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A Conditioning Lesion Promotes in Vivo Nerve Regeneration in the Contralateral Sciatic Nerve of Rats
Biochemical and Biophysical Research Communications 267, 715–718 (2000) doi:10.1006/bbrc.1999.2017, available online at http://www.idealibrary.com on
A Conditioning Lesion Promotes in Vivo Nerve Regeneration in the Contralateral Sciatic Nerve of Rats Koji Ryoke, Mitsuo Ochi, 1 Atsushi Iwata, Yuji Uchio, Soichiro Yamamoto, and Hidetoshi Yamaguchi Department of Orthopaedics, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane-ken 693-8501, Japan
Received December 6, 1999
A conditioning lesion in the sciatic nerve increases in vivo axonal regeneration in the nerve after a second transection. We studied whether this increased regeneration also occurs in the contralateral nerve. The left sciatic nerve was transected and sutured in Wistar rats; the nerve was exposed but not transected in controls. After 5 days, the right sciatic nerves of all rats were transected and sutured. Neuronal regeneration was measured at 0, 1, 3, 5, and 7 days with the pinch test and histological staining. IL-1 and TGF-1 expression was also measured. The initial delay in the experimental group was significantly shorter, but the regeneration rates were the same. The expression of IL-1 and TGF-1 in the right dorsal root ganglia was significantly higher in the experimental group. Nerve injury enhances cytokine expression in the contralateral dorsal root ganglion and promotes contralateral nerve regeneration in vivo by shortening the initial delay. © 2000 Academic Press Key Words: nerve regeneration; contralateral conditioning lesion; interleukin-1  (IL-1); transforming growth factor-1 (TGF-1); initial delay.
In rats with a previous, unilateral “conditioning” lesion in the sciatic nerve, axonal regeneration in the nerve after a second transection is more rapid than in nerves without such a conditioning lesion (1). Several experiments have shown that after the nerve cut, the mRNA of IL-1 (2– 4) and TGF-1 is elevated in the dorsal root ganglion (DRG) cells and in the anterior horn cells of the ipsilateral side, and that these cytokines are intimately involved in nerve regeneration (5, 6). However mechanism of this rapid regeneration has not been adequately described. Oka reported that a nerve injury can stimulate expression of TGF-1 in the contralateral DRG (7), indicating that a unilateral nerve injury also affects the To whom correspondence should be addressed. Fax: ⫹81 853-202236. E-mail: [email protected]. 1
metabolism of neurons in the normal, contralateral side. We hypothesized that regeneration might be promoted by a conditioning lesion not only in a second ipsilateral nerve injury but also in a contralateral nerve injury. The purpose of the present study was (1) to ascertain whether a previous conditioning injury to the sciatic nerve in the rats, followed by injury to the contralateral sciatic nerve would promote nerve regeneration in the contralateral nerve and (2) to determine whether the expression in IL-1 and TGF-1 in DRG were associated with nerve regeneration and, in fact, might explain this regeneration. MATERIALS AND METHODS Animals and surgery. We studied 8-week-old, male Wistar rats weighing 200 and 250 g (Clea Japan, Tokyo, Japan). Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (0.025 mg/g; Abbott Laboratories, North Chicago, IL). For the experimental group (n ⫽ 45), the left sciatic nerve was transected 5 mm distal to the biceps femoris branch and then re-anastomosed with 10-O nylon suture. Five days later, the procedure was repeated on the right sciatic nerve in each rat. The control group (n ⫽ 45) underwent a sham operation in which the left sciatic nerve was exposed but not transected. As in the experimental group, 5 days later, the right sciatic nerve was transected and sutured as above. On days 0, 1, 3, 5, and 7 after transection of the right sciatic nerve, 9 rats were sacrificed. Axonal regeneration in the right sciatic nerve was evaluated, and the level of expression of IL-1 and TGF-1 in the L 4-5 DRG was recorded. All animals were treated according to the guideline with University Regents’ Policy on Animal Care and Use, Shimane Medical University policy, Federal Animal Welfare Act, and other applicable standards. Evaluation of regeneration. On days 0, 1, 3, 5, and 7 after the right sciatic nerve transection and suture, a pinch test was performed for six rats in each group to determine the extent of nerve regeneration (8 –10). The extent of regeneration was defined as the distance between the most distal tip of the regenerated sensory neurons and the suture site on the right sciatic nerves (8 –11). For the pinch test, each rat was lightly anesthetized with a 0.2 ml intraperitoneal injection of solution containing sodium pentobarbital (60 mg/ml), saline, and diazepam (5 mg/ml, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) in 1:1:2 volume proportions. The right sciatic nerve was re-exposed, and the intact proximal branch to the biceps femoris was pinched to determine the appropriate level of
