Primary aldosteronism associated with subclinical Cushing syndrome.

BACKGROUND: Recently, it has been reported that the incidence of primary aldosteronism (PA) among patients with hypertension is much more frequent than previously reported. AIM: In the present study, we investigated the frequency and features of PA associated with subclinical Cushing syndrome (SCS). MATERIAL AND METHODS: Subjects included consecutive patients (no.=39) who were diagnosed as PA and performed adrenal venous sampling between 2003 and 2011 in our institute. RESULTS: In 39 subjects who were diagnosed as PA, 29 patients were operated and 5 cases (12.8%) showed no suppression in low-dose dexamethasone suppression test. Four cases of them were demonstrated to be associated with SCS, and one was associated with overt Cushing syndrome (CS). Post-operatively, 3 cases received replacement therapy of hydrocortisone, while others did not. Pathological findings indicated the diagnosis of aldosterone-producing adenoma in 4 cases associated with SCS, and of idiopathic hyperaldosteronismin in one case associated with overt CS. In all 5 cases, immunohistochemical analysis demonstrated the immunoreactivities of both 3ßHSD and P450c17 in the adrenocortical tumors, the marked cortical atrophy in the zona fasciculata and reticularis, the decreased dehydroepiandrosterone sulfotransferase expression, and suppression of hypothalamo- pituitary-adrenal axis indicating the autonomous secretion of cortisol from the tumor. CONCLUSIONS: The present study suggests that PA is frequently associated with SCS with prevalence of more than 10%, justifying the routine examinations for SCS in PA cases.

Improvement of insulin resistance following transsphenoidal surgery in patients with acromegaly: correlation with serum IGF-I levels.

AIM: The aim of this study was to determine the correlation between the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis and glucose intolerance in acromegaly during the early postoperative period. SUBJECTS AND METHODS: The study included 20 patients with acromegaly caused by GH-secreting pituitary adenoma who received transsphenoidal surgery in our hospital. Glucose tolerance was evaluated with oral glucose tolerance tests (OGTTs) performed during pre- and early postoperative periods (9 [7-18] days after surgery). Homeostasis model assessment of insulin resistance (HOMA-IR) and insulinogenic index (IGI) were calculated, and correlation analyses were performed between these values and the GH-IGF-I axis. Patients were divided according to postoperative changes of the axis, and glucose tolerance was compared between the groups. RESULTS: In preoperative OGTTs, nine patients had impaired glucose tolerance and two had diabetes mellitus patterns. Postoperatively, significant reduction was observed both in fasting plasma glucose levels (p<0.01) and in HOMA-IR (p<0.01), whereas IGI showed no significant change. HOMA-IR was significantly correlated with serum IGF-I levels both before (r=0.83, p<0.01) and after (r=0.57, p<0.01) surgery, although it was not correlated with serum GH levels. Patients who achieved more than 50% postoperative reduction in serum IGF-I levels showed significant improvement in OGTTs results (p<0.05). CONCLUSIONS: In patients with acromegaly, serum IGF-I levels, but not GH levels, were significantly correlated with insulin resistance. Early postoperative improvement of glucose tolerance is observed in patients who achieved postoperative reduction in serum IGF-I levels.

New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution.

The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.

Trichinella spiralis: ultrastructural localization of a target antigen recognized by a monoclonal antibody.

The localization of a 160-kDa molecule recognized by a monoclonal antibody of the IgG1-class (TS32D12) to Trichinella spirilis was demonstrated at the subcellular level. Mature muscle larvae recovered from infected mice were fixed with Zamboni solution, and embedded in LR white resin. Ultrathin sections were incubated with TS32D12 and subsequently stained using a secondary antibody-coated colloidal gold probe. TS32D12 reacted to constituents in the alpha-stichosome cells of the parasite; that is, alpha-granules and rough endoplasmic reticulum. Other structures were not recognized by the immunogold probe, but occasionally weak positive staining was seen on beta-granules, the cuticle, hypodermis and myofibrils.

