Differential toxicity expression of gentamicine in five-sixths nephrectomized rats assigned to three progressive stages of renal dysfunction--establishment of a new screening approach.

Progressive renal dysfunction in 5/6 nephrectomized (NX) rats can be physiologically divided into three stages, coinciding with morphological stages, after definition of physiological parameters for identification of stage. Now, for the establishment of a toxicity screening approach using 5/6 NX rats, our concept, "Differential toxicity synchronized with renal dysfunction process could be identified using 5/6 NX rats" was examined by dosing gentamicin. Firstly, electrophoretic fractional changes of urinary proteins during gentamicin treatment were clarified with determination of amino acid sequences and the three differential features were proven, revealing the unpredictable depression of urinary albumin with progression of the stages in NX rats. Secondly, marked elevation of urinary lactate dehydrogenase (LDH) and glucose (GLU) was evident, indicating the intensified hypoxic conditions and glycolysis in tubular cells synchronized with increased tubular damage. Thirdly, these transit metabolic changes were proven as intensive cause for the advancement of renal dysfunction by the reduction of FRelectrolytes and water at the end of each dosing period. These results indicate that toxicity studies of newly developed drugs using 5/6 NX rats have potentiality prior to clinical dosing to the patients.

Differential display.

The study of cancer genetics has focused primarily, until now, on changes at the genomic level. However, activating, or inactivating, mutations/deletions/rearran ments in DNA are not the only way in which alterations in gene expression can occur. Since the development of subtractive hybridization, which screens for differentially expressed genes between related populations at the RNA level, evidence has accrued showing that alterations in downstream gene expression might play a significant role in the biology and evolution of cancer. Establishment of the novel technique of differential display (DD) 1,2(1,2) has facilitated studies in this new field that has been termed "expression genetics" 3). Compared with traditional methods of gene expression analysis DD possesses the advantages of simplicity, sensitivity, reproducibility, and speed. It is the current method of choice in expression genetics not only in the cancer biology field but also in such diverse areas as embryology, developmental biology, and neurobiology. This novel technique involves the reverse transcription (RT) of mRNAs with oligo-dT primers anchored to the beginning of the poly(A) tail, followed by a poly merase chain reaction (PCR) in the presence of a second arbitrary random primer. The amplified cDNAs of 3' termini of mRNAs obtained (with the same primers) from different samples can then be run side by side on a sequencing gel to allow differentially expressed genes, either upregulated or downregulated, to be visualized (see Fig. 1). By changing primer pair combinations approx 15,000 individual mRNA species from a mammalian cell may be analyzed reasonably quickly, thus providing a "fingerprint" of the mRNA in any particular cell type. Differential cDNA fragments can easily be recovered and reamplified from the gel and cloned into vectors for future screening and characterization work. The DD technique can be subdivided into five steps fig. 1. Principal procedures of differential display technique.

Changes in plasma arginine vasopressin concentration during lactation in rats.

BACKGROUND AND PURPOSE: The objectives of the study reported here were to determine whether a change in the plasma arginine vasopressin (AVP) concentration occurred in lactating, compared with non-lactating rats and to examine the involvement of suckling with plasma AVP concentration. METHODS: Experiments were performed on 86 female Wister Imamichi rats, 12 weeks old at parturition, with fast lactation. On day 13 of lactation, AVP concentration and plasma osmotic pressure were measured in lactating and non-lactating rats. RESULTS: Plasma AVP concentration was always higher in rats of the lactating groups than in non-lactating controls (1.06 +/- 0.28 pg/ml), and a conspicuous increase in AVP concentration was seen during the postsuckling period (1.70-0.61 pg/ml before vs. 2.56 +/- 1.31 pg/ml after suckling, P < 0.05). Plasma osmotic pressure in lactating rats with 12 pups (296.6 +/- 5.2 mOsmol/kg x H2O) was lower than that in rats of the removed control groups (306.7 +/- 5.7 mOsmol/kg x H2O). CONCLUSION: On the basis of these results, it appears that "low plasma osmotic pressure-high AVP status" develops in the lactating period, similar to pregnancy, through resetting of the regulatory mechanism of the AVP system. It was concluded that suckling stimulation could release AVP, which could dilute the blood with water resulting in the increase in circulating blood volume.

Sequential alterations in clinical biochemical indicators of renal function in 5/6 nephrectomized rats--basic study for renal toxicity using 5/6 nephrectomized rats.

