| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
 
|
|
|
||
|
|
Anesth Analg 2004;98:841-845 © 2004 International Anesthesia Research Society doi: 10.1213/01.ANE.0000099718.51681.55
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Neurotoxicity of local anesthetics has been reported for both matured and growing neurons. In the present study, we examined if tetracaine increases Ca2+ concentration during growth cone collapse. Intracellular Ca2+ concentration was measured by fura 2/AM after exposure to tetracaine. Tetracaine (1–2 mM) induced increases in intra-growth cone Ca2+ concentration (P < 0.01). The Ca2+ hot spot was expanded into the neurite from the periphery towards the cell body. When tetracaine was applied to growth cones in Ca2+ free media, the increase was minor. However, tetracaine induced growth cone collapse even in the culture media, which did not contain Ca2+. Ni2+ (100 µM; a general Ca2+ channel inhibitor) and BAPTA-AM (5 µM; intracellular Ca2+ chelator) could not inhibit growth cone collapse induced by 1–2 mM tetracaine. Tetracaine (>1 mM) induces collapse and Ca2+ increase at growth cones simultaneously; however, these two phenomena might be provoked independently.
IMPLICATIONS: Tetracaine induced intracellular Ca2+ increases and growth cone collapse in dorsal root ganglion neurons. The Ca2+ hot spot in the growth cone expanded into the neurite from periphery towards the cell body.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Neurotoxicity of local anesthetics has been reported both in clinical and experimental studies (1). Several in vivo and in vitro studies have demonstrated that tetracaine and lidocaine directly injured matured neuronal tissues. Recently, Tan et al. (2) and Johnson et al. (3) demonstrated that these local anesthetics disrupted calcium homeostasis and induced cell death in cultured neuronal cell lines.
However, effects of local anesthetics on growing or regenerating neurons have not been fully understood. Growth cone is the highly motile structure at the end of growing axons and dendrites, and it has a crucial role in pathfinding and cytoarchitecture establishment in developing the nervous system (4). Substances that induce abnormal collapse of growth cones possibly provoke some teratogenicity when used for embryos or neonates or abnormal regeneration when applied to injured nervous tissues (5). In our previous studies, we demonstrated that local anesthetics induced growth cone collapse both concentration- and exposure time-dependently (6–8). Although clinically applied local anesthetics are quickly diluted by cerebrospinal or interstitial fluid, the concentrations tested in our previous studies were smaller than clinically prepared solutions and may be within the range encountered in clinical settings (e.g., local infiltration around injured tissues).
Because intracellular events during the growth cone collapse induced by local anesthetics have not been studied, we examined whether intra-growth cone Ca2+ concentration was changed by a local anesthetic, tetracaine. Although calcium homeostasis disruption in cell bodies has been observed in several studies using large concentrations of local anesthetics, it is possible that growth cones behave differently because of their unique calcium regulating mechanism and highly motile membrane structures. Also, we examined if Ca2+ channel inhibitor or calcium depletion prevents growth cone collapse induced by tetracaine.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
After approval by the Institutional Animal Care Committee, chick dorsal root ganglia were isolated from day 7 embryos and cultured in F-12 medium as described previously (6). Tetracaine (Sigma Co.; St. Louis, MO) was prepared in prewarmed fresh culture media and was gently added to the culture media after 20 h in culture. A growth cone collapse assay was conducted as described previously (9).
When the effects of calcium-free media, a general Ca2+ channel blocker, 1 mM NiCl2, or a intracellular calcium chelator, O,O'-Bis (2-aminophenyl) ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM; Sigma-Aldrich Co., St. Louis, MO), were examined, culture media was gently exchanged to calcium-free modified Eagle medium (Invitrogen Corp., Carlsbad, CA) 15 min before tetracaine application. NiCl2 or BAPTA-AM was gently added to the Ca2+ free solution. In a preliminary study, it was confirmed that neither culture media without Ca2+ nor the Ca2+ free media with Ni2+ or BAPTA-AM induced growth cone collapse up to 90 min.
