Populations of follicles in F344/N rats during aging.

Follicular populations were investigated in female F344/N rats to better understand the aging process of the rat ovary. Ovaries dissected at various ages (spanning 1-36 months old) were submitted for histological examination. The total number of primordial, growing (primary and secondary), tertiary, and atretic follicles as well as corpora lutea (CL) were counted in hematoxylin-eosin- and azocarmine-aniline-blue-stained ovarian sections. The number of healthy follicles including primordial, growing and tertiary follicles decreased rapidly between the first and third months and gradually thereafter. CL were found in 3-month-old rats, and their number remained unchanged until 18 months of age, at which point it decreased. The number of atretic follicles started to increase in rats older than 18 months, which corresponded to the cessation of estrous cyclicity. Several healthy follicles and CL were observed even in 36-month-old rats.

Immunohistochemical localization of aquaporin-4 in the rat pituitary gland.

Aquaporin-4 (AQP4) is an isoform of membrane water channel aquaporins. It is most abundant in the brain, and is believed to be expressed by astrocytes and ependymal cells. We have reported previously that rat pituitary glands express some aquaporin subtypes, including AQP4, but the precise distribution of AQP4 in the pituitary gland is not known. The present work investigated whether AQP4 immunoreactivity exists in various pituitary cell types in adult rats, using double immunofluorescent staining and confocal microscopy. In the adenohypophysis, co-labeling for S-100 protein indicated that folliculo-stellate (FS) cells and marginal layer cells in Rathke's residual pouch have extensive labeling for AQP4. Some AQP4-expressing cells also contained glial fibrillary acidic protein (GFAP) in parenchymal tissue of the anterior lobe, and cytokeratin in marginal layer cells. AQP4 was not coexpressed in any hormone-producing pituitary cells, however. In the neurohypophysis, AQP4 was expressed in some pituicytes, which have been identified as GFAP-positive. These results suggest that AQP4, which is expressed in FS cells, in marginal layer cells and in pituicytes, but not in hormone-producing cells, is important in water transfer within the pituitary gland; astrocytes and ependymal cells do the same in the brain.

Aging change of mandibular condyle in female F344/N rat.

To ascertain whether laboratory rats represent an adequate animal model for aging oral cavity research, this study focused on the morphology of the mandibular condyle. Aging changes of cartilaginous conformation and shape of the mandibular condyles were analyzed in female F344/N rats. In the condylar cartilage, articular, proliferative cell and hypertrophic cell layers were observed in 1-month-old (M) rat, whereas flattened cell layer was notable at 2M. A mature cell layer was observed in the condylar cartilage of rats at 7.7 M and over. Deranged cartilaginous layers and thinning articular layer were observed in 30.9 M rat. The sagittal length of the condyle decreased, whereas the frontal one increased with aging and/or age. There were three phases in the transition patterns of the size of the condyle, which seemed to correspond to the respective growing, aging and senescence phases in the rat. The results suggested that degenerative change of condylar cartilage in rat was similar to that in human, whereas change of the shape of the condyle was different between rat and human, caused by a different pattern of mastication.

Calcitonin gene-related peptide immunoreactive neurons innervating the soft palate, the root of tongue, and the pharynx in the superior glossopharyngeal ganglion of the rat.

We have examined whether calcitonin gene-related peptide immunoreactive (CGRP-ir) neurons in the glossopharyngeal ganglia innervate the soft palate, the root of tongue, and the pharynx of the rat. Immunohistochemical observations revealed that numerous CGRP-ir neurons are located in the superior glossopharyngeal ganglion located ventrolateral to the medulla oblongata in the cranial cavity, and that CGRP-ir neurons are also located in the inferior glossopharyngeal ganglion at the jugular foramen. When Fluorogold was injected into the soft palate, the root of tongue, or the pharyngeal constrictor muscles, many retrogradely Fluorogold-labeled neurons were found in the superior glossopharyngeal ganglion and the nodose ganglion, and several Fluorogold-labeled neurons were found in the inferior glossopharyngeal ganglion. Double labeling with immunohistochemistry for CGRP and Fluorogold showed that in every case of injections of Fluorogold into the soft palate, the root of tongue, or the pharynx, about 30% of the Fluorogold-labeled neurons in the superior glossopharyngeal ganglion expressed CGRP-like immunoreactivity, while no double-labeled neurons were found in the inferior glossopharyngeal ganglion or the nodose ganglion. These results indicate that nociceptive sensory information from the soft palate, the root of tongue, and the pharynx might be conveyed by the neurons in the superior glossopharyngeal ganglion to the nucleus tractus solitarii.

