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Objective: To evaluate the clinical practice and short-term efficacy of microwave endometrial ablation (MEA) to treat menorrhagia, and to identify prognostic factors for optimal outcomes. Methods: We performed MEA in 22 women with menorrhagia between October 2012 and December 2013. To evaluate efficacy, objective and subjective variables were measured using medical records and patients' pre- and postoperative responses to a written questionnaire with a visual analog scale (VAS) scored from 0 to 10 for each symptom. MEA outcome was evaluated 6 months after treatment. Patients with amelioration of menorrhagia and no anemia were defined as the effective group, and the others were defined as the noneffective group. Effective patients requiring no hormonal therapy were defined as the highly effective group. To identify prognostic factors, background factors were compared between the highly effective group and the other groups. Results: Uterine fibroids and adenomyosis were diagnosed in 68% and 32% of patients, respectively. The median VAS score of postoperative pain was 1.0, and that of satisfaction was 8.1. Hemoglobin concentration, menstrual bleeding volume, menstrual duration, menstrual pain, vaginal discharge, and fatigue were ameliorated in the postoperative period. The effective group, the highly effective group, and the noneffective group included 95%, 84%, and 5% of patients, respectively. The uterine corpus cavity was significantly shorter in the highly effective group than in the other groups. Conclusion: MEA was safe and effective. The short-term efficacy rate of MEA for alleviating menorrhagia symptoms was 95%. Optimal outcomes were correlated with a shorter uterine corpus cavity. Copyright © 2015, The Asia-Pacific Association for Gynecologic Endoscopy and Minimally Invasive

To determine the role of cytokines in the nervous system, we examined the effect of interleukin-12(IL-12) on the nerve regeneration of mouse superior cervical ganglion cells (SCG). IL-12 enhanced the neurite outgrowth in a concentration-dependent manner. Immunocytochemical studies demonstrated the expression of IL-12 receptors in neuronal bodies and neurites. The mRNA expression of IL-12 receptors in SCG cells was confirmed by reverse transcription-polymerase chain reaction. Our data demonstrated the presence of IL-12 receptors in sympathetic neurons and suggest that IL-12 plays an important role in neuronal regeneration.

We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.

The effects of acetyl-L-carnitine (ALCAR) on fast axoplasmic transport were studied in cultured dorsal root ganglion (DRG) neurons of diabetic rats. Three-month-old male rats were used 7 days after streptozotocin injection. Neurons obtained from ganglia were cultured with a high concentration of glucose. The amount and the mean velocity of retrogradely transported particles, reduced in the diabetic animal, were transiently recovered by 1 mM ALCAR. The number of particles moving at 0.8-1.2 microm/s, considered to be lysosomes, increased in the velocity distribution. ALCAR did not modify the amount and mean velocity of anterograde particles which were unaffected by diabetes, or of bidirectional particles in neurons of control rats. This study suggests that diabetic neuropathy may be relieved by ALCAR via recovering retrograde axoplasmic transport.

The abundance of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive nerve fibers in the carotid body was compared between normoxic and chronically hypoxic rats (10% O 2 and 3.0–4.0% CO 2 for 3 months). The immunoreactive fibers appeared as thin processes with many varicosities, and were distributed mainly around the vasculatures. In the normoxic control carotid body, NPY fibers were more numerous than VIP, CGRP, and SP fibers. In the chronically hypoxic rats, the carotid body was enlarged several fold, and the mean absolute number of VIP and NPY fibers was 3.88 and 2.22 times higher than in the normoxic carotid body, respectively, although that of SP and CGRP fibers was not changed. When expressed as density per unit area of the parenchyma, the density of SP and CGRP fibers in the chronically hypoxic carotid body decreased significantly to under 50%, the density of VIP fibers increased significantly 1.80 times, and the density of NPY fibers were unchanged. Immunoreactivity for four neuropeptides was not found in the glomus cells of normoxic or chronically hypoxic carotid bodies. These results suggest that altered peptidergic innervation of the chronically hypoxic carotid body is one feature of hypoxic adaptation. Because these neuropeptides are vasoactive in nature, altered carotid body circulation may contribute to modulation of the chemosensory mechanisms by chronic hypoxia.

