Search

Purpose: To determine if the corneas of naive mice contain resident CD4+ and CD8+ T cells., Methods: The presence of T cells in the corneas of naive BALB/c, C57BL/6, and SCID mice was determined by immunostaining with anti-CD4 (clone RM4-5) and anti-CD8 (clone 5H10-1) monoclonal antibodies. Immunostained corneal sections were examined by light microscopy, and immunostained intact corneas were examined by confocal microscopy. The levels of CD4 and CD8 mRNA transcripts in the corneas were determined by TaqMan reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and compared with the expression of these transcripts in the corneas of HSV-1 infected mice. Finally, the number of CD4+ and CD8+ T cells in the cornea of BALB/c, C57BL/6, and ICR mice was determined by cell sorting., Results: Both light microscopic examination of corneal sections and confocal microscopic examination of intact corneas revealed the presence of CD4+ and CD8+ cells in the central and peripheral regions of the corneas of BALB/c and C57BL/6 mice. Stained cells were not detected in corneas of control SCID mice. CD4 and CD8 mRNA transcripts were detected in corneas of BALB/c and C57BL/6 mice while there were markedly lower levels of transcripts in SCID mice. The number of CD4 transcripts was lower than the number of CD8 transcripts in the corneas of both BALB/c and C57BL/6 mice. Finally, cell sorting showed the presence of both CD4+ and CD8+ T cells in corneas of BALB/c, C57BL/6, and ICR mice., Conclusions: CD4+ and CD8+ T cells are present in corneas of naive C57BL/6, BALB/c, and ICR mice.

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2-5-day-old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express mu-, delta-, and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5-bromo-2'-deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP(-)/S100beta(-). When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors., (Copyright 2001 Wiley-Liss, Inc.)

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.

A lectin angiography method was applied to identify ovarian vasculature in 32-34 days old mice treated with gonadotropins. Blood was washed out by perfusion until the inferior vena cava became translucent. Horseradish-peroxidase (HRP)-conjugated Concanavalin A (Con A) solution (10-15 ml) was perfused into the systemic circulation via the left ventricle at the rate of 2-3 ml/min. The animals were left for a 30 min reaction interval. The lumina of the blood vessels were flushed with 5-10 ml of phosphate buffered saline (pH 7.4). The ovaries were excised and fixed by immersion in 4% paraformaldehyde and sectioned serially at thickness of 100, 200 or 400 microm using a Microslicer. The binding of HRP-conjugated-Con A to endothelial cells was visualized by using 3,3'-diaminobenzidine-4 HCl (DAB-4 HCl) reaction. By examining various sections the three-dimensional architecture of the vascular networks of the preovulatory Graafian follicles and corpora lutea can be established. Capillary networks of preovulatory Graafian follicles were identified in sections of ovaries removed 11 h after hCG injection. Then, capillary networks of the Graafian follicles increased due to the hypertrophic growth of the theca interna and extension of capillary branches into the follicles. Well-developed capillary networks of corpora lutea were found in ovaries removed 24 h after hCG injection. For these observations the 200 microm-sections were the most useful. The present modified lectin angiography is a useful method for visualizing the microvasculature of mouse ovaries.

Recent studies of the urethral glands in the male mouse and rat have suggested that they are testosterone-dependent glands that may be potential sites for secretory immunity in the male genital tract. In the present study we describe the ultrastructural features of these glands in normal mice and provide quantitative data on the sizes of the acinar cells and their organelles in sham-, oil-, and testosterone-treated castrated mice. Acinar cells in urethral glands from normal mice contain numerous secretory granules, prominent Golgi complexes, elongated mitochondria, and an abundance of rough endoplasmic reticulum (RER) with large and dilated cisternae, all of which are features characteristic of secretory cells. In some acinar cells the cisternae of the RER were filled with closely packed, unbranched, straight, tubular structures that were oriented parallel to one another, that radiated from aggregates of dense material, or that were randomly arranged. In other acinar cells the cisternae of the RER showed a network of branching and anastomosing vesicular-like structures whose limiting membranes were occasionally seen in continuity with the membranes of the RER. Secretory acini showed large, unbranched tubules in the acinar lumen. When cut at right angles the large tubules exhibited a distinct fuzzy outer coat with fine projections radiating outwards. The ultrastructure of the acinar cells and the presence of tubules in the lumen suggests that they are engaged in secretion of a tubular protein. Morphometric analysis of acinar cells in the urethral glands showed that the mean volumes of nuclei, cytoplasm, secretory granules, vacuoles, and mitochondria were significantly reduced in castrated mice in comparison to either normal or testosterone-treated castrated mice. This confirms earlier observations that the urethral glands are targets of testosterone.

