Changes in peptidergic fiber density in the synovium of mice with collagenase-induced acute arthritis.

The effect of acute osteoarthritis (OA) on peripheral nerve fibers (NFs) in synovial tissue, and their association with histological changes were investigated in collagenase-induced OA mice. Collagenase (10 U in 5 µL saline) was injected into the right knee, and the same volume of saline was injected into the left knee as the control. Mice were sacrificed 1, 2, 3, and 4 weeks after the collagenase injection. Histopathological changes in the knee joints were evaluated. The numbers of protein gene product (PGP) 9.5-, calcitonin-gene-related peptide (CGRP)-, and substance P (SP)-positive NFs in the synovial tissue were determined, and their densities in the tissue were calculated. The densities of PGP 9.5- and CGRP-positive NFs in the synovium were drastically decreased 1 week after the collagenase injection. However, by week 4, the density of PGP 9.5- and CGRP-positive NFs had recovered to 84% and 79% of their normal levels, respectively. Despite the poor correlation between the synovitis score and the density of CGRP- or SP-positive NFs in the synovium, the ossification rate of chondrophytes in chondro/osteophyte lesions correlated strongly with the density of CGRP-positive NFs (R = 0.855). These results suggest that the ossification of chondrophytes occurred in parallel with the increase in CGRP-positive fiber density in the synovium during the acute phase of collagenase-induced OA.

An ATF4-Signal-Modulating Machine Other Than GADD34 Acts in ATF4-to-CHOP Signaling to Block CHOP Expression in ER-Stress-Related Autophagy.

Cells respond to ER-stress via ER-stress sensors, leading to the UPR and subsequent apoptosis; however, occasionally, they activate autophagy without subsequent apoptosis in response to ER-stress. We previously showed that the induction of apoptosis by ER-stress was related to the presence or absence of CHOP expression; nevertheless, how ATF4 expression is elicited without downstream CHOP expression is unknown. We studied the role of GADD34 on the induction of autophagy and/or apoptosis by NaF- or tunicamycin-induced ER-stress in HepG2 cells transfected with GADD34 siRNA. Although NaF and tunicamycin both induced PERK activation followed by eIF2α phosphorylation and ATF4 expression, CHOP expression was only induced by tunicamycin. Concomitant with the signaling change, autophagy was activated both by NaF and tunicamycin, and apoptosis was induced only by tunicamycin. After 4 h, GADD34 mRNA expression was also increased by NaF and tunicamycin. Suppression of GADD34 by GADD34 siRNA increased ATF4 expression in both NaF- and tunicamycin-treated cells. The GADD34 siRNA increased CHOP expression, which corresponded to increased ATF4 in tunicamycin-treated cells; however, the increased ATF4 did not induce CHOP expression in NaF-treated cells. In concert with signal changes, siRNA treatment additively increased the autophagic activity of both NaF- and tunicamycin-treated cells; however, apoptosis was produced and accelerated only for tunicamycin-treated cells. These findings indicate that GADD34 expression induced by ER-stress delays CHOP expression and retards apoptotic cell death, and that an ATF4-signal-modulating machine other than GADD34 acts on ATF4-to-CHOP signaling to block ATF4-induced CHOP expression in ER-stress related autophagy.

Zyxin modulates the transmigration of Haemophilus influenzae to the central nervous system.

The mechanism by which Haemophilus influenzae causes meningitis is unclear. Previously, we established murine meningitis by intranasal instillation of H. influenzae as a cell-bound organism (CBO). In this study, we aimed to identify the molecules associated with inhibiting the transmigration of cells across the blood-brain barrier (BBB). Two-dimensional difference gel electrophoresis and protein identification by mass spectrometry were used for proteomic analysis. Analysis of the membranous extract from a tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cell (HBMEC) monolayer revealed 41 differentially expressed proteins. Zyxin, which is thought to be essential for tight cell-to-cell junctions, decreased 1.8-fold in TNF-α-treated HBMECs. In addition, zyxin transcript levels decreased 1.5-fold in cells derived from TNF-α-treated HBMECs. Intranasal instillation of CBOs in zyxin-deficient mice resulted in a significant higher mortality rate than in wild-type mice. Transmigration of CBOs across a HBMEC monolayer pretreated with TNF-α (1 ng/mL), interleukin (IL)-1ß (10 ng/mL), or lipopolysaccharide (LPS; 10 ng/mL) was assayed by counting CBOs that migrated from an upper chamber into a lower chamber. HBMEC pretreated with TNF-α exhibited significantly greater migration (P<0.01) than did control cells or cells treated with IL-1ß or LPS. Our findings highlight that zyxin is an important protein protecting the tight junction of the BBB against cell transmigration across the BBB. Finally, TNF-α produced in respiratory infection when the primary infection reached the BBB caused decreased zyxin levels in BBB cell membranes. Furthermore, H. influenzae reaching the BBB as CBOs could transmigrate into cerebrospinal fluid across the zyxin-decreased BBB.

