Morphological evaluation of cranial and maxillary shape differences of the brachymorphic mouse with spontaneous malocclusion using three-dimensional micro-computed tomography.

OBJECTIVES: The aim of this study was to determine whether significant cranial and maxillary deformity exists in BALB/c-bm/bm (brachymorphism) mouse with spontaneous malocclusion using three-dimensional (3D) images. MATERIALS AND METHODS: Thirty female mice were divided into the following three groups: control group (BALB/c mice, n = 10), Norm group (BALB/c-bm/bm mice with normal occlusion, n = 10), and Mal group (BALB/c-bm/bm mice with malocclusion, n = 10). Nine points in the skull were selected, and transverse and antero-posterior distances were measured using three-dimensional images of micro-computed tomography (CT). Moreover, 3D images were superimposed at the median plane to visualize the skull shape asymmetry. RESULTS: The transverse distances at the posterior cranial and maxillary region and the antero-posterior distances in the Norm and Mal groups were significantly shorter than those in the control group. The nasal septum of the Mal group was significantly shorter than that of the Norm group. Morphological measurements and superimposed 3D images showed that lateral deviation occurred at the anterior cranial and maxillary region in the Mal group. CONCLUSION: The 3D micro-CT images revealed that the antero-posterior length and posterior transverse width at the cranium and maxilla in BALB/c-bm/bm mice were significantly smaller than those in BALB/c mice. It was quantitatively and morphologically clear that BALB/c-bm/bm mice show a spontaneous transverse crossbite owing to lateral deviation of the maxilla and nasal bone.

Melatonin Its intracellular and genomic actions.

Besides its actions via membrane receptors on cells in specific areas of the brain and peripheral tissues, melatonin also has direct intracellular actions. The neurohormone melatonin is a potent free radical scavenger in vitro and a powerful antioxidant in vivo. Studies to date indicate that it is a better scavenger of the highly toxic hydroxyl and peroxyl radicals than some other known compounds. Melatonin also prevents the toxicity of singlet oxygen and stimulates the antioxidative enzyme, glutathione peroxidase. Considering its varied and potent antioxidant capability, it is possible that melatonin is an essential element of the antioxidant defense system of organisms. Besides these direct intracellular actions of melatonin, it was recently discovered that the indole also has binding sites in the nucleus of many cells. Melatonin's genomic actions are believed to follow its binding to these nuclear loci.

Muscarinic cholinergic and glutamatergic reciprocal regulation of expression of hippocampal cholinergic neurostimulating peptide precursor protein gene in rat hippocampus.

Hippocampal cholinergic neurostimulating peptide, an undecapeptide originally isolated from the hippocampus of young rats, enhances acetylcholine synthesis in rat medial septal nucleus in vitro. Hippocampal cholinergic neurostimulating peptide is derived from the N-terminal region of its 21-kmol.wt precursor protein. The highest expression of the hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA is in hippocampal pyramidal neurons. In an in vitro rat hippocampal slice, preparation in which electrical stimulation could be delivered to the Schaffer collateral-CA1 pyramidal cell synapse, semi-quantitative non-radioisotopic in situ hybridization, demonstrated that expression of the hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA is regulated by neuronal activity. Selective inhibition with pharmacological agents revealed that the constitutive hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA level can be up-regulated by D-(-)-2-amino-5-phosphono-valeric acid, and that activity-dependent transcription can be inhibited by tetrodotoxin, nifedipine, 6-cyano-7-nitroquinoxaline-2,3-dione, and scopolamine, but not by mecamylamine. These results indicate that septal cholinergic neurons and hippocampal glutamatergic neurons exert a reciprocal influence over the expression of hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA in the hippocampus, and that the activity-dependent and constitutive expressions of hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA may be regulated by different routes, involving calcium influx via L-type Ca(2+) channels and N-methyl-D-aspartate receptors.

A new histochemical method for detection of sialic acids using a physical development procedure.