Mouse embryos were explanted at 11.5 days of gestation and cultured for periods of 6, 12, 18, 24, 30, 36, 42 and 48 hours using the whole embryo culture system. A continuous flow of gas (5% CO2+95%O2) was maintained through the culture bottles during the culture periods. The cultured embryos were morphologically and histologically examined at the end of the designated periods. The growth of the embryos was depressed after about 36 hours, and differentiation was retarded as the culture time increased. General development had ceased almost completely by 42-48 hours. At 18-36 hours, the external auditory canal and pinna, which are not developed in 11.5 day embryos, could be observed clearly on the surface of the head. After 42 hours, the facial features including the external ear were similar to those of 13 day in vivo embryos. Serial sections of the ear region of cultured embryos showed that development of the inner and middle ear structures up to 42 hours was the same as that seen from 11.5 to 13 days in vivo. In the inner ear, the otocyst had developed into the vestibule, cochlea and semicircular canals by 12-30 hours. In the middle ear, the stapedial anlage appeared and its circular shape became clear between 0 and 12 hours. Subsequently, the medial portion of the stapedial anlage grew until it was adjacent to the developing otic capsule, and the anlages of other ossicles were also recognizable after 18 hours. Differentiation of the horizontal portion of the facial nerve was noted at the same time that stapedial anlage and otic capsule were developing.(ABSTRACT TRUNCATED AT 250 WORDS)

The intestinal Peyer's patches (PP) were significantly different in distribution and fine morphology between Mongolian gerbils and mice. The PP of gerbils were evenly distributed from the duodenum to ileum, whereas the PP of mice were more densely distributed in the lower ileum than other parts of the intestine. In gerbils, each PP had a much greater number of lymphoid follicles than in mice, although the total number of PP was smaller. Electron microscopy revealed that the PP dome of gerbils was covered with two types of epithelial cells, one with shorter microvilli and the other with longer ones, whereas the epithelial cells of the murine PP dome was uniform-shaped with numerous medium-sized microvilli. Some dome absorptive cells of gerbils were morphologically similar to poorly differentiated crypt cells of mice, suggesting to be immature M cells.

This study provides new information concerning the distribution of cartilage collagens in neonatal mammalian condylar cartilage. It became apparent that young cartilage cells contain pro-Type I collagen as well as pro-Type II collagen. The mature molecule of Type I collagen appears only in the extracellular matrix of the mineralisation zone close to the ossification front. Type II collagen, on the other hand, is apparent throughout the extracellular matrix as soon as the chondroprogenitor cells have differentiated into chondroblasts. In addition, Type II collagen is noticed in the core of the newly formed bone trabeculae within the primary spongiosa. Type IX collagen was found to be co-distributed with Type II collagen in cartilage and bone. Hypertrophic chondrocytes within the mineralisation zone, but in no other zone, demonstrate an intense reactivity for Type X collagen. Mild reactivity throughout the condylar process is encountered with regard to Type VI collagen. Perichondrium but not cartilage reacts positively for Type III collagen.

The length and weight of gastrointestinal tracts including contents of ten week old male pikas (Ochotona rufescens rufescens) and suncus (Suncus murinus) were measured and they were investigated and compared with those of millardias, ICR-mice and Wistar-rats. The length from the duodenum to the anus of pikas was much longer and the weight from the stomach to the anus was about 16g per 100g of body weight. The weight of cecum was about 7g per body weight and they were heavier than those of other species. The length from the duodenum to the anus of the suncus was short but the weight of the small intestine plus colon and rectum per body weight did not differ from that of other species. The suncus has no cecum but the weight from the stomach to the anus was almost the same as that of rats.

20 ICR mice (adult females) were used for analyzing the axonal projection of the trigeminal mesencephalic tract neurons and 10 Japanese shrew-moles for analyzing that of the snout proprioceptive neurons. The HRP-labeled axons were found to be ipsilaterally terminated in the trigeminal motor nucleus, supratrigeminal nucleus and trigeminal main and spinal tract nuclei, lateral pontine-medullary reticular formation, vagal dorsal motor and hypoglossal nuclei and the lamina V of the C2 spinal cord segments. No HRP-labeled axons were found in the facial and solitary nuclei and the cerebellum. Also, the functional localization of the trigeminal mesencephalic tract neurons was analyzed with the retrograde tracers of fluorescent compounds injected into the jaw-closing muscles having spindles. The fluorescent-labeled neurons were found to be intermingled throughout the nucleus and clustered at the caudal level of the nucleus. Also, double or triple fluorescent-labeled neurons were not observed in the nucleus. The HRP-labeled axon bundle of the facial proprioceptive neurons are divided rostro-caudally into the shorter ascending and the longer descending roots, both running closely dorsal to the trigeminal spinal tract nucleus. The ascending root lies adjacently dorsal to the spinal tract nucleus, giving off the terminal fibers to it, to the wider area of the dorso-medial pontine nuclei and finally to the cerebellar nuclei. At the level of the facial inner genu, it turns medially to meet the facial nerve root, giving off the terminal fibers to the facial motor nucleus and to the raphe nuclei and to the opposite nuclei bilaterally. The HRP-labeled descending root takes the course caudally at least down to the C3 segment of the spinal cord, giving off the terminal fibers to the spinal tract nucleus, the nuclei of the IXth, Xth and XIIth cranial nerves and the pontine-medullary reticular formation. In the spinal cord, it descends bilaterally through the posterior fascicles, giving off the terminal fibers to the dorsal and ventral horns.

