Negative Effect of High Calcium Levels on Schwann Cell Survival

Transcription

1 Neurophysiology, Vol. 44, No. 4, September, 2012 Negative Effect of High Calcium Levels on Schwann Cell Survival J.-G. Yan, 1 M. Agresti, 1 L.-L. Zhang 1, H. S. Matloub, 1 and J. R. Sanger 1 Received April 6, 2012 Our previous studies showed that the functional recovery of an injured nerve correlates with calcium absorption, and that acceleration of this absorption can greatly improve regeneration. In our study, we examined effects of the calcium concentration on cultured Schwann cells obtained from the rat sciatic nerve. Using published methods and simple calculations, we measured the calcium concentration inside normal and injured (crushed) nerve fibers and found that the injured-nerve internal calcium concentration is 16.8 mm, on average. The cells, including Schwann cells, were isolated from an intact sciatic nerve and cultured with various Ca 2+ concentrations of calcium-spiked media, from the normal (1.8 mm) to mm. The cells were fixed, blocked, and incubated with Anti-S-100, Goat-Anti-Mouse, and Propidium Iodide. They were viewed under fluorescent conditions to identify the Schwann cells. All the cells in the control dishes were viable, and there were large numbers of Schwann cells present. All the experimental dishes showed the profound concentration-dependent harmful effect of excessive calcium on the number and type of cells present. The high level of calcium corresponding to nerve injury severely affected Schwann and all other cell survival and growth in this cell culture study. Thus, Schwann cell survival and growth are very sensitive to and negatively affected by higher calcium levels than that in the normal media. Keywords: Schwann cells, calcium, peripheral nerve injury, culturing. INTRODUCTION Many studies, both in vitro and in vivo, support the association between excessive calcium influx and damage to neural tissues [1-3]. High levels of Ca 2+ are a direct result of a compromise to the cell membrane due to mechanical insult [4-6] or the interruptions of blood flow and oxygen supply [7]. Very little research has been conducted on injured and recovering peripheral nerves with regard to Ca 2+ concentrations. Our previous study indicated that, after nerve crush injury, the functional recovery significantly correlates with the calcium absorption [8]. Recently, we reported that accelerated (within certain limits) calcium absorption can greatly improve nerve regeneration [9]. It is well known that Schwann cells are the most important dynamic cells in the process of nerve regeneration. This study will further determine the effect of different calcium concentrations on cultured Schwann cells. 1 Medical College of Wisconsin, Milwaukee, Wisconsin, USA. Correspondence should be addressed to Ji-Geng Yan ( jyan@mcw.edu). METHODS Estimation of Calcium Concentra tions Corresponding to Nerve Crush Injury. A reasonable calcium concentration for this study should be the same as the concentration inside injured nerve fibers. A crush-injury rat model was used in our experiments. As in our previously published paper [8], eight adult male Sprague- Dawley rats were anesthetized with i.p. injection of sodium pentobarbital (35 mg/kg body mass) and prepared for aseptic surgery. The right-side sciatic nerve was crushed at the mid-point with a 4-mm-wide smooth-jaw needle holder. The needle holder was clicked twice for 4 sec producing 135 N (13.8 kg force) pressure, as measured by a pressure film. At the postoperative 4-week time point, the nerve segment distal to the crushed site was harvested on the injured side. The Ca 2+ concentration inside nerve fibers was measured using the following technique. The nerve segment was immediately placed in a Dulbecco s-modified Eagles medium (DMEM) without phenol red (Gibco/Invitrogen, USA). Under an operating microscope, the epineurium and /12/ Springer Science+Business Media New York

