Impaired blood-brain barrier function in angiotensinogen-deficient mice.

Astrocytes in the central nervous system have physiologically important roles in the response to brain injury. Brain damage results in disruption of the blood-brain barrier (BBB), producing detachment of astrocyte endfeet from endothelial cells. The resultant leakage of serum proteins from loosened tight junctions between endothelial cells produces brain edema. At the same time, reactive astrocytes migrate to the injured area, where they proliferate and produce extracellular matrix, thereby reconstituting the BBB. As astrocytes are known to express angiotensinogen, which is the precursor of angiotensins (AI to AIV), we have investigated a possible functional contribution of angiotensinogen or one of its metabolites to BBB reconstitution. The astrocytes of angiotensinogen knockout mice had very attenuated expression of glial fibrially acidic protein and decreased laminin production in response to cold injury, and ultimately incomplete reconstitution of impaired BBB function. Although these abnormalities were rescued by administration of AII or AIV, the restoration of BBB function was not inhibited by AII type 1 and 2 receptor antagonists. These findings provide evidence that astrocytes with angiotensins are required for functional maintenance of the BBB.

Hypertension induced in pregnant mice by placental renin and maternal angiotensinogen.

Maternal hypertension is a common complication of pregnancy and its pathophysiology is poorly understood. This phenomenon was studied in an animal model by mating transgenic mice expressing components of the human renin-angiotensin system. When transgenic females expressing angiotensinogen were mated with transgenic males expressing renin, the pregnant females displayed a transient elevation of blood pressure in late pregnancy, due to secretion of placental human renin into the maternal circulation. Blood pressure returned to normal levels after delivery of the pups. Histopathologic examination revealed uniform enlargement of glomeruli associated with an increase in urinary protein excretion, myocardial hypertrophy, and necrosis and edema in the placenta. These mice may provide molecular insights into pregnancy-associated hypertension in humans.

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity.

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

Assay for mutagenicity of bile in Sprague-Dawley rats treated subcutaneously with intestinal carcinogens.

To investigate the mode of action of sc injected intestinal carcinogens, the mutagenicity assay of bile collected from noninbred Sprague-Dawley rats treated sc with carcinogens was conducted in the presence and absence of beta-glucuronidase. The bile samples from rats inoculated with 4-aminobiphenyl were mutagenic for Salmonella typhimurium TA100 only in the presence of beta-glucuronidase, whereas those from the 3,2'-dimethyl-4-aminobiphenyl-treated rats did not require the enzyme for mutagenicity toward strain TA100. On the contrary, the assays with S. typhimurium G46 and TA100 of bile from rats inoculated with 1,2-dimethylhydrazine, azoxymethane, or methylazoxymethanol acetate failed to reveal mutagenicity whether beta-glucuronidase was added or not, though these carcinogens were highly mutagenic for strain G46 in the Salmonella-microsome mutagenicity test and/or in the host-mediated assay.

Inhibitors for the mutagenicities of colon carcinogens, 1,2-dimethylhydrazine and azoxymethane, in the host-mediated assay.

Inhibitory effects of several chemicals on the mutagenicities of 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) for Salmonella typhimurium G46 in the host-mediated assay were investigated. They were carbon disulfide (CS2), tetraethylthiuram disulfide (disulfiram, DSF), sodium diethyldithiocarbamate (SDDC), ethylene-bis(dithiocarbamato) manganese (Maneb), pyrazole (PZ), aminoacetonitrile hydrogen sulfate (AAN), and sodium selenite (SE). All the compounds, except for SE, inhibited the mutagenicities of DMH and AOM.

Activation of the nuclear oncogenes N-myc and c-jun in carcinoid [correction of cartinoid] tumors of transgenic mice carrying the human adenovirus type 12 E1 region gene.