The distribution of nitric oxide synthase (NOS) immunoreactive nerve fibers in the carotid body was compared between normoxic and chronically hypoxic rats (10% O2 and 3.0–4.0% CO2 for 3 months). NOS immunoreactive fibers appeared as thin processes with many varicosities. They were distributed predominantly around small arteries and arterioles, and around clusters of glomus cells. When expressed by the density of varicosities per unit area in the parenchyma, the density of NOS fibers associated with the vasculature and with the glomus cells in the chronically hypoxic carotid bodies was significantly decreased. Because nitric oxide (NO) is an inhibitory neuronal messenger in the normoxic carotid body, the present findings suggest that the sensory mechanisms in the hypoxic carotid body may be involved in `disinhibition' resulting from reduced NO synthesis.

The distribution of substance P-immunoreactive and calcitonin gene-related peptide-immunoreactive nerve fibers in the nasal mucosa was compared between normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). In the normoxic nasal mucosa, substance P- and calcitonin gene-related peptide-immunoreactive nerve fibers were found within and under the epithelium and around the glands and blood vessels in the lamina propria. These immunoreactive fibers have many varicosities. In the chronically hypoxic nasal mucosa, the relative density of intra- and subepithelial substance P-immunoreactive and calcitonin gene-related peptide-immunoreactive fibers and those in the lamina propria was higher than in normoxic mucosa. The length of substance P-positive fibers within the chronically hypoxic olfactory and respiratory epithelium was 1.66 and 2.45 times higher than within the normoxic epithelium, respectively. The length of calcitonin gene-related peptide-immunostained fibers within the chronically hypoxic olfactory and respiratory epithelium was 1.56 and 1.84 times higher, respectively. Because substance P and calcitonin gene-related peptide are the predominant signal peptides of primary sensory neurons, the increased number of these fibers may represent enhanced sensory mechanisms in the hypoxic nasal mucosa. In addition, considered together with the findings in chronically hypoxic tracheal mucosa, the increased density of intraepithelial fibers containing substance P and calcitonin gene-related peptide suggests that this is a predominant feature of hypoxic adaptation throughout the upper and lower respiratory tracts.

Neuron requires a continual supply of materials synthesized in the cell body, for example a wide range of soluble proteins, membranous components, and various organelles. The transported materials are needed to replace constituents that turn over in the membrane and organelles of the fiber and also are needed to bring substances participating in energy metabolism. Other transported components are neurotransmitters or transmitter-related components supplied to the nerve terminals for the release and subsequent excitation of postsynaptic cells. Moreover, neurotropic substances and modulators are released from the nerve terminals to affect the functional state of the neuron. Conversely, some materials are conveyed back to the cell body. These include organelles, lysosomes, nerve growth factor, and selected small molecules such as adenosine, Ca2+, and some neurotransmitters. Axoplasmic transport is thought to be fundamental for a variety of neuronal cell functions. Thus it may be considered that axoplasmic transport relates to the dynamic physiological activity of neurons; in other words, axoplasmic transport is supposed to express the physiological activity of neurons. In turn, as in the case for many other physiological functions, axoplasmic transport is possibly controlled by neuronal, hormonal, and immunological systems. Since axoplasmic transport supplies neuron materials toward the synapses and back to the cell body, a feedback system of regulatory mechanisms of a variety of neuronal functions might be operated through axoplasmic transport pathways. Although axoplasmic transport is the important neuronal function, its regulation is poorly understood. In this review, we focus on the dynamics of organelle transport and its regulatory mechanisms mediated by neurotransmitters.

We studied neurite regeneration in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus, using a culture system to investigate the influences of immunological abnormalities on neurons. The regeneration of cultured dorsal root ganglion (DRG) neurons from MRL-lpr/lpr mice was delayed compared with control MRL-+/+ mice. This modification of regeneration was age-dependent. MRL-lpr/lpr mice older than 16 weeks of age exhibited less neurite regeneration than controls but those younger than 6 weeks of age showed equal regeneration. Regeneration was improved by adding nerve growth factor (NGF) to culture medium. Following immunocytochemical staining, we counted the low affinity NGF receptor p75-positive DRG neurons in MRL mice. The percentage of p75-positive neurons in MRL-lpr/lpr mice older than 16 weeks of age was higher than that in MRL-+/+ mice. These neuronal abnormalities were thought not to be directly dependent on the genetic defect of Fas antigen, which is related to apoptosis in MRL-lpr/lpr mice, but to be the result of immunological abnormalities. The present study is the first to demonstrate a modification of neurite regeneration by immunological dysfunction in autoimmune mice.