2 Negative Effect of High Calcium Levels 275 perineurium were removed, and nerve fiber bundles were gently separated using micro-forceps to expose the nerve fibers with minimum fiber damage. These nerve fibers were put in a staining solution containing Calcium Green-1 acetoxymethyl (AM) ester, which showed bright green fluorescence under a fluorescent microscope. The details of the technique were described in our previous paper [8]. The fluorescence intensity was measured, and the Ca 2+ concentration was calculated and expressed in relative fluorescent units (RFUs) using MetaVue computer software (Universal Imaging, USA). The areas between normal nerve fibers containing the matrix corresponding to the outside of the axon (outside cell environment) demonstrated a standard Ca 2+ concentration, which was ± 1.34 RFUs, or 1.8 mm (Fig. 1A) [8]. At the same time, the mean Ca 2+ concentration index inside the nerve fibers located distal to the injured site was ± RFUs, which equals 16.8 mm according to simple algebraic proportions (B). These quantitated Ca 2+ concentrations were used to establish a baseline value for the future cell culturing experiments. Culturing of Schwann Cells. In the above rats, the left sciatic nerves were harvested. The outer membranes were peeled away, and the nerves were minced into small pieces. The latter were placed into 24 dry well plates and allowed to adhere in an incubator for about 30 min. The media were then carefully added so as not to disturb the tissues. The tissues were watched for fibroblast migration; the media and plates were changed every 3 days and once per week, respectively. The tissues were incubated for 2 weeks. The pieces were then placed in 15-ml tubes with 2 ml of 0.25% trypsin and 0.03% collagenase in DMEM. The tubes were incubated at 37 o C (5% CO 2 ) for 90 min and then vortexed; the debris were strained and spun at 1,500 min 1 for 2 min. The pellet was resuspended in a normal medium (DMEM, 10% FCS, 1% Pen/Strep, 1% NEAA, and 1% L-Glut). The cells were plated on poly-lysine-coated (100 mg/ml in saline, room temperature, RT, for 20 min, rinsed with distilled H 2 O, and air-dried) T-25 flasks. When the cells became confluent, they were split and grown for 2 additional weeks. Next, the cells were trypsinized and combined into a single tube. The tube was vortexed until a single-cell suspension was achieved. The exact same amount of suspension (5 ml) was added to 20 small Petri dishes (60 mm) coated with poly-lysine, as above, and grown for 1 day. On the following day, the dishes were randomly assigned as controls or experiments, so as not to prejudice the results, and normal or calciumspiked media were added to each dish. These media were 16.8, 10.0, 7.5, and 5.0 mm, while the normal was 1.8 mm Ca 2+. Four dishes were cultured with each different Ca 2+ concentration. The media were carefully changed every 3 days, and the cells were allowed to grow for 10 days. Identification of Schwann Cells using S-100 Antibody Staining. After 10 days, the dishes were washed with PBS, and the cells were fixed with 4% paraformaldehyde/pbs (Sigma, USA) for 10 min at 100 μm A B 100 μm F i g. 1. Measurements of the calcium concentration in an intact nerve segment (A) and in a segment distal to the crushed site 4 weeks after injury (B). Arrows point to the areas of calcium staining used for the calcium concentration calculations. For details, see Methods.

3 276 J.-G. Yan et al. A D B E C F i g. 2. Photomicrographs of the Schwann and other cells cultured in a normal medium (A, 1.8 mm Ca 2+ ) and in media with increased Ca 2+ concentrations (B E, 5.0, 7.5, 10.0, and 16.8 mm Ca 2+, respectively). Schwann cells and fibroblasts possess yellow and red nuclei, respectively.

4 Negative Effect of High Calcium Levels 277 RT. The dishes were washed with PBS blocked with 5% goat serum/pbs (Sigma, USA) for 60 min also at RT, and then incubated with anti-s-100/pbs (1:1000; Sigma, USA) with 1% goat serum overnight at 4 o C. The dishes were washed with PBS and incubated with goat-anti-mouse/pbs (1:100; Sigma, USA) for 60 min at 37 o C. Then, the dishes were washed again with PBS and incubated with propidium iodide dissolved in distilled H 2 O (100 mg/ml; Sigma, USA) for 10 min at RT in the dark. Finally, the dishes were washed with PBS, covered with glass coverslips, and viewed (200 ) under fluorescent conditions through FITC and Texas Red filters. Axiovision (Zeiss, USA) and MetaVue software (Universal Imaging, USA) were used to acquire the pictures and to combine both the green and red images to identify the yellow Schwann cells, while the fibroblasts and other cells howed the red nuclei. RESULTS Under magnification 200, the total numbers of Schwann cells within the central field of vision were counted for each culture dish. The average numbers of Schwann cells were obtained for each calcium concentration, as shown in Table 1. In the control, all the cells in the dishes with normal media were viable, and there were large numbers (more than two hundred per field of vision, on average) of Schwann cells present (Table 1 and Fig. 2A). The addition of extra Ca 2+ to the culture media only increased the Ca 2+ levels to the designed concentration without any changes to other media components. All the dishes showed profound harmful effects of high Ca 2+ on the number and type of cells present. In the dishes with 5.0 mm Ca 2+, the number of overall cells was only slightly (insignificantly) smaller than in the control, but the number of Schwann cells dramatically (about eight times) dropped (Fig. 2B). When the Ca 2+ level was increased to 7.5 mm, the overall cell number fell substantially (about three times), and Schwann cells were only sporadically present (C). At the highest Ca 2+ concentrations (10.0 and 16.8 mm, В and E, respectively), the overall numbers of cells were about ten and thirty times smaller, respectively, than in the control (i.e., survival of the cells tended to zero). Only exceptional (single) Schwann cells (not in all dishes) could be identified with difficulty. DISCUSSION It is well known that Schwann cells are the most important dynamic cells involved in nerve regeneration. Many complex factors affect Schwann cell growth after various types of nerve injuries. An adequate way to test the effect of high levels of Ca 2+ on these factors is using of Schwann cell culture. As was found in the control, we have successfully grown and identified Schwann cells in culture following established protocols. The only reason for the progressive reduction of cell numbers was the increasing Ca 2+ concentrations in the media. We have shown unequivocally that a Ca 2+ level even moderately higher (5.0 mm) than that in the normal medium (1.8 mm) exerted a strong negative effect on the survival of the cells in culture. Even when there is no injury, and the cells look otherwise healthy, there is a fine line between a tolerable level of Ca 2+ and one that will kill most (if not all) the cells in culture. As was stated previously, there are very few (if any) published data on the harmful effect of abovenormal levels of Ca 2+ on Schwann cells in culture. Mimicking the clinical situation is important for studying Schwann cells under the latter conditions. In this study, we used a crush injury animal model, and the corresponding calcium concentration in the T a b l e 1. Average Numbers and Standard Deviations (M ± s.d.) of Schwann and Other Cells in Cultures with Various Calcium Concentrations Used Cell types Ca 2+ level in the medium, mm 1.8 (control) Schwann cells 219 ± 24.1 (100) 26.5 ± 7.0 (12.1) 9.8 ± 3.4 (4.5) 2.5± 0.6 (1.1) 0.75 ± 0.5 (0.3) Cells of all types (including fibroblasts, etc.) 473 ± 79.5 (100) ± 57.8 (97.0) ± 79.8 (32.8) 50.8 ± 10.2 (10.7) 14.3 ± 9.3 (3.0) Footnote. Normalized cell numbers (%) are shown in brackets; the numbers in the control are taken as 100%.