The adenovirus (Ad) E1 region genes, E1A and E1B, are well known cooperatively to transform primary rodent cells and activate a number of cellular promoters, including nuclear oncogenes such as N-myc and c-jun, in transfected cell lines. However, there is still less information available on the in vivo mechanism(s) by which the E1 region gene, when chromosomally integrated in the living animals, exerts its effect on nuclear oncogene activation coupled with transformation. To investigate such in vivo activity of E1A we have used a series of microinjection experiments into fertilized eggs to generate three transgenic mice carrying the Ad12-type E1A/E1B genes under the control of the human renin gene. This transgene caused an early onset of bowel cartinoid tumors that express neural cell adhesion molecules, but do not metastasize to any region. Northern blot analysis revealed that the transgenes were considerably expressed in the tumors, but not in other tissues at detectable levels. Interestingly, the levels of N-myc and c-jun mRNAs in the cartinoid tumors were elevated 19- and 8-fold, respectively, as compared with those found in the control intestine. In contrast, the major histocompatibility complex (MHC) class I mRNA level was not altered between the tumor and control intestines, suggesting that this unchanged expression may reflect the loss of tumor metastasis. These findings provide the first in vivo evidence that the expression of the Ad12 E1 region gene induces cartinoid tumors associated with the activation of the nuclear oncogenes N-myc and c-jun.

The Tsukuba hypertensive mouse (transgenic mouse carrying human genes for both renin and angiotensinogen) as a model of human malignant hypertension: development of lesions and morphometric analysis.

The renin-angiotensin system has a pivotal role in hypertension. The Tsukuba hypertensive mouse (THM; a transgenic mouse carrying human genes for both renin and angiotensinogen) was generated to allow further examination of the renin-angiotensin system in a variety of pathologic conditions. We evaluated the development of renal lesions in these mice and in controls by morphometric, immunohistochemical and ultrastructural methods. Blood pressure was significantly higher in THM than in control mice; 1 year after birth, it was approximately 40 mmHg higher. The kidney-to-body weight ratio was also higher in THM than in control. Morphometrical analysis revealed that the glomerular sclerosis index was significantly elevated in THM with 10% of the glomeruli sclerotic at 18 months. The grade of vascular lesion and the frequency of fibronoid arteritis of the kidney exhibited the same tendency as the glomerular sclerosis index. Murine renin was located exclusively in the juxtaglomerular apparatus, whereas human renin was expressed not only in the juxtaglomerular apparatus, but also in periarteriolar smooth muscle cells and in mesangial and epithelial cells of the glomeruli. Light and electron microscopy revealed significant fibrinoid arteritis of the kidney in THM and also "onion skinning", both pathognomonic for malignant nephrosclerosis. THM may be an excellent model of human malignant hypertension.

Intraocular pressure in genetically distinct mice: an update and strain survey.

BACKGROUND: Little is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP. RESULTS: Based on over 30 studied mouse strains, average IOP ranges from approximately 10 to 20 mmHg. Gender does not typically affect IOP and aging results in an IOP decrease in some strains. Most tested strains exhibit a diurnal rhythm with IOP being the highest during the dark period of the day. Homozygosity for a null allele of the carbonic anhydrase II gene (Car2n) does not alter IOP while homozygosity for a mutation in the leptin receptor gene (Leprdb) that causes obesity and diabetes results in increased IOP. Albino C57BL/6J mice homozygous for a tyrosinase mutation (Tyrc-2J) have higher IOPs than their pigmented counterparts. CONCLUSIONS: Genetically distinct mouse strains housed in the same environment have a broad range of IOPs. These IOP differences are likely due to interstrain genetic differences that create a powerful resource for studying the regulation of IOP. Age, time of day, obesity and diabetes have effects on mouse IOP similar to those in humans and other species. Mutations in two of the assessed candidate genes (Lepr and Tyr) result in increased IOP. These studies demonstrate that mice are a practical and powerful experimental system to study the genetics of IOP regulation and disease processes that raise IOP to harmful levels.

Expression analysis of the 5'-upstream region of mouse P/Q-type Ca(2+) channel alpha( lA) subunit gene fused to Escherichia coli lacZ reporter gene in the spinal cord using transgenic mice.

