN)] divergent in STaH (DA = FHH >; BN). Rats from each strain were randomized to three treatment groups (n=5/group): controls without surgery (C); surgically catheterized rats (Cath); and catheterized and hemorrhaged rats (Bled). Surgeries were conducted ~ 24 hr prior to hemorrhage and tissue collections. Tissues were collected 30 min after start of a 26 min ~47% hemorrhage, or 30 min post handling in C and Cath rats. CARD9 mRNA was increased 3 to 25‐fold in DA vs BN or FHH rats (P < 0.01). Hemorrhage had no effect on CARD9 mRNA in any strain, but in DA CARD9 mRNA decreased in Cath vs C rats (P<0.05). These data indicate inbred rat strain‐dependent differences in cardiac CARD9 mRNA. High CARD9 mRNA in DA rats could influence cardiac responses to hemorrhage although not altered by hemorrhage itself.

Cardiac mitochondrial proteomic expression in inbred rat strains divergent in survival time after hemorrhage

We have previously identified inbred rat strains differing in survival time to a severe controlled hemorrhage (StaH). In efforts to identify cellular mechanisms and ultimately genes that are important contributors to enhanced STaH, we conducted a study to characterize potential differences in cardiac mitochondrial proteins in these rats. Inbred rats from three strains [Brown Norway/Medical College of Wisconsin (BN); Dark Agouti (DA), and Fawn Hooded Hypertensive (FHH)] with different StaH (DA = FHH > BN) were assigned to one of three treatment groups (n = 4/strain): nonoperated controls, surgically catheterized rats, or rats surgically catheterized and hemorrhaged 24 h postsurgery. Rats were euthanized 30 min after handling or 30 min after initiation of a 26 min hemorrhage. After euthanasia, hearts were removed and mitochondria isolated. Differential protein expression was determined using 2D DIGE-based Quantitative Intact Proteomics and proteins identified by MALDI/TOF mass spectrometry. Hundreds of proteins (791) differed among inbred rat strains (P ≤ 0.038), and of these 81 were identified. Thirty-eight were unique proteins and 43 were apparent isoforms. For DA rats (longest STaH), 36 proteins increased and 30 decreased compared with BN (shortest STaH). These 81 proteins were associated with lipid (e.g., acyl CoA dehydrogenase) and carbohydrate (e.g., fumarase) metabolism, oxidative phosphorylation (e.g., ubiquinol-cytochrome C reductase), ATP synthesis (F1 ATPase), and H2S synthesis (3-mercaptopyruvate sulfurtransferase). Although we cannot make associations between these identified mitochondrial proteins and StaH, our data do provide evidence for future candidate proteins with which to consider such associations.

Abstract 127: Identification of a Region of Rat Chromosome 1 That Impairs the Myogenic Response and Cerebral Blood Flow Autoregulation in Fawn Hooded Hypertensive Rats.

This study examined the effect of transfer of a 2.4 Mbp region of Brown Norway (BN) rat Chr 1 into the Fawn Hooded Hypertensive (FHH) genetic background on autoregulation (AR) of cerebral blood flow (CBF) and the myogenic response of isolated middle cerebral arteries (MCA). Autoregulation of CBF measured by laser Doppler flowmetry was poor in FHH rats (AR index (AI), 0.8±0.1) and in FHH.1BN congenic strains which excluded the critical region (AR-: AI, 0.9± 0.1). In contrast, autoregulation of CBF was completely restored by transfer of the region of BN Chr 1 between 258.8 to 261.2 Mbp in AR+ FHH.1BN congenic strains (AI, 0.3 ± 0.1). The diameter of MCA of FHH rats and AR- congenic strains increased by ∼10% (140 ± 1 to 157± 2 μm), when transmural pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA in AR+ congenic strain fell from 127 ± 2 to 65 ± 1 μm. Whole-cell patch-clamp of cerebral VSM cells revealed a 4.3-fold increase in BK channel current densities at depolarized potentials in FHH versus AR+ rats (105 ± 2 versus 24 ± 4 pA/pF at +80mV). Using single channel analysis we found that the increase in BK channel current was largely due to a marked increase in the NPo of BK channel in FHH as compared to AR+ rats (0.9 ± 0.1 and 0.2 ± 0.1 at +80mV). To explore the significance of the impaired myogenic response, we compared changes in CBF and infarct size following transient occlusion and reperfusion of the MCA in FHH rats and the AR- and AR+ congenic strains. Occlusion of MCA reduced CBF similarly in all the strains. However, the hyperemic response following reperfusion in FHH and AR- strains was significantly greater and more prolonged than that seen in AR+ rats (AR-: 173 ± 1%, 45 min versus AR+: 124 ± 5%, 15 min). Moreover, infarct size and edema formation was significantly greater in the AR- congenic strain (39 ± 3 % and 12 ± 2 %) in comparison to that seen in the AR+ strain (28 ± 2 %; 7 ± 1%). These results indicate that there is a gene that plays a critical role in the regulation of the myogenic response of the cerebral vasculature by altering BK channel activity in the critical 2.4 Mb region of Chr 1 containing just 15 genes and that transfer of this region from BN to FHH rats restores autoregulation of CBF, vascular reactivity and reduces infarct size following ischemia/reperfusion injury.

