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Rats have been used as important animal models in biomedical research since over 100 years. The advantage of using rats for cardiovascular disease related studies includes their size, a larger blood volume than that of mice, and lower cost compared to larger animals. Inbred rat lines have been used especially for hypertension related research such as spontaneously hypertensive rats. In addition since sequencing of the rat genome rat gene maps have been well-developed and maps are available for polymorphic marker and quantitative trait loci. Furthermore, transgenic rats have been generated for several cardiovascular disease entities, especially hypertension. Currently, no established rat lines with molecular biological technique induced gene deletions have been described. In view of the wealth of experimental data, especially related to hypertension research, rats will continue to be used as an excellent model for cardiovascular disease related studies.

Production efficiency of transgenic rats was compared directly between the routine pronuclear microinjection of exogenous DNA solution (PNMI-Tg method) and the ooplasmic injection of sperm cells exposed to exogenous DNA solution (ICSI-Tg method) using six DNA constructs. The overall production efficiency per treated oocyte in the ICSI-Tg method (mean 1.1%, range 0.2 to 3.1%) was similar to that in the PNMI-Tg method (mean 1.1%, range 0 to 2.4%). An advantage of the ICSI-Tg method in the production of transgenic rats is noted in cases in which a low yield of pronuclear zygotes is an inevitable fate of the rat strain.

Aim: Obesity is a risk factor for the development of cardiovascular diseases. Recently it was shown that overexpression of the Mas-receptor antagonist angiotensin(1-7) could prevent from diet-induced obesity. However, it remained unclear whether diet-induced obesity and angiotensin(1-7) overexpression might also have effects on the cardiovascular system in these rats. Methods: Twenty three male Sprague Dawley rats were fed with standard chow (SD+chow, n = 5) or a cafeteria diet (SD+CD, n = 6) for 5 months. To investigate the effect of angiotensin(1-7) transgenic rats, expressing an angiotensin(1-7)-producing fusion protein in testis were used. These transgenic rats also received a 5 month's feeding period with either chow (TGR+chow, n = 6) or cafeteria diet (TGR+CD, n = 6), respectively. Hemodynamic measurements (pressure-volume loops) were carried out to assess cardiac function and blood pressure. Subsequently, hearts were explanted and investigated according to the Langendorff technique. Furthermore, cardiac remodeling in these animals was investigated histologically. Results: After 5 months cafeteria diet feeding rats showed a significantly increased body weight, which could be prevented in transgenic rats. However, there was no effect on cardiac performance after cafeteria diet in non-transgenic and transgenic rats. Moreover, overexpression of angiotensin(1-7) deteriorated cardiac contractility as indicated by impaired dp/dt. Furthermore, histological analysis revealed that cafeteria diet led to myocardial fibrosis in both, control and transgenic rats and this was not inhibited by an overproduction of angiotensin(1-7). Conclusion: These results indicate that an overexpression of circulating angiotensin(1-7) prevents a cafeteria diet-induced increase in body weight, but does not affect cardiac performance in this experimental rat model of obesity. Furthermore, overexpression of angiotensin(1-7) alone resulted in an impairment of cardiac function.

On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Professor Paul R. Sanberg (Department of Neurosurgery, College of Medicine, University of South Florida, FL, USA), Executive Editor of Cell Medicine, for providing us such an excellent opportunity to publish the data that were presented at the annual meeting of JSOPMB. I also thank Dr. David Eve, Associate Editor of Cell Medicine, for editing our papers in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to newly join this organization. Cell-based therapy is one of the most important medical fields in JSOPMB because this form of therapy can be useful for a wide range of diseases. Addressing the current problem of severe human donor organ shortage for cell therapies is a big challenge. Research on adult and embryonic stem cells and artificial cell development, in addition to the recent and rapidly evolving invention of induced pluripotent stem (iPS) cells, encourages us to address the problems confronting cell transplantation. Therefore, JSOPMB has now importantly focused on regenerative medicine in collaboration with cell biologists. One of the extremely important missions of the annual meeting of JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 400 members and is run under the direction of Dr. Takehide Asano, the current President of JSOPMB. Excellent presentations conducted at the 38th annual meeting of JSOPMB held on November 25–26, 2011, in Miyagi, Japan, under the supervision of Dr. Takashi Kondo (Professor, Department of Thoracic Surgery, Tohoku University, Miyagi, Japan) were selected and given an opportunity to be published in this special issue of Cell Medicine. Nine of these presentations are herein published in this special JSOPMB issue. Teratani and Kobayashi reviewed results from various groups using their transgenic rats for direct visualization of their tissues at the bench level and in cell transplantation research. This included studies on renal and liver injury, parkinsonian models, and nerve injury and islet isolation. Diabetes continued to be a major focus with two review papers evaluating different aspects of islet transplantation. Kataoka and Noguchi reviewed the relationship between ER stress and the pathogenesis of type 1 and type 2 diabetes and the association between islet transplantation and ER stress. Maruyama et al. reviewed autologous islet transplantation. Stem cell research was a major topic of interest. There were two papers regarding pancreatic stem cells. One by Noguchi et al. explored the culture conditions for mouse pancreatic stem cells, while the other by Kuise et al. the isolation efficiency of mouse pancreatic stem cells. In addition, Kasahara et al. reported that protein fractions secreted by mesenchymal stem cells (MSCs) were capable of activating preserved islets. There were three papers regarding hepatocytes. Dosen et al. reported that hepatic ischemic reperfusion injury could be reduced using saline exposed to electron discharge in a rat model. Miyamoto et al. synthesized positively charged magnetic nanoparticles and observed these nanoparticles inside three-dimensional models of HepG2 spheroids, which mimic tissues. Finally, Hsu et al. showed that citrate phosphate dextrose-supplemented Euro-Collins solution improved the recovery of hepatocytes isolated from warm ischemic rat liver. The theme of this JSOPMB issue is “Long-Expected New Start.” The board members and I are looking forward to seeing further progress in JSOPMB in conjunction with Cell Medicine.

