BIOMEDICAL AND ENVIRONMENTAL SCIENCES 23, 244-249 (2010) www.besjournal.com
1This research was supported by Natural Science Foundation of Beijing, China; Grant number: 5072020.
2Correspondence should be addressed to Xin LIN and Jia-Mou LI. Tel: 86-010-67016611. E-mail: email@example.com
Biographical note of the first author: Cong REN, male, born in 1980, MD. Beijing Tiantan Hospital, Capital Medical University,
majoring in surgery.
Copyright © 2010 by China CDC
LIPUS Enhance Elongation of Neurites in Rat Cortical Neurons
through Inhibition of GSK-3β1
CONG REN, JIA-MOU LI*, AND XIN LIN*
Department of Orthopedics, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China
Objective Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance proliferation and to alter protein
production in various kinds of cells. In the present study, we measured the neurites length after LIPUS treatment to define the
effectiveness of LIPUS stimulation on neurons, and then we examined the acticity of GSK-3β to study the intracellular
mechanism of neurite’s outgrowth. Methods LIPUS was applied to cultured primary rat cortical neurons for 5 minutes
every day with spatial- and temporal average intensities (SATA) of 10 mW/cm2, a pulse width of 200 microseconds, a
repetition rate of 1.5 KHz, and an operation frequency of 1 MHz. Neurons were photographed on the third day after LIPUS
treatment and harvested at third, seventh, and tenth days for immunoblot and semi-quantitative RT-PCR analysis. Results
Morphology change showed that neurite extension was enhanced by LIPUS. There was also a remarkable decrease of proteins,
including p-Akt, p-GSK-3β, and p-CRMP-2, observed on the seventh and tenth days, and of GSK-3β mRNA expression,
observed on the seventh day, in neurons treated with LIPUS. Conclusion LIPUS can enhance elongation of neurites and it
is possible through the decreased expression of GSK-3β.
Key words: LIPUS; Rat cortical neurons; GSK-3β
In recent years, data on the therapeutic effects of
ultrasounds have been accumulating. So far, it has
been reported that LIPUS enhances cell proliferation
and alters protein production in various kinds of cells
such as endothelial cells, osteoblasts, chondrocytes,
and fibroblasts[1-3], but there is little information on
the response of neurons to LIPUS irradiation. Some
studies have indicated that LIPUS has positive effects
on axonal regeneration during in vivo peripheral
nerve injury trials[4-5] and that its stimuli on the
injured sciatic nerve can increase the number of nerve
fibers compared to that of untreated injured nerves in
rats. Thus, treatment with LIPUS is likely to be
advantageous to a curative effect on the regeneration
of neuronal axons. However, the mechanism of such
events is unknown.
There are two main types of ultrasound effects:
thermal and nonthermal. Both types are thought to
first “injure” the cells, resulting in their growth
retardation, and then to initiate a cellular recovery
response characterized by an increase in protein
production. Compared to high-intensity continuous
ultrasound, LIPUS is much lower in intensity (10
mW/cm2, SATA) and has unique characteristics such
nonthermogenic and nondestructive.
Intracellular mechanisms that enhance neurite
outgrowth evidently require the reorganization of the
neurite cytoskeletons including the microtubules and
actin filaments. Recently, a cytoskeletal-related
signaling pathway: PI
synthase kinase (GSK-3)/collapsin response mediator
protein (CRMP-2) was reported to be important for
the outgrowth of neurite, with GSK-3 being a central
serine/threonine kinase found ubiquitously in
eukaryotes and it plays key roles for various
biological processes, such as the canonical Wnt
signaling pathway, microtubule dynamics, and
astrocyte migration[12-13]. GSK-3 phosphorylates at
least have four types of microtubule-associated
proteins (MAPs), CRMP-2, tau, adenomatous
polyposis coil gene product[14-15] (APC), and
MAP1B[16-17]. It modulates axial orientation during
the development, differentiation,
outgrowth in neurons through phosphorylation of
these MAPs[10-11,18-19]. Some research have proved
that the local inhibition of GSK-3 effectively enhance
neurite/axon elongation whereas overexpression of
GSK-3 could impair neurite/axon elongation.
pulsed waves, which are regarded as
is a multifunctional
LIPUS ENHANCE ELONGATION OF NEURITES IN RAT CORTICAL NEURONS THROUGH INHIBITION OF GSK-3β
18. Chen Z L, Yu W M, Strickland S (2007). Peripheral
regeneration. Annu Rev Neurosci 30, 209-233.
19. Conde C, Caceres A (2009). Microtubule assembly,
organization and dynamics in axons and dendrites. Nat Rev
Neurosci 10, 319-332.
20. Kim W Y, Zhou F Q, Zhou J, et al. (2006). Essential roles for
GSK-3s and GSK-3-primed substrates in neurotrophin-induced
and hippocampal axon growth. Neuron 52, 981-996.
21. Munoz-Montano J R, Lim F, Moreno F J, et al. (1999).
Glycogen Synthase Kinase-3 Modulates Neurite Outgrowth in
Cultured Neurons: Possible Implications for Neurite Pathology
in Alzheimer's Disease. J Alzheimers Dis 1, 361-378.
22. Dent E W, Kalil K (2001). Axon branching requires
interactions between dynamic microtubules and actin filaments.
J Neurosci 21, 9757-9769.
(Received January 27, 2010 Accepted June 9, 2010)