Abstract 353: Rapid Gene Editing in Rat Models of Human Disease Using Targeted Nucleases

Previous investigations into mechanisms of genes in animal models have primarily used the mouse because of the availability of technologies for targeted manipulation of its genome in embryonic stem (ES) cells. To accelerate functional mechanistic studies in other species where ES technology is not yet widespread, we recently developed an alternative and rapid method for generating targeted mutations in rat genes by the application of Zinc Finger Nucleases (ZFNs). When introduced into an embryo, ZFNs target and induce a chromosome double strand DNA break at the investigator-specified locus, stimulating cellular responses which can result in knock out or knock in modifications at the locus. Using commercially available reagents, we targeted and disrupted more than 100 protein-coding genes across 13 different inbred, consomic, and outbred genetic backgrounds with a 97% (102/105) success rate in approximately 30 months. ZFNs can therefore access nearly every gene in the rat genome and can induce multiple different alleles at the target locus. The majority of mutations, 81% (355/437) were simple deletions ranging from 1-228 base pairs (median 13-bp) and upon breeding, 94% (178/189) of mutations were transmitted to the next generation with little evidence of germline mosaicism. Many of these strains are now available for functional mechanistic studies of candidate human disease genes for hypertension and chronic kidney disease. Co-introduction of a homologous gene template with the ZFNs could stimulate knockin at three different loci in the rat genome. Gene knock into the rat Rosa26 locus allows for uniform expression of a gene in all cells and tissues of the adult animal as well as germline-competent ES cells derived from the Fawn Hooded Hypertensive strain. This knockin technology in embryos and ES cells offers many new opportunities to make very precise modifications to the rat genome and is a key step toward creating conditional gene alleles in the rat. Transcriptional activator-like effector nucleases (TALENs) are also active in the rat embryo and we have disrupted 70% (7/10) genes with this new technology. In sum, targeted nuclease technology is enabling many new genetic approaches to the rat and other model systems to create even better human disease models.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

Novel Medication Targets for the Treatment of Alcoholism: Preclinical Studies

Alcoholism is a complex heterogeneous disease and a number of neurotransmitter and neuromodulator systems have been implicated in its manifestation. Consequently, it is unlikely that existing medications such as disulfiram (Antabuse®), naltrexone (ReVia®), acamprosate (Campral®)) can be efficacious in every individual. Thus, the development of novel therapeutic agents with greater selectivity and less unwanted effects for the treatment of this disease is one of the major objectives of alcohol research. This review summarizes the findings of five novel compounds with different neuronal targets for treating alcoholism. These compounds include sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors; carisbamate, a novel anti-epileptic agent; JNJ5234801, a novel anxiolytic agent; GS-455534, a highly selective inhibitor of mitochondrial aldehyde dehydrogenase; and JNJ-39220675, a selective histamine H3 antagonist. Inbred alcohol-preferring rats (iP), Fawn-Hooded (FH) rats, and P rats were used to evaluate the compounds. Naltrexone was used as a positive control in some experiments. All five compounds reduced alcohol consumption and preference. The mechanisms thought to underlie these effects suggest that, in addition to dopaminergic and opioidergic systems, other neuronal systems such as sodium channels (carisbamate), mitochondrial aldehyde dehydrogenase (GS-455534), 5-HT2 receptors (JNJ-5234801), histamine H3 receptors (JNJ-39220675), and α4β2 nicotinic receptors (sazetidine-A) can be involved in alcohol drinking. Further work is necessary to confirm the exact mechanisms of action of each drug and to determine any viable targets for putative treatment of alcohol-use disorders. The article presents some promising patents on novel medication targets for the treatment of alcoholism.