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anesthesia that allowed the animal respond. Beginning 15 mm distal from the suture site, the nerve was carefully pinched with a forceps at 0.5-mm intervals in a proximal direction. The most distal site at which the pinch test elicited a contraction of muscles on the back and a movement of the right leg was marked with 10-O nylon. The distance from this point to the suture site was the nerve regeneration distance. Immunocytochemistry and assessment of immunostaining. Immediately after the pinch test, the rats were euthanized with sodium pentobarbital (0.08 mg/g) and perfused with 4% paraformaldehyde (PFA) in 0.05 M phosphate buffered saline (PBS). In all rats, the right sciatic nerve, together with the right L4 and L5 DRG, were removed and fixed for 72 h at room temperature with 4.0% PFA in 0.05 M PBS. The fixed DRGs were embedded in paraffin and then cut longitudinally into 6-m-thick sections. The sections were placed on aminopropyl-triethoxysilane-coated glass slides (Matsunami Glass Ind., Ltd., Japan) and, after deparaffinization, were washed three times with 0.05 M PBS (pH 7.4). The sections were then pretreated with hydrogen peroxide to deplete endogenous peroxidase, incubated with polyclonal rabbit anti-human TGF-1 antibody (King Brewing Co., Kakogawa, Japan), and diluted 1000-fold with 1% bovine serum albumin (BSA) in PBS for 24 h at room temperature. A 500-fold diluted solution of biotinylated goat anti-rabbit immunoglobulin (Daco Japan Co., Tokyo, Japan) was left to react at room temperature for 30 min and was then reacted with a 500-fold diluted solution of peroxidase-labeled streptavidin (Daco Japan Co., Tokyo, Japan). Peroxidase was visualized using a solution of 3,3⬘-diaminobenzidine 4HCl, H 2O 2, CO 2⫹, Ni 2⫹ according to the method of Adams (12). The sections were then stained with 0.5% fast green (Wako Pure Chemical Industries, Ltd., Osaka, Japan). In place of the primary antibody, 1% BSA in PBS was added and used as a negative control. In the primary antibody used in this study, the active type TGF-1 has an affinity of about 27-fold compared to the inactive type TGF1. In addition, using the same technique, immunocytochemical analysis was performed using a polyclonal rabbit anti-human IL-1 antibody (Genzyme, Boston, MA). All sections were stained at the same time. The expression of IL-1 and TGF-1 in DRGs of each group and at each data collection point was determined by using quantitative estrogen-progesterone analysis software in the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). The mean value of the immunoreactive cell ratio was obtained for quantitative analysis with following formula: the immunoreactive cell ratio ⫽ (the number of IL-1 or TGF-1 stained cell)/(the number of total cell) in visual field from 20 randomly selected visual fields per glass slide. To confirm the accuracy of the pinch test, the longitudinal section of the right sciatic nerve was cut and prepared at a thickness of 15 m. After routine deparaffinization, immunocytochemical analysis was performed by using 70 kDa and 200 kDa monoclonal mouse anti-human neurofilament antibody (Daco Japan Co., Tokyo, Japan). The results of the immunocytochemical analysis (the length of the stain) were compared with those of the pinch test. Statistical analysis The results of the pinch test are reported as the regeneration distance, in millimeters, from the suture to the point along the nerve that elicited a pinch reaction. For both groups, the regeneration distances at each data collection period were plotted against the number of days after right nerve transection, and a regression line was calculated by the least squares method. The slope of the line represents the speed of regeneration, and the point where the line crosses the X axis is the initial delay. The results of the pinch test and the immunoreactive cell ratio of IL-1 and TGF-1 at each time point were expressed as mean and standard deviations (SD). Statistical comparisons were made with two-way ANOVA and Scheffe`’s test with the Stat-View 5.0 statistical software program. The correlation between the results of the pinch test and the distance from the suture site to the point where neurofilament staining was positive was assessed with the Spearman’s
FIG. 1. Results of the pinch test on the right sciatic nerve of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve, by number of days since transection and repair of the right sciatic nerve. The slope of the regression line represents the regeneration rate (mm/day) and the intersection of the regression line with the X axis represents the initial delay (arrowhead, experimental group; arrow, control group).
rank correlation coefficient. Differences in which P was less than 0.05 were regarded as statistically significant.
