Characterization of an in vivo model for the study of eyelash biology and trichomegaly: mouse eyelash morphology, development, growth cycle, and anagen prolongation by bimatoprost.

BACKGROUND: Hypertrichosis or alopecia of the eyelashes is associated with various diseases or may be drug induced. Although neither increase nor loss of eyelashes is life threatening, eyelash disorders can be psychologically disturbing. However, as control of eyelash growth and the underlying mechanisms of eyelash hypo- or hypertrichosis are largely obscure, available therapy is limited. OBJECTIVES: To improve this situation, we sought to establish a pragmatic, well-defined mouse model for the study and pharmacological investigation of eyelash follicle biology. METHODS: We took a morphometric approach to establish an eyelash model using female C57BL/6J mice by comparing with pelage hairs and highlighting the differences. We next applied a hypertrichosis-triggering agent and investigated its effect using the model. RESULTS: In eyelashes, a synchronized growth cycle was observed after morphogenesis but was completed earlier than pelage hairs. Exogen was strictly regulated and occurred in every cycle in the eyelash. Otherwise, general morphological features of mouse eyelashes (shafts, follicles, morphogenesis and growth cycle) were comparable with those of pelage hairs. The eyelash growth-stimulatory agent in humans, bimatoprost, significantly extended the duration of anagen, resulting in more and longer eyelashes, but there was no evidence of follicle neogenesis. CONCLUSIONS: This study shows that mouse eyelashes offer an excellent in vivo model for the quantitative and qualitative analysis of eyelash morphology, development, growth cycle, exogen and pharmacological modulation. This model will help to elucidate the unknown molecular controls of eyelash growth, and to develop novel drugs to treat eyelash disorders.

Role of the nrf-2 gene in protection and repair of gastric mucosa against oxidative stress.

Helicobacter pylori infection, as well as NSAIDs induce oxidative stress on gastric mucosa, thereby causing mucosal damage and retarding mucosal repair. Cells can survive against chronic oxidative stress by enhancing activities of anti-oxidant enzymes, thereby protecting cells from DNA damage. Recent studies have clearly shown that the gene encoding Nrf-2 (NF-E2 p45-related factor-2) plays an important role in the induction of antioxidant enzymes against oxidative stress. In this paper, we will describe the cellular mechanisms by which the nrf-2 gene stimulates anti-oxidant enzyme activities during exposure to oxidative stress. Secondly, we will also mention the beneficial effects of sulforaphane, an isothiocyanate family which is abundantly included in broccoli sprouts, on gastric mucosa. Sulforaphane stimulates nrf-2 gene-dependent anti-oxidant enzyme activities, thereby protecting cells from oxidative injury. Finally, we will state our perspective on the efficacy of sulforaphane in protection and repair of gastric mucosa against oxidative stress during H. pylori infection.

A prospective randomized trial of either famotidine or omeprazole for the prevention of bleeding after endoscopic mucosal resection and the healing of endoscopic mucosal resection-induced ulceration.

BACKGROUND: It has been reported that inhibitors of gastric acid secretion prevent bleeding after endoscopic mucosal resection for mucosal gastric neoplasm. However, uncertain whether an histamine2-receptor antagonist or proton-pump inhibitor is more effective. AIM: To evaluate prospectively the effectiveness of famotidine or omeprazole for ulcer management after endoscopic mucosal resection. METHODS: From July 2003 to October 2004, 57 patients were randomly assigned to famotidine or omeprazole for the management of endoscopic mucosal resection. Both drugs were given intravenously for the first 2 days, thereafter by mouth. The bleeding rates after endoscopic mucosal resection, the effects on the healing of endoscopic mucosal resection-induced ulceration, and cost-benefits were compared. RESULTS: Twenty-eight patients received famotidine and 29 received omeprazole. No significant difference was observed between the two groups in patient characteristics. The bleeding rates after endoscopic mucosal resection were not significantly different (18% vs. 14%) between the groups. Similarly, no differences were seen in the size of the endoscopic mucosal resection-induced ulceration at 1, 30 and 60 days after resection between groups. The total costs of anti-secretory agents demonstrated a significant cost-benefit to those treated with famotidine (10,420 yen vs. 17,782 yen). CONCLUSIONS: Famotidine is suggested as a better alternative to omeprazole for the management of endoscopic mucosal resection, as it showed a clear cost-benefit, and the healing results after endoscopic mucosal resection were similar for the two treatment strategies.