Bone mineral analysis through dual energy X-ray absorptiometry in laboratory animals.

To determine how to eliminate species difference in animal bone experiment, bone mineral content (BMC) was measured using dual energy X-ray absorptiometry (DXA) on the femurs of laboratory mice (Mus musculus) and rats (Rattus norvegicus), and common marmosets (Callithrix jacchus). Measures were taken on femurs in situ, detached from the body, skinned and defleshed, or dried completely. When the BMC of the bone measured in the intact limb attached to the trunk was set at 100%, the actual BMC of the dry bone was 58.7 +/- 11.5% in mice and 103.2 +/- 3.2% in rats. Similarly, the bone area (Area) and bone mineral density (BMD) of the dried femur was significantly lower in the mouse femurs than intact limb. Thus, soft limb tissue such as skin and muscle modified the BMC, Area, and BMD only in mouse but not in those from rats or marmosets. The bone mineral ratio (BMR; BMC divided by dry bone weight) was nearest to the human bone value in the rat femurs, whereas the mouse femur BMR was the most different. The BMR was proved to be a practical index in evaluating bone characteristics in laboratory animals, but the mouse femur might not be suitable as an animal model for research into the aging of human bone.

Body and major organ weights of A/J-Chr 11(SM) consomic mice.

The body and major organ weights of A/J-Chr 11(SM) consomic mice were compared to those of the progenitor strains, A/J and SM/J. The weights of the body and organs, except for those of the brain and uterus, were significantly greater in A/J mice than in SM/J mice. However, those of consomic mice were highly variable. For example, the average body weight of consomic mice was significantly greater than that of SM/J mice and nearly equal to that of A/J mice. Chromosome 11 of SM/J mice induced various significant changes of the organ weights of A/J mice, especially those of the brain, lung, kidney, adrenal, and ovary, demonstrating the importance of this chromosome in determining the phenotypes.

Distribution of vagal CGRP-immunoreactive fibers in the lower esophagus and the cardia of the stomach of the rat.

We have examined whether the smooth muscle fibers in the lower esophagus and the cardia of the stomach of the rat are innervated by calcitonin gene-related peptide-immunoreactive (CGRP-ir) fibers coming from the nucleus ambiguus. Immunohistochemical observations revealed that there were many CGRP-ir fibers and free endings in all external muscular layers of the lower esophagus and the cardia. Occasionally, bundles of CGRP-ir fibers were found in the inner oblique muscle layer of the cardia. There were also many CGRP-ir fibers in the mucous membrane in the lower esophagus and the cardia. When Fluorogold was injected into the junction of the lower esophagus and the cardia, many retrogradely labeled neurons were found in the compact formation of the nucleus ambiguus and the dorsal motor nucleus of the vagus nerve. Double labeling with immunohistochemistry for CGRP and the retrograde tracer Fluorogold showed that almost all of neurons (more than 90%) in the nucleus ambiguus that project to the lower esophagus or the cardia contained CGRP, while no CGRP-ir neurons were found in the dorsal motor nucleus of the vagus nerve. These results indicate that the vagal motor neurons of the nucleus ambiguus that contain CGRP project not only to the striated muscle fibers of the esophagus but also to the smooth muscle fibers of the external muscle layers of the lower esophagus and the cardia.

A morphometric study of the adrenal cortex of the female DDD mouse.

The aim of this study was to determine whether the thickness of the adrenocortical zone is associated with age in virgin and parous female DDD mice. The zona reticularis and zona glomerulosa of parous mice tended to be thicker than those of virgin mice at all ages. The zona fasciculata lactating parous mice was significantly thicker than that of virgin mice at 20 weeks of age (P<0.01). Age did not affect the thickness of the three outer adrenocortical zones in either group. However, in virgin mice, the X zone consisted of vacuolated and nonvacuolated cells at 5 weeks of age and only of vacuolated cells at 10 weeks of age; the number of vacuolated cells and the thickness of the zone decreased at 40 weeks of age. In contrast, parous mice of all ages lacked an X zone. The decrease in X zone thickness with age was most evident when expressed relative to organ weight. In conclusion, the thickness of the outer three adrenocortical zones is affected by endocrine changes associated with pregnancy and lactation but not by age. The thickness of the X zone is an index of growth and maturation in nulliparous female DDD mice less than one year of age.

Occlusal tooth wear in male F344/N rats with aging.

