Fukumi Hiragami

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.

cAMP and calcium ionophore induce outgrowth of neuronal processes in PC12 mutant cells in which nerve growth factor-induced outgrowth of neuronal processes is impaired

During continuous culturing, PC12 cells are subject to spontaneous mutations. We obtained PC12m3 cells, clone cells in which outgrowth of neuronal processes (dendrites and axons) under the condition of nerve growth factor (NGF) treatment was highly stimulated by various inducers, such as cyclic adenosine monophosphate (cAMP), calcium ionophore, steroid and high osmolarity. The number of cells with neuronal processes in the presence of cAMP was approximately twenty-fold greater than PC12 parental cells and other PC12 mutant cells. In PC12m3 cells, NGF-induced outgrowth of neuronal processes was reduced by cytotoxic solanine, whereas the effect of NGF was unaffected by hyaluronic acid. In PC12m3 cells, various inducers of neurite outgrowth, such as cAMP, calcium ionophore and high osmolarity, activated mitogen activated protein (MAP) kinase, whereas solanine and hyaluronic acid did not cause any significant activation of MAP kinase. However, PC12m3 cells, in which NGF-induced outgrowth of neuronal processes were impaired, had strong NGF-induced MAP kinase activity as PC12 parental cells had. These findings suggest that cAMP, calcium influx and high osmolarity induce outgrowth of neuronal processes in PC12m3 cells through activation of the downstream target of MAP kinase or through a novel pathway independent of NGF activation.

Osmotic shock-induced neurite extension via activation of p38 mitogen-activated protein kinase and CREB.

Although it is known that sustained activation of classical mitogen-induced protein kinase (MAPK, also known as ERK) induced by nerve growth factor (NGF) plays an important role in the induction of neurite outgrowth, the role of p38 MAPK in neural cell function is still not clear. We developed two neuronal cell lines from PC12 cells, PC12m3 and PC12m32, in which NGF-induced neurite outgrowth is impaired and that show neurite outgrowth in response to hyperosmotic shock. The frequencies of neurite outgrowth of PC12m3 and PC12m32 cells induced by osmotic shock were approximately 10- and 12-fold greater, respectively, than that in PC12 parental cells. The p38 MAPK pathway inhibitor SB203580 but not the ERK pathway blocker U0126 inhibited the ability of PC12m3 and PC12m32 cells to induce neurite outgrowth in response to osmotic shock. Furthermore, expression of a nonactivable form of p38 but not that of wild-type p38 significantly blocked neurite outgrowth induced by osmotic shock. The extent of phosphorylation of p38 MAPK induced by osmotic shock in PC12m32 cells was much greater than that in PC12 parental cells. The upstream kinases MKK3 and MKK6, which phosphorylate and activate p38 MAPK, also showed higher levels in PC12m32 cells than in PC12 parental cells when treated with osmotic shock. Inhibition of p38 MAPK by SB203580 resulted in inhibition of the activity of the transcription factor CREB, which is activated by osmotic shock. These findings indicate that activation of CREB mediated by a p38 pathway distinct from the NGF signaling pathway may be required for neurite outgrowth.

Immunosuppressant FK506 Induces Neurite Outgrowth in PC12 Mutant Cells with Impaired NGF-Promoted Neuritogenesis via a Novel MAP Kinase Signaling Pathway

We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.

Chinese Medicine Induces Neurite Outgrowth in PC12 Mutant Cells Incapable of Differentiation

During continuous culture of neural PC12 cells, we obtained a drug-hypersensitive PC12 mutant cell that showed high stimulation of neurite outgrowth by various drugs. When several Chinese medicines such as shu-jing-huo-xie-tang and Wu-Ling-San were provided to these PC12 mutant cells, the frequency of nerve growth factor (NGF)-induced neurite outgrowth increased approximately 30-fold compared to NGF alone. Neurite outgrowth induced by NGF in PC12 cells is accompanied by sustained activation of mitogen-activated protein kinase (MAPK); however, these Chinese medicines did not induce MAPK activity. The findings thus indicate that certain Chinese medicines may induce neurite outgrowth by a novel mechanism which is distinct from the NGF-activated pathway in PC12 mutant cells.

Formation of hydroxyapatite-mediated three-dimensional structures by mouse fibroblasts in response to physical stimulation.