We have established an efficient histochemical method for demonstration of sialic acids in light microscopy. The method consists of a selective periodate oxidation-phenylhydrazine blockade and a thiocarbohydrazide-silver protein sequence followed by a physical development procedure. From the results obtained by the present experimental and control studies in tissue sections from a series of rat and mouse organs, such as stomach, duodenum, colon, liver, sublingual gland, lung, and kidney, the specificity and sensitivity of the method were sufficient. In the tissues tested, sialic acids were visualized as distinct brownish and blackish reaction products. Comparisons of the new method with the periodic acid-phenylhydrazine-Schiff (PA-P-S) or selective periodate oxidation-Schiff (PA*-S) method employed hitherto have confirmed that the new method reported here is higher in efficiency and visibility of reaction products than the latter two methods.

A method for histochemical demonstration of protein-bound amino groups by light microscopy.

We have established an efficient method for the histochemical demonstration of protein-bound amino groups by light microscopy, using a ninhydrin or alloxan-thiocarbohydrazide-silver proteinate (NHY or ALX-TCH-SP) sequence followed by a physical development (PD) procedure. As a result of the present experimental studies on Carnoy's solution-fixed paraffin sections of a series of rat tissues, including three types of major salivary glands, liver, pancreas, stomach, duodenum, jejunum, colon, kidney, prostate, and spleen, the sensitivity and specificity of the new method were found to be sufficient. In the tissues tested, protein-bound amino groups were visualized by distinct brownish or blackish reaction products. Comparisons of the particular method with the NHY or ALX-Schiff methods employed hitherto have substantiated the fact that the former method leads to apparently higher visibility of reaction products than the latter.

Distribution of hippocampal cholinergic neurostimulating peptide (HCNP) immunoreactivity in the central nervous system of the rat.

Hippocampal cholinergic neurostimulating peptide (HCNP), an undecapeptide isolated from the hippocampal tissue of young rats, enhances the cholinergic development in explant cultures of medial septal nuclei. This report concerns the distribution of HCNP immunoreactivity in the central nervous system (CNS) of 11- and 28-day-old Wistar rats; two affinity-purified anti HCNP antibodies were used. Immunoblot analyses of extracts of different regions of the brain revealed a single 23 kDa band that corresponded to the presumed HNCP precursor protein. Immunostaining of the various CNS structures of the 28-day-old rats was more intense than in those of 11-day-old animals. HCNP immunoreactivity was detected in neurons as well as in glia cells, particularly oligodendroglia. The perikarya of neurons in the cerebral cortex, hippocampus, limbic cortex, caudate, putamen, arcuate nucleus of hypothalamus, trigeminal subnuclei, rostroventrolateral reticular nucleus and dorsal horn of the spinal cord were positively stained. In addition, nerve fibers and terminals in the hypothalamic subnuclei, zona incerta, thalamic subnucleus, caudate, putamen, locus coeruleus, trigeminal subnuclei, dorsal motor nucleus of the vagus, dorsal horn of the spinal cord and intermediolateral column also displayed HCNP immunoreactivity. These observations would suggest that HCNP and its related molecules may have multifunctional roles in the CNS.

Age-dependent changes in HCNP-related antigen expression in the human hippocampus.

Hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from the young rat hippocampus, enhances the cholinergic phenotype development of the medial septal nucleus in vitro. In this study, we examined the HCNP-antigen distribution and the age-related changes in the number of positive cells in the hippocampus (obtained at autopsy from 74 subjects with no known neurological disorders). Immunohistochemical assay revealed that the immunopositive cells were GABAergic neurons and oligodendrocytes. They were first identified in the fetus at around 25 to 30 weeks and their number increased rapidly with advancing postconceptional age to reach maximal at the perinatal stage and in early postnatal life; it then decreased to the adult level by 10 years old. These results suggest that HCNP-related antigen may play important roles in the development and/or differentiation of the human hippocampus.

Microencapsulation of pancreatic islets. A technique and its application to culture and transplantation.