Although rats are preferred over mice as an animal model, transgenic animals are generated predominantly using mouse embryos. There are limitations in the generation of transgenic rat by embryo manipulation. Unlike mouse embryos, most of the rat embryos do not survive after male pronuclear DNA injection which reduces the efficiency of generation of transgenic rat by this method. More importantly, this method requires hundreds of eggs collected by killing several females for insertion of transgene to generate transgenic rat. To this end, we developed a noninvasive and deathless technique for generation of transgenic rats by integrating transgene into the genome of the spermatogonial cells by testicular injection of DNA followed by electroporation. After standardization of this technique using EGFP as a transgene, a transgenic disease model displaying alpha thalassemia was successfully generated using rats. This efficient method will ease the generation of transgenic rats without killing the lives of rats while simultaneously reducing the number of rats used for generation of transgenic animal.

Background: In the present study we evaluated plasma and kidney angiotensin II (ANG II) levels in female and male Ren-2 transgenic rats (TGR) in comparison to age-matched female and male normotensive Hannover Sprague-Dawley rats. Methods: The rats were maintained on a normal sodium (NS) diet (0.6% NaCl) or fed a high sodium (HS) diet (2% NaCl) for 4 days or were sodium depleted by administration of 40 mg furosemide per liter drinking water overnight followed by 3 days of low sodium diet (0.01% NaCl) (LS + F). ANG II levels were determined by radioimmunoassay. Results: Female TGR at the age of 38 days were already hypertensive and had developed cardiac hypertrophy, whereas male TGR at this age still exhibited a normotensive phenotype. HS diet increased the blood pressure (BP) but did not alter the ANG II levels in TGR at any age. LS + F decreased the BP without significant change in ANG II concentrations in TGR. Female TGR responded to salt loading and salt depletion by more pronounced changes in BP than male TGR. Conclusions: Female TGR develop hypertension more rapidly and the salt-sensitive component of hypertension is more pronounced in female than in male TGR.

Study objective: We tested the hypothesis where the combined renin-angiotensin system (RAS) and endothelin 1 (ETA) receptor blockade could be more effective in the slowing of progression of CKD wit...

Previous studies have demonstrated that chronic blockade of PDGF receptors prevents the decreases in renal hemodynamic function and renal injury that occur in Cyp1a1-Ren2 transgenic rats with angio...

In this study, we aimed to investigate the adaptation of blood pressure (BP), heart rate (HR), and locomotor activity (LA) circadian rhythms to light cycle shift in transgenic rats with a deficit in brain angiotensin [TGR(ASrAOGEN)]. BP, HR, and LA were measured by telemetry. After baseline recordings (bLD), the light cycle was inverted by prolonging the light by 12 h and thereafter the dark period by 12 h, resulting in inverted dark-light (DL) or light-dark (LD) cycles. Toward that end, a 24-h dark was maintained for 14 days (free-running conditions). When light cycle was changed from bLD to DL, the acrophases (peak time of curve fitting) of BP, HR, and LA shifted to the new dark period in both SD and TGR(ASrAOGEN) rats. However, the readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. The LA acrophases changed similarly in both strains. When light cycle was changed from DL to LD by prolonging the dark period by 12 h, the reentrainment of BP and LA occurred faster than the previous shift in both strains. The readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. In free-running conditions, the circadian rhythms of the investigated parameters adapted in TGR(ASrAOGEN) and SD rats in a similar manner. These results demonstrate that the brain RAS plays an important role in mediating the effects of light cycle shifts on the circadian variation of BP and HR. The adaptive behavior of cardiovascular circadian rhythms depends on the initial direction of light-dark changes.

