Search

The neurotrophic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on rat sensory neuronal cell line ND7/23 cells were investigated. PACAP caused a concentration-dependent increase in the number of neurite-bearing cells and the expression of the substance P precursor (PPT) mRNA in 24 h. The effects of PACAP were mimicked by vasoactive intestinal polypeptide with lower potency and dibutyryl-cyclic AMP, and inhibited by inhibitors of protein kinase A, ERK kinase or p38 kinase, KT5720, U0126, or SB203580, respectively. In a PPT promoter luciferase reporter assay, the increase of PPT mRNA was the result of an increase in PPT gene transcriptional activity by PACAP. The increasing effects of PACAP on PPT mRNA were similarly observed in primary cultured rat dorsal root ganglion cells. Thus, PACAP could induce differentiation-like phenomena in sensory neurons in a cAMP-, protein kinase A-, ERK kinase-, and p38 kinase-dependent manner. These results provide evidence of the neurotrophic action of PACAP, which may function to rescue damaged neurons or to switch the neuronal phenotype in injured or inflamed sensory neurons.

The effect of high molecular weight chitosan supplement (HMCS), a natural polymer derived from chitin, on indices of oxidative stress was investigated in normal volunteers. The use of HMCS for 8 weeks resulted in a significant decrease in total cholesterol levels and atherogenic index, and increased levels of high density lipoprotein (HDL) cholesterol. HMCS treatment also resulted in a lowered ratio of oxidized to reduced albumin and an increase in total plasma antioxidant activity. A good correlation between the atherogenic index and oxidized albumin ratio was found. The results suggest that the ratio of oxidized to reduced albumin ratio represents a potentially useful marker of the metabolic syndrome. In in vitro studies, HMCS slightly reduced the levels of two stable radicals in a dose- and time-dependent manner. The strong binding capacity of indoxyl sulfate and low density lipoprotein (LDL) cholesterol was also observed with HMCS. These results suggest that HMCS reduces significant levels of pro-oxidants such as cholesterol and uremic toxins in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation in humans.

To estimate the extent of use of over-the-counter (OTC) drugs and health food, we administered a questionnaire to the parents of children in a nursery school and to elderly people in a daycare center in Fukuyama city. The aim of the questionnaire was to determine the percentage of children and elderly people who use OTC drugs and health food, the purpose of using them, and the types of OTC drugs and health food used. Other questions concerned the person advising them on the use of OTC and health food, the side effects of OTC drugs and health food, and the awareness of children and elderly people regarding possible interactions between prescription drugs and OTC drugs. In children, the most frequently consumed OTC drugs were cold medicines (32.1%), followed by topical creams (22.6%) and eye lotion (14.3%). In elderly people, the most frequently consumed OTC products were eye lotion (18.0%), followed by laxatives (14.8%) and fomentation agents (13.1%). The purchase ratio of health food for children and elderly people were 4.8% and 11.5%, respectively. These results suggest that the need for OTC drugs and health food in children are very different from those in elderly people. In addition, in promoting self-medication, the demand for the opinion of a specialist occupied about 80% or 70% of the total specialist time among children and elderly people, respectively. Therefore, when providing information on health food and OTC drugs, the needs of each generation should be taken into account. The information obtained from the responses received will allow us to provide better pharmaceutical care for both children and elderly people in Fukuyama city.

Since the six-year education system for pharmacy students was established in 2006,educational programs to cultivate humanism in pharmacists have been an important part of the curriculum in each year.In Fukuyama University,a program providing contact with elderly people in day-care centers and children in nursery schools was introduced for second-year under graduate pharmacy students in 2007,in order to cultivate students’ability to communicate and empathize with them.Analysis of the results of a questionnaire survey on the effects of the exchange education program that we conducted for 3 years revealed the following.On the first visit,many students could not communicate well with either the elderly people or the children but from the 2nd visit onwards,almost all students were able to communicate and control the level of their voice better,and have more eye contact.As the communication program went on,students were able to build close relationships with their partners.The results of the questionnaire also showed that almost all students were satisfied with the communication education program at both facilities and their partners were also satisfied with it.In addition,good cooperation with the facilities produced a stepwise increase in student satisfaction rates over the three years.These results suggest that communication education plays an important role by cultivating hospitality skills and having students think about their behavior as medical professionals.