The effect of dibutyryl cyclic AMP (dbcAMP) on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95 +/- 1.01 microm/s (n = 55) to 2.66 +/- 1.26 microm/s (n = 58) and from 1.94 +/- 0.85 (n = 57) to 2.39 +/- 0.93 (n = 57). Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport.

The occurrence and distribution of several neuropeptides were studied in human von Ebner's glands. Immunoreactivity for substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin, and somatostatin was found in the nerve fibers around the acini, ducts, and blood vessels. VIP-immunoreactive varicose fibers were numerous compared with the other five neuropeptides. Most NPY fibers were associated with the vasculature in the gland. These findings suggest that the neuropeptides may regulate the secretion and vascular tone in human von Ebner's glands.

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase-antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin-positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (+/- SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 +/- 7.25%, 70.52 +/- 9.33% and 41.76 +/- 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 +/- 6.19% and 59.62 +/- 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 +/- 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow.

The effects of primary sensory neurons in the dorsal root ganglia (DRG) on immunoglobulin (Ig)-secreting activity of spleen cells were investigated in culture. The conditioned medium (CM) of normal spleen cells stimulated DRG neurons to release neurokinin A which increased the number of Ig-secreting spleen cells. In contrast, the CM of concanavalin A-induced suppressor spleen cells induced the release of vasoactive intestinal peptide, an inhibitor of Ig secretion of spleen, from DRG neurons. These findings indicate that sensory neurons can help and suppress Ig secretion, and these bidirectional activities are controlled by the factors released from immune cells.

An application of double-immunolabelling in combination with a multiple dye filter system demonstrated new findings regarding the distribution pattern of peptidergic fibers in the carotid labyrinth to addition to our previous findings shown by the individual filter system. In high magnification images of about 10% of the yellowish fibers which represent the coexistence of two neuropeptides, there was a definite difference in localization between the fluorescence originating from rhodamine (substance P fibers) and from FITC (vasoactive intestinal polypeptide and neuropeptide Y fibers), but it was clear that they are intertwined within a single nerve bundle. This combination method was able to discriminate two different peptidergic fibers which run side by side. The coexistence suggested previously by the individual filter system may actually be due to the phenomenon described above. This means that it is necessary to apply the multiple dye filter system for reliable evidence of coexistence of different two substances in a single nerve fiber.

We modified the method of somatic cell fusion for neurons to improve the efficiency of hybrid production. C1300 neuroblastoma cells were incubated with emetine and actinomycin D before fusion. After fusion between C1300 cells and adult mouse dorsal root ganglia neurons with polyethyleneglycol, we were able to select the hybrids from non-fused cells. We obtained hybrid clones at high efficiency (7.2 clones/ 10 4 neurons)

Nitric oxide synthase (NOS)-immunofluorescence techniques were applied to the mouse vomeronasal organ. Immunoreactivity for NOS was found in the nerve fibers distributed in the receptor-free epithelium, and around the blood vessels and glands in the cavernous tissue. No NOS fibers were seen in the receptor area. A combination of double immunofluorescence labeling and multiple dye filter revealed that a part of the substance P (SP)-immunoreactive nerve fibers in the cavernous tissue contained NOS and that all the vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers around the blood vessels and glands in the cavernous tissue contained NOS. A few SP-immunoreactive cell bodies in the trigeminal ganglion showed coexistence with NOS, and almost all VIP-immunoreactive cell bodies in the sphenopalatine ganglion showed coexistence with NOS. Immunoreactivity for NOS without VIP in the cell bodies in the sphenopalatine ganglion was also found. These results suggest that NOS-immunoreactive nerve fibers in the mouse vomeronasal organ originate from the trigeminal and the sphenopalatine ganglia, and may modulate the vascular tone and the glandular secretion. In addition, these functions may be controlled in part by the interaction of nitric oxide and neuropeptides.

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine- and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.

Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy.