Plasma cholesteryl ester transfer protein (CETP) has a profound effect on neutral lipid transfers between HDLs and apolipoprotein B (apoB)-containing lipoproteins when it is expressed in combination with human apoA-I in HuAI/CETP transgenic (Tg) rodents. In the present study, human apoA-I-mediated lipoprotein changes in HuAI/CETPTg rats are characterized by 3- to 5-fold increments in the apoB-containing lipoprotein-to-HDL cholesterol ratio, and in the cholesteryl ester-to-triglyceride ratio in apoB-containing lipoproteins. These changes occur despite no change in plasma CETP concentration in HuAI/CETPTg rats, as compared with CETPTg rats. A number of HDL apolipoproteins, including rat apoA-I and rat apoC-I are removed from the HDL surface as a result of human apoA-I overexpression. Rat apoC-I, which is known to constitute a potent inhibitor of CETP, accounts for approximately two-thirds of CETP inhibitory activity in HDL from wild-type rats, and the remainder is carried by other HDL-bound apolipoprotein inhibitors. It is concluded that human apoA-I overexpression modifies HDL particles in a way that suppresses their ability to inhibit CETP. An apoC-I decrease in HDL of HuAI/CETPTg rats contributes chiefly to the loss of the CETP-inhibitory potential that is normally associated with wild-type HDL.

Objective : Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) but increased function of sarcolemmal Na+/Ca2+ exchanger (NCX), leading to decreased SR Ca2+ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca2+ handling. Methods : Therefore, we characterized TG ( n = 35) vs. wild-type (WT) control rats ( n = 39) under physiological conditions (37 °C, stimulation rate

The article discusses the importance of rats in regenerative medicine research. It touches on wound healing using bone marrow derived cells and the artifical dermis.

We have developed an efficient and selective radioligand, the [35S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B1 receptor.

Growth hormone (GH) is essential in the development and growth of the skeleton and for the maintenance of bone mass and density, and its secretion is known to decline with aging. We have previously produced transgenic rats with low circulating GH that represent several age-associated phenotypes such as obesity, insulin-resistance and leptin-resistance. In the present study, the cross-sectional area, bone mineral density, and strength indexes of the hind leg skeletons of the transgenic rats were examined by an X-ray computed tomography scanning. The mean cross-sectional area of the transgenic rats showed no increase after 2 months old up to 8 months old and the strength indexes were significantly lower than their non-transgenic siblings at all ages examined. The trabecular bone mineral density in the transgenic rats drastically decreased at 8 months old, while the cortical bone mineral density was comparable to the non-transgenic rats, suggesting the onset of osteoporosis at this period. The results obtained in this study indicate that the transgenic rats could be useful model to gain insight into the complex mechanism leading to osteoporosis with aging.

A five-step synthesis of α-methylspermidine (1,8-diamino-5-azanonane), the first polyamine analogue preventing pathological consequences of spermidine depletion in transgenic rats overproducing spermine/spermidine N1-acetyltransferase, from ethyl 3-aminobutyrate was achieved in a high overall yield.

The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.

Objective and background: HLA-B27 transgenic (TG) rats exhibit severe colitis, arthritis and other inflammatory lesions. Previous studies in female TG rats indicate that they develop severe alveolar bone loss (ABL). Lack of data on male TG rats has left open the question of possible hormonal/sex dependence for the observed ABL. The purpose of the present study was to assess the natural history of ABL in male HLA-B27 rats, compared to age- and sex-matched wild-type Fischer 344 (WT) rats. Materials and methods: Fourteen WT and 11 TG male rats, aged 7-8 weeks, were used. Sacrifice times occurred at 10, 22 and 35 weeks. Animal heads were defleshed and treated to remove organic material, and skulls were stained to locate the cemento-enamel junction. ABL was measured as exposed molar root surface area (mm 2 ) on the right maxilla and right mandible. Blinded measurements were performed using a computer-assisted image analysis system. Results: ABL for the entire TG group was significantly different from the WT group (p < 0.05). There was no significant difference in ABL between WT and TG rats at 10 weeks of age. At 22 and 35 weeks of age TG rats experienced 23% and 37% greater ABL than WT rats, respectively; these differences were statistically significant (p < 0.015). For both TG and WT animals, ABL was significantly different between the three age groups. Conclusions: These results, consistent with previous findings in female TG rats, suggest that the accelerated ABL found in TG rats is an adult-onset, age-dependent, and sex-independent process.