The effect of hemin-induced oxidative stress on erythropoietin production in HepG2 cells.

Erythropoietin (EPO) and iron are both indispensable hematopoietic factors and are often studied in humans and rodents. Iron activates prolyl hydroxylases (PHDs) and promotes the degradation of the α-subunit of hypoxia inducible factor (HIF), which regulates EPO production. Iron also causes oxidative stress. Oxidative stress leads to alterations in the levels of multiple factors that regulate HIF and EPO production. It is thought that iron influences EPO production by altering two pathways, namely PHDs activity and oxidative stress. We studied the differential effect of varying concentrations of hemin, an iron-containing porphyrin, on EPO production in HepG2 cells. Hemin at 100 µM reduced EPO mRNA expression. The hemin-induced reduction of EPO mRNA levels was attenuated at concentrations greater than 200 µM and EPO production increased in the presence of 500 µM hemin. In comparison, protoporphyrin IX, iron-free hemin did not influence EPO mRNA expression. Additionally, malondialdehyde (MDA) concentrations and superoxide dismutase (SOD) activity significantly increased with 300 µM hemin. Importantly, the antioxidant tempol inhibited the hemin-induced (500 µM) increase in EPO mRNA levels. In conclusion, these results suggest that restraint of EPO production by hemin was offset by the promotion of EPO production by hemin-induced oxidative stress at hemin concentrations greater than 300 µM.

Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression.

Cells exposed to ER-stress undergo the Unfolded Protein Response (UPR) to avoid apoptosis, but may also activate autophagy. However, the signal for selection of one of these two protective responses is unknown. To clarify the key switch between autophagy and apoptosis, we examined the correlation of UPR-related signals with autophagy and/or apoptosis inductions in HepG2 cells exposed to three ER-stress inducers (NaF, tunicamycin, and thapsigargin) with time, including the effect of small interfering RNA on the cell responses. Thapsigargin-induced ER-stress caused only apoptosis after ∼2 hr with Ire1 phosphorylation, and Grp78, ATF4, and CHOP expressions. On the other hand, NaF- and tunicamycin-induced ER-stress caused only autophagy in the early stage by ∼8 hr with ATF4 expression and without CHOP expression. ATF4-siRNA completely inhibited the autophagy induced by NaF or tunicamycin with suppressed ATF4 protein and mRNA expressions, and also inhibited apoptosis by thapsigargin with suppression of both ATF4 and CHOP. CHOP-siRNA had no effect on autophagy activation by NaF and tunicamycin. On the other hand, CHOP-siRNA activated autophagy in thapsigargin-induced ER-stress with significant ATF4 expression, and suppressed apoptosis with CHOP suppression. These results showed that ATF4 is the key signal for autophagy induced by ER-stress, and that autophagy is switched to apoptosis by subsequent CHOP upregulation, suggesting that the changeover switch between autophagy and apoptosis is located between ATF4 to CHOP in the PERK pathway.

Sar1 translocation onto the ER-membrane for vesicle budding has different pathways for promotion and suppression of ER-to-Golgi transport mediated through H89-sensitive kinase and ER-resident G protein.

ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G(i2) protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G(i/o) protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G(i/o) protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways.

ER-resident Gi2 protein controls sar1 translocation onto the ER during budding of transport vesicles.

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.

Differential effects of Trypanosoma brucei gambiense and Trypanosoma brucei brucei on rat macrophages.