The effect of adrenaline on the axoplasmic transport of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Adrenaline increased the axoplasmic transport reversibly in both anterograde and retrograde directions. A beta 2-antagonist, butoxamine, antagonized the increasing effects of adrenaline, but alpha-antagonists and beta 1-antagonists did not. A beta 2-agonist, albuterol, mimicked the adrenaline effect, but beta 1-, alpha 1-, alpha 2-agonists did not. The adrenaline receptor may be a beta 2-receptor. Dibutyryl cyclic AMP and forskolin increased the axoplasmic transport. Therefore, adrenaline increases the axoplasmic transport by raising the cyclic AMP level. In light of our former report that acetylcholine suppresses the axoplasmic transport, neurotransmitters control axoplasmic transport and this neurotransmitter control reflects the activity of the nerve cell.

1. We analyze bradykinin-sensitive cells of the mouse dorsal root ganglion in culture from the viewpoints of cell size, electrical responses, and Ca2+ concentration change due to bradykinin and immunocytochemistry of substance P. 2. Sixteen percent of cells in the cell group 26-30 microns in diameter fired in response to 10 microM bradykinin. None of other cell groups showed a firing response to bradykinin. 3. We measured a cytosolic Ca2+ change due to bradykinin using a Ca(2+)-sensitive fluorescent dye, Fura 2. The rapid rise (peak time, 20 sec) in the Ca2+ concentration was ascribed to Ca2+ release from intracellular Ca2+ stores. The profound change in the Ca2+ concentration was observed again in the cell group 26-30 microns in diameter. Seventeen percent of cells in this group increased the Ca2+ concentration by approximately seven times that at resting level. 4. Among cells which increase Ca2+ concentration responding to bradykinin, 83% of them contain substance P (an immunocytochemical study). 5. We conclude that 16-17% of the cell group 26-30 microns in diameter of the dorsal root ganglia in culture are polymodal nociceptors and respond to bradykinin.

Dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice were exposed to three different hypotonic environments ( 1 2 , 1 4 , and 1/8 osmolar solutions). After rapid applications of these hypotonic solutions to the neurons, the cell volume autoregulatory mechanism operated in 1 2 osmolar solution but was disrupted in superhypotonic solutions below 1 4 osmolar in both kinds of mice. None of the neurons could survive 12 h after treatment with superhypotonic solutions. On the other hand, a gradual reduction of osmolarity of the culture medium enabled neurons in the normal mice to survive in 1 2 and 1 4 osmolar solutions as well as i an isotonic solution. However, this reduction of osmolarity increased neuronal cell death in the diabetic mice. These results suggest that the ability of DRG neurons to survive in hypotonic environments in culture may be lost in diabetes.

The distribution of substance P (SP)-immunoreactive nerve fibers in the rabbit carotid body was studied in combination with catecholamine autofluorescence images of sections where SP immunoreactivity was confirmed. Immunoreactivity for SP was found in nerve fibers distributed in the parenchyma of the carotid body. No glomus cells with SP immunoreactivity were observed in the carotid body. On comparing the distribution of SP-immunoreactive fibers with the catecholamine autofluorescence image in a single section, most SP fibers appeared associated with the fluorescent glomus cells, and were located around clusters of them. These results support the suggestion that SP fibers in the cat and rat carotid bodies are involved in chemosensory mechanisms. Furthermore, a survey of the present results and previous ones reported by other workers indicates that SP may be an essential neuropeptide in chemoreceptor organs in most vertebrates from amphibians on upwards evolutionally. In addition, the courses of some catecholaminergic fibers precisely agreed with those of some SP fibers. This suggests that certain sympathetic nerve fibers also contain SP.

Attachment of dorsal root ganglion (DRG) neurones from 3-month-old (young adult) and 24-month-old (aged) mice to the following substrates was evaluated after 6 h in culture: (1) poly-L-lysine (PL), (2) PL + type I collagen (CL-I), (3) PL + type IV collagen (CL-IV), (4) PL + laminin (LM) and (5) PL + fibronectin (FN). In the young adult mice, each substrate coated onto PL, significantly increased the ratio of attachment, compared with PL alone. In the aged mice, CL-I and LM, but not CL-N or FN improved the attachment. There was no difference in cell survival or neurite extension after 48 h in culture between the two groups of mice. These results suggest that ageing causes selective changes of the neuronal cell-surface receptors respective to extracellular matrix proteins such as CL-IV and FN.