Intracellular-free Ca2+ concentrations were measured using a Ca2+ sensitive dye, fura-2/AM (Dojindo Inc., Masuki, Japan). Fura-2/AM was loaded by an incubation for 30 min at 37°C. After loading, the cells were incubated for 1 h at 37°C for recovery. The fura-2 fluorescence at 510 nm elicited by excitation at 340 or 380 nm was measured using an inverted Diaphot microscope equipped with a cooled CCD camera and an image processing system (Aquacosmos; Hamamatsu Photonics, Hamamatsu, Japan). The F340/F380 ratio was measured to estimate the internal Ca2+ concentration. Absolute [Ca2+]i was calculated by the method of Grynkiewicz et al. (10) using a Kd value of 224 nM. The area of measurement was approximately 20 µm2. Tetracaine was gently added to the culture media from the opposite side of the focused growth cone (so as to let the tetracaine reach the focused growth cone later than the other parts of cells). After the completion of the measurement, ranges of interest were manually adjusted to a growth cone and neural shafts (Fig. 1A).
|
The data are expressed as the mean ± SD of six independent measurements. The time-dependent change of growth cone collapse in the time-course study was analyzed by one-way analysis of variance for repeated measurements. Each result of the growth cone collapse assays was statistically analyzed by two-way analysis of variance with the Scheffé method using StatView 5.0. P values <0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
After a temporal and minor morphological change immediately after the fura-2/AM loading, growth cone shape was recovered within an hour. In the experiment where 0.5 mM (0.015%) tetracaine was used, Ca2+ hot spot did not appear anywhere in the viewing field within 60 min. When 1 mM (0.03%) or 2 mM (0.06%) tetracaine was used, the first Ca2+ hot spot was observed at 25 ± 8 or 8 ± 4 min after the application, respectively. Ca2+ hot spot was first observed at the growth cone, and then the area expanded into the neurite from the periphery towards the cell body (Fig. 1A-B). Approximately estimated [Ca2+]i at growth cones increased from 100.5 ± 4.7 nM to 653.0 ± 38.4 nM after exposure to 1 mM tetracaine and from 93.8 ± 2.3 nM to 571.4 ± 56.1 nM after exposure to 2 mM solution. When 2 mM tetracaine was used, growth cones and neurites started to detach from the dish floor at 30 min after the exposure and observation became impossible.
Tetracaine induced growth cone collapse even in the culture media, which did not contain Ca2+ (Fig. 2A-B). In the presence of Ni2+ or BAPTA, growth cone collapse percentage became larger than the corresponding pre-exposure value at 40 min after the exposure to 1 mM tetracaine, whereas in the absence of these blockers, the value became larger than the corresponding preexposure value at 30 min after the exposure (Fig. 2A). Growth cone collapse percentage became higher than the corresponding pre-exposure value at 20 min after the exposure to 2 mM tetracaine, regardless of the presence of Ni2+ or BAPTA-AM. (Fig. 2B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The concentration of free calcium ions in the cytosol influences many components of growth cone behavior, including gross collapse and retraction of the growth cone (11). Several studies have demonstrated that externally applied substances induced growth cone collapse by altering intracellular Ca2+ concentration (12–13). In the present study, we observed an increase of Ca2+ concentration in growth cones exposed to tetracaine. However, the manner of increase was different from that observed in spontaneous increase or induced by neurotrophic factors (11). When Ca2+ flows in through the channel, the fluorescence ratio sharply increases and then decreases to the original level, showing brief spikes. The duration of each increase is milliseconds or seconds. The increase is observed within a limited area in the growth cone. Tetracaine induced a slow increase that took approximately 10 minutes to reach the maximal value. The increased value did not return to the original value. The area of increase was not restricted to the growth cone but expanded into the neurite from distal to proximal. The manner of Ca2+ increase observed in this study resembles the pattern demonstrated by Johnson et al. (3). In their experiment using ND7 cells, 2.5%–5% lidocaine induced plasma membrane lysis and toxic increase of cytoplasmic Ca2+.
In the present study, growth cone collapse and Ca2+ increase were observed almost simultaneously. Also, in a Ca2+ free media, a general calcium channel blocker (Ni2+) and intracellular Ca2+ chelator (BAPTA-AM) made Ca2+ increase insignificant but could not prevent growth cone collapse induced by tetracaine. It is possible that growth cone collapse and Ca2+ increase could be induced independently by different biochemical mechanisms. Several reports demonstrated that local anesthetics disrupted membrane integrity. Kanai et al. (14) showed that lidocaine disrupted axonal membrane of mature rat sciatic nerve at 80 mM. Tan et al. (2) revealed that tetracaine (0.5 and 1 mM) induced a morphological change of PC12 cell membrane and leakage of lactate dehydrogenase. In their experiments, cytoplasmic Ca2+ increase was observed; however, EGTA and BAPTA-AM only partially inhibited the increase. It is possible that tetracaine disrupts membrane integrity of growth cones.