RESULTS Agreement between Measures of Regeneration The results of neurofilament staining correlated well with those of the pinch test (r 2 ⫽ 0.67, P ⫽ 0.001). Pinch test results show that nerve regeneration was significantly greater in the experimental group at all time points (P ⬍ 0.05 in all cases) and that the initial delay was shorter (Fig. 1). The regeneration speed of the experimental group was 0.99 mm/day and did not differ significantly from the 0.79 mm/day observed in the control (P ⫽ 0.21; Fig. 1). However, the initial delay of the experimental group, 1.03 days, was significantly shorter than that of the control group, 2.31 days (P ⬍ 0.001). Thus, contralateral nerve regeneration was associated with a shortening of the initial delay without acceleration of the regeneration speed. Expression of IL-1 in Dorsal Root Ganglia The expression of IL-1-positive cells was significantly higher in the experimental group from day 0 and peaked on day 1, with the value being significantly higher than that in the control group (Fig. 2). Thereafter, on days 3, 5, and 7, it decreases over time but remained higher than that in the control group (P ⫽ 0.53, P ⫽ 0.17, and P ⫽ 0.37, respectively). No immunoreactive cell was observed in the negative control. Expression of TGF-1 in Dorsal Root Ganglia The expression of TGF-1 in the experimental group was more predominant than that in the control group,
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FIG. 2. Changes over time in the immunoreactive cell ratio of IL-1 in the right dorsal root ganglion of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve.
even at day 0. Thereafter, the expression of TGF-1 slowly increased in both groups, and peaked at day 5 (Fig. 3). Using a CAS 200 image analyzer for the quantitative analysis, we recorded a positive expression ratio of TGF-1 of 12.7 ⫾ 2.5% at day 0 in the experimental group, which was significantly higher than the 2.9 ⫾ 0.8% in the control group (P ⬍ 0.001). No immunoreactive cell was observed in the negative control. DISCUSSION We found that a conditioning lesion accelerated nerve regeneration in the contralateral nerve in vivo by shortening the initial delay and not by increasing the regeneration speed. Although a number of reports have verified the accuracy of the pinch test (13–16), we also stained the neurofilament at the same time, which verified both the accuracy of the pinch test and the fact that a conditioning lesion was accompanied by histological evidence of accelerated nerve regeneration on the contralateral side. We found that accelerated nerve regeneration appears to be primary the results of a shortening of the initial delay. These results is consistent with our previous study of a culture system in vitro (17), which showed that the conditioning effect is not merely restricted to the ipsilateral neurons but also affects the undamaged sensory neurons of the contralateral DRG. The mechanism for promoting in vivo nerve regeneration must be clarified so that new procedure promoting nerve regeneration can be developed to improve clinical results after nerve repair. It is generally agreed that an ipsilateral conditioning lesion induces several responses within the cell bodies that promote the synthesis of various proteins necessary for nerve regeneration. These proteins appear to increase the capacity for axonal growth after the second nerve injury (1). Gutman et al. reported that an ipsilateral conditioning
lesion shortened the initial delay in the rabbit (8). On the other hand, McQuarrie et al. documented an increase in the regeneration speed in the rat sciatic nerve after an ipsilateral conditioning lesion (18). However, whether nerve injury can affect the metabolism of contralateral nerve cells is not known. To clarify the mechanism, in which the contralateral conditioning lesion promotes nerve regeneration, we investigated the chronological changes of the expression of IL-1 and TGF-1 in the contralateral DRG. Our results also clearly show that nerve injury enhances the expression of IL-1 and TGF-1 in nerve cells of the contralateral DRG. Several studies have revealed that IL-1 has many physiological effects on nerve regeneration. For instance, IL-1 indirectly promotes the production of NGF (3) and the mRNA of amyloid precursor protein (APP), which has a neurotrophic effect (4) and which directly promotes the survival of neurons in vitro in the nervous system (2). IL-1 also promotes nerve regeneration in the DRG culture system (3). In addition, IL-1 promoted the expression of TGF-1 in astrocytes in the central nervous system of the rat (19). In the present study, the expression of IL-1 was higher in the experimental group, reaching its peak expression on day 1 and thereafter gradually declining. This cytokine not only has a favorable direct effect on nerve regeneration but also stimulates the expression of TGF-1 in the nerve cell body, which promotes nerve regeneration (2– 4, 19). In the nervous system, TGF-1 has the capacity both to promote growth and to protect motor and sensory neurons against damage (6, 20, 21), and it is important in nerve regeneration. In 1997, Oka reported that in a crushed unilateral sciatic nerve, TGF-1 expression peaked at day 5 after crush not only in the ipsilateral DRG but also in the normal contralateral DRG (7). In
FIG. 3. Changes over time in the immunoreactive cell ratio of TGF-1 in the right dorsal root ganglion of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve.