A technique for isolation of adult Schwann cells (ScC) from dorsal root ganglia (DRG) is described. Decapsulated DRG explants embedded into type I collagen gels were cultured for 3 days in serum-free medium during which ScC migrated from the explant. These explants were then grown in serum-supplemented medium to allow ScC proliferation. On day 10 the number of ScC isolated from DRG explants per mouse was about 2.5 x 10(5), and the purity was greater than 95%. This culture system provided sufficient numbers of highly purified adult ScC in a shorter culture period (2-3 times) than other methods. We used ScC from this method to determine the age-related changes in attachment, growth, and survival of ScC cultured in serum-free medium. The attachment capacity of adult ScC on type I collagen or polylysine was similar to that of newborn ScC. However, the collagen promoted growth and survival of adult ScC but not that of neonatal ScC, indicating age-related differences of ScC properties in vitro.

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca2+ concentration.

Immunoreactivities for FMRFamide and substance P (SP) in the carotid labyrinth of the bullfrog were detected using the peroxidase-antiperoxidase method, and the results compared with corresponding differential interference-contrast (Nomarski) images. Colocalization of both peptides was determined by the indirect double immunofluorescence method. Immunoreactivities for FMRFamide and SP were found in nerve fibers distributed in the intervascular stroma of the labyrinth. The FMRFamide-immunoreactive fibers were less numerous than the SP-immunoreactive fibers. In the Nomarski image, FMRFamide-fibers were recognized in relief, with most of them located near the walls of blood vessels. All FMRFamide-fibers coexisted with SP. The results suggest that FMRFamide-immunoreactive fibers are also involved in local vascular regulation of the carotid labyrinth.

Dorsal root ganglia neurons from streptozotocin-induced diabetic and normal C57BL mice were cultured in serum-containing and serum-free media. The ratio of dead cells was higher in diabetic neurons than in controls in the early stages of culture. The effect of glucose concentration on survival in the culture medium was also measured for 1 week. Treatment with high glucose concentrations improved the survival of diabetic neurons, which was enhanced by duration of diabetes in the animal. These results indicate that exposure to hyperglycemia in vivo might adapt neurons to a high glucose environment in vitro.

The purpose of this study was to examine the effect of graded conditioning contractions of the antagonist knee flexor muscles on the output characteristics of knee extensor muscles in healthy humans. Eight male university students performed maximum isometric contractions of knee extensors, preceded by isometric conditioning contractions of the antagonist knee flexors. The developed force and electromyographic (EMG) amplitudes of the knee extensors after the conditioning contraction were measured and compared with those of simple knee extension without conditioning. The forces of the conditioning flexor contraction were set at three levels: low (20% of maximum voluntary contraction: MVC), moderate (60% of MVC), and high (100% of MVC). The EMG amplitudes of the vastus medialis, vastus lateralis, and rectus femoris muscle were recorded and the root mean square amplitudes were calculated. The strongest enhancement of the extension force was obtained by moderate intensity conditioning contraction (108.95+/-1.87% of simple knee extension), although high intensity conditioning also induced a significant increase (105.41+/-2.69%). Low intensity conditioning did not cause a significant enhancement of the contraction force (103.17+/-2.99%). Similarly, the EMG amplitudes were significantly increased by moderate and/or high conditioning. These results suggest that antagonist conditioning contraction of moderate intensities is sufficient and may be optimal to potentiate knee extensor contraction.