The P/Q-type Ca(2+) channel alpha(1A) subunit is expressed in spinal cord including ventral motor neurons and interneurons and dorsal horn. To identify the transcriptional mechanisms of the mouse alpha(IA) subunit gene in spinal cord, transgenic mice carrying a 0.5, 1.5, 3.0 or 6.3-kb 5'-upstream region fused to the Escherichia coli lacZ reporter gene were examined. Transgenic mice carrying the 3.0-kb region expressed the reporter gene in dorsal horn and interneurons of ventral horn, although those with the 0.5-kb, 1.5-kb or 6.3-kb region did not. No transgenic mice expressed the reporter gene in motor neurons of ventral horn. These results suggest that in spinal cord, the expression mechanisms of the alpha(1A) subunit gene are complex, involving both positive and negative cis-regulatory elements, and the 6.3-kb 5'-upstream region alone is not sufficient for the expression.

Reduction of depressive-like behavior in mice lacking angiotensinogen.

We made use of targeted disruption of the mouse angiotensinogen (ATN) gene to examine the functional role of the ATN in the central nervous system. Both male and female ATN-deficient mice displayed the reduction of depressive-like behavior in the behavioral despair swim tests and spontaneous locomotor activity diminished. However, both male and female ATN-deficient mice showed no anxiogenic-like or memory-deficit behavior and there was no change in the pain threshold. We propose that endogenous ATN in the central nervous system may regulate the depressant state in the brain.

Anti-apoptotic action of angiotensin fragments to neuronal cells from angiotensinogen knock-out mice.

The morphological analysis in a congenic line of angiotensinogen knock-out mice (AgKO) revealed the decreased density in granular layer cells of hippocampus and cerebellum, suggesting neuronal cells of AgKO susceptible to apoptotic cell death. This phenomenon was further studied by culture of the hippocampal neurons with decreased concentration of serum. AgKO neuronal cells, which showed apoptosis by lower concentration of the serum within several hours, however, survived much longer in the presence of angiotensin II (AII) and IV (AIV). This anti-apoptotic action was not interfered by AII receptor antagonists, CV11874 and PD123319. These results suggest that the renin-angiotensin system could play a critical role in central nervous system, preventing neuronal cells from apoptosis not only by AII but also AIV.

Expression analysis of Escherichia coli lacZ reporter gene in transgenic mice.

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of beta-galactosidase activity.

Pathologic characterization of hypotensive C57BL/6J-agt: angiotensinogen-deficient C57BL/6J mice.

a fpreviously produced angiotensinogen-deficient mice, i.e. mice with deleted renin-angiotensin system (RAS), with a genetic background on C57BL/6J - C57BL/6J-agt (-/-) -, but no C57BL/6J-agt (-/-) which survived long enough to be weaned. In the present study, we attempted to prevent neonatal death and analyzed pathological development in C57BL/6J-agt (-/-). We indicate that mortality in C57BL/6J-agt (-/-) derived from C57BL/6J-agt (+/-) can be reduced by hypodermic saline injection in the 7 days following birth, that hydronephrosis developed by day 14 in association with polydiplasia and polyuria by day 30, and that chronic hypotension occurs. Hydronephrosis is less damaging to electrolyte resorption in younger mice, but not in adults. We also observed that C57BL/6J-agt (-/-) derived from C57BL/6J-agt (-/-) frequently develop fetal hydronephrosis and die of respiratory failure at birth. These results suggest that maternal RAS is associated with structural maturation of kidney and lung in late fetus and that postnatal RAS plays important roles in structural and functional maintenance of the kidneys.

Mutagenicity screening of feed additives in the microbial system.

18 feed additives were tested for DNA-modifying effects by the repair test named "rec-assay" with Bacillus subtillis H17 (rec+) and M45 (rec-), and for mutagenicity with Escherichia coli WP2 hcr and 5 Salmonella typhimurium tester strains with the use of a top-agar overlay method. Carbadox, furazolidone, panazon and zoalene were positive in both assays. The former 3 were mutagenic for TA100, TA98 and WP2 hcr, while zoalene was mutagenic for all strains. These 4 compounds did not require a metabolic activation for their mutagenic activities. Nicarbazin was weakly mutagenic for TA1538 and TA98 with and without S9 mix. Amprolium and caprylohydroxamic acid also showed very weak mutagenicities only for TA100 with S9 mix and for WP2 hcr with and without S9 mix, resp. The mutagenic activities of carbadox, furazolidone and panazon for TA100 were reduced only by the addition of S9 mix, but not by S9 fraction or blood, whereas that of zoalene was decreased by any of the 3 factors.