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the present study, a similar time course of TGF-1 expression was detected in the contralateral DRG after the second transection in rat with a conditioning lesion. Furthermore, our results revealed that TGF-1 expression in the experimental group was significantly enhanced at day 0, compared to that in the control. When compared to the control group, nerve regeneration from the suture site in the experimental group was enhanced from day 1 after the second transection and suture, with a shortening of the initial delay. This shortened delay may be related to higher expression of IL-1 and TGF-1 in the nerve cell bodies in the contralateral DRG, indicating the preparatory process for nerve regeneration. After the initial injury on the contralateral side, the proteins necessary for nerve regeneration may start to be synthesized more vigorously within the nerve cell body, and some of them may be already transported in advance to the peripheral region by axonal transport. At the time of second transection and suture, the proteins for nerve regeneration may be already present near the transection site, resulting in a shortened initial delay. Two mechanisms may promote regeneration in the neuron. One mechanism may be the effect mediated by local synaptic communication between right and left neurons. Rotto-Percelay et al. showed that transneural transport occurred not only between sensory neurons of both sides but also between motor neurons (22). The other mechanism may be the delivery of neurotrophic cytokines through the general circulation. Consistent with this mechanism is evidence indicating that NGF m RNA is increased in cervical DRG and bilateral lumbar DRG after unilateral crushing of the sciatic nerve (23, 24). Although further investigations on other cytokines and for more prolonged observation period are necessary to clarify the mechanism, the present study showed that the conditioning lesion enhances cytokines expression in the contralateral DRG and shortens the initial delay of nerve regeneration in vivo. In the sciatic nerves of rats, a conditioning lesion promotes contralateral nerve regeneration by shortening the initial delay and enhances the expression of cytokines IL-1 and TGF-1 in the contralateral nerve cell body.
ACKNOWLEDGMENT This work was supported in part by a grant to M. Ochi, M.D., from the Japanese Ministry of Education Science and Culture (No. 09771089).
REFERENCES 1. Mcquarrie, I. G., and Grafstein, B. (1973) Arch. Neurol. 29, 53–55. 2. Brenneman, D. E., Schultzberg, M., Bartifai, T., and Gozes, T. (1992) J. Neurochem. 58, 454 – 460. 3. Horie, H., Sakai, I., Akahori, Y., and Kadoya, T. (1997) NeuroReport 8, 1955–1959. 4. Ohyagi, Y., and Tabira, T. (1993) Mol. Brain Res. 18, 127–132. 5. Heine, U. I., Munoz, E. F., Flandes, K. C., Ellingworth, L. R., Peter Lam, H-Y., Thompson, N. L., Roberts, A. B., and Sporn, M. B. (1987) J. Cell. Biol. 105, 2861–2876. 6. Henrich-Noack, P., Prehn, J. H. M., and Krieglstein, J. (1994) J. Neural Transom. 43, 33– 45. 