Neuropeptide Y (NPY) plays a modulatory role in processing nociceptive information. The present study investigated the effects of NPY on axonal transport of particles in neurites of cultured adult dorsal root ganglion (DRG) cells using video-enhanced microscopy. Application of NPY decreased the number of particles transported in both the anterograde and retrograde directions. This effect was persistently observed during NPY application and was reversed after washout. The inhibitory effect of NPY was concentration dependent between 10(-9) M and 10(-6) M. The instantaneous velocity of individual particles moving in anterograde and retrograde directions was also reduced by NPY. Both the NPY Y1 receptor agonist [Leu31,Pro34]-NPY and NPY Y2 receptor agonist NPY(13-36) mimicked the effect of NPY on the number of transported particles. An immunocytochemical study using an antiserum against the NPY Y1 receptor protein revealed that the Y1 receptor was expressed in the majority (85.9 %) of cultured adult mouse DRG cells. Pre-treatment of cells with pertussis toxin, a GTP-binding protein (G protein) inhibitor, completely blocked the inhibitory effect of NPY. Each application of SQ-22536, an adenylate cyclase inhibitor, and H-89, a protein kinase A inhibitor, mimicked and occluded the effect of NPY. In contrast, dibutyryl cAMP (dbcAMP), a membrane permeable cAMP analogue, and forskolin, an activator of adenylate cyclase, produced a transient increase in axonal transport. The application of dbcAMP and forskolin in combination with NPY negated the effect of NPY alone. These results suggest that NPY, acting at Y1 and Y2 receptors, inhibits axonal transport of particles in sensory neurones. The effect seems to be mediated by a pertussis toxin-sensitive G protein, adenylate cyclase, and protein kinase A pathway. Therefore, NPY may be a modulatory factor for axonal transport in sensory neurones.

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.

1 Department of Otorhinolaryngology Department of Anatomy 7Department of Physiology Yokohama City University School of Medicine, Yokohama 236-0004, Japan Department of Systems Physiology University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan Department of Medicine and Department of Neurosciences University of California San Diego, La Jolla, CA 92093, USA Department of Physiology Kitasato University School of Medicine, Sagamihara 228-8555, Japan

The carotid bodies are enlarged by several fold in rats exposed to chronic hypoxia; their volume increases and endothelial cells multiply (Barer et al. 1976; Heath et al. 1973; Laider and Kay 1975a, b). Recently, we examined the peptidergic innervation in the carotid body of the rats exposed to chronically isocapnic hypoxia (Kusakabe et al. 1998). The density of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive fibers in the chronically hypoxic carotid body decreased significantly to under 50%, the density of vasoactive intestinal polypeptide (VIP) immunoreactive fibers increased significantly 1.80 times, and the density of neuropeptide Y (NPY) immunoreactive fibers was unchanged in comparison with the controls. These findings suggest that altered peptidergic innervation of the chronically hypoxic carotid body is one feature of hypoxic adaptation, which may involve modulation of chemosensory mechanisms by modification of carotid body circulation. As far as we are aware, however, there are no immunohistochemical studies on the peptidergic innervation in the carotid body during deacclimatization after chronic hypoxia is terminated. In the present study, the distribution and abundance of neuropeptide

The effects of prostaglandin E 2 on axonal transport in cultured mouse dorsal root ganglion neurons were investigated by analysing the number of axonally transported particles under video-enhanced microscopy. Application of prostaglandin E 2 increased the number of particles transported in anterograde and retrograde directions. The EP 2 prostaglandin receptor agonist butaprost mimicked the effect of prostaglandin E 2 , but the EP 1 /EP 3 prostaglandin receptor agonist 17-phenyl trinor prostaglandin E 2 and the EP 3 prostaglandin receptor agonist M&B 28767 had no effect. The membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP and the adenylate cyclase activator forskolin mimicked the effect of prostaglandin E 2 . The protein kinase A inhibitor H-89 reversibly reduced the number of particles in both anterograde and retrograde directions. The effects of prostaglandin E 2 and dibutyryl cyclic AMP were blocked by H-89. Taken together with previous biochemical studies showing that prostaglandin E 2 increases cyclic AMP levels, the present results suggest that prostaglandin E 2 enhances axonal transport via the EP 2 receptor and cyclic AMP-dependent protein kinase A pathway. We further investigated the role of prostaglandin E 2 in neurite growth. Prostaglandin E 2 increased both the number of cells exhibiting neurites and the neurite growth rate, operating by a similar mechanism to stimulation of axonal transport. Prostaglandin E 2 may modulate axonal transport to supply materials for morphogenesis as well as other functions in sensory neurons.