Background/Aims: A growing body of evidence suggests that the interplay between the endothelin (ET) and the renin-angiotensin systems (RAS) plays an important role in the development of the malignant phase of hypertension. The present study was performed to evaluate the role of an interaction between ET and RAS in the development of hypertension and hypertension-associated end-organ damage in homozygous male transgenic rats harboring the mouse Ren-2 renin gene (TGRs) under conditions of normal-salt (NS, 0.45% NaCl) and high-salt (HS, 2% NaCl) intake. Methods: Twenty-eight-day-old homozygous male TGRs and age-matched transgene-negative male normotensive Hannover Sprague-Dawley (HanSD) rats were randomly assigned to groups with NS or HS intake. Nonselective ETA/B receptor blockade was achieved with bosentan (100 mg/kg/day). Systolic blood pressure (BP) was measured in conscious animals by tail plethysmography. Rats were placed into metabolic cages to determine proteinuria and clearance of endogenous creatinine. At the end of the experiment the final arterial BP was measured directly in anesthetized rats. Kidneys were taken for morphological examination. Results: All male HanSD fed either the NS or HS diet exhibited a 100% survival rate until 180 days of age (end of experiment). The survival rate in untreated homozygous male TGRs fed the NS diet was 41%, which was markedly improved by treatment with bosentan to 88%. The HS diet reduced the survival rate in homozygous male TGRs to 10%. The survival rate in homozygous male TGRs on the HS diet was significantly improved by bosentan to 69%. Treatment with bosentan did not influence either the course of hypertension or the final levels of BP in any of the experimental groups of HanSD rats or TGRs. Although the ET-1 content in the renal cortex did not differ between HanSD rats and TGRs, ET-1 in the left heart ventricle of TGRs fed the HS diet was significantly higher compared with all other groups. Administration of bosentan to homozygous male TGRs fed either the NS or HS diet markedly reduced proteinuria, glomerulosclerosis and attenuated the development of cardiac hypertrophy compared with untreated TGR. Conclusions: Our data show that nonselective ETA/B receptor blockade markedly improves the survival rate and ameliorates end-organ damage in homozygous male TGRs without significantly lowering BP.

Background/Aims: Since there are no data available so far on the role of renal cyclooxygenase-2 (COX-2) in hypertensive Ren-2-transgenic rats (TGR), in the present study we evaluated renal cortical COX-2 protein expression and prostaglandin E2 (PGE2) concentrations as well as renal functional responses to acute COX-2 inhibition in male heterozygous TGR and in normotensive Hannover Sprague-Dawley (HanSD) rats fed either a normal-sodium (NS) or a low-sodium (LS) diet. Methods: In rats fed either the NS or the LS diet for 12 days and prepared for clearance experiments with left ureteral catheterization, the renal functional responses of the left kidney were evaluated after intrarenal COX-2 inhibition with DuP-697 or NS-398. In renal cortical tissue, COX-2 protein expression was assessed by immunoblotting, and the concentration of PGE2 as a marker of COX-2 activity was determined by enzyme immunoassay. Mean arterial pressure in the right femoral artery was monitored by means of a pressure transducer. Results: In heterozygous TGR, to our surprise, the LS diet normalized the mean arterial pressure. Despite significantly higher renocortical expression of COX-2 and PGE2 concentrations as well as urinary PGE2 excretion in TGR as compared with HanSD rats kept on the NS diet, selective intrarenal COX-2 inhibition did not influence renal function either in TGR or in HanSD rats. The LS diet increased renocortical COX-2 expression and PGE2 concentrations as well as urinary PGE2 excretion significantly stronger in TGR than in HanSD rats. Regardless of these increases, the intrarenal COX-2 inhibition caused comparable decreases in glomerular filtration rate, in absolute and fractional sodium excretion, as well as in urinary PGE2 excretion in TGR and HanSD rats kept on the LS diet. Conclusions: The present data show that a LS diet normalizes the mean arterial pressure in heterozygous male TGR. This first study on the role of renal COX-2 in TGR also demonstrates that COX-2-derived vasodilatory prostanoids do not act as renal compensatory vasodilator and natriuretic substances in this model of hypertension.

For comparison of mammary gland whole mounts with examination of 2 histologic sections of mammary gland, 56 Hras128 rats were intravenously injected with 50 mg/kg body weight of N-methyl- N-nitrosourea at 50 days of age and then sacrificed at days 5, 10, 15, 20, 25, and 56. Comparison of detection sensitivity between the whole mounts and histologic sections revealed no lesions apparent in whole mounts on day 10, although intraductal proliferation was clearly detected in histologic sections in 44% of treated rats. Proliferative lesions were first detected in whole mounts at a 44% incidence on day 15, while intraductal proliferations and atypical hyperplasias were apparent in the sections at 89% and 44% incidences, respectively. On day 20, atypical hyperplasias and small adenocarcinomas in histologic sections were found in almost all animals. In conclusion, examination of 2 histologic sections from mammary tissues was found to be practical for detection of small malignant lesions as early as 15 days after MNU injection, and suppressive effects of soy isoflavones were clearly evident within 20 days after carcinogen exposure. These results suggest that this model has practical utility for short-term screening of chemopreventive agents for mammary carcinogenesis.