Mammalian immune responses to Trypanosoma brucei infection are important to control of the disease. In rats infected with T. brucei gambiense (Wellcome strain; WS) or T. brucei brucei (interleukin-tat 1.4 strain [ILS]), a marked increase in the number of macrophages in the spleen can be observed. However, the functional repercussions related to this expansion are not known. To help uncover the functional significance of macrophages in the context of trypanosome infection, we determined the mRNA levels of genes associated with an increase in macrophage number or macrophage function in WS- and ILS-infected rats and in cultured cells. Specifically, we assayed mRNA levels for macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage migration inhibitory factor (MIF). Upregulation of GM-CSF and MIF mRNA levels was robust in comparison with changes in M-CSF levels in ILS-infected rats. By contrast, upregulation of M-CSF was more robust in WS-infected rats. The phagocytic activity in macrophages harvested from ILS-infected rat spleens, but not WS-infected spleens, was higher than that in macrophages from uninfected rats. These results suggest that macrophages of WS-infected rats change to an immunosuppressive type. However, when WS or ILS is cocultured with spleen macrophages or HS-P cells, a cell line of rat macrophage origin, M-CSF is upregulated relative to GM-CSF and MIF in both cell types. Anemia occurs in ILS-, but not WS-infected, rats. Treatment of spleen macrophages or HS-P cells cocultured with ILS with cobalt chloride, which mimics the effects of anemia-induced hypoxia, led to downregulation of M-CSF mRNA levels, upregulation of GM-CSF and MIF, and an increase in phagocytic activity. However, the effect of cobalt chloride on spleen macrophages and HS-P cells cocultured with WS was restricted. These results suggest that anemia-induced hypoxia in ILS-infected rats stimulates the immune system and activates macrophages.

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats.

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of ß-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.

Masseteric nerve injury increases expression of brain-derived neurotrophic factor in microglia within the rat mesencephalic trigeminal tract nucleus.

The distribution of brain-derived neurotrophic factor was examined in the rat mesencephalic trigeminal tract nucleus after transection and crush of the masseteric nerve. In the intact mesencephalic trigeminal tract nucleus, brain-derived neurotrophic factor was detected in small cells with fine processes. These cells and processes were occasionally located adjacent to tyrosine kinase B receptor-immunoreactive sensory neurons. The transection and crush of the masseteric nerve increased expression of brain-derived neurotrophic factor in the nucleus. The number and size of brain-derived neurotrophic factor-immunoreactive cells and processes were dramatically elevated by the nerve injury. As a result, the density of brain-derived neurotrophic factor-immunoreactive profiles in the mesencephalic trigeminal tract nucleus at 7 days after the injury was significantly higher compared with the intact nucleus. Double immunofluorescence method also revealed that brain-derived neurotrophic factor-immunoreactive cells were mostly immunoreactive for OX-42 but not glial fibrillary acidic protein. In addition, the retrograde tracing method demonstrated that brain-derived neurotrophic factor-immunoreactive cells and processes surrounded retrogradely labeled neurons which showed tyrosine kinase B receptor-immunoreactivity. These findings indicate that the nerve injury increases expression of brain-derived neurotrophic factor in microglia within the mesencephalic trigeminal tract nucleus. The glial neurotrophic factor may be associated with axonal regeneration of the injured primary proprioceptor in the trigeminal nervous system.

Effects of extracellular acidic-alkaline stresses on trigeminal ganglion neurons in the mouse embryo in vivo.

Acidic-alkaline stresses caused by ischemia and hypoglycemia induce neuronal cell death resulting from intracellular pH disturbance. The effects of acidic-alkaline disturbance on the trigeminal ganglion (TG) neurons of the embryonic mouse were investigated by caspase-3-immunohistochemistry and Nissl staining. TG neurons exhibited apoptosis in 3.08 ± 0.55% of neurons in intact embryos at day 16. Intraperitoneal injection of alkaline solution (pH 8.97; 0.005-0.1 M K2HPO4 or 0.01-0.04 M KOH) into the embryo at embryonic day 15 significantly increased the number of apoptotic neurons in the TG at embryonic day 16 with dependence on concentration (3.40-6.05 and 2.93-5.55%, respectively). On the other hand, acidic solutions (pH 4.4; 0.01-0.2 M KH2PO4 slightly, but not significantly, increased the number of apoptotic cells (3.64-5.15%, without dependence on concentration). Neutral solutions (pH 7.4; 0.01-0.2 M potassium phosphate buffer) had no effect on neuronal survival in the TG (2.89-3.48%). The results indicated that alkaline stress significantly increased apoptosis in the developing nervous system, but acidic stress did not.