Immunohistochemical survey of the gut endocrine cells in the common tree shrew (Tupaia belangeri).

Regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the common tree shrew (Tupaia belangeri) were studied immunohistochemically. Ten types of immunoreactive endocrine cells were localized in the gastric mucosa, i.e., chromogranin-, serotonin-, gastrin-, somatostatin-, bovine pancreatic polypeptide (BPP)-, enteroglucagon-, pancreatic glucagon-, peptide tyrosine tyrosine (PYY)-, motilin-, and substance P (SP)-immunoreactive (IR) cells. In the intestine, 13 types of immunoreactive cells were observed, i.e., chromogranin-, serotonin-, somatostatin-, gastrin-, BPP-, enteroglucagon-, PYY-, secretin-, cholecystokinin (CCK)-, gastric inhibitory peptide (GIP)-, motilin-, neurotensin-, and SP-IR cells. The regional distribution and relative frequency of the cell types varied along the gastrointestinal tract. Basically, the types, distribution, and relative frequency of the gut endocrine cells were similar to those reported in other mammalian species. However, some characteristic findings were noted in the present study: (1) the considerably large number of gastrin-IR cells in the pyloric region; (2) numerous serotonin-IR cells in the stomach; (3) appreciable number of BPP-IR cells in the transitional region of the stomach; and (4) wide distribution of PYY- and motilin-IR cells in the gut.

Immunohistological studies of metabotropic glutamate receptor subtype 6-deficient mice show no abnormality of retinal cell organization and ganglion cell maturation.

Immature retinal ganglion cells (RGCs) initially show a multistratified dendritic pattern, and, during the postnatal period, these dendrites gradually monostratify into ON and OFF sublaminae. The selective agonist of group III metabotropic glutamate receptors (mGluR), L-2-amino-4-phosphonobutyrate (L-AP-4), hyperpolarizes ON bipolar cells and reduces glutamate release. On the basis of L-AP-4-evoked inhibitory effects on ON-OFF segregation of developing RGCs, it has been hypothesized that glutamate-mediated synaptic activity is crucial for formation of the ON-OFF network. Gene-targeted ablation of mGluR6 specifically expressed in ON bipolar cells blocks normal ON responses but has been predicted to enhance glutamate release from ON bipolar cells. The mGluR6 knock-out mouse therefore provides a unique opportunity to investigate whether glutamate release and ON responses are important factors in the development of ON-OFF segregation. The combination of several different morphological analyses indicates that ON bipolar cells, as well as several distinct amacrine cells, in mGluR6 knock-out mice are normally distributed and correctly extend their terminals to defined retinal laminae. Importantly, both alpha and delta RGCs in adult mGluR6 knock-out mice are found monostratified into cell type-specific layers. Furthermore, no difference between wild-type and mGluR6 knock-out mice is observed in the maturation and dendritic stratification of developing RGCs. Hence, despite a deficit in normal ON responses, mGluR6 deficiency causes no abnormality in the retinal cellular organization nor in the stratifications of both ON bipolar cells and developing and mature RGCs. Based on these findings, we discuss several possible mechanisms that may underlie ON-OFF segregation of RGCs.

Retinal neurones specific for centrifugal modulation of vision.

The avian retina receives centrifugal projections from a midbrain nucleus, the isthmo-optic nucleus. We labelled target cells for the isthmo-optic fibres by intracellular injections of Lucifer Yellow in fixed retinal slices. The isthmo-optic recipient (IOR) cells had no major dendrites extending into the inner plexiform layer, but had a thin axon-like process running horizontally in the junction between the inner nuclear layer and the inner plexiform layer. The IOR cells were morphologically similar to the association amacrine cells of Cajal. Immunohistochemical localization of aspartate and glutamate in the IOR cells suggested that they may use these excitatory amino acids as neurotransmitters.