The effects of glucagon on the axoplasmic transport of cultured mouse superior cervical ganglion cells were analyzed with the video-enhanced DIC microscope system. Glucagon increased the rate of fast axoplasmic transport by 30% in both anterograde and retrograde directions. The average velocity was increased from 1.36 +/- 0.48 microns/s to 1.74 +/- 0.43 microns/s (anterograde, n = 60) and from 1.37 +/- 0.48 microns/s to 1.62 +/- 0.39 microns/s (retrograde, n = 60). The stimulatory effect of glucagon on the axoplasmic transport was reversed in a glucose-free medium, whereas blocking the citrate cycle by pretreating neuronal cells with malonate did not alter the effect of glucagon. Together with our previous findings, our data suggest that neurotransmitters and hormones play a major role in the regulation of fast axoplasmic transport.

Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.

The occurrence and distribution of neuropeptide-containing nerve fibres in the human circumvallate papillae were examined by the peroxidase-antiperoxidase immunolocalisation method using surgical specimens that had not been subjected to radiotherapy, and the abundance of neuropeptide-containing fibres was expressed as the percentage of total nerve fibres demonstrated by protein gene product (PGP) 9.5 immunoreactivity for a quantitative representation of these peptidergic fibres. Substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (IR) nerve fibres were densely distributed in the connective tissue core of the circumvallate papillae, and some SP and CGRP-IR fibres were associated with the taste buds. A moderate number of vasoactive intestinal polypeptide (VIP)-IR fibres and a few galanin (GAL)-IR fibres were also seen in the connective tissue core and subepithelial layer. There were, however, no VIP-IR or GAL-IR fibres associated with the taste buds. Neuropeptide Y (NPY)-IR fibres were few and were associated with the blood vessels. Within the epithelium of the circumvallate papillae, no peptidergic fibres were found, although a number of PGP 9.5-IR fibres were detected. The abundance of SP, CGRP, VIP, and GAL-IR fibres expressed as the percentage of total PGP 9.5 IR fibres was 25.35+/-3.45%, 22.18+/-3.26%, 10.23+/-1.18%, and 4.12+/-1.05%, respectively. The percentage of NPY-IR fibres was below 3%. In a deeper layer of the papillae, a few VIP, GAL, and NPY-IR ganglion cells were found, and VIP immunoreactivity was detected in a few cells of the taste buds. There was no somatostatin, leucine enkephalin, or methionine enkephalin immunoreactivity in the circumvallate papillae. These results suggest that the dense SP and CGRP-IR fibres within the connective tissue core of the human circumvallate papillae may be involved in the deep sensation of the tongue.

We established a hybrid cell line which extended neurites in the presence of nerve growth factor by fusion between adult mouse sensory neurons and neuroblastoma C1300 cells using emetine and actinomycin D. The serine-phosphorylated 200 and 160 kDa neurofilament proteins were detected in the hybrid cells. In comparison, C1300 cells expressed both subunit of non-phosphorylated neurofilaments at serine residues.

Chick collapsin-1, a member of the by a mechanism distinct from that causing growth semaphorin family, has been implicated in axonal cone collapse. Pertussis toxin (PTX) but not its B pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which oligomer blocks collapsin-induced growth cone col- may include CRMP-62 and heterotrimeric G proteins. lapse. The holotoxin does not affect collapsin-stimu- CRMP-62 protein is related to UNC-33, a nematode lated axoplasmic transport. Mastoparan and a myelin neuronal protein required for appropriately directed protein NI-35 induce PTX-sensitive growth cone col- axonal extension. Mutations in unc-33 affect neural lapse but do not stimulate axoplasmic transport. microtubules, the basic cytoskeletal elements for axo- These results provide evidence that collapsin has a plasmic transport. Using computer-assisted video-en- unique property to activate axonal vesicular transport hanced differential interference contrast microscopy, systems. There are at least two distinct pathways we now demonstrate that collapsin-1 potently pro- through which collapsin exerts its actions in motes axoplasmic transport. Collapsin-1 doubles the developing neurons. q 1997 John Wiley & Sons, Inc. J number of antero- and retrograde-transported organ- Neurobiol 33: 316-328, 1997 elles but not their velocity. Collapsin-1 decreases the Keywords: collapsin; axoplasmic transport; nerve number of stationary organelles, suggesting that the growth cone; pertussis toxin; G proteins fraction of time during which a particle is moving is