Immunization with amyloid-beta (Abeta) peptides, a therapeutic approach in Alzheimer's disease (AD), reduces brain Abeta, and microglial Abeta phagocytosis has been proposed as an Abeta-lowering mechanism. We transplanted rat microglia into the rat lateral ventricle just after intra-hippocampal Abeta injection, and then investigated the contribution of exogenous microglia to Abeta clearance. Migration of exogenous microglia from the lateral ventricle to Abeta plaque was detected by magnetic resonance imaging and histochemical analysis, and the clearance of Abeta was increased by transplantation. These results suggest the possible usefulness of exogenous microglia to the therapeutic approach in AD.

The actin cytoskeleton plays a critical role in the cellular morphological changes. Its organization is essential for neurite extension and synaptogenesis under the processes of neuronal development. Recently, neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE) have been identified as key molecules, which specifically participate in regulation of actin cytoskeleton through small GTPases. The functions of these factors have been investigated using cultured cells; however, in vivo developmental changes in these factors are not fully understood. In this study, we examined the expression levels and distributions of N-WASP, WAVE and their related proteins in the rat cerebral cortex and hippocampus during postnatal development. Protein levels of these factors were progressively increased during development, and actin was accumulated in membranous fractions. Immunoreactivities for these factors were widely but differentially observed in entire brain. In the developing brain, N-WASP and WAVE seemed to exist in the synapse-rich areas, such as stratum radiatum of hippocampal CA1 subfield. A similar tendency in the distributions of these factors was observed in the mature brain. Taken together, N-WASP, WAVE and their related proteins may participate in normal brain development and synaptic plasticity by regulating the actin cytoskeleton.

DJ-1 has recently been shown to be responsible for onset of familial Parkinson’s disease (PD), PARK7. DJ-1 has been shown to play roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to trigger onset of PD. In this study, a recombinant DJ-1 protein was administrated into the brain of PD model rats that had been injected to 6-hydroxydopamine (6-OHDA) in the left substantia nigra. PD phenotypes, including dopaminergic neuron death in the substantia nigra, decrease in dopamine, and dopamine transporter levels in the striatum, and motor abnormality, were dramatically improved by wild-type DJ-1 but not L166P DJ-1, a mutant form of DJ-1 found in PD patients. Furthermore, production of reactive oxygen species and cell death induced by 6-OHDA in SH-SY5Y cells and mesencephalic neurons were inhibited by addition of the recombinant DJ-1. These findings suggest that DJ-1 is a therapeutic target for PD.

Presenilin 1 (PS1) is a multifunctional protein, and its mutations are highly related to familial Alzheimer’s disease (AD). In this study, we examined the effects of PS1 overexpression on neuronal morphology using SH-SY5Y cells. Overexpression of dominant-negative D385A PS1 induced morphological change and impairment of neurite formation, while those of wild-type and pathogenic P117L mutant PS1 did not change cellular morphology compared with native cells. Moreover, filopodium-formation-related proteins were decreased only in cells overexpressing D385A PS1. Therefore, PS1 may be involved in neuritogenesis and morphological change in SH-SY5Y cells, and P117L mutation may linked to AD by different mechanisms. Keywords:: presenilin 1, SH-SY5Y cell, neuritogenesis

Usefulness of the in vitro and in vivo generation of neural precursors from embryonic stem (ES) cells has been widely discussed, but functional recovery in animal models of Parkinson's disease (PD) is not fully understood. The aim of this study was to investigate a transplantation strategy for PD by assessing whether double-transplants in the striatum (ST) and substantia nigra (SN), or ST and subthalamic nucleus (STN) induce functional recovery in 6-hydroxydopamine-lesioned rats. Methamphetamine-induced rotation was significantly reduced by transplantation of mouse ES cell-derived neurons into the ST, but not the STN or SN alone. Double-transplantation was also effective at recovering rotational behavior. Although immunoreactivity for tyrosine hydroxylase (TH) was almost completely lost in the ipsilateral striatum in hemiparkinsonian rats, TH immunoreactivity was detected in transplanted cells and sprouting fibers in the ST, STN and SN. These results suggest that both the involvement of ST as a place of transplantation and the number of ES cell-derived neurons are essential factors for efficacy on hemiparkinsonian behaviors.