Adrenal tissue characterization with 0.5-T MR imaging: value of T2*-weighted images.

The authors investigated the value of magnetic resonance (MR) imaging at 0.5 T for distinguishing adrenal adenomas from adrenal metastases. The series included 23 adrenal adenomas (18 nonhyperfunctioning, five hyperfunctioning) and 23 adrenal metastases from various organs. Adrenal tumor-liver signal intensity ratios on T1-, T2-, and T2*-weighted images were calculated for adrenal tissue characterization. Adrenal adenomas were more precisely distinguished from adrenal metastases on T2*-weighted images (21 of 23, 91%) than on T2-weighted images (15 of 23, 65%). T1-weighted images were not useful for this distinction. In conclusion, T2*-weighted images were better than routine T2-weighted images for distinguishing adrenal adenomas from adrenal metastases. It can be postulated that the total signal intensity of adrenal adenomas, which contain some fat components, decreased on T2*-weighted images because of an out-of-phase effect.

Trace elements and mineral requirements for very low birth weight infants in rickets of prematurity.

To establish mineral and trace element requirements for very low birth it is important to prevent bone mineral disorder. Those infants fed mother's milk only are thought to be at higher risk of this disorder. Both calcium and phosphorus supplementation were thought to be needed to prevent it. Copper and zinc are important as cofactors of major enzymes involved in the synthesis of collagen. These trace elements especially zinc may not be enough for very low birth weight infants fed mother's milk. At present however the relationship between these trace elements and minerals, and bone metabolic disease in preterm infants is not completely clear.

Morphological comparisons between outer and inner ramifying alpha cells of the albino rat retina.

The somato-dendritic morphologies of large ganglion cells were studied by intracellular injections of Lucifer yellow in perfused in vitro preparations of the albino rat retina. The ganglion cells were prelabeled with retrogradely transported granular blue or labeled with acridine orange dropped into the perfusate of in vitro preparations. After the dye injection, somato-dendritic morphologies were successfully studied for 210 cells, the majority of which had a large soma more than 20 microns in diameter and were identified as alpha cells. According to the level of dendritic extensions within the inner plexiform layer (IPL) these alpha cells were further classified into inner ramifying (inner) and outer ramifying (outer) cells. Both qualitative and quantitative observations led us to conclude the following: 1) The outer cells have a spherical soma with relatively few primary dendrites, while inner cells have a large polygonal soma with more primary dendrites. 2) The dendritic field of inner cells was always larger than that of outer cells at every retinal location. The dendritic field diameter tended to increase as a function of retinal eccentricity from the optic disk, the tendency being more clear among inner cells. 3) The dendrites of outer cells branch more frequently in the proximal part of the dendritic field while those of inner cells branch more distally. 4) Total dendritic length of outer cells increases linearly with eccentricity whereas that of the inner cells does not change much irrespective of retinal location.

Single cell shape and population densities of indoleamine-accumulating and displaced bipolar cells in Reeves' turtle retina.