International audience; Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

Background & Aims: Recently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD. Methods: Conventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RBhigh CD4+ T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7–12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4+ T cell outgrowth. Results: Neither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5–6 weeks compared with 8–12 weeks observed in conventionally reared mice. The outgrowth of CD4+ T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice. Conclusions: Among the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model. GASTROENTEROLOGY 2002;122:1346-1354

A transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene, including its own promoter region, has been established in our laboratory (Hras128 rats), and shown to be highly susceptible to induction of mammary and urinary bladder tumors. Mutation analysis of induced lesions indicated the majority to contain some but not all cells with transgene mutation. In the present study, the susceptibility of Hras128 rats to N-nitrosomethylbenzylamine (NMBA) induction of esophageal tumors was examined with a similar mutation analysis of the transgenes. Male 6-week-old Hras128 and wild littermate rats were treated with NMBA, 0.5 mg / kg subcutaneously, 3 times a week for 5 weeks and then maintained for 5 weeks without any further treatment. Multiple esophageal tumors, squamous cell papillomas and carcinomas, rapidly developed within this 10-week experimental period in Hras128 rats (11.05 +/- 7.83 / rat). In contrast, wild-type littermates had only small numbers of mostly benign tumors (1.67 +/- 2.06 / rat). The Hras128 rats had no other tumors or abnormalities. In their esophageal lesions, codon 12 GGC to GAC mutations of the transgene were frequently detected by restriction fragment length polymorphisms (RFLP) and subsequent direct sequencing analysis (19 / 25, 76%). In the endogenous rat c-Ha-ras gene they were less frequent (2 / 25, 8%), than in wild-type rats (8 / 14, 57.1%). The densities of mutated bands in the RFLP analysis indicated that mutated cells were major populations in tumors, in contrast to the case with mammary and urinary bladder lesions. Furthermore, activated ras protein, detected by binding to raf protein, was clearly increased in tumors as compared to surrounding epithelium or the normal esophagus of untreated rats. The results show that Hras128 rats are highly susceptible to NMBA esophageal carcinogenesis, as well as induction of mammary and urinary bladder tumors, but that tissue-specific characteristics exist for the roles of transgene ras mutations.

Adult stem cells have been well characterized in numerous organs, with the exception of the kidneys. Therefore, the present study aimed to identify and isolate kidney-derived stem cells. A total of 12 Fischer 344 transgenic rats expressing the human diphtheria toxin receptor in podocyte cells of the kidney, were used in the present study. The rats were administered 5-bromo-2'-deoxyuridine (BrdU) in order to detect cellular proliferation. After 60 days, the rats were treated with the diphtheria toxin (DT), in order to induce kidney injury. Immunohistochemical analysis indicated that the number of BrdU-positive cells were increased following DT treatment. In addition, the expression of octamer-binding transcription factor 4 (Oct-4), a stem cell marker, was detected and suggested that kidney-specific stem cells were present in the DT-treated tissue samples. Furthermore, tissue samples exhibited repair of the DT-induced injury. Further cellular culturing was conducted in order to isolate the kidney-specific stem cells. After 5 weeks of culture, the majority of the cells were non-viable, with the exception of certain specialized, unique cell types, which were monomorphic and spindle-shaped in appearance. The unique cells were isolated and subjected to immunostaining and reverse transcription-polymerase chain reaction analyses in order to reconfirm the expression of Oct-4 and to detect the expression of Paired box 2 (Pax-2), which is necessary for the formation of kidney structures. The unique cells were positive for Oct-4 and Pax-2; thus suggesting that the identified cells were kidney-derived stem cells. The results of the present study suggested that the unique cell type identified in the kidneys of the DT-treated rats were kidney-specific stem cells that may have been involved in the repair of DT-induced tissue injury. In addition, these cells may provide a useful cell line for studying the fundamental characteristics of kidney stem cells, as well as identifying kidney-specific stem cell markers.