One pathogenic characteristic of Alzheimer's disease (AD) is the formation of extracellular senile plaques with accumulated microglia. According to the amyloid hypothesis, the increase or accumulation of amyloid-beta (Abeta) peptides in the brain parenchyma is the primary event that influences AD pathology. Although the role of microglia in AD pathology has not been clarified, their involvement in Abeta clearance has been noted. High mobility group box protein-1 (HMGB1) is an abundant nonhistone chromosomal protein. We reported recently that HMGB1 was associated with senile plaques and the total protein level significantly increased in AD brain. In this study, diffuse HMGB1 immunoreactivity was observed around dying neurons in the kainic acid- and Abeta1-42 (Abeta42)-injected rat hippocampi. HMGB1 also colocalized with Abeta in the Abeta42-injected rats but not in transgenic mice, which show massive Abeta production without neuronal loss in their brains. Furthermore, coinjection of HMGB1 delayed the clearance of Abeta42 and accelerated neurodegeneration in Abeta42-injected rats. These results suggest that HMGB1 released from dying neurons may inhibit microglial Abeta42 clearance and enhance the neurotoxicity of Abeta42. HMGB1 may thus be another target in the investigation of a therapeutic strategy for AD.

In Alzheimer's disease (AD) brain, amyloid-β (Aβ) peptides accumulate to form amyloid plaques. However, the balance of production and clearance of Aβ peptides has been unclear. Microglia in the brain are likely to participate in maintaining this balance. Many heat-shock proteins (HSPs) are known as intracellular molecular chaperones, but their function in microglia is still unknown. In this study, we examined the effects of exogenous HSPs on microglial function. In microglial culture, Aβ peptides were phagocytosed, suggesting that microglial cultures may be useful as the in vitro model of AD pathology. In this culture, HSPs markedly induced the production of interleukin-6 and tumor necrosis factor-α, and enhanced the phagocytosis and clearance of Aβ peptides. We also examined the mechanism of microglial activation by exogenous HSPs, and found the possible involvement of the nuclear factor-κB and p38 mitogen-activated protein kinase pathways. Activation of these pathways might be mediated through Toll-like receptor-4. These observations suggest that HSP-induced microglial activation may serve a neuroprotective role by facilitating Aβ clearance and cytokine production.

One of the principal pathological features of Alzheimer's disease (AD) is the extracellular deposition of fibrillar amyloid-β (Aβ) associated with reactive microglia. However, microglial pathogenesis in AD is still unclear. In this study, we examined the relationship between microglial activation and Aβ deposition on trasgenic mice and Aβ1–42 (Aβ42)-injected rats. In the brain of a double-transgenic mouse carrying mutations in both the presenilin-1 and Aβ precursor protein (APP) transgenes, there was a marked production of Aβ peptides and reactive microglia were accumulated around Aβ deposits, but global neuronal loss was not detected. Intrahippocampal injection of Aβ42 induced microglial accumulation and then Aβ42 gradually decreased. Recent studies suggest that several cytokines produced by reactive microglia have neuroprotective effects. In addition, anti-Aβ antibodies, produced by Aβ-vaccine therapy, induce microglial phagocytosis. Taken together, microglial activation may essentially contribute to compensatory neuroprotection by Aβ phagocytosis and production of cytokines.

