Analysis of circadian locomotor activity, Golgi-Cox impregnation, and immunohistochemistry were studied on the hereditary microphthalmic rat which congenitally lacked the optic nerve. These blind rats showed free-running circadian rhythms in their locomotor activities. Both the normal and microphthalmic rats had similar ultradian rhythms in addition to circadian rhythms. The neuronal cell population and volume of the hypothalamic suprachiasmatic nucleus (SCN) of the microphthalmic rats were 66% and 71% of those in normal rats, respectively. The number of SCN neurons containing vasoactive intestinal peptide-like immunoreactive substance was dramatically decreased to 35% of that in normal rats. Golgi-Cox impregnation revealed that three types of neurons in the SCN of the microphthalmic rats were consistently distinguished as observed in normal rats. Although there were no changes in the numbers of primary dendrites of the SCN neurons in the microphthalmic and normal rats, the number of secondary and tertiary dendrites in the SCN of the microphthalmic rats was smaller than that of normal rats. These observations suggest that the retinal input may be important for normal morphological formation of the SCN during development, but not for the generation of circadian rhythms and ultradian rhythms.

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The distribution of atrial natriuretic peptide (ANP)- and brain natriuretic peptide (BNP)-granules was examined immunohistochemically and ultrastructurally in the hearts of the chicken, Japanese quail, Japanese rat snake and bull-frog. Moreover, natriuretic peptide (NP)-granules in the cardiocytes were analyzed by ultrastructural morphometry. Immunohistochemically, ANP-immunoreactivity (IR) was not detected in any cardiocytes, but BNP-IR was detectable in most atrial and ventricular cardiocytes of both chicken and quail. In the snake, ANP-IR was seen in most atrial and ventricular cardiocytes, which showed traces and negative in BNP-IR, respectively. Both ANP- and BNP-IR were detected in the atrial and ventricular cardiocytes in the frog. Ultrastructurally, most of NP-granules were found in the perinuclear region in the chicken, quail and snake atrium, but the frog atrial cardiocytes had granules generally dispersing widely in the cell. By ultrastructural morphometry, the number of granules in the atrial cardiocyte was greatest in the frog, followedby the snake, and chicken or quail, in this order. The diameter of granules in the atrial cardiocyte was largest in the snake and reduced via the frog to the chicken or quail. In the ventricular cardiocytes of all species, the number and size of granules were significantly less than that in the atrial ones. These results indicated that the hearts of the chicken and quail contain only BNP, and that there are two different natriuretic peptides, ANP and BNP, in the snake and frog hearts.

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Atrial natriuretic peptide (ANP)-granules of auricular cardiocytes were examined by immunohistochemistry, transmission electron microscopy and ultrastructural morphometry in dehydrated and rehydrated mice. In addition, plasma ANP and tissue ANP mRNA levels were determined by radioimmunoassay and polymerase chain reaction, respectively. ANP immunoreactivity and the number of granules in the cardiocytes were increased with time in the dehydration group, while plasma ANP and tissue ANP mRNA levels were decreased on day 3 of dehydration. On day 3 of dehydration, the number of ANP-granules (153.2 ± 8.3, mean ± standard error) was significantly (p<0.05) greater than in the control (125.8 ± 6.7). In the rehydration group, the immunoreactivity and number of ANP-granules were less than those in the group on day 3 of dehydration. The plasma ANP level during rehydration for 12 hr was slightly elevated in comparison with the group on day 3 of dehydration. The tissue ANP mRNA level after 12 hr of rehydration was lower than that on day 3 of dehydration. The diameter of ANP-granules was significantly (p<0.01) smaller in all experimental groups than in the control. These findings suggest that synthesis and secretion of ANP are inhibited and ANP-granules are stored in auricular cardiocytes during dehydration.