It has been established that progression of renal lesions in 5/6 nephrectomized rats, in which the total right kidney was removed and two poles of the left kidney were excised surgically, and are used as an animal model of renal failure in man, can be morphologically divided into three stages. In the present study, for establishing a renal toxicity study using this animal model on the physiological side, changes of biochemical parameters were sequentially investigated in 20 male 5/6 nephrectomized Wistar rats until 26 weeks after nephrectomy. Creatinine clearance (CLcre) and water as well as electrolyte reabsorption (FRwater and FR Na, K, Cl) were reduced at weeks 2-4, then increased slightly from weeks 6 to 10, and reduced again thereafter. On the contrary, urinary protein was elevated throughout the experimental period, while albumin fraction was increased after week 2 and low-molecular tubular protein increased after week 6 by electrophoresis. Urinary LDH also demonstrated high levels throughout the observation period, but ALP only increased after week 18. The present study thus confirmed that renal function after 5/6 nephrectomy is indeed changed in three stages with clinical biochemical parameters, especially CLcre and FR Na, K, Cl being good indicators to distinguish the three stages of glomerular and tubular dysfunction, respectively. In addition, urinary protein-fractions by electrophoresis in this animal model were examined for the first time, proving useful approach to glomerular and tubular dysfunction.

Model of emotional stress in rats.

A new experimental rat model for suppression of release of arginine vasopressin (AVP) was evaluated. Release of AVP is potentiated by physiologic stimuli, but is suppressed by emotional stress. Rats were exposed to the aggressive behavior of other rats injected with 6-hydroxydopamine hydrochloride (6-OHDA) in both lateral ventricles and subjected to a pain stimulus applied to the tail. To ascertain whether emotional stress is induced by use of this method, plasma corticosterone, glucose, and AVP concentrations were measured in the stressed group of rats (n = 8) placed near a group of aggressive (fighting) rats. Characteristic changes in behavior, significant increases in plasma corticosterone and glucose values, and lack of increase in plasma AVP concentration in stressed rats confirmed that they experienced emotional stress. This new model meets the experimental requirements for suppression of AVP release in rats.

Blood sampling technique for measurement of plasma arginine vasopressin concentration in conscious and unrestrained rats.

A new technique for collecting blood from conscious and unrestrained rats is reported. Plasma arginine vasopressin (AVP) concentration has been known to be influenced by various procedures used for blood collection (e.g., manual contact and anesthesia). In the study reported here, the improvement of the routine jugular vein cannula method was undertaken by extending the connection tubing to allow use of a swivel mechanism. Plasma AVP, glucose, and corticosterone concentrations in virgin rats were measured in blood samples collected by use of the new method and of two generally accepted methods (decapitation and routine jugular vein cannula methods). All values of the three parameters obtained were the lowest when the new method was used, suggesting that this new swivel method induced the least stress in the rats.

Para-chloroamphetamine (PCA)-induced ejaculation in aged rats, semen analysis and artificial insemination.

The present study was undertaken to determine sperm motility and counts in semen yielded by para-chloroamphetamine (PCA)-induced ejaculation of aged rats which had lost their reproductive ability, and to attempt artificial insemination with suspensions of spermatozoa obtained in this way. The semen yielded by PCA-induced ejaculation from aged (75-week-old) rats had average sperm counts of 0.82 +/- 0.69 x 10(7), which were much lower than the average counts (9.42 +/- 1.65 x 10(7)) for semen spontaneously ejaculated by young adult rats (14 weeks old). However, 77.5% of the spermatozoa contained in the PCA-induced semen were rated as showing the most active movement. Spermatozoa collected in this way were injected into the upper parts of both uterine horns or into both ovarian bursae. Both methods made the females pregnant, but the results were better after injection into the ovarian bursae. The offspring born to these females showed no abnormalities.

Effects of streptozotocin-induced diabetes on receptive and proceptive behaviors in female rats.

Sexual dysfunction in diabetic men is thoroughly recognized, while not yet done in diabetic women. Recently, Streptozotocin (STZ)-induced male rats showed a significantly depressed copulatory behavior, compared with normal animals. We investigated whether STZ-induced diabetic female rats would produce observable deficits in sexual behavior. Results in the present study are the first to show that STZ-induced diabetic female rats have a depressed sexual behavior.

Effects of bromocriptine (CB-154) treatment on copulatory behavior in hyperprolactinemic adult male rats.

Adult male rats received pituitary transplantations to induce hyperprolactinemia. Some of those had bromocriptine (CB-154) treatment, and the other had vehicle (saline) as control. The grafted animals with vehicle exhibited significantly more suppression than sham operated animals. While the hyperprolactinemic rats treated with CB-154 showed no significantly more suppression of copulatory behavior than sham operated control. In the grafted animal without CB-154 treatment, the concentration of serum PRL was significantly higher than in sham operated controls. There were no significant differences in adrenal weight and serum levels of gonadotropin between grafted and control groups. These results may imply that PRL has direct actions on central nervous system (CNS) and inhibits copulatory behavior in hyperprolactinemic male rats.

[Copulatory behavior after ectopic pituitary grafting in male rats].