The brains of patients with Parkinson's disease (PD) show evidence that mitochondrial complex I is suppressed. Therefore, it is considered that rotenone, a specific inhibitor of mitochondrial complex I, is a useful tool in animal models of PD. However, the mechanism of rotenone-induced neuronal death is not fully understood. In human SH-SY5Y cells, rotenone induced the degradation of caspases-9 and -3, followed by cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and cell death. At that time, we detected a caspase-12-like protein (casp-12LP) that has a molecular mass (about 60 kDa) similar to that of mouse caspase-12, and it was degraded by rotenone, like an endoplasmic reticulum (ER) stress. Pretreatment with phorbol-12-myristate-13-acetate inhibited the rotenone-induced decrease in caspases-9 and -3, but not that in casp-12LP. In contrast, nonselective caspase inhibitor z-VAD.fmk inhibited the decrease in casp-12LP, but not those in caspases-9 and -3 in this study. These results suggest that rotenone-induced apoptosis may be mediated through both mitochondria- and ER-dependent pathways in human SH-SY5Y cells.

Embryonic stem (ES) cells have many of the characteristics of an optimal cell source for cell-replacement therapy. Although the usefulness of the in vitro generation of dopamine (DA)-neural precursors from ES cells has been widely discussed, functional recovery in animal models of Parkinson’s disease is not fully understood. In 6-hydroxydopamine-lesioned rats, apomorphine markedly induced contralateral rotation. Apomorphine-induced rotation was significantly reduced by transplantation of neuron-like cells that had differentiated from mouse ES cells using nicotinamide, but not L-lysine. In addition, methamphetamine-induced ipsilateral rotation was significantly reduced. On the other hand, picrotoxin did not inhibit apomorphine-induced rotational asymmetry. Fluoxetine alone and fenfluramine alone induced slight contralateral rotation and rotation in both directions, respectively, and these effects were similar in transplanted rats. Although immunoreactivity for tyrosine hydroxylase (TH) was almost completely lost in the ipsilateral striatum in hemiparkinsonian rats, TH immunoreactivity was detected in transplanted cells and sprouting fibers. In contrast, immunoreactivities for γ-aminobutyric acid (GABA) and serotonin (5-HT) neurons were not changed. These results suggest that improvement of rotational behavior may be induced predominantly by transplantation of nicotinamide-treated ES cell-derived DA neurons, rather than by changes in the activities of GABA or 5-HT neural systems, in hemiparkinsonian rats. Keywords:: mouse embryonic stem cell, dopaminergic differentiation, transplantation, rat striatum, Parkinson’s disease

One of the pathological characteristics of Alzheimer's disease (AD) is the formation of dystrophic neurites accompanied by aberrant neuronal sprouting. Although a number of studies have focussed on the formation of amyloid plaques and neurofibrillary tangles, the mechanism of neuronal sprouting in AD is not fully understood. The protein levels of neural Wiskott-Aldrich syndrome protein (N-WASP), WASP interacting SH3 protein (WISH) and WASP family verprolin-homologous protein (WAVE) were significantly increased in AD brains. In addition, N-WASP, WISH and WAVE were co-localized with filamentous actin in abnormal dendrite-like processes sprouting from staurosporine-treated human SH-SY5Y cells. These results suggest that N-WASP, WISH and WAVE may participate in the neurodegenerative aberrant sprouting in AD neurons.

In Alzheimer's disease, fibrillar amyloid-beta (Abeta) peptides form senile plaques associated with microglia. However, the relationship between Abeta peptides and microglia is not fully understood. In this study, the incubation of Abeta1-40 (Abeta40) produced small oligomers, while incubation with Abeta1-42 (Abeta42) caused large molecular aggregates. Microglial production of nitrite, interleukin-6 and tumor necrosis factor-alpha was induced by Abeta40, but not Abeta42. This production was significantly reduced by 15-deoxy-Delta(12,14) prostaglandin J(2), and it was completely suppressed by beta-sheet breaker peptide, Leu-Pro-Phe-Phe-Asp. These results suggest that small oligomers, rather than large molecular aggregates, mediate microglial activation induced by Abeta peptides.