Neurotropism of mouse-adapted haemagglutinating encephalomyelitis virus.

The propagation of a mouse-adapted strain (67N) of haemagglutinating encephalomyelitis virus in infected mice and murine cells was examined by viral re-isolation and immunostaining. Viral propagation was strictly limited to the neurons and to an established line of neuroblastoma cells in in-vivo and in-vitro experiments. These results provide adequate evidence that this virus is neurotropic.

Development of genetically engineered mice with hypertension and hypotension.

Human essential hypertension is generally recognized as a multifactorial disease involving the interplay of environmental factors based on genetic diathesis. Spontaneously hypertensive rats (SHR) and Dahl rats are widely used as animal models for human essential hypertension and salt-sensitive hypertension, respectively. The definitive genetic factor ruling the development of hypertension in these strains remains unclear, but recently advances in embryonic engineering and molecular biological techniques may make it possible for transgenic mice and gene-targeted mice to become important pathological models defined genetically and functionally for human disease. The author developed two types of genetically engineered mice that may serve as such models: a transgenic mouse with hypertension caused by enhancing the renin-angiotensin system and a gene-targeted mouse with hypotension caused by disrupting the renin-angiotensin system.

Chromosomal mapping of human angiotensinogen gene and human renin gene by fluorescence in situ hybridization (FISH) in transgenic mice.

We attempted to apply FISH to chromosomal mapping of the human angiotensinogen (hAG) and human renin (hRN) transgenes which are carried by the parental strains of the Tsukuba hypertensive mouse. We report here that the hAG gene is mapped in the C2 region of Chr 19 and the hRN gene in the A1 region of Chr 6.

Vertical transmission to embryo and fetus in maternal infection with rat virus (RV).

The influence of maternal rat virus (RV) infection on rat embryogenesis and fetus was examined by viral reisolation, immunostaining and PCR analysis. Vertical transmission caused by the UT-1 strain of RV depended on the stage of gestation when maternal infection occurred. When females were infected at the pre-mating point, the number of fetuses was smaller than that normally obtained, possibly due to infection at the stage of the hatched blastocyst, but almost all of the fetuses obtained were free from infection and developed normally. The incidence of transplacental infection was the highest when pregnant females were infected in the middle of the gestation stage, and some of the fetuses died. In pregnant females which were infected late in the gestation stage, all fetuses developed normally. Some of them were infected transplacentally and harbored the infectious virus. Much attention should be paid to performing reliable rederivation of RV-infected rat colonies by hysterectomy and embryo transfer.

Prevalence of "orphan" parvovirus infections in mice and rats.

"Orphan" parvovirus (OPV) infection in laboratory mice and rats was serologically surveyed for 465 mouse sera and 271 rat sera collected from 1986 to 1987 and from 1993 to 1996 in Japan. The results suggest that parvovirus infection is rare in mice but common in rats (positive rate: 13-22%) and that most putative viruses were OPVs. OPV is therefore considered to already have been harbored for at least ten years in Japan.

Vascular remodeling in hypertensive transgenic mice.

We physiologically and histopathologically analyzed vascular damage due to hypertension and vascular remodeling in hypertensive transgenic mice (Tsukuba hypertensive mice; THM). Pubertal (6-week-old) THM already had hypertension similar to blood pressure in adult THM due to an enhanced renin angiotensin system (RAS). They progressively developed remarkable vascular hypertrophy composed of dedifferentiation of vascular smooth muscle cells (VSMCs) and extracellular matrix accumulation in the thoracic aorta, and VSMC hyperplasia was predominant in the abdominal aorta. THM are therefore a useful animal model for studying vascular remodeling mediated by enhanced RAS.