Hydroxyapatite (HAP) ceramics are widely used as implant materials for periodontal bone defects because of their excellent biocompatibility. We demonstrated that physical stimulation, that is, (1). mechanical stimuli or (2). laser irradiation, causes HAP-mediated C3H10T1/2 mouse fibroblasts to form three-dimensional tissue-like structures. Trypsinized 10T1/2 cells were cultured simultaneously with 200 HAP granules on a rotator for 7 days in mechanical stimulation experiments. The cells were later transferred to a regular incubator. Cell reactions were observed by phase-contrast microscopy. The formation of three-dimensional structures around the HAP granules was observed in the third week of cultivation after stimulation. In laser irradiation experiments, trypsinized cells were irradiated with 1, 5, and 16 J/cm(2) at a wavelength of 1000 nm and cultured with 200 HAP granules for 10 weeks. The formation of three-dimensional structures, like those observed in the mechanical stimulation experiments, was observed in the third week after irradiation. The formation of these structures was most frequent at 1 J/cm(2), and the frequency of formation of these structures gradually decreased as the irradiation dose was increased. These results indicate that physical stimuli may stimulate cell proliferation, leading to the repair of damaged tissue. These results also indicate that mouse fibroblasts do not form these three-dimensional structures without HAP and that HAP alone is not sufficient to stimulate the formation of three-dimensional structures.

The utility of a care model to individualize rehabilitation in adults aged over 80 years

Abstract Objective: The objectives of the present study were to assess the complexity and multidimensionality of rehabilitation needs of very old stroke patients aged ≥ 80 years and report how rehabilitation interventions are customized to meet the complex needs of patients at a hospital with a majority of old patients. Methods: The complex problems faced by 18 post-stroke patients (age, range: 80–92 years) were characterized in terms of the following multiple dimensions: (1) clinical features, (2) functional (motor/cognitive) impairment features, (3) psychological aspects, and (4) environmental aspects. We then evaluated the rehabilitation interventions designed to address the problems identified in these different dimensions in detail. Results: The needs of very old stroke patients were extremely complex and unique. To cope with this complexity, rehabilitation interventions were customized in a flexible manner, considering the different dimensions of the needs of these patients. Although the interventions were customized, the complex problems experienced by patients could be divided into stroke conditions on the basis of some invariant patterns in rehabilitation intervention. Conclusions: We obtained empirical data that illustrated the necessity of considering not only clinical features, but also multiple dimensions of problems faced by very old stroke patients during rehabilitation interventions.

Heat-shock-induced three-dimensional-like proliferation of mouse fibroblasts mediated by hydroxyapatite.

The aim of the present study was to determine both the minimal and the optimal conditions under which heat treatment is effective for enhancing 3D (three‐dimensional)‐like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or 45°C for 2, 10, 15, 20, 30, 45, 60, 90, 180 and 360 min. Then minimal and optimal conditions of temperature and duration of heat treatment for induction of 3D‐like proliferation of cells were determined. The minimal conditions of heat treatment to induce 3D‐like proliferation were 43°C for 2 min and the optimal conditions were 43°C for 10 min. The mean rates of formation of 3D‐like proliferation patterns by cells heat‐treated at 43°C for 2 and 10 min were significantly higher (1.7‐ and 3.7‐fold respectively) than that by untreated cells (P<0.05). We also observed significantly greater effects of heat treatment on 3D‐like proliferation at 40°C for 90 or 180 min and at 41.5°C for 15 min and 44°C for 10 min. We found that apoptosis had occurred in 7.5 and 87.0% of the cells at 1 week after heat treatment at 43°C for 10 min and 30 min respectively. Western‐blot analysis demonstrated that phosphorylation of p38 MAPK (mitogen‐activated protein kinase) was markedly increased by heat treatment at 43°C for 10 min. These findings suggest that activation of p38 MAPK by heat shock is associated with 3D‐like cell proliferation. The results of the present study should be useful for further studies aimed at elucidation of the physiological mechanisms underlying thermotherapy and hyperthermia.

A Process of Multidisciplinary Team Communication to Individualize Stroke Rehabilitation of an 84-Year-Old Stroke Patient.

Previously, we have used a multidisciplinary team (MDT) approach to individualize rehabilitation of very old stroke patients as a means to establish intervention points for addressing impaired activities of daily living (ADL). However, this previous study was limited because of a lack in describing the communication process over time. This case study characterized the MDT communication process in the rehabilitation of an 84-year-old patient over the course of 15 weeks. The MDT consisted of 3 nurses, 1 doctor, 6 therapists, and the patient/families. Meetings (15 minutes each) were held at 4, 6, 8, and 15 weeks following the patient’s admission. To individualize the rehabilitation, the communication process involved gaining knowledge about ADL impairments, sharing assessments, providing treatment options, and reflecting on desired treatment outcomes—a process termed KATR. The knowledge, assessment, treatment, and reflection (KATR) process established intervention points focusing on specific ADL impairments. The team members focused the interventions on the impaired ADL identified in the KATR process, and individualized rehabilitation was generated from the MDT information-sharing knowledge. In the initial meeting (Week 4), intervention points derived from the KATR process focused on rehabilitation of self-care impairments. These impairments improved by Week 15. By the last meeting, the MDT intervention points focused on mobility impairments. Having an organized communication process (i.e., KATR) facilitates individualization of rehabilitation without lengthy and frequent MDT meetings and enhances the quality of rehabilitation after a stroke.