Recently, it has been shown that endoplasmic reticulum (ER) stress causes apoptosis. However, the mechanism of the ER stress-dependent pathway is not fully understood. In human neuroblastoma SH-SY5Y cells, we detected a caspase-12-like protein that has a molecular mass (approximately 60 kDa) similar to that of mouse caspase-12. Thapsigargin, an inhibitor of ER-associated Ca2+-ATPase, induced the degradation of caspase-12-like protein. In addition, the degradation of caspases-9 and -3, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and cell death were also observed. Pretreatment with phorbol-12-myristate-13-acetate, which induces the expression of antiapoptotic Bcl-2, inhibited thapsigargin-induced degradation of caspases-9 and -3, but not caspase-12-like protein degradation. A caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2F, inhibited the degradation of caspase-12-like protein, but not that of caspases-9 and -3. These results suggest that thapsigargin may induce the activation of both ER- and mitochondria-dependent pathways in human SH-SY5Y cells.

The effect of chitosan on oxidative stress and chronic renal failure was investigated using 5/6 nephrectomized rats. The ingestion of chitosan over a 4-week period resulted in a significant decrease in total body weight, glucose, serum creatinine and indoxyl sulfate levels ( P = 0.0011, P = 0.0006, P = 0.0012, and P = 0.0005, respectively), compared with the non-treated nephrectomized group. The ingestion of chitosan also resulted in a lowered ratio of oxidized to reduced albumin ( P = 0.003) and an increase in biological antioxidant potential ( P = 0.023). Interestingly, the oxidized albumin ratio was correlated with serum indoxyl sulfate levels in vivo . These results suggest that the ingestion of chitosan results in a significant reduction in the levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation.

Recently, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium ion (MPP+) and rotenone have been shown to be dopaminergic neurotoxins. However, their neurotoxicities in rat brains in vivo are not fully understood. In the present study, we compared the in vivo neurotoxicities of 6-OHDA, MPP+ and rotenone using a single intranigral injection. The injection of 6-OHDA caused the greatest loss of dopaminergic neurons in both the substantia nigra and striatum, and apomorphine induced contralateral rotation. In contrast, apomorphine-induced rotational behavior was in the ipsilateral direction with MPP+, and was not observed with rotenone. Although MPP+ and rotenone caused a loss of dopaminergic neurons in the substantia nigra, striatal neurodegeneration varied. These results suggest that intranigral injections of these neurotoxins produce different degrees of dopaminergic neurotoxicity in rats in vivo.

The effect of high and low molecular weight chitosans (HMC; 1000 kDa, LMC; 30 kDa) on oxidative stress and hypercholesterolemia was investigated using male 6-week-old Wistar Kyoto rats as a normal model (Normal-rats) and spontaneously hypertensive rat/ND mcr-cp (SHP/ND) as a metabolic syndrome model (MS-rats), respectively. In Normal-rats, the ingestion of both chitosans over a 4 week period resulted in a significant decrease in total body weight (BW), glucose (Gl), triglyceride (TG), low density lipoprotein (LDL) and serum creatinine (Cre) levels. The ingestion of both chitosans also resulted in a lowered ratio of oxidized to reduced albumin and an increase in total plasma antioxidant activity. In addition to similar results in Normal-rats, the ingestion of only HMC over a 4 week period resulted in a significant decrease in total cholesterol levels in MS-rats. Further, the ingestion of LMC resulted in a significantly higher antioxidant activity than was observed for HMC in both rat models. In in vitro studies, LMC caused a significantly higher reduction in the levels of two stable radicals, compared to HMC, and the effect was both dose- and time-dependent. The findings also show that LDL showed strong binding in the case of HMC. These results suggest that LMC has a high antioxidant activity as well as antilipidemic effects, while HMC results in a significant reduction in the levels of pro-oxidants such as LDL in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation in metabolic model rats.