The synaptic structure of PACAP immunoreactive axons in the intermediolateral nucleus of the rat.

Immuno-electronmicroscopic studies were performed to detect the presence and features of synaptic contacts between pituitary adenylate cyclase activating polypeptide immunoreactive (PACAP-ir) axons and cholera toxin B-horseradish peroxidase labeled preganglionic sympathetic neurons (PSNs) in the intermediolateral nucleus of the rat thoracic spinal cord. PACAP-ir axon varicosities, which contained small clear and large core synaptic vesicles, were found to form asymmetric type of synaptic contacts with dendrites and infrequently with somata of labeled preganglionic neurons. The present study provides ultrastructural evidence of PACAP-ir synaptic contacts with PSNs, raising the possibility that the peptide may function as a transmitter/modulator to these neurons.

Beneficial effects of nitric oxide synthase inhibition on the recovery of neurological function after spinal cord injury in rats.

We have demonstrated recently that treatment with N(G)-monomethyl-L-arginine (L-NMMA) accelerates electrophysiological recovery after transient spinal cord ischaemia in anaesthetized cats. To determine whether nitric oxide synthase (NOS) inhibition in the acute phase of spinal cord injury results in better functional recovery in the chronic phase, we evaluated the influence of L-NMMA on the time course of changes of neurological function and the histopathological changes after spinal cord compression in rats. Experimental spinal cord injury was produced in anaesthetized rats by short-term (5 min) compression with a thread placed around the spinal cord at T13. The recovery of motor function was assessed by a treadmill test 10, 20 and 30 days after spinal cord compression. The latency of potentials evoked by hindlimb stimulation was measured at the funiculus posterior at C1 10 days after the spinal cord injury in anaesthetized rats. Histological examinations were also performed at the same time. The compression-induced spinal cord injury resulted in motor dysfunction of hindlimbs, an increase in the latency of the evoked potentials and neuronal degeneration in funiculus posterior at T13. Repeated administration of L-NMMA for 1 day significantly accelerated the recovery of the motor function, shortened the latency of the evoked potentials and attenuated the myelin vacuolization in the spinal cord. These beneficial effects of L-NMMA on neurological function and histopathological changes were abolished by coadministration of L- but not D-arginine. These results suggest that NOS inhibition during the early stage of spinal cord injury has beneficial effects on the recovery of neurological function and the histopathological changes in the chronic stage.

Effect of 1.5 T steady magnetic field on neuroconduction of a bullfrog sciatic nerve in a partially active state within several hours after extraction.

The bullfrog sciatic nerve within six hours after extraction was determined from the change with time in the action potential and the impedance to be a partially active nerve. Steady magnetic fields have been determined to be ineffective in the neuroconduction in the fresh nerve just after extraction. Although ion motions involved in the neuroconduction in the partially active nerve are not normal, it was confirmed that a 1.5 T steady magnetic field is ineffective in the neuroconduction in the partially active nerve. The obtained results suggest that the neuroconduction in damaged nerves is not affected by the 1.5 T steady magnetic field used in MRI.

Behavioral and morphologic studies of the chronically compressed cauda equina. Experimental model of lumbar spinal stenosis in the rat.

STUDY DESIGN: An experimental model in rats of chronically compressed cauda equina was produced, and behavioral and morphologic changes were examined. OBJECTIVES: To provide a useful model for analyzing the pathophysiologic changes of the cauda equina by chronic compression and to examine behavioral and morphologic changes in this model. SUMMARY OF BACKGROUND DATA: Several animal models have been reported in which various materials were used to compress the cauda equina. However, the pathophysiology of the cauda equina by chronic compression is not yet well understood. Studies in which rats were used are scarce. METHODS: A silicone sheet was applied to the spinal canal at L4 in the rat. Walking durations on treadmill tests and paw-withdrawal latencies to thermal stimuli were measured before and after the operation for 24 weeks. Histologic changes also were examined. RESULTS: Walking durations decreased after chronic compression. However, paw-withdrawal latencies were not significantly changed. Histologically, the number of large-diameter myelinated axons decreased after compression, whereas the number of small-diameter myelinated axons increased. Electron microscopic observation indicated that the continuous degeneration and regeneration of axons occurred throughout the chronic compression experiment. CONCLUSIONS: The current model and behavioral assessments may be useful in analyzing the pathophysiology of chronically compressed cauda equina.