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As a possible step in examining the difference between pathological models and most popular and ‘normal’ models, the mammary gland and some parameters were compared in SHN mice, a high mammary tumor strain, and ICR mice. SHN was lower than ICR in almost all urinary component levels as indicators of general metabolic activity in both the female and the male, and glucose tolerance in the female. Body weight and several organ weights including liver and kidneys were lower in SHN than in ICR. Normal and preneoplastic mammary gland growth and TGFα mRNA expression in the glands of the female were much higher in SHN than in ICR. These findings stress the importance of having a right knowledge of the parameters of ‘normal’ animals, especially for researchers using pathological models.

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This study of 28 CLAWN miniature pigs (male 17, female 11, mean weight 29 kg) was undertaken to investigate the coronary arterial branching patterns and the ischemic area induced by surgical occlusion of the left anterior descending artery (LAD) and change in the ischemic area over time. These results were compared with those in dogs, which have frequently been used in myocardial ischemic research. Regarding the coronary arterial branching pattern, there were fewer ventricular branches from the right and left coronary arteries than in dogs. The septal branches arose from only the LAD and the posterior descending artery (PD). The largest septal artery branched from the LAD. There were two types of septal artery branching patterns. In approximately 80% of the CLAWN miniature pigs, the PD arose from the right coronary artery (Right dominance). The peculiarity of the coronary arterial branching pattern in the CLAWN miniature pigs was more similar to human beings than to dogs. The ischemic area induced by occlusion at three-fifths distal section of the LAD was 12.1% to 22.6% (mean 17.1%) of the left ventricle. The ischemic area in all animals that died of global left ventricular malfunction and hemodynamic instability after LAD occlusion was more than 25% of the left ventricle.

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Histochemical, lectin-histochemical and morphometrical studies were carried out on intestinal goblet cells of 8-week-old germfree (GF) and conventional (CV) mice of the BALB/c strain. Except for the reactivity of cecal goblet cells to Dolichos biflorus agglutinin (DBA) and Ulex europeus-I agglutinin (UEA-I), there was no difference between GF and CV mice in histochemical and lectin-histochemical properties. In the cecal mucosa, DBA stained the goblet cells strongly in CV mice but not in GF mice and UEA-I stained the goblet cells strongly in the lower part of crypts in CV mice but only faintly in GF mice. These findings suggest that terminal residues of cecal goblet cell mucin were different in GF and CV mice. Morphometrically, cecal goblet cells were fewer in number and smaller in size in GF mice than in CV mice. In addition, high iron diamine-alcian blue staining made a very clear border between the cecum and colon, because cecal goblet cells were exclusively positive for sulfomucin and colonic goblet cells were predominantly positive for sialomucin.

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In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats. Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats. In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg. The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment. In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats. Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.

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Magnetic resonance imaging (MRI) has potential as an imaging technique in fetal anatomy. In this article are presented images obtained from mouse fetuses at 16 days of pregnancy by means of the ultraconductive MRI system (JEOL AIM270). When fetuses removed from the uterus were fixed with 10% neutral formalin, various organs including heart, liver, lungs and bones were clearly seen, and a clear outline was obtained of the fetal skin and subcutaneous tissue by varying the setting. The same specimens were able to be used for histological evaluation. This MRI system will allow wider application in fetal anatomy and provide additional information about the developing mouse fetus.

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Levels of brain histamine (HA) and pituitary luteinizing hormone (LH) were determined in female rats anesthetized with ether in the afternoon of proestrus. The rats after 6-hr ether anesthesia had an average HA concentration higher than that of non-anesthetized rats in the hypothalamus, but not in the cortex or diencephalon. Ether-anesthetized rats also showed a higher LH level in the pituitary than that of non-anesthetized rats. These findings agree well with our previous observation of an inhibited ovulation associated with a decrease in serum LH in female rats anesthetized with ether in the afternoon of proestrus. Furthermore, it is suggested that the release of pituitary LH into the circulating blood is regulated by the level of HA in the hypothalamus.

