Kenji Kawamura

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.

Changes in rupture formation and zonary region stained with evans blue during the recovery process from aluminum toxicity in the pea root apex

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. Elongation and re-elongation of the root during the recovery process from Al injury occurred only in the apical 5-mm region of the pea root. With the model system of the pea root for recovery from Al injury, images of the root characterized by zonal staining with Evans blue showed the existence of two regions in the root apex consisting of rupture and zonary stained regions. Ruptures enlarged by increase in their depth but without widening of the intervals among zonary stained regions in the roots treated with Al continuously. On the other hand, intervals of the zonary stained regions were widened due to re-elongation of the root and were narrow in the rupture region in the recovery root.

Microwave irradiation induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.

Electromyographic analysis of a modified maneuver for quadriceps femoris muscle setting with co-contraction of the hamstrings.

A „quadriceps femoris muscle setting”︁ is isometric quadriceps femoris exercise which can be widely used in early knee rehabilitation. However this exercise cannot obtain enough co‐contraction of the hamstrings. Isolated quadriceps femoris contraction in knee extension imposes severe strain to anterior cruciate ligament. We succeeded in developing a simple training maneuver that is effective in obtaining co‐contraction of the hamstrings—a modified maneuver for the quadriceps femoris muscle setting with the contralateral lower limb raised (MQS). In this study, we analyzed the effect of this maneuver by EMG quantification. Twenty‐eight healthy young adult men performed sequential trials consisting of normal quadriceps femoris muscle setting (NQS) and MQS. Electromyographic activity was recorded from surface electrodes on the gluteus maximus, vastus medialis, rectus femoris, vastus lateralis, semitendinosus and biceps femoris (long head), and normalized to values derived from maximal isometric trials. The % maximal voluntary isometric contraction (%MVIC) of the vastus medialis, vastus lateralis and rectus femoris did not vary in the each maneuver. However, the %MVIC of the hamstrings varied significantly in the MQS. This study suggests that effective co‐contraction of the hamstrings can be obtained in MQS by adjusting the load to the raised lower limb.

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.

A survey of wheelchair use by paraplegic individuals in Japan. Part 1: Characteristics of wheelchair cushions

The characteristics of wheelchair cushions used by 218 paraplegic patients who lived independent lives were surveyed to clarify the present state of wheelchair cushioning for pressure sore prevention in Japan. Out of 586 cushions surveyed, 91.0% were ready-made and the rest were custom-made. The outstanding popularity of polyurethane foam ready-made cushions (76.3%) suggested that insufficient consideration was taken in the selection of cushions. Custom-made cushions displayed unique modifications to relieve contact pressure or to stabilize sitting posture, which should be systematically provided for all patients. The variety of cushion types and the frequent dissatisfaction with cushions seen in patients with current pressure sores suggested a strong demand for the effective prescription of cushions. Furthermore, 30% of all cushions had had an excessively prolonged use, indicating insufficient follow-up. A medical system including deliberate prescription and regular follow-up of wheelchair cushions should be established for the effective prevention of pressure sores.

Factors for Assessing the Effectiveness of Early Rehabilitation after Minimally Invasive Total Knee Arthroplasty: A Prospective Cohort Study

The effectiveness of current rehabilitation programs is supported by high-level evidence from the results of randomized controlled trials, but an increasing number of patients are not discharged from the hospital because of the schedule of the critical path (CP). The present study aimed to determine which factors can be used to assess the effectiveness of early rehabilitation. We enrolled 123 patients with medial knee osteoarthritis (OA) who had undergone unilateral minimally invasive total knee arthroplasty for the first time. The following factors were assessed preoperatively: the maximum isometric muscle strength of the knee extensors and flexors, maximum knee and hip joint angle, pain, 5-m maximum walking speed, sex, age, body mass index, exercise habits, Kellgren-Lawrence grade, femorotibial angle, failure side (bilateral or unilateral knee OA), and functional independence measure. We re-evaluated physical function (i.e., muscle strength, joint angle, and pain) and motor function (5-m maximum walking speed) 14 days postoperatively. Changes in physical function, motor function (5-m maximum walking speed), and number of days to independent walking were used as explanatory variables. The postoperative duration of hospitalization (in days) was used as the dependent variable in multivariate analyses. These analyses were adjusted for sex, age, body mass index, exercise habits, Kellgren-Lawrence grade, femorotibial angle, failure side, and functional independence measure. The duration of hospitalization was significantly affected by the number of days to independent walking (p < 0.001, β = 0.507) and a change in the 5-m maximum walking speed (p = 0.016, β = -0.262). Multiple regression analysis showed that the radiographic knee grade (p = 0.029, β = 0.239) was a significant confounding factor. Independent walking and walking speed recovery were considered to reduce the duration of hospitalization. Therefore, these indices can be used to assess the effectiveness of early rehabilitation.