H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled ß-tubulin.

ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was ß-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that ß-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled ß-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of ß-tubulin regulates Sar1 translocation.

Tunicamycin-induced cell death in the trigeminal ganglion is suppressed by nerve growth factor in the mouse embryo.

The effect of nerve growth factor (NGF) on tunicamycin (Tm)-treated neurons in the trigeminal ganglion was investigated by use of caspase-3 immunohistochemistry. In intact embryos at embryonic day 16.5, only a few caspase-3-immunoreactivity were detected in the ganglion neurons. Mean +/- SE of the density of the immunoreactivity was 0.22 +/- 0.03%. In contrast, the number of the immunoreactive neurons was increased at 24 h after injection of 0.5 microg Tm in 1 microl of 0.05 N NaOH solution into mouse embryos at embryonic day 15.5. The density of immunoreactivity was also increased (mean +/- SE = 1.44 +/- 0.11%) compared to intact and 0.05 N NaOH-treated embryos (mean +/- SE = 0.35 +/- 0.03%). The Tm treatment caused increase of the number of trigeminal neurons representing apoptotic profiles (intact, mean +/- SE = 79.3 +/- 8.5; 0.05 N NaOH, mean +/- SE = 132 +/- 11.5; 0.5 microg Tm, mean +/- SE = 370.2 +/- 64.8). In addition, NGF significantly prevented the increase of density of the immunoreactivity (mean +/- SE = 0.54 +/- 0.16%) and the number of apoptotic cells (mean +/- SE = 146.2 +/- 11.3). Saline application (without NGF) had no effect on Tm-induced increase of the immunoreactivity (mean +/- SE = 1.78 +/- 0.23%) or the apoptotic profiles (mean +/- SE = 431.9 +/- 80.5). These results indicate that Tm-induced cell death in the trigeminal ganglion is suppressed by NGF in the mouse embryo.

Effect of polyamine-deficient chow on Trypanosoma brucei brucei infection in rats.

Polyamines are essential for proliferation of Trypanosoma brucei brucei, and feeding rats polyamine-deficient chow (PDC) decreases their blood polyamine concentrations. Proliferation of T. b. brucei (IL-tat 1.4 strain) (IL) is not restrained within PDC-fed rats. However, symptoms of IL-infected rats such as anemia decrease by PDC feeding. We reported cytokine and nitric oxide (NO) production of T. b. gambiense (Wellcome strain [WS])-infected rats were affected by PDC feeding, and WS proliferation was restrained. Therefore, we investigated whether the change in production of cytokines and NO by PDC feeding affects IL proliferation and decreases symptoms in vivo. In IL-infected PDC-fed rats, NO, interleukin (IL)-12, and tumor necrosis factor-alpha production increased while interferon-gamma and IL-10 decreased compared to normal chow-fed rats. IL proliferation was restrained by NO production when it was co-cultured with spleen cells harvested from uninfected rats. In contrast, IL proliferation in infected rats was not changed by PDC feeding, although NO production was increased. The results suggest that changes in cytokines and NO production in IL-infected rats by PDC feeding have little influence on IL proliferation. However, they may serve to decrease symptoms.

ER-stress caused by accumulated intracistanal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride.

In rat exocrine pancreas cells, fluoride treatment causes autophagy resulting from intracisternal granule accumulation. Excessive autophagy might promote a type of programmed cell death different from apoptosis. To clarify how fluoride-induced autophagy and subsequent cell death occurs, we investigated morphological and biochemical changes in exocrine pancreas cells of rats subcutaneously injected with NaF saline solution at 20 mg/kg dose twice daily for 4 days. Intracisternal granule, excessive autophagy and ribosomal degranulation were observed in fluoride-exposed cells, occasionally with necrotic changes. Fluoride-induced rER-stress increased eIF-2alpha phosphorylation and CHOP expression, but did not affect GRP78. Spliced XBP-1 expression was decreased in damaged cells. These findings indicate that rER-stress by intracisternal granule accumulation lead to autophagy in exocrine pancreas cells without UPR, suggesting that signal process of autophagy differs from that of UPR-apoptosis. It is likely that intense degranulation is a turning point that damaged cells change over from autophagy, cell-protective process, to cell-death process.