Hamster pancreatic islets were encapsulated by a biocompatible membrane composed of the molecular sequence of alginate-polylysine-alginate. The encapsulated islets released insulin into the culture medium in response to secretagogues in short-term incubation. In long-term culture, the encapsulated islets maintained their insulin-releasing capacity for 28 days at a level similar to that of the unencapsulated islets. No overgrowth of fibroblastic cells was observed inside the capsule even after 70 days of culture. Further, the encapsulated hamster islets were xenotransplanted to streptozotocin-induced diabetic rats intraperitoneally. Some of the encapsulated islets, which were recovered from a recipient 27 days after transplantation, were found to be viable, although prolonged normalization of fasting plasma glucose levels of the recipients could not been achieved. On the contrary, the unencapsulated islets were replaced by massive connective tissue elements and insulin-positive B cells were hardly detected within the grafts 22 days after transplantation. The results of this study seem to confirm the potential of the application of the encapsulating technique to primary culture of parenchymal cells and to transplantation of pancreatic islets.

Human scalp hair as evidence of individual dosage history of haloperidol: a possible linkage of haloperidol excretion into hair with hair pigment.

We report a method for determining haloperidol concentration in human scalp hair and discuss a possible linkage of haloperidol excretion into hair with the hair pigment melanin. First, an animal study was conducted to support the idea that hair contains amounts of haloperidol corresponding to the doses given and pigmented hair contains much more drug than does unpigmented hair. The haloperidol concentration was measured using a radioimmunoassay technique after hairs were dissolved in 2.5 N NaOH solution and the drug extracted. Pigmented and albino rats, whose hair from an area on the back had been removed beforehand by plucking, were administered either 1, 3, or 10 mg of haloperidol (i.p.) per kg body weight every day for 3 weeks. At the end of the administration period hair which had newly grown on the denuded area was plucked and collected. In each of the two groups classified by hair color the drug levels in the hair correlated with the doses given; however, the concentrations in the hair from the albino rats were much lower than those in the hair from the pigmented rats (which was less than 8.5%). Second, black and white hair was collected from each of seven human subjects with grizzled hair, who were receiving or had been administered haloperidol at fixed daily doses for more than 1 month, and the concentration of haloperidol in each type of hair was measured. In the same subject the concentration in the white hair was found to be much lower than that in the black (less than 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin histochemistry of the canine anal glands.

The distribution and selectivity of complex carbohydrates in the canine anal glands were studied by means of lectin histochemistry, using PO-labeled lectins. The secretory epithelium of the anal glands and the excretory duct system exhibited large amounts of mainly neutral glycoproteins with various terminal sugars (alpha-D-mannose, beta-N-acetyl-D-glucosamine, alpha-N-acetyl-D-galactosamine, alpha-D-galactose, alpha-L-fucose, N-acetyl-neuraminic acid). Distinctly prominent in the secretion were alpha-L-fucose residues. This relatively hydrophobic sugar may in particular modify or control the viscoelastic properties of the anal gland mucus, so that a stable mucous coat of the rather dry faeces can be formed. In addition, it was obvious that the major part of the excretory duct system is also involved in secretion production, and that the essential function of the saccular dilatations of the excretory ducts is to ensure secretion maturation.

Effects of a triple enzyme digestion method on a diamine reaction for glycosaminoglycans of the rat aorta in electron microscopy.

Effects of a triple enzyme digestion method using glycosaminoglycans-degrading enzymes upon a diamine reaction have been tested for electron microscopic histochemical detection of glycosaminoglycans in extracellular matrix of the rat aorta. The triple enzyme digestion method consists of a sequence of chondroitinase B, testicular hyaluronidase and heparitinase. The results obtained by the present experimental and control studies indicated that dermatan sulfates, chondroitin sulfates (A and/or C) or heparan sulfates were apparently observed in various ultrastructural features of aortic extracellular matrix, such as bundle of collagen fibers and soluble matrix of interstitial space. Particularly, we found that both heparan sulfates and chondroitin sulfates (A and/or C) were detected in association with the basal lamina of smooth muscle cells and the external surface of elastic lamina, and in the latter heparan sulfates were frequently recognized as a mass, whereas chondroitin sulfates (A and/or C) were found intermittently along the external surface of elastic lamina. This suggests that the triple enzyme digestion method which combines the glycosaminoglycans-degrading enzymes with the diamine reaction can be postulated to represent efficient and useful technique for precise electron microscopic histochemical detection of the glycosaminoglycans in the extracellular matrix of the rat aorta.