With the aim of clarifying the aging properties of an animal model, the progress of occlusal tooth wear (OTW) of molars in male F344/N rats was monitored. Dried maxilla and mandible specimens from 61 male F344/N rats, aged 7 to >30 months, were used. The levels of OTW of all molars were monitored with aging. The cuspis dentis of molar teeth were worn out by 7 months (M) of age, and the occlusal surface became flat. As for each molar tooth (M(1-3), numbered in accordance with its position), OTW of M(1) was more severe in the lower than in the upper jaw, whereas M(3) was more severe in the upper than the lower jaw. OTW of M(2) in both the upper and the lower jaws progressed rapidly after 27M. OTW in male F344/N rats progressed faster than in females. However, when compensated for life span, both genders had similar profiles in OTW progress with aging. This study suggested that male rats were more convenient than females as a model for gerodontological research because of the earlier course of OTW progress.

Direct synaptic projections to the myenteric ganglion of the rat subdiaphragmatic esophagus from the dorsal motor nucleus of the vagus.

We have examined the ultrastructure of the myenteric ganglion of the subdiaphragmatic esophagus and determined whether the ganglion neurons receive direct projections from the dorsal motor nucleus of the vagus (DMV) using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) as an anterograde tracer. The neurons (22.2 microm x 13.3 microm) of myenteric ganglion in the esophagus contained dark cytoplasm having many free ribosomes, mitochondria, and an oval nucleus, and received only a few axon terminals contacting somata. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Approximately 85% of the axon terminals contacting dendrites and about 50% of the axon terminals contacting somata contained pleomorphic vesicles, while the rest contained round synaptic vesicles. When WGA-HRP was injected into the DMV, anterogradely labeled fibers and terminals were found in the myenteric ganglia. The WGA-HRP labeled terminals were large (1.97 microm) and contained round clear vesicles and small granular vesicles. These labeled terminals contacted exclusively the small dendrites, but not the somata. These results suggest that the DMV neurons project directly to the myenteric ganglion neurons and regulate the esophageal muscles via the ganglion neurons.

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum.

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold-silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62+/-0.07microm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20-80nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99+/-0.07microm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.

Expression of aquaporin water channels in the rat pituitary gland.

We investigated the expression of aquaporin (AQP) subtypes (AQP1-AQP9) in the rat pituitary gland by the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Specific amplification by RT-PCR was observed in AQP1, AQP3, AQP4 and AQP5 but not in the others. AQP1 immunoreactivity was detected in the endothelial cells of blood vessels. No positive reaction to AQP3 was observed by immunohistochemistry, in spite of the detection of mRNA. AQP4 was localized in the supporting cells of the adenohypophysis and neurohypophysis. AQP5 was expressed in parts of the marginal cells in Rathke's residual pouch. These observations suggest that several subtypes of the AQP family are involved in regulation of water homeostasis in the rat pituitary gland.

Morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid of the rat.

The cornea is sensitive to nociceptive stimuli and receives dense sensory innervations from the trigeminal ganglion, which also innervates the upper eyelid. We investigated the morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid. We injected the retrograde tracer Fluoro-Gold (FG) into the cornea and the retrograde tracer cholera toxin subunit b (CTb) into the upper eyelid of the same animal. Less than 10% of the FG-labeled neurons were also labeled with CTb. The FG-labeled neurons were small (29.6+/-0.6microm), while the CTb-labeled neurons were large (36.1+/-0.5microm). We also characterized the neurons in the trigeminal ganglion with the retrograde tracer FG following its injection into the cornea or the upper eyelid, and immunohistochemical double-labeling with nociception-related neuronal markers, such as calcitonin gene-related peptides (CGRP), transient receptor potentiated vanilloid 1 (TRPV1), and substance P (SP). About 27% of the neurons innervating the cornea were double-labeled with CGRP, about 23% with TRPV1, and about 8% with SP. About 4% of the neurons innervating the upper eyelid were double-labeled for CGRP, about 11% for TRPV1, and 3% for SP. Thus, the percentages of double-labeled neurons for the neurons innervating the cornea were higher than those for the neurons innervating the upper eyelid. These results indicate that the cornea and the upper eyelid receive innervations mainly from different neurons of the trigeminal ganglia. The cornea is innervated by many characteristic sensory neurons containing nociception-related neuronal markers.

Occlusal tooth wear in female F344/N rats with aging.