Dissection of the Renal Failure‐3 QTL

Rf‐3, a quantitative trait locus (QTL) on the rat chromosome 3, affects renal disease susceptibility in the FHH (Fawn Hooded Hypertensive) rat. Synergistic interaction between Rf‐3 and other loci such as Rf‐1 had shown to be important in the disease development. Therefore, we used a triple congenic strain, in which Rf‐1, Rf‐3, and Rf‐4 were introgressed onto the genetic background of the renal disease resistant strain ACI (August Copenhagen Irish), to elucidate the genetic control of Rf‐3. The aim of this study was to reduce the Rf‐3 congenic region and identify the disease‐causing gene(s). By developing set of congenic strains we reduced the Rf‐3 region from 110 Mbp to 3.2 Mbp. The congenic strain carrying the reduced Rf‐3 region showed 2 and 3.5‐fold increase in proteinuria and albuminuria, respectively, compared to the control group. Kidney histology also revealed significantly increased protein casting and glomerular sclerosis in the minimal congenic strain. Sequence analysis of the congenic region revealed 2995 sequence variants between the FHH and ACI. We prioritized the variants based on sequence conservation across species and their location. Our analysis found 42 variants in 13 different genes. Two of them, Defb28 and Bcl2l1, are present in the syntenic mouse and human renal function QTL, which makes them potential candidates for future study. Research support: NHLBI‐5R01HL069321 to H.J.J.

Liver heme oxygenase‐1 (HO‐1) protein expression in inbred rat strains divergent in survival to severe hemorrhage

We have previously identified inbred rat strains that are different in survival time to severe controlled hemorrhage (Brown Norway/Medical College of Wisconsin < Fawn Hooded Hypertensive = Dark Agouti; Klemcke et al., Physiol Genomics 43: 758, 2011). In efforts to identify cellular mechanisms associated with enhanced survival, we measured protein expression of liver HO‐1, an inducible, cytoprotective, anti‐inflammatory, and anti‐apoptotic protein. Inbred rats from these three strains were randomly assigned to one of three treatment groups (n = 4– 5/group): non‐operated controls, surgically catheterized rats, or catheterized rats bled (~ 46% of blood volume) 24 h post‐surgery. Rats were euthanized 30 min after handling or after initiation of a 26 min hemorrhage. Western blot analyses were conducted using a primary antibody specific for HO‐1. β‐actin, a “housekeeping” protein that was unaffected by strain or treatment, was used for normalization of HO‐1 expression. At the time intervals measured, liver HO‐1 protein expression did not differ among inbred rat strains (P = 0.51), but in all strains increased 28% (P = 0.02) after surgery with no further changes after hemorrhage. Such data suggest liver HO‐1 may be involved in cytoprotective effects after the stressor of surgery, but do not provide evidence for a strain‐dependent involvement in liver function post hemorrhage that might affect differential survival.

Abstract 3142: Exaggerated K Ca Channel Current Activity Accounts for Reduced Myogenic Constriction in the Cerebral Vasculature of the Fawn Hooded Hypertensive (FHH) rats

The ionic mechanism by which pressure induces myogenic arterial constriction is not completely understood. In the present study we compared the Fawn Hooded Hypertensive (FHH) rat strain bred that does not autoregulate CBF and the FHH.1 BN rat strain created by transfer of a 2.6 Mb region of a Brown Norway (BN) rat chromosome 1 and introgressed into the FHH genetic background and recovered the myogenic phenotype, with respect to K Ca channel activity and pressure-induced myogenic constriction. We investigated the functional role of Ca 2+- activated K + channel in the impaired pressure-induced myogenic response in cannulated and pressurized middle cerebral arterial segments of FHH and compared with that of the FHH.1 BN rat strain. The cannulated arterial segments of FHH rat strain elicited significantly reduced pressure-induced myogenic constriction, whereas those of the FHH.1 BN exhibited more than 40-50 % constriction or reduction in diameter during step increases in intravascular pressure from 40 to 140 mmHg. Pretreatment of the he FHH rat cerebral arterial segments with the K Ca channel inhibitor iberiotoxin (100 nM) rendered the FHH rat cannulated arterial segments elicit near equal level of myogenic constriction as that of the FHH.1 BN . The magnitude and density (pA/pF) of a macroscopic whole-cell K Ca channel current recorded from FHH and FHH.1 BN rat cerebral arterial muscle cells during a 10 mV step depolarization from -70 mV to +80 mV was 2- to 3-fold greater in the FHH than that in the FHH.1 BN rat cerebral arterial muscle cells. Similarly, the NPo of the K Ca single-channel currents recorded at +40 mV using symmetrical KCl solution was significantly higher in the FHH (0.0035 ± 0.0002) than in the FHH.1BN (0.0013 ± 0.0003, n =5), albeit the unitary conductances were not different. These findings indicate that exaggerated K Ca channel current activity in the FHH rat cerebral arterial muscle cell membrane could account for the impaired pressure-induced myogenic constriction in the FHH rat strain and may have implications for stroke susceptibility in the FHH rat model.