5 278 J.-G. Yan et al. nerve segment distal to the crushed site was found to be about 17 mm. Other (diluted) Ca 2+ concentration levels were also used in our Schwann cell culture study, because different nerve injuries could be tested, and the injury conditions can be less severe. We found that at Ca 2+ concentrations much lower than 16.8 mm, other cells (such as fibroblasts) were alive, but Schwann cells practically did not survive (Figs. 2B, C). This is indicative of the fact that Schwann cells are very sensitive to even moderate increases in the calcium concentration. We found that there is an influx of Ca 2+ after a peripheral nerve injury, and it remains high for over 8 weeks [8]. These facts are reflected in the clinical cause of slow progress in nerve regeneration. Schwann cells and nerve sprouts cannot actively grow until the excessive Ca 2+ has been effectively absorbed. This study suggests that the Ca 2+ level has to be less than 5 mm for this to occur. We also found that if the Ca 2+ level was greater than 7.5 mm, the number of fibroblasts and other cells dramatically decreased. These cells are also very important for supporting nerve regeneration. It is beyond doubt that accelerating calcium absorption is a major challenge for decreasing the injury cascade, thus preventing more severe injuries and improving nerve regeneration after peripheral nerve injuries. Therefore, the high levels of calcium corresponding to the conditions of nerve injury severely affected Schwann and all other nerve cell survival and growth in this cell culture study. Schwann cell survival and growth are very sensitive and negatively affected by higher calcium levels than those in the normal media. REFERENCES 1. M. P. Goldberg, R. G. Giffard, M. C. Kurth, and D. W. Choi, Role of extracellular calcium and magnesium in ischemic neuronal injury in vitro, Neurology, 39, Suppl., 217 (1989). 2. R. M. LoPachin and E. J. Lehning, Mechanism of calcium entry during axon injury and degeneration, Toxicol. Appl. Pharmacol., 143, No. 2, (1997). 3. H. Manev, M. Favaron, A. Guidotti, and E. Costa, Delayed increase of Ca 2+ influx elicited by glutamate: Role in neuronal death, Mol. Pharmacol., 36, (1989). 4. J. H. Lucas, G. W. Gross, D. G. Emery, and C. R. Gard ner, Neuronal survival or death after dendrite transection close to the perikaryon: Correlation with electrophysiologic, morphologic, and ultrastructural changes, Cent. Nerv. Syst. Trauma, 2, (1985). 5. R. Y. Shi, J. H. Lucas, A. Wolf, and G. W. Gross, Calcium antagonists fail to protect mammalian spinal neurons after physical injury, J. Neurotrauma, 6, (1989). 6. A. F. Strautman, R. J. Cork, and K. R. Robinson, The distribution of free calcium in transected spinal axons and its modulation by applied electrical fields, J. Neurosci., 10, (1990). 7. S. Rehncrona, L. Mela, and B. K. Siesjo, Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia, Stroke, 10, (1979). 8. J. G. Yan, H. S. Matloub, Y. Yan, et al., The correlation between calcium absorption and electrophysiological recovery in crushed rat peripheral nerves, Microsurgery, 30, No. 2, (2010). 9. Z. Wang, J. G. Yan, J. Sanger, et al., Accelerating calcium absorption significantly improves peripheral nerve regeneration an experiment in rats, in: Proc. of the ASPN Annu. Meet. (Jan. 15, 2012), Chicago (2012), p. 71.