OBJECTIVES: This study was conducted to ascertain whether laboratory rats are an adequate animal model for aging oral cavity research, especially on occlusal tooth wear (OTW), which progresses with aging and causes abnormal occlusions. Mastication has been reported to relate to cognition in the elderly. Thus, it is important to care for the oral cavity, especially in the frail elderly, for the maintenance of all-round quality of life. Adequate and appropriate animal models are essential for basic and clinical research on the oral cavity. METHODS: Dried maxilla and mandible specimens from 98 young, aging or aged female F344/N rats were used. RESULTS: The levels of OTW of all molars were monitored with aging. The molar tooth began to wear at 1-month old (M) and progressed rapidly till 12M. Subsequently, OTW progressed slowly till 30M, and then rapidly again after 35M. CONCLUSIONS: This study showed that progress of OTW is well correlated with the entire life span of the rat, and suggested that the rat aged over 12M would be an adequate animal model for research on OTW in middle-aged and elderly people.

Changes of estrous cycles with aging in female F344/n rats.

Changes of estrous cycles with aging of F344/N rats between 1 and 30 months of age (M) were monitored by vaginal smear cytology. The vaginal opening and first cornified cell phase were identified at 1.3 +/- 0.1 M and 1.5 +/- 0.2 M, respectively. Thereafter, estrous cycles showed about 5-day intervals, and ceased at 16.4 +/- 1.2 M. Thereafter irregular appearance of single cornified cell phases without the preceding of nucleated cell phases interspersed with a predominant leukocyte phase was seen in vaginal smears until 26.9 +/- 0.5 M. Growing and mature follicles as well as corpora lutea persisted until at least 30 M, and characterized the post reproductive aging of F344/N females. The F344/N rats seem to resemble humans in that the cessation of estrous cycles occurs at approximately half their entire lifespan. However, other aging characteristics are unknown in postmenopausal women. Therefore, we must be careful when extrapolating the aging changes of reproduction in F344/N rats to human beings.

Morphology and ultrastructure of the sympathetic celiac ganglion neurons projecting to the cardia and pylorus of the rat stomach.

The stomach receives sympathetic projections from the celiac ganglion. To determine what kinds of neurons in the celiac ganglion project to the cardia or the pylorus of the stomach, we injected the retrograde tracer Fluoro-Gold into the cardia and the retrograde tracer cholera toxin subunit b into the pylorus of the same animal. A few neurons (about 10%) innervating the cardia sent collateral projections to the pylorus. Ultrastructural observations revealed that the celiac ganglion contained oval, medium-sized to large neurons. They had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum, mitochondria, lysosomes, several Golgi apparatuses, and an oval nucleus. The axon terminals were small and usually contacted thin processes extending from the dendrites or the soma. About half of the terminals contained round vesicles, while the rest contained pleomorphic vesicles. Both types of terminals made asymmetric synaptic contacts. We then retrogradely labeled the neurons projecting to the cardia and the pylorus with wheat germ agglutinin conjugated horseradish peroxidase to examine their ultrastructural characteristics. The neurons projecting to the cardia (33.3x22.4 microm) were similar to the neurons projecting to the pylorus (33.4x24.7 microm) in their size and ultrastructural appearance. The neurons not projecting to the stomach (40.4x28.0 microm) were significantly larger than the neurons projecting to the cardia or the pylorus. Only a few axosomatic terminals were found on the neurons projecting to the cardia (1.6 per somatic profile), the pylorus (1.3) or the neurons not projecting to the stomach (0.9). These results provide morphological bases for the sympathetic motor neurons innervating the stomach.

Natural dental caries in molars of osteogenic disorder Shionogi rats.

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.

Comparison of rat mandible bone characteristics in F344 substrains, F344/Du and F344/N.

The characteristics of the mandible bone were compared through DXA methods between two major substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc at around 60 days of age. Since these two substrains are clearly different in survival and mandible morphology, some genetic differences are supposed to exist. In contrast to a previous microsatellite analysis, clear and significant differences were detected in the body and mandible weights, the mandible bone mineral contents (BMC), bone area (AREA), bone mineral density (BMD) and bone mineral ratio (BMR), between F344/DuCrlCrlj and F344/NSlc, with the mandible molar teeth intact in the bone. Thus, care is needed in the experimental use of these substrains, as results may differ between them. The newly proposed parameter, BMR, may especially contribute to the comparison of bone characteristics among species.

Morphometric comparison of the rat mandible in F344 substrains, F344/Du and F344/N.

The shape of the mandible was compared by morphometric methods to ascertain the genetic differences between two substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc. Since these two substrains are clearly different in survival and the incidence of age associated disorders; thus, some genetic differences are suggested to be present between them. Although previous microsatellite analysis did not detect any differences between the two F344 substrains, the present study clearly detected interesting differences in the mandible morphology. At 2 months of age, the F344/Du mandible was characterized by a larger size, especially in length, than the F344/N mandible. The shape of the mandible seemed to be more variable in F344/N. This clear substrain difference suggests the importance of the substrain recognition in F344 rats, especially in experimental usage.