Vestibular sensory epithelia in patients with acoustic neurinoma.

The human vestibular sensory epithelia of the macula utriculi were examined ultrastructurally in 13 cases with acoustic neurinoma. The sensory epithelia were fairly well preserved, with several morphological changes such as lipofuscin granules, intraepithelial cysts, pyknosis, vacuoles, laminated structures, banded structures and degeneration of subepithelial myelinated nerves evident. The pathological significance of these findings was discussed. There were thickened irregular membrane formations in spaces between the nerve calyces and type I hair cells which might be related to acoustic neurinoma. Some type I hair cells were incompletely surrounded by nerve calyces. The neck regions of these cells received direct contact from the efferent nerve endings.

Atypical innervation pattern of human vestibular hair cells.

Human vestibular sensory epithelia of macula utriculi were examined in 3 cases with acoustic neurinoma by intermediate voltage electron microscope. The innervation pattern of vestibular hair cells was studied by means of computer aided three-dimensional reconstruction technique. The sensory epithelia were fairly well preserved. Most of type I and all of type II hair cells appeared normal. However, some type I hair cells were incompletely surrounded by nerve calyces and received direct contact from the efferent nerve endings. These type I hair cells were also innervated by a few neighboring afferent nerve calyces. These atypical type I hair cells constituted 5-8% of the total number of hair cells.

An ultrastructural study of the nasal mucosa by rapid freezing and freeze-substitution.

We demonstrated the ultrastructures of the nasal mucosa of the rat by rapid freezing and freeze substitution with OsO4. Liquid helium was used for the rapid freezing. The depth of the good preservation was approximately 20 micron below the cut surface of the tissue. The characteristic features of the nasal mucosa obtained here are as follows: 1) The cross-section profiles of the cilium were round and smooth. 2) Mitochondrial matrix was electron dense and a few dense granules were observed in it. 3) Secretory granules of the goblet cells were enclosed by a limited membrane. 4) Extracellular space was of variable widths. 5) Cytoplasmic filaments were well preserved.

Distribution of vasoactive intestinal polypeptide immunoreactive nerve fiber in the rat nasal mucosa.

The distribution of vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fiber of the respiratory nasal mucosa of the rat was examined by use of immunohistochemistory with light, laser confocal scanning and electron microscopy. On light microscopy, it was observed that VIP immunoreactive nerve fibers distributed under and within the epithelium in addition to around the blood vessels and glands. The laser confocal scanning microscope revealed the network of VIP-immunoreactive nerve fibers at glands, but it was denser around glands than within glands. The VIP-immunoreactive nerve fibers formed fine varicosities and was distributed below the middle portion of the epithelium. Some fibers were observed as if they were surrounding goblet cells. Electron microscopy showed that the VIP-immunoreactive nerve fibers within the epithelium were terminated as free nerve endings. Some of them were in close contact with goblet cells.

Ultrastructural changes of afferent nerve terminals in the cat medial geniculate body after destruction of the inferior colliculus.

Three to seven days after destruction of the inferior colliculus, changes of the afferent nerve terminals of the cat medial geniculate body were examined morphometrically by thin sectioning and freeze fracture. The results were compared with those of the control. Three and five days after the operation, postsynaptic densities (PSD) of synapses of the afferent nerve terminals increased their width significantly (p less than 0.01) compared with the control. The packing density of intramembrane particles (IMPs) in aggregates at the synaptic site on the postsynaptic E-face decreased significantly (p less than 0.01) during this period. IMPs of 13 nm in diameter within these aggregates decreased. Ring-shaped IMP aggregates having an IMP free area at the center increased. Width of dense materials of the filamentous contact, which is a non-synaptic contact, decreased significantly (p less than 0.01) 3 and 5 days after the operation as compared with the control, but 7 days after the operation they recovered to the level of the control. No significant change was observed in the intervals of dense materials of the filamentous contact after the operation.