It is known that suppression of copulatory behavior observed in aged male animals is due to elevate prolactin levels, the so-called hyperprolactinemia, found in these animals. The present study was done to determine whether hyperprolactinemia can modify the copulatory behavior of young adult male rats. Hyperprolactinemia was induced by grafting one or two pituitary glands of adult males of the same strain under the kidney capsules of each sexually experienced male. Grafted animals exhibited suppressed copulatory behavior patterns when tested one month after pituitary transplantation. The animals given two pituitary grafts differed from the sham-operated controls in latency to intromit (P < 0.05), as well as in the numbers of intromission (P < 0.05) and ejaculation (P < 0.01). After tests of copulatory behavior, serum concentrations of LH, FSH, prolactin and testosterone were measured by radioimmunoassay. Serum levels of prolactin were significantly increased (P < 0.05) in the animals given 2 grafts, compared with sham-operated controls. From these results, suppression of copulatory behavior in the young adult animals grafted was similar to that in aged adults. The findings suggest that decline of copulatory behavior with increasing age results from chronic elevation of prolactin.

Changes of plasma osmotic pressure during lactation in rats.

It is known that blood and plasma volume increase during lactation. The present paper examines whether an increase in plasma volume is accompanied by the change in plasma composition or attributed to hydro-dilution. Six dam-nursed pups and six dam-removed pups housed individually were designated as lactating rats and control rats, respectively. The plasma osmotic pressure and hematocrit value (Ht) were measured in the rats on days 3, 5, 7, 10, 13 and 18 of lactation. The total plasma protein (TP) and serum sodium concentration were also measured as they are factors affecting the plasma osmotic pressure. In addition, milk yield was estimated by the Morag technique. On day 5 and after day 10, the osmotic pressure of the lactating rats was found to be significantly lower than that of the control rats. The serum sodium concentration (days 5 and after day 10) and Tp values (days 3, 10 and 18) of the lactating rats were significantly lower than those of the control rats. Except on day 5, the Ht values of the lactating rats were significantly lower than those of the controls. During the period between days 3 and 10, milk yield was increased and it become steady (18 g/12 hr) on days 10 and 18. On and after day 10 when rats secreted a large amount of milk, it is considered that a decrease in the plasma osmotic pressure was mainly attributed to the reduction of sodium concentration by hydro-dilution. The Ht values indicate that an increase in blood volume is mainly through plasma volume rather than blood cell volume in lactating rats.

[Comparison of sexual behavior patterns in small laboratory animals].

It is known that in the reproductive systems, aged-related changes are prominent. Similarly, in normal nonhuman primates and other mammals such as rats, various sexual activities of male and female decline with aging. The purpose of the present study was to clarify the pattern of sexual behavior in matured mice, voles, Syrian hamsters, rats, guinea pigs, rabbits and house musk shrews of both sexes, and to select the appropriate animals for studies of aged-related changes in sexual behavior. There were species differences among laboratory animals in the pattern of sexual behavior. In summary, hamsters and rats ejaculated about three times during 30 min, while mice did once during 10 hrs. The former animals showed the intromission and ejaculation within a few min and 10 min, respectively, the latter did the ejaculation a few hrs after introduction of a heat female. In female animals, rats displayed receptive and proceptive behaviors, while hamsters and guinea pigs did only receptive behavior. From the above results, rats of both sexes may be recommended for studies of aged-related changes in sexual behavior.

Production of transgenic rats using pregnant and pseudopregnant rats prepared at a breeding farm.

We used pregnant and pseudopregnant Wistar-Imamichi rats, prepared at a breeding farm, for production of transgenic rats. The donor and recipient rats were transported to our facility for ova manipulation and embryo implantation, respectively. As a foreign gene, 1.9 kb of a hybrid gene was constructed with the rat renin promoter and chloramphenicol acetyltransferase-coding gene. Fertilized oocytes were collected from the donor and the foreign DNA was microinjected into the pronuclei of 539 oocytes by the method of Hochi. The manipulated oocytes were cultured to the 2-cell stage. One hundred seventeen 2-cell embryos were implanted in the recipient rats. Of 67 newborns, 55 rats grew up to be 3-week-old weanlings. Genomic DNA was extracted from the tail of these weanlings and examined by polymerase chain reaction analysis. Six transgenic rats were found to have been generated by the present method. In this way, transgenic rats were produced by an efficient combination of breeding farm utilization and laboratory research.

[Fertility of male Syrian hamsters paired with one, two and four proestrous females each].

In order to improve the productivity of Syrian hamsters, we investigated whether a male could impregnate overnight more than one proestrous female. Twenty-one males and 49 females aged 8 to 10 weeks were used in this experiment. The mating systems were as follows: one male was paired with one female, one male was paired with two females, and one male was paired with four females. Six out of 7, 7 of 14 and 4 of 28 females became pregnant and gave birth under these three systems, respectively. We conclude that it is possible for a male to impregnate overnight about one female under the above mating systems.