Electron microscopic cytochemical studies of anionic sites in the rat spleen.

The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endothelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde -4% paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170-250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage.

Carbohydrate cytochemistry of the endothelium lining the splenic blood vessels in the rat.

In the endothelial cells lining the rat splenic blood vessels, neutral carbohydrates were studied by means of combined periodic acid-thiocarbohydrazide-silver protein (PA-TCH-SP) and alpha-amylase digestion methods. In the endothelial cells lining the central and follicular arteries of the spleen, the neutral glycoconjugate-containing surface coat of the luminal plasma membrane and related pinocytotic invaginations and vesicles in the apical cytoplasm were strikingly distinguished, as compared with those in the cells lining the splenic sinuses. In contrast, cytoplasmic glycogen particles in the sinus endothelial cells were apparently larger in amount than those in the arteriolar endothelial cells. Such cytochemical variations of neutral carbohydrates with the arteriolar and venous vessels of the rat spleen were discussed with special reference to varying cytophysiological functions of the endothelial cells with the different segments of the splenic blood vessels.

Lectin-binding glycoconjugates in the subfornical organ of the rat.

In the rat subfornical organ (SFO), lectin-binding glycoconjugates were histochemically examined by 9 biotinylated lectins using a streptavidin-biotin peroxidase system. The strong or moderate binding of Canavalia ensiformis (Con A), Lens culinaris (LCA) and Phaseolus vulgaris erythroagglutinin (ePHA) indicated overall distributions of high mannose, intermediate, hybrid N-linked, and non-bisected, bi/triantennate N-linked complex oligosaccharides in the rat SFO. Nonsialylated terminal N-acetylglucosamines were detected throughout this organ, as revealed by its stainabilities with Triticum vulgaris (WGA) and Limax flavus (LFA). In this organ, Ricinus communis (RCA-1) specifically bound to vessel-associated structures, whereas Arachis hypogaea (PNA) reacted with selected neurons in the central and rostral regions of this organ. Dolichos biflorus (DBA) and Ulex europaeus (UEA-1) did not stain any histologic structures in the rat SFO. The results obtained in this study provide a basis for comprehensive analyses of glycoconjugates in the rat SFO.

The use of periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) procedure for the histochemical detection of neutral carbohydrates in the circumventricular organs of the rat.

A periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) procedure was applied to the rat circumventricular organs (CVOs), which are known to be specialized structures in the brain. In the rat CVOs, vivid PA-TCH-SP-PD reactions were obtained in the apical surface and cytoplasm of the ependymal cells of the subcommissural organ (SCO) and the epithelial cells in the choroid plexuses (CPs) examined, and similar positive reactions were detected in the vascular walls and perivascular connective tissues in all the CVOs tested. Further, varying intensities of PA-TCH-SP-PD reactions were noted in the neuronal and glial networks of the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO) and area postrema (AP). The results obtained in the present study indicate that in the rat CVOs the histologic structures mentioned contain varying amounts of neutral carbohydrates and possible histophysiological significances of these carbohydrates in these organs have been discussed with references to their functions.

Effects of iodine pretreatment of sections upon immunogold-silver staining in light microscopic immunohistochemistry.