Freeze-fracture study of the lateral-line canal organ of the Japanese sea eel, Lincozymba nystromi.

Specializations of apical surfaces of hair cells, supporting cells and marginal cells in the lateral-line canal organ of Japanese sea eel, Lincozymba nystromi, were examined with a freeze-fracture technique. Apical surfaces of hair cells have a lower density of intramembrane particles (IMP) than those of the surrounding supporting cells. Density of IMP on the stereocilia is almost the same as that on the apical surface of hair cells. Junctions between hair and supporting cells were tighter than those between two supporting cells; those between supporting and marginal cells were tighter than those between hair and supporting cells, and those between two marginal cells were the tightest in the lateral-line canal organ.

Thin section and freeze-fracture studies on the synapses in the ventral nucleus of the cat medial geniculate body.

Ultrastructure of the synapses in the ventral nucleus of the cat medial geniculate body was examined with freeze-fracture and thin section techniques. Two types of synapses were recognized in this nucleus. One was the asymmetrical type such as the afferent nerve fiber to the principal cell dendrite or to the process of Golgi type 2 cell. The other was the symmetrical type such as the process of Golgi type 2 cell to the principal cell dendrite. These synapses made conglomerate on the principal cell dendrite called the glomerulus, which was ensheathed by thin sheets of the astroglia and easily recognized by freeze-fracturing. A random distribution of intramembrane particles (IMP) and synaptic vesicle attachment sites were found on the presynaptic P-face of both type synapses. Asymmetrical synapses had distinct aggregates of IMP on the postsynaptic E-face and the area of IMP aggregates on this face is often larger than 0.07 micrometer2. Similar aggregates of IMP occurred on the same face of symmetrical synapses. However, the area of IMP aggregates of this type synapses is frequently smaller than 0.05 micrometer2. IMP composing the aggregate on the postsynaptic E-face showed bimodal distribution in size, having peaks at 10 nm and 13 nm in diameter. The ratio of large IMP (13 nm) to the total number of IMP increased as the synaptic contact on the postsynaptic membrane widened in area. Non-synaptic contacts, having a complex of dense materials and neurofilaments at the subjunctional area and being similar to the filamentous contact in the lateral geniculate body, were frequently observed on the dendrite of the principal cell in thin sections. Clusters of IMP were scattered on the dendritic P-face near around the postsynaptic area and may represent the area being attached with dense materials of the contacts.

Postsynaptic arch in the frog neuromuscular junction: paramembranous protuberances coating the inner surface of the postjunctional membrane.

The frog neuromuscular junction was fixed and processed for electron microscopy according to the method of rapid freezing followed by freeze-substitution. The synaptic structures, including cleft material, paramembranous cytoplasmic coating on the postsynaptic membrane, and subsynaptic cytoplasmic elements, were examined in thin sections. The basal lamina, about 50 nm thick, was seen to bisect a synaptic cleft approximately 100 nm wide. The lamina consists of two parts: the central dense line and the fine filaments protruding from it in the direction of the apposing postjunctional membrane. Present on the cytoplasmic surface of the postjunctional membrane are electron-dense protuberances, 41 +/- 5 nm in width and 27 +/- 5 nm in height (top to membrane centre). They are arranged in regular parallel rows, at 54 +/- 4 nm intervals (centre to centre). The paramembranous protuberance coats the inner surface of the postjunctional membrane at its apex as well as at the middle portion of the junctional process, pointing to its probable hairpin-like course in a transverse plane of the process. From its location and three-dimensional arrangement, this protuberance was termed the 'postsynaptic arch'. A filamentous meshwork is present just beneath the postjunctional membrane and extends into the cell interior. The submembranous meshwork appears to connect to the underlying bundles of cytoskeletal filaments. The possibility is discussed that the postsynaptic, electron-dense arch corresponds to the 43-kDa protein, a major alkaline-extractable protein thought to be associated with the cholinergic receptor molecules in the postsynaptic membrane.