Distribution of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, galanin (GAL)-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the human submandibular gland was examined by the peroxidase-antiperoxidase method with attention to high-quality fixation and the condition of patients. NPY-, VIP-, and GAL-immunoreactive varicose fibers were densely distributed around the acini and ducts. Some of these fibers extended between acinar cells. The density of SP- and CGRP-immunoreactive fibers was relatively low. The number of NPY-, VIP-, and GAL-immunoreactive fibers around the mucous acini was significantly higher than around the serous acini. In the perivasculature, NPY-immunoreactive fibers were more numerous than other immunoreactive fibers. No somatostatin-, leucine-, or methionine-enkephalin-immunoreactive fibers were detected. Our findings suggest that a large number of periacinar VIP-, NPY-, and GAL-immunoreactive fibers may participate in regulating the synthesis of saliva and its secretion. Since the VIP-, NPY-, and GAL-immunoreactive fibers are more numerous around the mucous acini than around the serous ones, these fibers may take part more actively in regulating the secretory mechanisms in the mucous acini than in the serous ones. The relatively low number of CGRP- and SP-immunoreactive fibers suggests that they are less involved in the function of the human submandibular gland. Perivascular peptidergic fibers, especially NPY-immunoreactive fibers, may be involved in controlling local blood flow in this gland.

During metamorphic stages (stages XX–XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NPY) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP, and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation.

The first appearance of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), leucine-enkephalin (1-ENK), and methionine-enkephalin (m-ENK) in the male mouse submandibular glands were different for each. VIP immunoreactive fibers first appeared on embryonic day 15 (E15), SP on E16, and CGRP fibers on E18. GAL, 1-ENK, and m-ENK fibers appeared in the early postnatal period, and NPY fibers occurred on postnatal day 21 (P21). From P0 to P21, VIP fibers rapidly increased in number, but SP and CGRP fibers increased only slightly. After P21, VIP, SP, and CGRP fibers decreased in number. ENK fibers were found only from P0 to P14. The number of these immunoreactive fibers in the adult phase was low in comparison with that in early postnatal phase. Around the blood vessels, SP, VIP, CGRP, NPY, and GAL fibers appeared by at least P7. These findings suggested that the transient high activity of VIP, CGRP, SP, and GAL and the transient appearance of ENKs in the nerve fibers may be related to the cell proliferation and differentiation of the functionally important structures of the mouse submandibular glands, and that the peptidergic innervation around the vasculature is probably involved in controlling local glandular circulation.

1. Our previous study demonstrated that cultured macrophages release neurotrophic factors spontaneously. In a histological study of Wallerian degeneration, macrophages phagocytosed myelin debris and expressed activated markers. 2. To investigate the role of myelin-stimulated macrophages on neurite regeneration, we prepared conditioned media from cultured mouse peritoneal macrophages which had phagocytosed a myelin fraction. This conditioned media enhanced both neurone survival and neurite regeneration of adult dorsal root ganglia (DRG) neurons compare to conditioned media from macrophage cultures without myelin. 3. The production of the neurotrophic supernatant was dose-dependent on myelin fraction and specific for myelin because supernatants from macrophages incubated with LPS (lipoplysaccharide), MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) or latex beads were not neurotrophic. 4. The neurotrophic factors from myelin-stimulated macrophages were different from spontaneously released macrophage factors as they differed in heat-sensitivity. 5. These results suggest that myelin-stimulated macrophages contribute to axon regeneration after Wallerian degeneration.

Transduction mechanism for modulation of axoplasmic transport by neurotransmitters was studied using cultured mouse superior cervical ganglion cells. The transported particles were analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Acetylcholine depressed and adrenaline increased axoplasmic transport. GTP-binding proteins linked with both receptors activate or inactivate adenylyl cyclase, thereby altering the intracellular concentration of cyclic AMP. The cyclic AMP activates protein kinase A, which phosphorylates certain enzymes and the enzymes in turn phosphorylate motor proteins. An inhibitor of protein kinase A, KT5720, decreases the number of the transported particles. In a stable state the cyclic AMP level stays at a normal level. Treatment with neurotransmitters causes a change in this level, which changes the activity of protein kinase A and thus decreases or enhances the phosphorylation of motor proteins. These changes are involved in the modulation of axoplasmic transport.