Thermogenesis in brown adipose tissue (BAT) contributes to substantial increases in body temperature evoked by threatening or emotional stimuli. BAT thermogenesis also contributes to increases in body temperature that occur during active phases of the basic rest-activity cycle (BRAC), as part of normal daily life. Hypothalamic orexin-synthesizing neurons influence many physiological and behavioral variables, including BAT and body temperature. In conscious unrestrained animals maintained for 3 days in a quiet environment (24–26°C) with ad libitum food and water, we compared temperatures in transgenic rats with ablation of orexin neurons induced by expression of ataxin-3 (Orx_Ab) with wild-type (WT) rats. Both baseline BAT temperature and baseline body temperature, measured at the onset of BRAC episodes, were similar in Orx_Ab and WT rats. The time interval between BRAC episodes was also similar in the two groups. However, the initial slopes and amplitudes of BRAC-related increases in BAT and body temperature were reduced in Orx_Ab rats. Similarly, the initial slopes and amplitudes of the increases in BAT temperatures induced by sudden exposure to an intruder rat (freely moving or confined to a small cage) or by sudden exposure to live cockroaches were reduced in resident Orx_Ab rats. Constriction of the tail artery induced by salient alerting stimuli was also reduced in Orx_Ab rats. Our results suggest that orexin-synthesizing neurons contribute to the intensity with which rats interact with the external environment, both when the interaction is “spontaneous” and when the interaction is provoked by threatening or salient environmental events.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT(1) receptors. However, the role of myocardial AT(1) receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT(1) receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT(1) receptor under the control of the alpha -myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT(1) receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma -S and increased sensitivity of intracellular Ca2+ [Ca2+](i) to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT(1) receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Abstract —Angiotensin II recruits transforming growth factor β 1 (TGFβ 1 ) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFβ 1 expression blunts fibrosis and improves outcome in angiotensin II–dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg · kg −1 · d −1 ), or tranilast (a nonspecific TGFβ inhibitor; 400 mg · kg −1 · d −1 ) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1±0.16 versus 2.1± 0.06 mg/g body wt; P P 1 mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFβ 1 ( r =0.62; P =0.019). In situ hybridization demonstrated increases in TGFβ 1 mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats ( P =0.029). In conclusion, TGFβ 1 mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II–dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFβ 1 expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

Angiotensin TI recruits transforming growth factor beta (1) (TGF beta (1)) and is related to left ventricular fibrosis, However, it is unclear whether chronic in vivo reduction in left ventricular TGF beta (1), expression blunts fibrosis and improves outcome in angiotensin II-dependent hypertension. Four week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg.kg(-1).d(-1)), or tranilast (a nonspecific TGF beta inhibitor; 400 mg.kg(-1).d(-1)) (n = 10 For each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1 +/- 0.16 versus 2.1 +/- 0.06 mg/g body wt; P

Background-We evaluated the role of the cardiac endothelin (ET) system in compensated hypertensive left ventricular (LV) hypertrophy (LVH) and after the transition toward LV dysfunction. Methods and Results-Hypertensive transgenic rats overexpressing the Ren2 gene (Ren2 rats) were investigated between the ages of 10 and 30 weeks (Ren2-10 and Ren2-30 groups, respectively) and compared with age-matched normotensive Sprague-Dawley (SD) rats (SD-10 and SD-30 groups, respectively). Systolic blood pressure and LV weight were elevated in both Ren2 groups compared with their age-matched SD control groups (P Conclusions-Activation of the cardiac ET system accounts at Least in part for the LV dysfunction that gradually develops in LVH. The protective effect of ETA antagonism can be attributed to the improvement of diastolic LV function that is due to normalization of impaired SR Ca2+ uptake.

Occupational and environmental exposures to bitumen fumes during road paving or roofing activities represent a safety issue since this complex mixture of volatile compounds and particles contains carcinogenic polycyclic aromatic hydrocarbons (PAH). However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning bitumen fumes toxicity. In order to gain better insights on their genotoxic potential, we used an experimental device able to generate bitumen fumes at 170°C (upper range used during road paving operations) with a total particulate matter of 112 ± 13 mg/m3 and a mass median aerodynamic diameter of 4.6 μ m. The nose-only exposure of Big Blue® transgenic rats was performed 6 h/day for 5 consecutive days. Biological exposure to this aerosol was monitored by measuring the rat urinary levels of 1-hydroxypyrene and lungs were collected at various time-points after the end of treatment for further analysis. As determined by the 32P post-labeling method, no DNA bu...