Genetic influences on survival time after severe hemorrhage in inbred rat strains

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Genetic basis of altered myogenic response and renal injury in FHH rats

This study examined the effect of substitution of a 2.6Mb segment from Brown Norway (BN) rat Chr 1 into the Fawn Hooded Hypertensive (FHH) rat genetic background on autoregulation of renal blood flow (RBF) and development of renal injury. FHH rats exhibited poor autoregulation of RBF and GFR rose by 60% when renal perfusion pressure (RPP) was increased from 100 to 150 mmHg while the FHH.1BN congenic strain exhibited perfect autoregulation of RBF. Protein excretion rose from 51±5 to 259±41 mg/day in 9 to 21 week FHH rats but only increased to 147±24 mg/day in the FHH.1BN congenic strain. Diameter of isolated perfused afferent arterioles from FHH rats did not change significantly when transmural pressure was increased from 60 to 140 mmHg. In contrast, diameter of the congenic decreased 19.4% with the same pressure increase. Patch clamping experiments indicate that large conductance Calcium activated K+ channel (BK) current was 5 fold greater in vascular smooth muscle (VSM) cells isolated from renal vessels of FHH rats than the congenic strain. This data indicates that the gene responsible for restoring autoregulation of RBF lies within this 2.6 Mb region containing 11 genes and transfer of this region restores the myogenic response and slows the progression of renal disease. The impaired myogenic response is likely due to hyperpolarization of VSM cells due to increased BK channel activity. NIH HL36279, DK079306 and HL69321.

Genetic basis of impaired myogenic response and cerebral blood flow autoregulation FHH rats

This study examined the effect of transfer of a small region from chromosome 1 (Chr 1) of the Brown Norway (BN) rat (FHH.1BN AR+ strain) into the Fawn Hooded Hypertensive (FHH) rat genetic background on autoregulation of cerebral blood flow (CBF) and the myogenic response of isolated perfused middle cerebral arteries (MCA). FHH rats exhibited poor autoregulation measured by laser Doppler flowmetry and CBF rose by 50% when perfusion pressure was increased from 100 to 150 mmHg. Autoregulation of CBF was completely restored in the AR+ strain. The diameter of isolated middle cerebral artery (MCA) of FHH rats increased by 10% when transmural pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA fell from 127±16 to 65±14 μm in the AR+ strain when pressure was increased over this range. FHH rats exhibited a much more prolonged hyperemia and larger infarct than that seen in the AR+ strain following ischemia/reperfusion. These results indicate that a gene that plays a critical role in the myogenic response of cerebral arteries lies within this critical 2.6 Mb region containing just 11 genes and that transfer of this region restores autoregulation of CBF and reduces infarct size following ischemia/reperfusion injury. The impaired myogenic response is likely due to hyperpolarization of VSM cells due to increased BK channel activity. NIH HL36279, DK79306, HL59996.

Diabetic rats are unable to elicit cardioprotection by isoflurane: role of mitochondrial dysfunction

We investigated whether differences in mitochondrial genome play a role in anesthetic-induced preconditioning (APC) in the diabetic heart model with “swapped” mitochondria. These rats have identical nuclear genome, but some bear Wistar (T2DNmtWistar) while others bear Fawn Hooded Hypertensive (T2DNmtFHH) mitochondrial genome. The production of reactive oxygen species (ROS) and changes in flavoproteins (FPs) fluorescence were monitored from ventricular myocytes in the presence of 1 MAC isoflurane by laser scanning confocal microscopy. Opening of mitochondrial permeability transition pore (mPTP) was detected by tetramethylrhodamine dye. During isoflurane, ROS were increased by 165% and 107% in T2DNmtFHH and T2DNmtWistar cardiomyocytes, respectively. In addition, isoflurane induced oxidation of mitochondrial FPs in T2DNmtFHH by 94% and by 35% in T2DNmtWistar myocytes. Compared to respective controls, APC increased the mPTP opening time in T2DNmtWistar but not in T2DNmtFHH myocytes. Our findings suggest that mitochondrial genome-related differences in the function of respiratory chain complexes have an impact on anesthetic sensitivity of the mitochondria. This in part could explain that APC protection against mPTP opening is compromised in T2DNmtFHH but not in T2DNmtWistar cardiomyocytes. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