Middle cerebral artery occlusion (MCAO) caused behavioral dysfunction with massive neuronal loss. Cell transplantation may recover this deficit by replacing damaged brain cells. In this study, we examined the effects of transplantation of mouse embryonic stem (ES) cells or ES cell-derived neuron-like (ES-N) cells on behavioral function in ischemic rats. Seven days after MCAO, ES or ES-N cells were transplanted into ipsilateral striata (but not the substantia nigra) of ischemic rats. Transplanted rats exhibited a gradual reduction in the number of rotations induced by methamphetamine compared to vehicle-injected rats. These rats also showed a significant improvement in rota-rod performance. At 15 weeks after transplantation, immunoreactivities for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum were significantly recovered in rats grafted with ES or ES-N cells compared to vehicle-injected rats. These results suggest that intrastriatal-transplantation of ES or ES-N cells improved the dopaminergic function and subsequently recover behavioral dysfunction in focal ischemic rats.

Serofendic acid was recently identified as a neuroprotective factor from fetal calf serum. This study was designed to evaluate the neuroprotective effects of an intranigral microinjection of serofendic acid based on behavioral, neurochemical and histochemical studies in hemi-parkinsonian rats using 6-hydroxydopamine (6-OHDA). Rats were injected with 6-OHDA in the presence or absence of serofendic acid, or were treated with serofendic acid on the same lateral side, at 12, 24 or 72 h after 6-OHDA lesion. Intranigral injection of 6-OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta (SNpc). Either simultaneous or 12 h post-administration of serofendic acid significantly prevented both dopaminergic neurodegeneration and drug-induced rotational asymmetry. Immunoreactivities for oxidative stress markers, such as 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE), were markedly detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-administration of serofendic acid, similar to the results in vehicle-treated control rats. In addition, serofendic acid inhibited 6-OHDA-induced alpha-synuclein expression and glial activation in the SNpc. These results suggest that serofendic acid protects against 6-OHDA-induced SNpc dopaminergic neurodegeneration in a rat model of Parkinson's disease.

The function of microglia in the brain parenchyma is not fully understood. Occlusion of the middle cerebral artery (MCA) and reperfusion caused behavioral dysfunction with massive neuronal loss in the rat cerebral cortex and striatum. When exogenous microglia were microinjected into the intracerebroventricle (i.c.v.) during MCA occlusion, focal ischemia-induced behavioral dysfunction was significantly inhibited. At that time, many microglia migrated into the ischemic lesion, and microglia-derived neuron-like cells were barely detectable. These results suggest that exogenous microglia protect against focal ischemia-induced neurodegeneration and improve behavioral dysfunction. Keywords:: behavioral recovery, microglia, focal brain ischemia

Microglia are macrophage-like phagocytic cells in the brain parenchyma. However, microglial function after neurodegeneration is not fully understood. In this study, occlusion of the middle cerebral artery (MCA) and reperfusion caused massive neuronal loss in the rat cerebral cortex and striatum after 3 days. When exogenous microglia were microinjected into the intracerebroventricle during MCA occlusion, neurodegenerative areas significantly decreased. At that time, migrated microglia were detected in the ischemic lesion. These results suggest that exogenous microglia can migrate into brain parenchyma and then protect against neurodegeneration induced by MCA occlusion and reperfusion. Keywords:: microglia, neuroprotection, focal brain ischemia

Alzheimer's disease is characterized by the accumulation of extracellular amyloid-beta (A beta) fibrils with microglia. In an in vitro microglial culture, we recently found that heat-shock protein-90 (Hsp90) enhanced the microglial phagocytosis and clearance of A beta (1-42) (A beta 42). In this study, we examined the microinjection of A beta 42 in the presence or absence of Hsp90 into the rat hippocampus in vivo. Intrahippocampal injection of A beta 42 alone induced microglial accumulation, and the amount of A beta 42 then gradually decreased. In addition, simultaneous injection with Hsp90 significantly reduced the amount of A beta 42 and increased the production of cytokines. These results suggest that Hsp90 may facilitate microglial A beta 42 clearance in rat brain in vivo.