Two types of bipolar cell in the Geoclemys reevesii retina were studied quantitatively by means of specific cell labelling with an indoleamine derivative (5,6-dihydroxytryptamine, 5,6-DHT), a nucleic acid stain (4,6-diamidino-2-phenylindole, DAPI) and Lucifer yellow CH. Indoleamine-accumulating (IA) bipolar cells were selectively labelled with 5,6-DHT applied intraocularly. After the cells accumulated 5,6-DHT, the indoleamine fluorescence was photoconverted to diaminobenzidine products to allow observation of morphological details. Close examination of many cells (cell number; n = 120) showed that the IA bipolar cells consist of a single morphological type whose axon collaterals ramify sublaminae 1, 4 and 5 respectively. This terminal branching pattern corresponds to cells that hyperpolarize when their receptive field centres are illuminated (Weiler 1981). The density of IA bipolar cells was highest in the visual streak (4130 cells mm-2) and lowest at the peripheral margin (1970 cells mm-2). By applying a small amount of DAPI to the eye, nuclei located in the most proximal row of the outer nuclear layer were labelled selectively. By using selective intracellular dye injection into DAPI-labelled cells under fluorescence microscope (Tauchi & Masland 1984, 1985), these cells were found to have Landolt's clubs and single descending axons. Dye injections into more than fifty DAPI-labelled somata showed that they belonged exclusively to displaced bipolar cells. These comprised at least two subtypes that differ in the ramification pattern of their axon terminals within the inner plexiform layer: one was monostratified, whereas the other was bistratified. The displaced bipolar cell density was as high as 9400 cells mm-2 in the central retina, falling to 2000 cells mm-2 in the superior margin. In vitro Lucifer labelling revealed that the overall bipolar cell density in the central retina was as high as 39,300 cells mm-2. Both the conventionally located and displaced bipolar cells were included in this population. About 11% of the total bipolar cell population consisted of IA bipolar cells. Assuming that one half of the conventionally located bipolar cells are the centre-hyperpolarizing type, IA bipolar cells represent approximately 28% of the total. As displaced bipolar cells represent almost one quarter of the total bipolar population, the dislocation of their somata stands out morphologically, inviting investigation of possible functional correlates.

Shapes and distributions of the catecholamine-accumulating neurons in the rabbit retina.

Rabbit retinas were fixed with mixed aldehydes and examined for the fluorescence of catecholamines. Labeled cell bodies were present in the layer of the amacrine cells. A band of fluorescent processes was present in layer 1 of the inner plexiform layer. Weaker labeling was present in two deeper strata, one near the middle of the inner plexiform layer (presumably layer 3) and one at the junction of layers 4 and 5. Immunohistochemistry showed tyrosine hydroxylase (TH) to be present in the same cells and the same strata of the inner plexiform layer as the endogenous catecholamines. Exposing the retina to exogenous dopamine or norepinephrine resulted in stronger labeling in the middle and deep levels of the inner plexiform layer. At the same time a second population of amacrine cell bodies became visible. Catecholamine fluorescence contained in the amacrine cell bodies was used as a guide to their injection with Lucifer yellow CH. The filled dendritic arbors revealed two main types of cells. The type 1 cells are monostratified at the most distal level of the inner plexiform layer. They have relatively uncomplicated, radially branching dendritic trees. They are the cells densely stained by immunohistochemistry with antibodies against TH. The type 2 cells are tristratified, with minor branching in layer 1 of the inner plexiform layer and major branching in the two deeper sublayers. The descending dendrites follow a complicated course, and it is not uncommon for intermediate dendrites to cross between strata more than once. The relationship of the cells to their dendritic plexuses was further studied in retinas in which the aldehyde-induced fluorescence of catecholamines was photoconverted to a diaminobenzidine product. The type 1 cells were found to dominate the plexus of dendrites in layer 1 of the inner plexiform layer. The catecholaminergic plexuses in the middle and deep levels of the inner plexiform layer are formed by dendrites of the type 2 cells. The position of every type 1 cell was mapped in retinas stained with antibodies directed against TH. In one retina we counted 5,613 type 1 cells, distributed evenly across the retina. In another retina, all of the catecholamine-accumulating cells were counted. There were 9,058 with a distribution that peaks in the visual streak. The type 1 cells appear to be the dopaminergic cells previously studied by others and thought to regulate the flow of information from rod bipolar cells to ganglion cells. The low density and wide spread of type 2 cells suggests that they, too, perform a generalized control function, presumably a novel one that dictates their intricate, tristratified shape.

Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat.

The coexistence of immunoreactivities for choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in some brain regions of the rat, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for ChAT and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the retina, cerebral cortex, basal forebrain and spinal cord, colocalization of ChAT-like and GAD-like or GABA-like immunoreactivities was observed in some cell types, whereas no such colocalization was observed in cells in the striatum or brainstem. In the retina, the majority of ChAT-like immunoreactive (ChAT-LI) amacrine cells contained GABA-like or GAD-like immunoreactivity. About half of the ChAT-LI neurons in the cerebral cortex showed GABA-like immunoreactivity. In the basal forebrain only a small proportion of ChAT-LI neurons (0.6%) contained GAD-like immunoreactivity. In the spinal cord, about one-third of ChAT-LI central canal cluster cells and about half of ChAT-LI dorsal horn cells showed GAD-like and/or GABA-like immunoreactivities. These observations indicate the possible coexistence of two classical transmitters, GABA and acetylcholine, in various brain regions and spinal cord of the rat.