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The effect of insulin on in vitro development of early stage tetraploid mouse embryos was examined. Tetraploid embryos were produced at the 2-cell stage by treatment with cytochalasin B for 12 hr, then they were cultured with M16 medium containing 1 μM insulin. Although insulin had no effect on the embryos until 72 hr after hCG injection, at 96 hr the number of cells of insulin (+) tetraploid blastocysts increased 20.2% more than insulin (-) tetraploid embryos. The increase in the number of cells mainly depended on the inner cell mass (ICM), in which insulin (+) cells increase 76.3% more than insulin (-) cells. These effects of insulin on tetraploid embryos were comparable with those on diploid embryos. The present results suggested that the effect of insulin on the cell proliferation of early stage embryos was not affected by tetraploidy, but the increased number of cells in tetraploid blastocysts was significantly smaller than that in diploid embryos.

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Hepatitogenicity of 3 plaque purified variants of hepatotropic mouse hepatitis virus, MHV-2 were examined in athymic BALB/c-nu/nu mice up to 9 weeks post infection (9WPI). All of the MHV-2S- and MHV-2M-infected mice died with severe acute hepatitis in 3WPI. On the other hand, MHV-2L-infected mice did not die until 9WPI and showed signs of slow-developing chronic hepatitis with persistent infection under low serum virus neutralizing antibody titers. This suggests that MHV-2L-infected athymic nude mice may be useful as a new model of chronic viral hepatitis.

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Methods for electrophoresis for the analysis of biochemical marker genes, which are used widely for genetic monitoring of inbred straines of rats, have been complicated by the variation of the gel and electrode buffer and electrophoretic conditions with the enzyme or the protein to be examined. To simplify the methods, we performed electrophoresis under fixed conditions of 200 V and 30 min using cellulose acetate membrane as the gel and veronal solution as the gel and electrode buffer. Good results were obtained concerning 12 loci, namely, Amy1, Cs1, Esl, Es2, Es3, Es4, Fh1, Gc, Hbb, Ldr1, Mup1, and Svp1. This method was applied to 8 inbred strains of rats and confirmed to be practical.

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To investigate the validity of ETCO₂ in porcine neonates, which have been frequently used as an experimental model for human neonates, the relationship between arterial (PaCO₂) and end-tidal PCO₂ (ETCO₂) in porcine neonates was examined under different respiratory conditions by regulating both inspiratory flow and positive end expiratory pressure (PEEP). The difference between PaCO₂ and ETCO₂ widened significantly, according to the significant decrease in tidal volume/body weight ratio (TV/BW) caused by the increase of PEEP. A lower correlation between PaCO₂ and ETCO₂ was observed in <6 ml/kg than in ≥ 6 ml/kg TV/BW. It therefore seems reasonable to conclude that, in porcine neonates, the valid ETCO₂ measurements corresponding to PaCO₂ would be obtained at ≥ 6 ml/kg TV/BW.

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To investigate the role of apolipoprotein E (apo E) in amyloidoses of cynomolgus monkeys, the localization of apo E in cerebral amyloid, including senile plaques and cerebrovascular amyloid, and in islet amyloid was examined immunohistochemically. Mature types of senile plaques with amyloid deposits and cerebrovascular amyloid showed intense immunoreactivity to both antisera to apo E and amyloid β protein (Aβ). In contrast, diffuse plaques without obvious Congophilic amyloid showed weak immunoreactivity to antiserum to apo E, but intense reactivity to antiserum to Aβ. In addition, the number of these apo E-positive diffuse plaques was small compared with that of Aβ-positive plaques. On the other hand, diabetic islet amyloid that was negative with Aβ, reacted intensely with antiserum to apo E. These findings suggest that apo E plays an important role in amyloid fibril formation in several types of amyloidoses.

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Male retired breeder F344/DuCrj rats of 17 months of age were purchased in three lots and maintained for aging studies until 25 months of age. These rats were compared with male virgin rats of the same strain for survival percentage, body weight and food consumption. In the retired breeders, decrease in body weight and low food consumption were noted after delivery, and one or two months were required for these parameters to return to the delivery level. After recovery, the body weight and food consumption as well as survival percentage in the retired breeders were similar to those in virgins. From our results, we consider that it takes one to two months to acclimatize aged rats.

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