7. Oka, S. (1997) J. Hiroshima Med. Sci. 45, 1–12. 8. Gutmann, E., Guttmann, L., Medawar, P. B., and Young, J. Z. (1942) J. Exp. Biol. 19, 14 – 44. 9. Young, J. Z., Holmes, W., and Saunders, F. K. (1940) Lancet 2, 128 –130. 10. Young, J. Z., Medawar, P. B. (1940) Lancet 2, 126 –128. 11. Sjoberg, J., and Kanje, M. (1990) Brain Res. 529, 79 – 84. 12. Adams, J. C. (1981) J. Histochem. Cytochem. 29, 775. 13. Kerns, J. M., Danielsen, N., Holmquist, B., Kanje, M., and Lundborg, G. (1993) Exp. Neurol. 122, 28 –36. 14. Rusovan, A., and Kanje, M. (1991) Exp. Neurol. 112, 312–316. 15. Rusovan, A., and Kanje, M. (1992) NeuroReport 3, 813– 814. 16. Rusovan, A., Kanje, M., and Mild, H. K. (1992) Exp. Neurol. 117, 81– 84. 17. Yamaguchi, H., Ochi, M., Mori, R., Ryoke, K., Yamamoto, S., Iwata, A., and Uchio, U. (1990) NeuroReport 10, 1359 –1362. 18. Mcquarrie, I. G., Grafstein, B., and Gershon, M. D. (1977) Brain Res. 132, 443– 453. 19. Dacunha, A., Jefferson, J. A., Jacksonn, R. W., and Vitkovic, L. (1993) J. Neuroimmunol. 42, 71– 86. 20. Martinou, J-C., Thai, A. L. A., Vallete, A., and Weber, M. J. (1990) Dev. Brain Res. 52, 175–181. 21. Prehn, J. H., Peruche, B., Unisicker, K., and Krieglstein, J. (1993) J. Neurochem. 60, 1665–1672. 22. Rotto-Percelay, D. M., Wheeler, J. G., Osorio, F. A., Platt, K. B., and Loewy, A. D. (1992) Brain Res. 574, 291–306. 23. Hermann, R., Lindholm, D., Bandtlow. C., Mayer, M., Radeke, J., Misko, T. M., Shooter, E., and Thoenen, H. (1987) Proc. Natl. Acad. Sci USA 84, 8735– 8739. 24. Wells, M. R., Vaidya, U., and Schwartz, J. P. (1991) Exp. Brain Res. 101, 53–58.
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A Conditioning Lesion Promotes in Vivo Nerve Regeneration in the Contralateral Sciatic Nerve of Rats Koji Ryoke, Mitsuo Ochi, 1 Atsushi Iwata, Yuji Uchio, Soichiro Yamamoto, and Hidetoshi Yamaguchi Department of Orthopaedics, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane-ken 693-8501, Japan
Received December 6, 1999
A conditioning lesion in the sciatic nerve increases in vivo axonal regeneration in the nerve after a second transection. We studied whether this increased regeneration also occurs in the contralateral nerve. The left sciatic nerve was transected and sutured in Wistar rats; the nerve was exposed but not transected in controls. After 5 days, the right sciatic nerves of all rats were transected and sutured. Neuronal regeneration was measured at 0, 1, 3, 5, and 7 days with the pinch test and histological staining. IL-1 and TGF-1 expression was also measured. The initial delay in the experimental group was significantly shorter, but the regeneration rates were the same. The expression of IL-1 and TGF-1 in the right dorsal root ganglia was significantly higher in the experimental group. Nerve injury enhances cytokine expression in the contralateral dorsal root ganglion and promotes contralateral nerve regeneration in vivo by shortening the initial delay. © 2000 Academic Press Key Words: nerve regeneration; contralateral conditioning lesion; interleukin-1  (IL-1); transforming growth factor-1 (TGF-1); initial delay.