Effects of stimulus intensity and duration on photoresponses of single rods in the frog retina.

Electrical responses to light flashes were recorded from single red rods in dark-adapted retinas of the bullfrog, Rana catesbeiana. When the flashes were less than or equal to 50 ms in duration, plots of the peak amplitude of the responses as a function of log stimulus intensity were found to be parallel, and the stimulus intensity and duration required to elicit criterion-amplitude responses showed a linear relation with a negative unit slope. Furthermore the waveforms of equal-amplitude responses to flashes of different intensities and durations were superimposable. With increases in the stimulus duration beyond 50 ms, however, the slopes of the response-log intensity curves for the higher stimulus intensities decreased, the slope of curves describing the intensity required to elicit criterion responses became less negative, and the responses of the rods decayed more slowly. These results indicate that within 50 ms after the onset of flash stimuli, the effect of incident photons summate linearly to evoke rod responses of certain sub-saturating amplitudes when assessed in terms of the peak response amplitude as well as the response waveform.

Specificity of naturally occurring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus: determination by an enzyme linked immunosorbent assay.

Serum autoantibodies to poly(ADP-ribose), single-stranded (ss) DNA and double-stranded (ds) DNA in 145 patients with systemic lupus erythematosus (SLE) were measured by an enzyme-linked immunosorbent assay (ELISA). The specificities of the antibodies for poly(ADP-ribose) or for ssDNA in 14 serum samples from different patients, who had relatively high antibody titers to either or both these antigens, were tested by competitive ELISA with poly(ADP-ribose) and ssDNA as inhibitors. The IgG class anti-poly(ADP-ribose) antibodies of 4 serum samples (cases 9, 11, 13 and 14) preferred poly(ADP-ribose) and those of 2 samples (cases 2 and 4) cross-reacted preferentially with ssDNA, while the IgG class anti-ssDNA antibodies of 2 serum samples (cases 9 and 11) significantly cross-reacted with poly(ADP-ribose). Hence, the nature of the antibodies to poly(ADP-ribose) in SLE patients seemed to be different from that of the anti-poly(ADP-ribose) antibodies in autoimmune MRL/Mp-lpr/lpr (MRL/l) mice, which seem to be subpopulations of anti-ssDNA antibodies and react equally well with poly(ADP-ribose) and ssDNA.

Local order among the dendrites of an amacrine cell population.

The cholinergic amacrine cells of the rabbit retina branch within a narrow stratum of the retina's inner synaptic layer, and their dendritic fields overlap as much as 70-fold. Because each cell's dendrites have many branches, the overlap must create a dense meshwork of cholinergic dendrites. To learn how the overlapping dendrites are positioned with respect to each other, we filled the dendrites of groups of neighboring cells with Lucifer Yellow CH. The cholinergic amacrine cells were selectively stained by intraocular injection of the fluorescent molecule 4,6-diamidino-2-phenylindole. The retinas were then fixed with 2% paraformaldehyde and 0.01% glutaraldehyde. The stained cells were penetrated under visual control by Lucifer Yellow-filled micropipettes. A systematic arrangement of the dendrites was observed. When a pair of cells was injected, their dendrites were often seen to lie alongside each other. In the terminal dendritic region, there are virtually no dendrites that do not end in apposition to a dendrite of a neighboring cholinergic amacrine cell. When small clusters of nearby cells were injected, an ordered microstructure appeared. The dendrites of the cells join together to form curving bundles, which enclose spaces that rarely contain any cholinergic dendrites: the appearance of the dendritic mosaic is that of a lattice with a repeating unit roughly 10 microns in diameter. The significance of this ordering is not certain, but it is possible that the repeating structural unit participates in a modular functional arrangement.