Vascular responses in aortic rings of a consomic rat panel derived from the Fawn Hooded Hypertensive strain

The present experiments, utilizing the high-throughput vascular protocol of PhysGen (Program for Genomic Applications) characterized the responses of aortic rings to vasoconstrictor (phenylephrine) and vasodilator (acetylcholine, sodium nitroprusside, and reduced tissue bath Po(2)) stimuli in consomic rat strains derived from a cross between the Fawn Hooded Hypertensive rat (FHH/EurMcwi) and the Brown Norway normotensive (BN/NHsdMcwi) rat. The effects of substituting individual BN chromosomes into the FHH genetic background were determined in animals that were maintained on a low-salt (0.4% NaCl) diet or switched to a high-salt (4% NaCl) diet for 3 wk. Sex-specific differences were evaluated in male and female consomic rats on similar dietary salt intake. Multiple chromosomes affected various vascular reactivity phenotypes in the FHH × BN consomic panel, and substantial salt-dependent changes in vascular reactivity and sex-specific differences in aortic reactivity were observed in individual consomic strains. However, compared with earlier studies of consomic rats derived from a cross between the BN rat and the Dahl salt-sensitive (SS) rat, only 3-7% of the vascular phenotypes were affected in a similar manner by substituting specific BN chromosomeschromosomes into the FHH genetic background versus the SS genetic background. The findings of the present study stress the potential value of consomic rat panels in gaining insight into genetic factors influencing vascular reactivity and suggest that the chromosomes that appear to be involved in the determination of aortic ring reactivity in different rodent models of hypertension are highly strain- and sex specific.

Brain stem heme oxygenase 1 (HO‐1) mRNA expression in inbred rat strains after severe hemorrhage

Previously Klemcke et al. showed 8‐fold difference in survival time among inbred rat strains after severe hemorrhage (Shock 29:748, 2008). For Brown Norway (BN/Mcwi) rats we also found a 2.7‐fold increase in cardiac HO‐1 mRNA expression in catheterized plus hemorrhaged rats (Bled) compared to unmanipulated controls (CN; FASEB J. 23:801.2, 2009). To further examine HO‐1 as a possible intermediary of cellular responses to hemorrhage, in the same study we measured HO‐1 mRNA expression in brain stem of DA, Fawn Hooded Hypertensive (FHH), and BN/Mcwi strains. Using real time PCR, the relative quantity (RQ) of HO‐1 mRNA was measured with multiple housekeeping genes (Genome Biology 8:R19, 2007). Across all treatments (CN, catheterized only, and Bled) RQs were greater (P&lt;0.05) in BN/Mcwi (0.47 ± 0.02; mean ± SEM) and FHH (0.48 ± 0.02) than in DA rats (0.41 ± 0.02). When considering all strains, HO‐1 mRNA RQ increased 12% (P&lt;.05) in Bled rats compared with CN. Within strains and treatments, BN/Mcwi CN rats had greater (P&lt;.05) RQs (0.46 ± 0.01) than did DA control rats (0.38 ± 0.03). The data imply that rat brain stem has minor strain‐dependent differences in HO‐1 mRNA expression, and that basal expression is modestly increased after the combined stressors of catheterization and hemorrhage.

Survival time after hemorrhage (STaH) in inbred rats with known blood volumes

Eight‐fold differences in STaH were detected among 15 inbred rat strains (Klemcke et al., Shock 29:748, 2008) suggesting genetic effects on STaH. In that study, blood volumes (BV) for all strains were assumed to be 5.83 % of body weight and 55% of that BV was removed. We also showed that differences in STaH might reflect strain‐dependent differences in BV (Klemcke et al., FASEB J 23: 801.1, 2009). To address this hypothesis, each rat of 5 inbred rat strains was hemorrhaged based on its own BV measured using Evans Blue dye 24 hr prior to hemorrhage and its STaH determination. BV varied (P &lt;0.01) among Brown Norway (BN/Mcwi; n = 14; 7.43 ± .10 ml/100 g body weight, mean ± SEM), Salt Sensitive (S; n = 10; 7.08 ± .15), Fawn Hooded Hypertensive (FHH; n=10; 6.74 ± .08), Lewis (LEW; n=10; 6.74 ± .08), and DA (n=10; 6.74 ± .08) rat strains. With equal hemorrhages (47.1 ± .05 % of measured BV), STaH did not differ among rat strains (155 ± 12 min; P = 0.95). When considering both studies it is apparent that: 1) Strain‐related differences in BV contributed to differences in STaH in our 1st study; 2) In FHH rats 1.3% more of total BV was removed in the current study, yet they survived 1.5 hr longer; and 3) In LEW rats identical BV were removed in both studies, yet currently LEW rats survived 1 hr longer. Such observations suggest that Evans Blue dye and/or blood sampling for BV determinations might be preconditioning stimuli that improve STaH in some rat strains.