Copulatory behavior of sexually inexperienced male guinea pigs paired with synchronized estrus females.

The copulatory behavior in sexually inexperienced male guinea pigs aged 12 weeks was observed under dim red illumination. The synchronized estrus females, which were used for receptive females, received a subcutaneous implantation of progesterone-filled tubing for 2 weeks, followed by 0.5mg progesterone injection. Females given these treatment exhibited the lordosis behavior at 19:00 and 21:00 h on 4th day, and had ovulated by 09:00 h on 5th day after removal of implant. During a 2-hrs test session, standard measures of copulatory behavior were recorded. The ejaculation could be observed within the first 30 min in 6 of 7 males. The mounts, intromissions and ejaculation in male guinea pigs were shown in a lower frequency and longer latency than those of rats and hamsters.

Effects of p-chloroamphetamine (PCA) on ejaculation in aged rats.

In general, sexual activity in normal animals declines with increasing age. In aged male rats, for example, no ejaculation occurs when paired with receptive females. The ejaculatory response induced by p-chloroamphetamine (PCA) in aged adult rats, 14 to 18 months of age, was observed. The animals were treated intraperitoneally with PCA. The ejaculation rates were 0.28.6, 57.1 and 100% at doses of 0.3125, 0.625, 1.25 and 2.5 mg/kg, respectively. These animals, with a few exceptions, showed an ejaculation within 2 hrs of PCA injection. These data for aged adults are similar to those for young adults.

[The change with age of the copulatory behavior of the male rats aged 67 and 104 weeks].

Changes with age in sexual fertility in male rats of the Wistar-Imamichi strain were investigated. Litter size and numbers of implantation sites in females mated with males aged 14 to 44 weeks tended to decrease with increased aging of males. These indicators in females mated with males aged over than 44 weeks decreased, compared with that aged 14 to 44 weeks (P < 0.05 to 0.001). In addition, all females mated with males aged 95 weeks were not pregnant. In male copulatory behavior with aging, incidences and numbers of mounting at 67 and 104 weeks of age were 100%, 42.6 +/- 15.3 (Mean +/- S.D.) and 42%, 1.8 +/- 2.8, respectively. The intromission and ejaculation were observed at 67 weeks, but not at 104 weeks of age. These incidences and frequencies were 86%, 10.3 +/- 10.2 for intromission, and 43%, 1.0 +/- 1.3 for ejaculation. Mount latencies (ML) at 67 and 104 weeks of age were 535.4 +/- 607.9 and 3,822.0 +/- 1,753.4 sec, respectively. There were significant differences in ML between two groups (P < 0.05). At 67 weeks of age, intromission and ejaculation latencies, and post-ejaculatory intervals were 2,563.0 +/- 2,216.3, 1,633.7 +/- 977.6, and 657.3 +/- 320.6 sec, respectively. The pregnant rate and numbers of implantation sites in females mated with males aged 67 weeks were 43% and 5.4 +/- 6.8, respectively. In females mated with males aged 104 weeks, all were not pregnant. The weights of tests, epididymides, ventral prostate and seminal vesicles at 104 weeks were light, compared with those at 67 weeks of age (P < 0.05). From these results, the copulatory behavior in male rats of the Wistar-Imamichi strain declined with aging. Sexual fertility in males aged 67 weeks decreased in less than 50%, and males aged 104 weeks showed no intromission and ejaculation.

[Effect of age on the copulatory behavior of the male rat].

Fifteen male SPF Wistar-Imamichi rats, aged 10, 27 and 44 weeks, that had been raised in the authors' laboratory, were used for the observation of copulatory behavior. Each male was given the opportunity to mate with a proestrous female. During a two hour period, standard measures of copulatory behavior, e.g., mounting, intromission and ejaculation, were documented by a video recording. The following results were obtained; the number of ejaculations in 10-week-old rats, over a two hour period, was 6 to 8. Males showed a subsequent post-ejaculatory interval. The males aged 27 weeks performed 3 to 4 times during the first hour, and then refrained from sexual activity. The copulatory behavior in males aged 44 weeks declined, as compared with that of their 10 and 27 weeks old counterparts.

Effects of naloxone and naltrexone on receptive and proceptive behaviors in female rats.

The sexual receptive and proceptive behaviors induced by opiate antagonists, naloxone and naltrexone in estrogen-primed ovariectomized rats were observed under the presence of sexually active males. The females were treated intraperitoneally with naloxone or naltrexone at doses ranging from 0.5 to 4.0 mg/kg and the sexual behavior of females was tested before and after the injection of drug. The results obtained suggest that the opiate antagonists play a role in the regulation of lordosis behavior, but not proceptive behavior in female rats.