332
Brain Research, 564 (1991) 332-335 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124927K
BRES 24927
Glia maturation factor 13 stimulates axon regeneration in transected rat sciatic nerve K a t h e r i n e H a r m a n 1, Julia Katnick 2, R a m o n Lim3, . Asgar Z a h e e r 3 and Jack C. de la Torre 2 1Physiotherapy Program, 2Division of Neurosurgery, University of Ottawa, Ottawa, Ont. (Canada) and 3Division of Neurochemistry and Neurobiology, Department of Neurology, University of lowa, Iowa City, IA (U.S.A.) (Accepted 13 August 1991) Key words: Sciatic nerve; Transection; Axon regeneration; Neurotrophic factor; Glia maturation factor fl
Rat sciatic nerves were bilaterally transected and repaired with an entubulation technique. The nerve interstump gap was filled with either collagen gel or collagen gel mixed with a putative neurotrophic factor (leupeptin, 4-aminopyridine, lipid angiogenicfactor or glia maturation factor fl (GMF-fl)). Six weeks after nerve transection, the myelinated distal stump axons were quantified for each nerve. Only the nerves treated with GMF-fl had significantly more axons than the control side. Peripheral nerves are known to regenerate after transection when adequate substrate and sufficient blood flow for the elongating axons are provided u'2°'25. Collagen gel has been used effectively as a substrate, supporting axonal regeneration 3'7'1° as well as blood vessel regrowth. Neurotrophic factors (NTF) have been found to modify peripheral nerve regeneration ~6"19"24, yet the key to understanding and improving functional outcome remains unclear. We hypothesized that by implanting a collagen/neurotrophic factor mixture (COL/NTF) into the interstump gap formed after transecting the rat sciatic nerve, the resultant axonal regeneration would be more abundant than with collagen only. Also, we believed that treatment with NTFs may provide clues with respect to events specifically occurring at the transection site. In this communication, we report that recombinant glia maturation factor fl (GMF-fl), a 17 kDa protein endogenous to the nervous system 9"13, appears to increase the number of regenerating axons when applied to the repair site after transection. Following anaesthesia with ketamine (60 mg/kg intramuscular) and pentobarbital (19 mg/kg intraperitoneal), both sciatic nerves of 16 female Sprague-Dawley rats (225-250 g) were exposed for transection. Each nerve was first anchored to polyethylene tubing (PE 280), then bathed in 15% polyvinyl alcohol:chlorpromazine solution 4. After flushing out the solution, the nerve was rapidly cooled with fluori-methane spray (15% dichlorodifluoromethane and 85% trichloromonofluoromethane
spray; Gebauer Chemical Co., Cleveland, OH) to render it firm 7, then quickly transected with an ultrafineedged razor blade. For each rat sciatic nerve, the 2 mm ( - 0.25 mm) interstump gap created was filled with either collagen (COL; collagen type I, 35 mg/ml; Collagen Corp., Palo Atlo, CA) or COL/NTF. Selection of NTF concentrations was based on previous reports showing the effectiveness of these compounds in protecting or stimulating axonal regeneration 1'2'5'13'~3-25. We then extrapolated these doses to make them appropriate for in situ application 6a6. The concentration of each NTF/ml collagen was: 4-aminopyridine 0.2 mg; lipid angiogenic factor 2.0 mg; leupeptin 0.2 mg; GMF-fl 20 ng/ml. Each preparation required 0.025 ml of collagen to fill the gap. Six weeks later, the rats were anaesthetized and perfused through the left cardiac ventricle with 500 ml heparinized saline and then 500 ml 10% buffered formalin. Both sciatic nerves were then removed and processed for semi-thin (0.5 #m) cross-sections. During surgery, a reference marker was placed at the distal stump:collagen interface and was later sighted when microtome sectioning. This marker served as a starting point from which we measured the distance each section was taken from the transection site. The collagen gel is mostly absorbed by the peripheral nerve tissue after 6 weeks but a few particles migrated for short distances into the distal nerve stump. Therefore, the first section that was completely free of collagen particles was chosen for analysis. A section from the treated sciatic nerve in the opposite leg
Correspondence: K. Harman, Physiotherapy Program, University of Ottawa, 451 Smyth Road, Room 2047, Ottawa, Ont. K1H 8M5, Canada. Fax: (1) (613) 738-3191.