In rats with a previous, unilateral “conditioning” lesion in the sciatic nerve, axonal regeneration in the nerve after a second transection is more rapid than in nerves without such a conditioning lesion (1). Several experiments have shown that after the nerve cut, the mRNA of IL-1 (2– 4) and TGF-1 is elevated in the dorsal root ganglion (DRG) cells and in the anterior horn cells of the ipsilateral side, and that these cytokines are intimately involved in nerve regeneration (5, 6). However mechanism of this rapid regeneration has not been adequately described. Oka reported that a nerve injury can stimulate expression of TGF-1 in the contralateral DRG (7), indicating that a unilateral nerve injury also affects the To whom correspondence should be addressed. Fax: ⫹81 853-202236. E-mail: [email protected]. 1
metabolism of neurons in the normal, contralateral side. We hypothesized that regeneration might be promoted by a conditioning lesion not only in a second ipsilateral nerve injury but also in a contralateral nerve injury. The purpose of the present study was (1) to ascertain whether a previous conditioning injury to the sciatic nerve in the rats, followed by injury to the contralateral sciatic nerve would promote nerve regeneration in the contralateral nerve and (2) to determine whether the expression in IL-1 and TGF-1 in DRG were associated with nerve regeneration and, in fact, might explain this regeneration. MATERIALS AND METHODS Animals and surgery. We studied 8-week-old, male Wistar rats weighing 200 and 250 g (Clea Japan, Tokyo, Japan). Each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (0.025 mg/g; Abbott Laboratories, North Chicago, IL). For the experimental group (n ⫽ 45), the left sciatic nerve was transected 5 mm distal to the biceps femoris branch and then re-anastomosed with 10-O nylon suture. Five days later, the procedure was repeated on the right sciatic nerve in each rat. The control group (n ⫽ 45) underwent a sham operation in which the left sciatic nerve was exposed but not transected. As in the experimental group, 5 days later, the right sciatic nerve was transected and sutured as above. On days 0, 1, 3, 5, and 7 after transection of the right sciatic nerve, 9 rats were sacrificed. Axonal regeneration in the right sciatic nerve was evaluated, and the level of expression of IL-1 and TGF-1 in the L 4-5 DRG was recorded. All animals were treated according to the guideline with University Regents’ Policy on Animal Care and Use, Shimane Medical University policy, Federal Animal Welfare Act, and other applicable standards. Evaluation of regeneration. On days 0, 1, 3, 5, and 7 after the right sciatic nerve transection and suture, a pinch test was performed for six rats in each group to determine the extent of nerve regeneration (8 –10). The extent of regeneration was defined as the distance between the most distal tip of the regenerated sensory neurons and the suture site on the right sciatic nerves (8 –11). For the pinch test, each rat was lightly anesthetized with a 0.2 ml intraperitoneal injection of solution containing sodium pentobarbital (60 mg/ml), saline, and diazepam (5 mg/ml, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) in 1:1:2 volume proportions. The right sciatic nerve was re-exposed, and the intact proximal branch to the biceps femoris was pinched to determine the appropriate level of
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anesthesia that allowed the animal respond. Beginning 15 mm distal from the suture site, the nerve was carefully pinched with a forceps at 0.5-mm intervals in a proximal direction. The most distal site at which the pinch test elicited a contraction of muscles on the back and a movement of the right leg was marked with 10-O nylon. The distance from this point to the suture site was the nerve regeneration distance. Immunocytochemistry and assessment of immunostaining. Immediately after the pinch test, the rats were euthanized with sodium pentobarbital (0.08 mg/g) and perfused with 4% paraformaldehyde (PFA) in 0.05 M phosphate buffered saline (PBS). In all rats, the right sciatic nerve, together with the right L4 and L5 DRG, were removed and fixed for 72 h at room temperature with 4.0% PFA in 0.05 M PBS. The fixed DRGs were embedded in paraffin and then cut longitudinally into 6-m-thick sections. The sections were placed on aminopropyl-triethoxysilane-coated glass slides (Matsunami Glass Ind., Ltd., Japan) and, after deparaffinization, were washed three times with 0.05 M PBS (pH 7.4). The sections were then pretreated with hydrogen peroxide to deplete endogenous peroxidase, incubated with polyclonal rabbit anti-human TGF-1 antibody (King Brewing Co., Kakogawa, Japan), and diluted 1000-fold with 1% bovine serum albumin (BSA) in PBS for 24 h at room temperature. A 500-fold diluted solution of biotinylated goat anti-rabbit immunoglobulin (Daco Japan Co., Tokyo, Japan) was