Objective: Treatment with the beta-blocker carvedilol leads to an improvement of outcome and ejection fraction in heart failure. These effects occur without affecting the number of beta-adrenergic receptors, as determined in right ventricular biopsies from patients with heart failure. This study was aimed at investigating the effects of carvedilol on beta-adrenergic signal transduction alterations in a model of left ventricular pressure overload, which is characterized by sympathetic activation and a desensitized beta-adrenergic signal transduction. Methods: Transgenic rats with overexpression of renin [TG(mREN2)27] were treated with carvedilol (30 mu g/kg) or held under control conditions and were compared with Sprague-Dawley rats. Myocardial beta-adrenoceptors (I-125-labeled iodocyanopindolol binding), Gi alpha (pertussis toxin labeling), Gs alpha-activity (reconstitution into cyc-S49 membranes) and adenylyl cyclase activity were measured. Blood pressure and heart rate, increase in heart rate during sacrifice and pressure rate products were determined. Results: beta-Adrenoceptors were downregulated and Gi alpha-protein levels were significantly increased, producing a desensitization of basal, isoprenaline- and guanine nucleotide-stimulated adenylyl cyclase activity compared to controls. Carvedilol reduced heart rate, blood pressure and pressure rate product in TG(mREN2)27. Carvedilol did not restore biochemical alterations, but even further reduced beta-adrenoceptor numbers and adenylyl cyclase. It exhibited a two affinity state, guanine nucleotide-sensitive binding to cardiac beta-adrenergic receptors similar to isoprenaline but different from metoprolol. Conclusions: Carvedilol did not restore beta-adrenergic signal transduction at concentrations producing antiadrenergic effects in vivo. This effect might be due to an atypical guanine nucleotide-dependent interaction with beta-adrenergic receptors. Thus, ancillary properties could explain the recently reported beneficial effects in patients with heart failure independent from an upregulation of beta-adrenergic receptors. (C) 1998 Elsevier Science B.V. All rights reserved.

Objective: Treatment with the beta-blocker carvedilol leads to an improvement of outcome and ejection fraction in heart failure. These effects occur without affecting the number of beta-adrenergic receptors, as determined in right ventricular biopsies from patients with heart failure. This study was aimed at investigating the effects of carvedilol on beta-adrenergic signal transduction alterations in a model of left ventricular pressure overload, which is characterized by sympathetic activation and a desensitized beta-adrenergic signal transduction. Methods: Transgenic rats with overexpression of renin [TG(mREN2)27] were treated with carvedilol (30 mu g/kg) or held under control conditions and were compared with Sprague-Dawley rats. Myocardial beta-adrenoceptors (I-125-labeled iodocyanopindolol binding), Gi alpha (pertussis toxin labeling), Gs alpha-activity (reconstitution into cyc-S49 membranes) and adenylyl cyclase activity were measured. Blood pressure and heart rate, increase in heart rate during sacrifice and pressure rate products were determined. Results: beta-Adrenoceptors were downregulated and Gi alpha-protein levels were significantly increased, producing a desensitization of basal, isoprenaline- and guanine nucleotide-stimulated adenylyl cyclase activity compared to controls. Carvedilol reduced heart rate, blood pressure and pressure rate product in TG(mREN2)27. Carvedilol did not restore biochemical alterations, but even further reduced beta-adrenoceptor numbers and adenylyl cyclase. It exhibited a two affinity state, guanine nucleotide-sensitive binding to cardiac beta-adrenergic receptors similar to isoprenaline but different from metoprolol. Conclusions: Carvedilol did not restore beta-adrenergic signal transduction at concentrations producing antiadrenergic effects in vivo. This effect might be due to an atypical guanine nucleotide-dependent interaction with beta-adrenergic receptors. Thus, ancillary properties could explain the recently reported beneficial effects in patients with heart failure independent from an upregulation of beta-adrenergic receptors. (C) 1998 Elsevier Science B.V. All rights reserved.

Systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR) and locomotor activity have been measured at 1-min intervals for 24 h in Sprague-Dawley (N = 5) and for 2x24 h in transgenic hypertensive (N = 4) rats. The animals were freely mobile and entrained to a 12:12 LD cycle (lights on at 0700). The endogenous circadian component of the cardiovascular variables was removed from the raw data, and then correlations between activity and the residual component (raw data minus the endogenous component) of SBP, DBP and HR were calculated. This calculation was performed twice, in the mid-light and mid-dark phases. We have investigated if the mean size of the correlation coefficients depended on cardiovascular variable (SBP, DBP or HR), phase (D or L) or strain (Sprague-Dawley, SPD, or Transgenic, TG, rats). Nearly all correlations were positive and ANOVA's showed a significant effect of cardiovascular variable for both strains, with correlations for HR being significantly higher than those for SBP and DB...