In the present study, Ca2+ flows first into growth cones. Growth cone membrane may be most sensitive to tetracaine toxicity in growing or regenerating neurons. This can be explained by the fact that the growth cone has unique biochemical characteristics. The lipid composition of the membrane, calcium-regulating system, and cytoarchitectures are unique to ensuring the highly motile characteristics (15). The results of the present study suggest that growing or regenerating nerves (especially growth cones) are vulnerable to the toxicity of local anesthetics and that calcium channel blockers do not antagonize the toxicity.
In the present study, we examined the effect of tetracaine on growth cones within 30–60 min after exposure. This was because fura 2 loading and exposure to tetracaine significantly affected weak adhesion of growth cones to the floor of culture dishes. Observation for a longer duration was technically difficult. Further study with different culture technique may be indispensable to clarify the effects of tetracaine on growth cone membrane at various concentrations. Also, further biochemical and morphological study may provide the precise mechanism of growth cone collapse induced by local anesthetics.
|
Acknowledgments |
|---|
Supported, in part, by a grant-in-aid for Scientific Research (No. 13671562) from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan.
|
Footnotes |
|---|
Presented, in part, at the annual meeting of the Society for Neuroscience, Orlando, Florida, November 5, 2002.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Hodgson PS, Neal JM, Pllock JE, Liu SS. The neurotoxicity of drugs given intrathecally. Anesth Analg 1999; 88: 797–809.
[Free Full Text] - Tan X, Dohi S, Chen J, et al. Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death. Anesthesiology 2002; 96: 1191–201.[Web of Science][Medline]
- Johnson M, Saenz J, DaSilva A, et al. Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model. Anesthesiology 2002; 97: 1466–76.[Web of Science][Medline]
- Letourneau PC, Kater SB, Macagno ER. The nerve growth cone. New York: Raven Press, 1992.
- Fawcett FW. Growth cone collapse: too much of a good thing? Trends Neurosci 1993; 16: 165–7.[Web of Science][Medline]
- Saito S, Radwan I, Obata H, et al. Direct neurotoxicity of tetracaine on growth cones and neurites of growing neurons in vitro. Anesthesiology 2001; 95: 726–33.[Web of Science][Medline]
- Radwan I, Saito S, Goto F. The neurotoxicity of local anesthetics on growing neurons: a comparative study of lidocaine, bupivacaine, mepivacaine, and ropivacaine. Anesth Analg 2002; 94: 319–24.
[Abstract/Free Full Text] - Radwan I, Saito S, Goto F. Growth cone collapsing effect of lidocaine on DRG neurons is partially reversed by several neurotrophic factors. Anesthesiology 2002; 97: 630–5.[Web of Science][Medline]
- Raper JA, Kapfhammer JP. The enrichment of a neuronal growth cone collapsing activity from embryonic chick brain. Neuron 1990; 4: 21–9.[Web of Science][Medline]
- Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50.
[Abstract/Free Full Text] - Gomez T, Snow D, Letourneau P. Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration. Neuron 1995; 14: 1233–46.[Web of Science][Medline]
- Wang Q, Zheng JQ. cAMP-mediated regulation of neurotrophin-induced collapse of nerve growth cones. J Neurosci 1998; 18: 4973–84.
[Abstract/Free Full Text] - Janevski J, Choh V, Stopper H. Diethylstilbestrol alters the morphology and calcium levels of growth cones of PC12 cells in vitro. Neurotoxicology 1993; 14: 505–11.[Medline]
- Kanai Y, Katsuki H, Takasaki M. Graded, irreversible changes in crayfish giant axon as manifestations of lidocaine neurotoxicity in vitro. Anesth Analg 1998; 86: 569–73.[Abstract]
- Bamburg JR. Introduction to cytoskeletal dynamics and pathfinding of neuronal growth cones. J Histochem Cytochem 2003; 51: 407–9.
[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
 |
|
  K. Sekimoto, S. Saito, and F. Goto Tetracaine at a small concentration delayed nerve growth without destroying neurites and growth cones. Anesth. Analg., September 1, 2006; 103(3): 608 - 614. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
