Elsevier

Brain Research

Volume 781, Issues 1–2, 19 January 1998, Pages 159-166
Brain Research

Research report
Transmural compression-induced proliferation and DNA synthesis through activation of a tyrosine kinase pathway in rat astrocytoma RCR-1 cells

https://doi.org/10.1016/S0006-8993(97)01226-2Get rights and content

Abstract

Gliosis results from abnormal proliferation of glial cells and often occurs in response to brain or spinal cord injury. There are many factors that trigger gliosis associated with such injuries, including ischemia, humoral factors produced by the injured tissue, and possibly mechanical compression itself. In the present study, the effects of mechanical compression on cell proliferation and DNA synthesis were examined in vitro with the rat astrocyte cell line RCR-1. Pressure was applied to cells by instilling compressed helium into sealed plates or flasks in which the partial pressure of oxygen were maintained constant. Compression resulted in time- and intensity-dependent increases in cell number and [3H]thymidine incorporation, with maximum effects apparent at 10 min and 120 mmHg. Compression-induced cell proliferation and DNA synthesis were not inhibited by gadolinium (Gd3+), a blocker of stretch-activated ion channels, or by inhibitors of protein kinase A, protein kinase C, or Ca2+/calmodulin-dependent protein kinases. However, the tyrosine kinase inhibitor genistein inhibited these effects of compression in a concentration-dependent manner. Conditioned medium from compressed cells also induced cell proliferation and DNA synthesis at atmospheric pressure in a genistein-sensitive manner. These results suggest that transmural compression triggers the release of a factor (or factors) that induces cell proliferation and DNA synthesis through a tyrosine kinase pathway in RCR-1 cells.

Introduction

Gliosis results from abnormal proliferation of glial cells and often occurs in response to brain or spinal cord injury 3, 7, 8. Factors associated with central nervous system (CNS) injury that trigger gliosis include ischemia, caused by compression-induced obstruction of blood vessels; humoral factors such as angiotensin II, adenosine, and various growth factors produced by the injured tissue; and, possibly, mechanical compression itself 9, 10, 18, 19, 29. Among these factors, however, it has remained unclear whether compression alone promotes glial cell proliferation.

Hishikawa et al. 12, 14have established a nonischemic system with which to investigate the effects of compression on cultures of rat aortic smooth muscle and endothelial cells. Compression is achieved by the introduction of helium into a sealed flask in which the partial pressure of oxygen and pH maintained constant. With this system, the researchers showed that compression alone promoted cell proliferation and DNA synthesis in cultured smooth muscle cells [13]. We have now applied this approach to investigate the effects of compression on glial cell proliferation with the use of RCR-1 cells, a rat glial cell line that shows astrocytic characteristics 15, 16. Mechanical compression induces the secretion of substances from cardiac cells that promote cell hypertrophy [23], and enhances the release of endothelin-1 [14]as well as that of interleukin-1 and interleukin-6 [27]from endothelial cells and neurotransmitters from motor nerve terminals [4]. We therefore also examined the effects on cell proliferation of conditioned medium prepared from compressed cells.

Section snippets

Materials

The rat astrocytoma cell line RCR-1 was obtained from Riken Cell Bank (Wako, Japan). Culture plates (24 and 96 wells) and 25-cm2 culture flasks were from Corning (New York, NY). 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and 2-[N-hydroxyethyl-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) were from Seikagaku Kogyo (Tokyo, Japan), staurosporine from Kyowa Hakko (Tokyo, Japan), and genistein and GdCl3 from Sigma (St. Louis, MO). The Abacus Cell

Effects of duration and intensity of compression on cell proliferation

Compression of cells at 120 mmHg resulted in a time-dependent increase in cell number, which was 140±7, 179±10, 174±12 and 183±24% of the control value at after 5, 10, 30, and 60 min compression, respectively (Fig. 1A). In the absence of compression, cell number did not change significantly after a 60-min incubation period.

Compression for 10 min promoted cell proliferation in a pressure-dependent manner from 0 to 120 mmHg; cell numbers 48 h after 60, 120, and 240 mmHg compression were 133±8,

Discussion

We have shown that compression alone increases cell number and the incorporation of [3H]thymidine in rat astrocytic RCR-1 cells. In CNS injury, neuronal, glial and other surrounding cells can be subjected to mechanical forces that produce transmural compression and result in gliosis. With an in vivo model, Balentine [2]showed that acute traumatic spinal cord compression induced by compression by falling weights results in gliosis. Although it is difficult to clarify how compression caused

Acknowledgements

This work was supported by grants from the University of Occupational and Environmental Health to Y.O. and Y.U., a grant from Japan Orthopaedics and Traumatology Foundation to Y.O. (No. 0090), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan to Y.O.

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