Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

Abstract

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.

Enhancement of Neurite Outgrowth by Warming Biomaterial Ultrasound Treatment

“…Through the ultrasonic treatment of the warming biomaterial with N2A cells, neurite outgrowth was induced, extending the axonal length by about twice that of the control group ( Figure 5). This result was similar to Kudo's study [9], which confirmed that the mechanism of neurite outgrowth was mainly due to heat induction; however, our AChE activity results are higher compared to theirs. This can be attributed to the shrinkage of the membrane due to acoustic overstimulation affecting the distribution of acetylcholinesterase, thereby promoting neurotransmission from the presynaptic to the postsynaptic neurons.…”

“…Thermally stimulated cells are confirmed to enhance neurite outgrowth through activation and signaling of the p38 mitogen-activated protein kinase (MAPK) pathway [9,25,26] in PC12 cells, and there is a lack of relevant studies on N2A cells but some studies have explored the MAPK pathway related to neurite outgrowth in N2A cells [27,28]. Heat stimulation activates phosphatidylinositol 3-kinase (PI3K)/Akt and the three MAPK signal transduction pathways.…”

“…Physiotherapists have been using therapeutic ultrasound or thermal therapy mostly to repair soft tissue injuries, such as damage to muscles or nerves [6][7][8]. Thermal stimulation of neural cells at 39.5-40 • C [9] can enhance neurite outgrowth, but therapeutic ultrasound can also induce nerve damage or cause local tissue injury due to the thermal effect of cavitation. Attenuation is determined by ultrasound frequency and tissue depth [10][11][12][13].…”

Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal

“…The requirement of p38 activation in Ngb-facilitated neurite regeneration was strengthened by the blocking effects of kinase-dead p38 (i.e., p38 K53A ). In PC12 cells, p38 MAPK is involved in nerve growth factor- and thermal-stimulated neurite outgrowth 9 , 34 , supporting a functional role of p38 MAPK pathway in axon/neurite regrowth under pathological conditions. We found that p-p38 was well co-localized with Ngb in ischemic neurons in the brain.…”

Fabrication of precisely aligned microwire and microchannel structures: Toward heat stimulation of guided neurites in neuronal cultures

“…Understanding the regulation of neurite outgrowth is crucial for developing therapies to promote axon regeneration after injury or in neurodegenerative diseases . Recently, it was shown that transient heat stimulation, such as mild hyperthermia (42.0–43.0 °C), can induce neurite outgrowth and influence neuronal networks . In order to investigate the changing behaviour of isolated neurites under the influence of thermal stress, it is desirable to grow neurons into specific regions where functional components are located.…”