left to react at room temperature for 30 min and was then reacted with a 500-fold diluted solution of peroxidase-labeled streptavidin (Daco Japan Co., Tokyo, Japan). Peroxidase was visualized using a solution of 3,3⬘-diaminobenzidine 4HCl, H 2O 2, CO 2⫹, Ni 2⫹ according to the method of Adams (12). The sections were then stained with 0.5% fast green (Wako Pure Chemical Industries, Ltd., Osaka, Japan). In place of the primary antibody, 1% BSA in PBS was added and used as a negative control. In the primary antibody used in this study, the active type TGF-1 has an affinity of about 27-fold compared to the inactive type TGF1. In addition, using the same technique, immunocytochemical analysis was performed using a polyclonal rabbit anti-human IL-1 antibody (Genzyme, Boston, MA). All sections were stained at the same time. The expression of IL-1 and TGF-1 in DRGs of each group and at each data collection point was determined by using quantitative estrogen-progesterone analysis software in the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). The mean value of the immunoreactive cell ratio was obtained for quantitative analysis with following formula: the immunoreactive cell ratio ⫽ (the number of IL-1 or TGF-1 stained cell)/(the number of total cell) in visual field from 20 randomly selected visual fields per glass slide. To confirm the accuracy of the pinch test, the longitudinal section of the right sciatic nerve was cut and prepared at a thickness of 15 m. After routine deparaffinization, immunocytochemical analysis was performed by using 70 kDa and 200 kDa monoclonal mouse anti-human neurofilament antibody (Daco Japan Co., Tokyo, Japan). The results of the immunocytochemical analysis (the length of the stain) were compared with those of the pinch test. Statistical analysis The results of the pinch test are reported as the regeneration distance, in millimeters, from the suture to the point along the nerve that elicited a pinch reaction. For both groups, the regeneration distances at each data collection period were plotted against the number of days after right nerve transection, and a regression line was calculated by the least squares method. The slope of the line represents the speed of regeneration, and the point where the line crosses the X axis is the initial delay. The results of the pinch test and the immunoreactive cell ratio of IL-1 and TGF-1 at each time point were expressed as mean and standard deviations (SD). Statistical comparisons were made with two-way ANOVA and Scheffe`’s test with the Stat-View 5.0 statistical software program. The correlation between the results of the pinch test and the distance from the suture site to the point where neurofilament staining was positive was assessed with the Spearman’s
FIG. 1. Results of the pinch test on the right sciatic nerve of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve, by number of days since transection and repair of the right sciatic nerve. The slope of the regression line represents the regeneration rate (mm/day) and the intersection of the regression line with the X axis represents the initial delay (arrowhead, experimental group; arrow, control group).
rank correlation coefficient. Differences in which P was less than 0.05 were regarded as statistically significant.
RESULTS Agreement between Measures of Regeneration The results of neurofilament staining correlated well with those of the pinch test (r 2 ⫽ 0.67, P ⫽ 0.001). Pinch test results show that nerve regeneration was significantly greater in the experimental group at all time points (P ⬍ 0.05 in all cases) and that the initial delay was shorter (Fig. 1). The regeneration speed of the experimental group was 0.99 mm/day and did not differ significantly from the 0.79 mm/day observed in the control (P ⫽ 0.21; Fig. 1). However, the initial delay of the experimental group, 1.03 days, was significantly shorter than that of the control group, 2.31 days (P ⬍ 0.001). Thus, contralateral nerve regeneration was associated with a shortening of the initial delay without acceleration of the regeneration speed. Expression of IL-1 in Dorsal Root Ganglia The expression of IL-1-positive cells was significantly higher in the experimental group from day 0 and peaked on day 1, with the value being significantly higher than that in the control group (Fig. 2). Thereafter, on days 3, 5, and 7, it decreases over time but remained higher than that in the control group (P ⫽ 0.53, P ⫽ 0.17, and P ⫽ 0.37, respectively). No immunoreactive cell was observed in the negative control. Expression of TGF-1 in Dorsal Root Ganglia The expression of TGF-1 in the experimental group was more predominant than that in the control group,
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FIG. 2. Changes over time in the immunoreactive cell ratio of IL-1 in the right dorsal root ganglion of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve.