The histochemical staining, labeling index and incorporation of [3H] thymidine [TdR] in large intestinal epithelium were compared in four anatomically distinct segments from ICR/Ha and C57Bl/Ha mice. This comparison was done because the incidence of 1,2-dimethylhydrazine (DMH)-induced carcinomas is different for different anatomic segments as well as for the two strains. Within each strain, the amount of [3H] TdR incorporated into mucosal DNA was found to vary less than 20% at each anatomic site of the large intestine. However, there were site-specific differences in the depth of the proliferative populations within the crypts. In autoradiograms from both strains, the crypts of the proximal colon showed maximal [3H] TdR labeling of nuclei in mid-crypt cells, some of which contained mucin. In contrast, the distal colon and rectum were characterized by maximal nuclear labeling in a population of undifferentiated cells near the base of each crypt. In distal ICR/Ha colon, the proportion of labeled nuclei at each crypt depth corresponded to the [3H] TdR labeling of DNA that had been isolated from frozen sections cut sequentially. As visualized with alcian blue-periodic acid Schiff (AB-PAS) and high iron diamine-alcian blue (HID-AB) stains, the epithelial mucin showed site-specific differences, but the differences between the two strains of mice were not remarkable. In contrast to the human and rat large intestine, the acidic mucin in the mouse large intestine was predominantly sialomucin. However in the cecum and mid-distal colon, there was a predominance of sulfomucin. In the various anatomic segments of both strains, the histochemical staining, labeling index and incorporation of [3H]TdR were remarkably similar considering the large differences in susceptibility to chemically-induced neoplastic change.

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increase. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.

Three hundred CD-1 HaM/ICR mice were observed for 2 years, and useful necropsies were done on 99 males and 102 females. Mortality was 50% at 16 months in the males and 18 months in the females. Among mice that came to autopsy, total tumor incidence was 54% for males and 75% for females, with most neoplasms occurring after 18 months. Adenomas or adenocarcinomas of the lung were the most frequent, followed by lymphoreticular tumors, vascular tumors, hepatomas, subcutaneous fibrosarcomas, and adenocarcinomas of the mammary glands. Some degree of amyloidosis was seen in half the mice of both sexes, beginning at 8 months in males and 12 months in females. Variability in tumor incidence among small groups if mice emphasized the need for adequate samples.

The heart weight of germfree mice has been reported to be lighter than that of conventional mice. The difference in heart function between these mice, however, has not been well investigated. In the present study, we recorded ECGs of germfree, ex-germfree and conventional mice anesthetized with pentobarbitone sodium and weighed the hearts of these animals. On ECGs, there was no significant difference between germfree and conventional mice except QT and PP intervals, which were shorter in male germfree mice than those in male conventional mice. It was found that the heart weight of germfree mice was significantly lighter than that of conventional mice in both sexes. It was concluded that although the heart weight between germfree and conventional mice was different, the heart function evaluated using the ECG was not.

Female germfree (Gf) and conventional (Cv) ICR mice aged 21-25 days were administered with hCG (50IU/head/day for 2 days). Six hours after the last administration, uterine weights of Gf and Cv mice were 175.8 mg% and 238.7 mg% respectively, the difference between Gf and Cv animals was statistically significant (p less than 0.05). Ovariectomized Gf and Cv mice aged 80-150 days were successively administered with estradiol benzoate (0.02 mg/head/day for 5 days). Six hours after the last administration, uterine weights of Gf and Cv mice were 358.8 mg% and 462.7 mg% respectively, being statistically significant between Gf and Cv animals (p less than 0.01). After administration of estradiol benzoate (0.02 mg/head/day for 2 days), 1.0 mg/head/day of progesterone for 3 days were given ovariectomized Gf and Cv mice aged 80-150 days. Six hours after the last administration, uterus of Gf and Cv mice were weight 337.6 mg% and 423.5 mg% respectively, the difference was statistically significant (p less than 0.01). Castrated Gf and Cv mice aged 80-150 days were administered with 1.0 mg of testosterone propionate (head/day for 5 days). Six hours after the last administration, weights of seminal vesicle and coagulating gland of Gf and Cv mice were 376.0 mg% and 611.8 mg% respectively, the difference was statistically significant (p less than 0.01).