The morphological differences of stereocilia and cuticular plates between type-I and type-II hair cells of human vestibular sensory epithelia.

The sensory epithelia of macula utriculi were examined by conventional and intermediate voltage transmission electron microscopy. The specimens were obtained from three cases of acoustic neurinoma who were operated on using the translabyrinthine approach. The mean diameter of the vestibular hair cell stereocilia was obtained and the cuticular plates of type-I and type-II hair cells were reconstructed three-dimensionally from the consecutive 0.5-micron-thick sections. The mean diameter of stereocilia of type-I hair cells was 488 +/- 59 nm (n = 13) and that of stereocilia of type-II hair cells was 373 +/- 21 nm (n = 14). Stereocilia of type-I hair cells numbered about 70 and those of type-II hair cells about 50. The cuticular plates of type-I hair cells were several times as thick as those of type-II hair cells. The cuticular plate of the type-I hair cell appeared to be an inverse cone and that of the type-II hair cell seemed to be a flat disc.

Characteristics of the myelin sheath membrane of the spiral ganglion cell and the cochlear nerve of guinea pig.

The membrane structures of the myelin sheath of guinea pig spiral ganglion cell and cochlear nerve were examined using freeze-fracturing, rapid freezing and freeze substitution, and deep-etching techniques. The intermediate dense lines of the myelin sheath were observed only in the outermost several layers using thin and thick (1 micron) section techniques. The spaces between the two adjacent major dense lines at any layer of myelin sheath were occupied by flocculent materials in the freeze-substituted specimen. Periods between the two adjacent major dense lines were 13.3 nm in conventional fixation and 11.7 nm in rapid freezing followed by freeze substitution, respectively. A freeze-fracturing study showed no differences in membrane structures at any layer of myelin sheath. Developed tight-junction strands were observed parallel to the long axis of the myelin sheath in the deep-etched specimen. These findings may show the characteristics of myelin sheath membrane in the spiral ganglion cell and cochlear nerve of the guinea pig.

Ultrastructural and immunocytochemical study of the subepithelial fiber component of the guinea pig inner ear.

The connective tissues of the lateral cochlear wall and semicircular canal of the guinea pig were investigated by transmission electron microscopy, intermediate voltage electron microscopy and immunocytochemistry. In the spiral ligament, numerous fibroblasts and fiber bundles were intermingled with each other. Fibroblasts and fibers were also observed in the semicircular canal. Ultrastructurally, immuno-gold particles, indicating the presence of type II collagen, were found on the fibers in the spiral ligament and the semicircular canals. The present results revealed that both stria vascularis and the membranous semicircular canal were supported by a network of fibroblasts and type II collagen fibers.

Observation of apical part and nerve terminals of human vestibular hair cells.

The human vestibular sensory epithelia of macula utriculi in 3 cases of acoustic neurinoma were examined by conventional and intermediate voltage electron microscopes. The apical part and the nerve terminals of hair cells were studied by means of a computer-aided three-dimensional (3-D) reconstruction technique. The sensory epithelia were fairly well preserved. Most type I and all type II hair cells appeared as those described in the other reports. However, some type I hair cells were incompletely surrounded by nerve calyces and received direct contacts from the efferent nerve endings. These type I hair cells were also innervated by a few neighbouring afferent nerve calyces. The stereocilia and the cuticular plate of type I hair cells differed from those of type II hair cells. The mean diameter of type I hair cell stereocilia was 488 +/- 59 nm and that of type II hair cells was 373 +/- 21 nm. The cuticular plate of type I hair cells resembled a cone and was about several times as thick as that of type II hair cells which was similar to a flat disc.