even at day 0. Thereafter, the expression of TGF-1 slowly increased in both groups, and peaked at day 5 (Fig. 3). Using a CAS 200 image analyzer for the quantitative analysis, we recorded a positive expression ratio of TGF-1 of 12.7 ⫾ 2.5% at day 0 in the experimental group, which was significantly higher than the 2.9 ⫾ 0.8% in the control group (P ⬍ 0.001). No immunoreactive cell was observed in the negative control. DISCUSSION We found that a conditioning lesion accelerated nerve regeneration in the contralateral nerve in vivo by shortening the initial delay and not by increasing the regeneration speed. Although a number of reports have verified the accuracy of the pinch test (13–16), we also stained the neurofilament at the same time, which verified both the accuracy of the pinch test and the fact that a conditioning lesion was accompanied by histological evidence of accelerated nerve regeneration on the contralateral side. We found that accelerated nerve regeneration appears to be primary the results of a shortening of the initial delay. These results is consistent with our previous study of a culture system in vitro (17), which showed that the conditioning effect is not merely restricted to the ipsilateral neurons but also affects the undamaged sensory neurons of the contralateral DRG. The mechanism for promoting in vivo nerve regeneration must be clarified so that new procedure promoting nerve regeneration can be developed to improve clinical results after nerve repair. It is generally agreed that an ipsilateral conditioning lesion induces several responses within the cell bodies that promote the synthesis of various proteins necessary for nerve regeneration. These proteins appear to increase the capacity for axonal growth after the second nerve injury (1). Gutman et al. reported that an ipsilateral conditioning
lesion shortened the initial delay in the rabbit (8). On the other hand, McQuarrie et al. documented an increase in the regeneration speed in the rat sciatic nerve after an ipsilateral conditioning lesion (18). However, whether nerve injury can affect the metabolism of contralateral nerve cells is not known. To clarify the mechanism, in which the contralateral conditioning lesion promotes nerve regeneration, we investigated the chronological changes of the expression of IL-1 and TGF-1 in the contralateral DRG. Our results also clearly show that nerve injury enhances the expression of IL-1 and TGF-1 in nerve cells of the contralateral DRG. Several studies have revealed that IL-1 has many physiological effects on nerve regeneration. For instance, IL-1 indirectly promotes the production of NGF (3) and the mRNA of amyloid precursor protein (APP), which has a neurotrophic effect (4) and which directly promotes the survival of neurons in vitro in the nervous system (2). IL-1 also promotes nerve regeneration in the DRG culture system (3). In addition, IL-1 promoted the expression of TGF-1 in astrocytes in the central nervous system of the rat (19). In the present study, the expression of IL-1 was higher in the experimental group, reaching its peak expression on day 1 and thereafter gradually declining. This cytokine not only has a favorable direct effect on nerve regeneration but also stimulates the expression of TGF-1 in the nerve cell body, which promotes nerve regeneration (2– 4, 19). In the nervous system, TGF-1 has the capacity both to promote growth and to protect motor and sensory neurons against damage (6, 20, 21), and it is important in nerve regeneration. In 1997, Oka reported that in a crushed unilateral sciatic nerve, TGF-1 expression peaked at day 5 after crush not only in the ipsilateral DRG but also in the normal contralateral DRG (7). In
FIG. 3. Changes over time in the immunoreactive cell ratio of TGF-1 in the right dorsal root ganglion of rats, with (experimental) and without (control) a previous conditioning lesion on the left sciatic nerve.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the present study, a similar time course of TGF-1 expression was detected in the contralateral DRG after the second transection in rat with a conditioning lesion. Furthermore, our results revealed that TGF-1 expression in the experimental group was significantly enhanced at day 0, compared to that in the control. When compared to the control group, nerve regeneration from the suture site in the experimental group was enhanced from day 1 after the second transection and suture, with a shortening of the initial delay. This shortened delay may be related to higher expression of IL-1 and TGF-1 in the nerve cell bodies in the contralateral DRG, indicating the preparatory process for nerve regeneration. After the initial injury on the contralateral side, the proteins necessary for nerve regeneration may start to be synthesized more vigorously within the nerve cell body, and some of them may be already transported in advance to the peripheral region by axonal transport. At the time of second transection and suture, the proteins for nerve regeneration may be already present near the transection site, resulting in a shortened initial delay. Two mechanisms may promote regeneration in the neuron. One mechanism may be the effect mediated by local synaptic communication between right and left neurons. Rotto-Percelay et al. showed that transneural transport occurred not only between sensory neurons of both sides but also between motor neurons (22). The other mechanism may be the delivery of neurotrophic cytokines through the general circulation. Consistent with this mechanism is evidence indicating that NGF m RNA is increased in cervical DRG and bilateral lumbar DRG after unilateral crushing of the sciatic nerve (23, 24). Although further investigations on other cytokines and for more prolonged observation period are necessary to clarify the mechanism, the present study showed that the conditioning lesion enhances cytokines expression in the contralateral DRG and shortens the initial delay of nerve regeneration in vivo. In the sciatic nerves of rats, a conditioning lesion promotes contralateral nerve regeneration by shortening the initial delay and enhances the expression of cytokines IL-1 and TGF-1 in the contralateral nerve cell body.
ACKNOWLEDGMENT This work was supported in part by a grant to M. Ochi, M.D., from the Japanese Ministry of Education Science and Culture (No. 09771089).
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