Effect of fluoroquinolones on body temperature of mice.

OBJECTIVES: The purpose of this study was to assess the effect of fluoroquinolones on rectal temperature of infected and non-infected mice. METHODS: The effect of fluoroquinolone administration on rectal temperature was investigated in a mouse model with and without infection. RESULTS: Hypothermia was induced by administration of gatifloxacin at doses of 1 and 10 mg/kg in non-infected mice, but not by cefditoren pivoxil. In addition, the rectal temperature was decreased in infected mice administered fluoroquinolones or cephems, a greater decrease being noted in animals receiving the former agents. CONCLUSIONS: This study shows that fluoroquinolones decrease the rectal temperature of mice regardless of infections.

Effect of BDNF depletion on the formation of Ruffini endings in vibrissa follicles and the survival of their mechanoreceptive neurons in trigeminal ganglion.

We examined the influence of BDNF depletion in peripheral tissues on the formation of Ruffini endings and their neuronal survival by injections of neutralizable anti-BDNF antibody into mouse mystacial pads for periods of 5 days at different developmental stages of the Ruffini endings (the pre-formation stage from the 2nd to 6th day after birth, the formation stage from the 4th to 8th, or the post-formation stage from the 10th to 14th). The treatment at the pre-formation and formation stages caused a significant decrease in the number of Ruffini endings in vibrissa follicles. This decrease in Ruffini endings was accompanied with a significant increase in neuron apoptosis in the trigeminal ganglion (TG) in both stages. However, at the post-formation stage, the anti-BDNF injection showed no effect on the formation of the mechanoreceptors nor their neuronal survival. In the post-formation stage, the axoplasmic spins of Ruffini endings were circumferentially embraced with the cytoplasmic processes of terminal Schwann cells. The present study indicates that target-derived BDNF is essential for survival of mechanoreceptive nerves in the pre-formation and formation stages, but not in the post-formation stages of their development. It seems that Schwann cells participate in this switch-over of neuronal dependency on brain-derived neurotrophic factor.

Therapeutic effects of beta-thujaplicin eardrops on canine Malassezia-related otitis externa.

An eardrop solution of beta-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of beta-thujaplicin solution of 100 microg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that beta-thujaplicin eardrops are effective for Malassezia-related otitis externa in dogs.

The development of myelinated nociceptors is dependent upon trks in the trigeminal ganglion.

Cell size of primary sensory neurons and distribution patterns of neurons that are immunopositive (ip) for VRL-1, a newly cloned capsaicin-receptor homologue, were examined in trigeminal ganglia (TGs) of knockout mice for trkA, trkB or trkC to determine the developmental dependency of myelinated nociceptors on expression of the genes. The number of TG neurons was strongly decreased in the knockout mice as compared to wildtype and heterozygous mice (82%, 39%, and 48% reduction for trkA, trkB and trkC, respectively). The absence of trkA and trkC reduced the number of TG neurons in all cell-size ranges. The number of medium-sized and large TG neurons was decreased in trkB-knockout mice, whereas that of small TG neurons was barely affected by trkB deficiency. TG contained abundant VRL-1-ip neurons in wildtype and heterozygous mice; 9% of TG neurons exhibited immunopositivity. In trkA-knockout mice, VRL-1-ip neurons almost disappeared (1% of TG neurons were VRL-1-ip). However, 13% and 9% of TG neurons in trkB- and trkC-knockout mice, respectively, were immunostained for the ion channel protein. In trkC-knockout mice, the proportion of large VRL-1-ip neurons decreased whereas that of small and medium-sized VRL-1-ip neurons increased. In addition, immunohistochemistry of the protein gene product 9.5 (PGP 9.5) demonstrated that trkA deficiency caused a marked reduction of varicose endings in the epithelium of the palatal mucosa. Loss of trkC diminished the number of PGP 9.5-ip varicose fibers in the deep layer of mucosal connective tissue of the palate. In tooth pulp, PGP 9.5-ip nerve fibers were absent in trkA-knockout mice but abundant in trkB- and trkC-knockout mice. The present study suggests that the development of myelinated nociceptors is dependent on trkA and trkC but not on trkB.