Inter‐ and intra‐strain variability for survival time after hemorrhage in inbred rats

In an effort to identify genes influencing survival time after hemorrhage (STaH) we earlier reported 8‐fold differences in STaH among 15 inbred rat strains, indicating a genetic basis for STaH (Klemcke et al., Shock 29:748, 2008). Subsequently, we identified that the inbred rat strains differed in blood volume (BV) which could account for differences in STaH. Thus, to re‐address the question of genetic influences on STaH, 5 inbred rat strains [Brown Norway (BN/Mcwi, n=9), Dark Agouti (DA, n=8), Fawn Hooded Hypertensive (FHH, n=10), Lewis (Lew, n=9) and Salt Sensitive (SS, n=9)] were bled using strain specific BVs. For each rat, 47% of its BV was removed and STaH was measured. Threefold differences for STaH among the strains (p&lt;0.0001) were observed (Table). Rats of an inbred strain are genetically very similar with a small degree of genetic variability. Thus, it was of interest to observe considerable intra‐strain variation for STaH (Table). Strain Mean STaH ± SEM (min) Range (min) Coefficient of Variation (%) BN/Mcwi 52 ± 5 35 – 79 28 DA 190 ± 29 42 – 240 43 FHH 113 ± 24 31 – 240 66 LEW 95 ± 19 62 – 240 60 SS 114 ± 17 70 – 235 44 Environmental conditions were highly controlled in this study, thus: 1) observed inter‐strain variability confirms the presence of a genetic component to STaH; and 2) intra‐strain variability may be explained by alternatives such as epigenetic mechanisms. (Funding provided by USAMRMC).

Renal vascular dysfunction precedes the development of renal damage in the hypertensive Fawn-Hooded rat

It is unknown whether generalized vascular dysfunction precedes the development of kidney disease. Therefore, we studied myogenic constriction and endothelium-mediated dilatory responses in two inbred Fawn-Hooded (FH) rat strains, one of which spontaneously develops hypertension, proteinuria, and glomerulosclerosis (FHH), whereas the other (FHL) does not. Small renal, mesenteric resistance arteries and thoracic aorta isolated from FH rats before (7 wk old) and after the development of mild proteinuria (12 wks old) were mounted in perfused and isometric set-ups, respectively. Myogenic response, endothelium-dependent relaxation, and the contribution of endothelium-mediated dilatory compounds were studied using their respective inhibitors. Myogenic reactivity was assessed constructing pressure-diameter curves in the presence and absence of calcium. At the age of 7 wk, renal arteries isolated from kidneys of FHH rats developed significantly lower myogenic tone compared with FHL, most likely because of excessive cyclo-oxygenase 1-mediated production of constrictive prostaglandins. Consequently, young FHH demonstrated reduced maximal myogenic tone (22 +/- 4.8 vs. 10.8 +/- 2.0%, P = 0.03) and the peak myogenic index (-6.9 +/- 4.8 vs. 0.6 +/- 0.8%/mmHg, P = 0.07 for FHL vs. FHH, respectively). Active myogenic curves obtained in mesenteric arteries isolated from 7-wk-old rats did not differ between either strain, demonstrating a similar level of systemic myogenic tone in FHL and FHH rats. Therefore, before any renal end-organ damage is present, myogenic response seems selectively impaired in renal vasculature of FHH rats. Aortic reactivity did not differ between FHL and FHH at the time points studied. The present study shows that vascular dysfunction in both small renal and systemic arteries precedes renal end-organ damage in a spontaneous model of hypertension-associated renal damage. These early vascular changes might be potentially involved in the increased susceptibility of FHH rats to renal injury.

Suppression of Heavy Drinking and Alcohol Seeking by a Selective ALDH‐2 Inhibitor

Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.