Prorenin, the precursor of renin, exists in circulating blood at concentrations that are ≈5 to 10× higher than those of renin. For many years, prorenin was considered to be an inactive form of renin with no physiological role. Then, in the mid-80s of the last century, Luetscher et al reported that the levels of circulating prorenin (but not renin) are increased in diabetic subjects.1 Subsequent studies revealed that these high levels correlated with the presence of microvascular complications, and it was proposed that prorenin might be used to predict the occurrence of microalbuminuria.2 Simultaneously, high prorenin levels were observed in pregnant women, and evidence was obtained for the local synthesis of prorenin at extrarenal tissue sites such as the ovary and eye. Until today, however, the exact function of prorenin remains unknown. An attractive concept is that in tissues not synthesizing renin locally, circulating prorenin, after its local conversion to renin, contributes to angiotensin (Ang) generation. This would not only provide a role for prorenin in vivo, but also explain why tissues, in contrast with plasma, contain predominantly renin.3 Studies in transgenic animals support this idea. Veniant et al4 described that transgenic rats expressing prorenin exclusively in the liver (resulting in a 400-fold rise in plasma prorenin) display cardiac hypertrophy and vascular damage in the absence of hypertension. Prescott et al5 detected increased cardiac Ang I levels in mice overexpressing human prorenin in the liver and human angiotensinogen in the heart. The question then arises how tissues sequester and activate circulating prorenin. Sequestration may involve diffusion into the interstitial fluid and/or binding to a receptor.3 …

The present study investigated whether functional, molecular, and biochemical alterations occurring in chronic heart failure can already be detected in compensated hypertensive cardiac hypertrophy. Force of contraction (isolated papillary muscle strip preparations), sarcoplasmic reticulum (SR) protein and myosin heavy chain isoform expression (Northern and Western blot analysis), myocardial fibrosis (collagen stains, hydroxyproline quantification), myocardial renin mRNA (RT-PCR), and angiotensin II levels and plasma aldosterone concentrations (radioimmunoassay) were studied in hypertrophied myocardium from transgenic rats harboring the mouse Ren-2d gene. Contraction and relaxation velocities of isolated papillary muscle strips were significantly reduced in cardiac hypertrophy. The beta-/alpha-myosin heavy chain ratio was significantly increased in the hypertrophied left ventricles, whereas SR Ca2+-ATPase (SERCA 2a) and phospholamban mRNA and protein levels were significantly decreased. The decrease in SERCA 2a was more pronounced than the decrease in phospholamban levels. There was no increased myocardial fibrosis. Left ventricular myocardial renin mRNA and angiotensin II concentrations, as well as plasma aldosterone levels, were higher in transgenic than in control rats. In hypertensive cardiac hypertrophy, myosin heavy chain isoform shift and reduction of SR protein levels are related to systolic and diastolic dysfunction, respectively. These alterations pre cede the development of myocardial fibrosis. Increased myocardial renin mRNA and angiotensin II concentrations suggest that an activated tissue renin-angiotensin system might contribute to these alterations. Since the alterations in compensated cardiac hypertrophy apparently precede those in chronic heart failure, they might accelerate the transition from hypertrophy to failure and could therefore be targets for pharmacological interventions.

Background Hypertensive cardiomyopathy is a major risk factor for the development of chronic heart failure,Objective To investigate whether treatment with an angiotensin converting enzyme inhibitor (ACEI) or with an angiotensin type 1 receptor antagonist (AT(1)-RA) is sufficient to prevent the development of hypertensive cardiomyopathy and cardiac contractile dysfunction. Special emphasis was placed on the effects of both treatments on sarcoplasmic reticulum Ca2+-ATPase (SERCA 20) gene expression as a major cause of impaired diastolic cardiac relaxation.Methods and results Eight-week-old rats harboring the mouse renin 2(d) gene [TG(mREN2)27] were treated for 8 weeks with 100 mg/kg captopril (Cap) in their food and 100 mg/kg of the AT(1)-RA Bay 10-6734 (Bay) in their food. Untreated TG(mREN2)27 and Sprague-Dawley rats (SDR) were used as controls. Both treatment regimens normalized the left ventricular weight, which was increased significantly (P Conclusions In renin-induced hypertensive cardiomyopathy, left ventricular diastolic dysfunction occurs at the stage of compensated myocardial hypertrophy, The decreased left ventricular relaxation velocity might be due to reduced SERCA 20 gene expression. In this model of hypertensive cardiomyopathy, AT(1)-RA and ACEI treatments are similarly effective at reducing the arterial pressure, preventing myocardial hypertrophy and diastolic contractile dysfunction. Normalization of SERCA 2a gene expression, either by AT(1)-RA or by ACEI treatment, might contribute to the improvement in diastolic function.

Transgenic rats were produced by pronuclear microinjection with 42 kinds of exogenous DNAs. The producing efficiency of transgenic rats ranged from 0.73 to 3.53% of the microinjected eggs. Promoter region, structural gene, chain length, and strand ends of the exogenous DNA was not involved in the producing efficiency of transgenic rats. The exogenous DNA can be prepared at 5 μg/ml with better efficiency than the lower and higher concentrations. Rat strain for the pronuclear eggs was a possible factor to produce transgenic rats. These results provide fundamental knowledge to produce transgenic rats effectively.