Effects of Lugol's iodine pretreatment upon immunogold-silver staining were examined in terms of the intensification of the reaction products obtained by physical development. Iodine pretreatment has diminished the duration necessary for the development, whereas the staining reaction of an appropriate intensity, could hardly be obtained by the pretreatment. In addition, essential staining reactions tended to be accompanied by marked nonspecific silver precipitations. It is concluded that iodine pretreatment should either be omitted or used with cautions in immunogold-silver staining procedures for the precise detection of antigenic sites.

A dual staining method for neutral carbohydrates and DNA (deoxyribonucleic acid) using periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) and Feulgen (Hot HCl-Schiff) techniques.

A dual staining method was established for the histochemical detection of neutral carbohydrates and deoxyribonucleic acid (DNA) in light microscopy. The method consisted of combined periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) and hot hydrochloric acid (H-HCl)-Schiff (Feulgen) procedures, which produced reaction products of blackish and magenta shades respectively. The present dual staining method is believed to be unusually useful for the light microscopic histochemical studies on neutral carbohydrate-containing cells, since it exhibits a high contrast between these two shades and reveals distinctly the localization of cytoplasmic neutral carbohydrates and nuclear DNA in one and the same tissue section.

Dual staining methods for carbohydrates using lectin-gold silver techniques.

Three dual staining methods were established for the histochemical detection of saccharide residues and acidic groupings of carbohydrates in light microscopy. The first method consisted of combined lectin-gold silver (LT-G-S) and alcian blue (AB) pH 1.0 techniques, whereas the second staining technique was composed of LT-G-S and AB pH 2.5 procedures. These two techniques were found to color saccharide residues and acidic groupings of carbohydrates in black and blue shades respectively, which exhibited a high contrast between the both. The third methods, LT1-G-S-LT2-PO-DAB techniques, yielded reaction products of blackish and yellowish brown shades, which represented the localizations of two different saccharide residues in one and the same section. According to the results of the experimental and control procedures, the present three dual staining methods are believed to be reliable, reproducible and unusually useful for light microscopic histochemical studies on acidic and non-acidic carbohydrates.

Lectin-binding glycoconjugates in the tongues of the rat and guinea pig as revealed by lectin-gold-silver methods.

Lectin-gold-silver (LT-G-S) procedures using two lectins (RCA-I and Con A) were applied to appropriately prepared paraffin sections of tongues in the rat and guinea pig. In the lingual mucous membranes of the rat and guinea pig, positive RCA-I-G-S and Con A-G-S reactions were obtained in the intercellular spaces and the cytoplasm of epithelial cells of the basal and intermediate layers respectively. Likewise, the LT (RCA-I and Con A)-G-S techniques gave rise to varying intensities of positive reactions in the serous and mucous gland acini, nerve and muscle fiber bundles and connective tissue elements. The results obtained in the present study indicate that in the rat and guinea pig tongues the histologic structures mentioned contain varying amounts of beta-D-galactose and alpha-D-mannose or alpha-D-glucose residues of glycoconjugates, and that such histochemical properties of some of the lingual structures can be correlated with their possible histophysiological functions.

HID-AB2.5-PAS and HID-PAS methods for the histochemical analyses of a variety of carbohydrates.

The high iron diamine (HID) staining procedure was combined with alcian blue pH 2.5 (AB2.5) and periodic acid-Schiff (PAS) methods for the simultaneous demonstration of carbohydrates containing sulfate esters, carboxyl groups and oxidizable vicinal diols, whereas it was sequentially employed with PAS alone to differentiate sulfate esters from oxidizable vicinal diols. A variety of rat tissues and the epidermis of four fish species were utilized to test the specificity or selectivity of these methods. The HID-AB2.5-PAS sequence largely coloured carbohydrates containing sulfate esters, carboxyl groups and oxidizable vicinal diols in brownish black, turquoise and magenta shades respectively. The HID-PAS sequence coloured sulfate esters brownish black and oxidizable vicinal diols magenta. On the basis of the experimental and control studies on the both staining techniques, these staining methods could be postulated to represent efficient and useful techniques for precise histochemical analyses and simultaneous differentiations of a variety of carbohydrates in light microscopy.