333 was then taken at a matching distance. W h e n matching of the distances in the two nerves could not be achieved, the rat preparation was rejected from the study. All sciatic nerve cross-sections were stained with Toluidine blue and their images were projected by camera lucida directly onto a digitizing tablet where the axons were quantified using our line-sampling technique 8. All nerve cross-sections were analyzed by comparing the axon counts at matching distances in nerves treated with C O L vs C O L / N T F in terms of percentage change (Table I). Also, Student's t-test and A N O V A were applied to the data. Each animal served as its own control. Table I lists the distance from the transection site and the number of axons in each nerve section. The nerves treated with GMF-fl were remarkably similar with a 4 8 52% increase in the number of axons as compared to their controls. This increase was statistically significant using a paired t-test at < 0.05 confidence level. In the other 3 groups we found significant variability, with increases and decreases in the number of distal stump axons as compared to their controls, and there was no statistical significance when comparing these groups. Our premise for this study was that after nerve transection, repair involving the application of a C O L / N T F mixture at the transection site would improve axonal regeneration when compared to nerves treated with C O L
only. Others have reported growth stimulation after application of NTFs at the transection site 17'18'24. Lipid angiogenic factor 5, extracted from the omentum, has been shown to strongly stimulate angiogenesis, an essential element in neuronal regeneration TM. GMF-fl is a brain protein that has been implicated in neuronal recovery following injury TM. These two substances have not previously been tested in the peripheral nerve following transection. 4-Aminopyridine is a potassium channel blocker that has been reported to provide temporary conduction improvement to heat-injured and demyelinated nerves, but has only been administered intravenously or in vitro 22'23. Leupeptin is a calcium-activated neutral protease inhibitor that has been reported to enhance primate peripheral nerve regrowth 1 and appears to protect axonal structure 21. Each N T F held the prospect of exerting a stimulatory or protective effect on regenerating axons when applied at the transection and repair site. We previously reported axonal elongation and angiogenesis when transected rat sciatic nerve was repaired with our entubulation technique containing collagen gel 3'7. Our linesampling technique has been shown to be highly accurate for quantifying myelinated axons in sciatic nerve cross-sections 8. Two factors prevented us from studying all nerve sec-
TABLE I Percent difference in axonal counts between collagen and COLINTF treated transected sciatic nerves
Four neurotrophic factors mixed with collagen (COL/NTF) were studied. The table indicates the axonal count at a specific distance distal to the transection site for each nerve section. The last column lists the percent difference between the axonal counts of nerves treated with COL/NTF mixture and treated with collagen only (COL). The percent difference was calculated as follows: % difference =
no. of axons (COL/N'IT) - no. of axons (COL) x I00 no. of axons (COL)
Collagen
Collagen/neurotrophic factor
Difference (%)
Axonal counts
Distance (l~m)
Axonal counts
Distance (~m)
4-Aminopyridine (n = 3)
7909 16,243 13,120
700 675 1200
19,309 9678 15,528
700 675 1200
144 --40 18
Lipid angiogenic factor (n = 3)
11,861 13,872 19,225
1200 975 1200
11,932 15,875 14,366
1200 975 1200
0.59 14 -25
Leupeptin (n = 3)
10,682 11,445 11,094
1800 1200 900
10,993 7995 11,166
1800 1200 900
2.9 -30 0.6
Glia maturation factor fl (n = 3)
18,489 10,895 15,186
900 1000 900
27,847 16,099 23,020
900 1000 900
* GMF-fl (COL vs COL/NTF) statistically different using a paired t-test, P < 0.05.
50* 48* 51"
334 tions at exactly the same site in the distal stump. First, small particles of collagen gel were displaced into the degenerated distal stump, presumably due to proximodistal pressure from migrating tissue elements (no such displacement was noted in the proximal stump). In order to have a clear section, we cut distally until no collagen was observed. The second factor was that 25% of the nerves trifurcated more proximally than others, limiting the extent distally we could cut on a single nerve trunk. These two factors resulted in matched pairs of nerve sections at different distances from the reference point. Despite this, all sections were taken within a range of 700 #m. Results were analyzed in pairs by comparing axonal counts in C O L vs C O L / N T F (left to right legs of the same animal) treated nerves. These comparisons were expressed in terms of percentage change of axonal counts. We have used axonal counts in the distal stump to measure the effect of each N T F because once they have crossed the interstump gap, the axons have overcome a primary obstacle in recovery after transection. Although we assume increased axon regeneration favours improved function, there is no evidence that this premise is valid. Studies involving the evaluation of the density of target
Treatment at the transection site with 20 ng/ml GMF-fl in collagen results in significantly more axons in the regenerate. O u r finding supports an earlier report of decreased post-traumatic brain atrophy with GMF-fl treatm e n t 12. Bosch et al.2 demonstrated that nerve injury induces
the
production
of
endogenous
GMF-fl
in
Schwann cells. O u r results suggest a role for GMF-fl in peripheral axon regeneration. A dose-response study should establish the optimal GMF-fl concentration. The absence of a positive relationship between axonal counts and treatment with the other NTFs may be due to suboptimal concentrations in the collagen gel, the inability of those NTFs to exert a focal effect on regenerating axons in this preparation or by some other unk n o w n cause. The implantation of a C O L / N T F mixture in our entubulation preparation provides a useful and quantifiable technique for screening potential enhancers of peripheral axon growth.
tissue reinnervation and functional improvement are necessary to establish this correlation.
This project was supported by the Easter Seal Research Institute (Ontario) to J.C.T. and by the following grants to R.L.: Department of Veteran's Affairs Merit Review Award, Grant BNS8917665 from the National Science Foundation, Grant DK-25295 from the Diabetes-Endocrinology Center, and Grant LA-9001-1 from the American Paralysis Association.
1 Badalamente, M.A., Hurst, L.C. and Stracher, A., Neuromuscular recovery using calcium protease inhibition after median nerve repair in primates, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5983-5987. 2 Bosch, E.P., Zhong, W. and Lim, R., Axonai signals regulate expression of glia maturation factor-beta in Schwann cells: immunohistochemical study of injured sciatic nerve and cultured Sehwann cells, J. Neurosci., 9 (1989) 3690-3698. 3 de la Torre, J.C., Karaca, M., Merali, Z., Fortin, T. and Richard, M., Laser of razor? A novel experimental peripheral nerve repair technique, Neurosurgery, 22 (1988) 531-538. 4 de Medinaceli, L. and Church, A.C., Peripheral nerve reconnection: inhibition of early degenerative processes through the use of a novel fluid medium, Exp. Neurol., 84 (1984) 396-408. 5 Goldsmith, H.S., Griffith, A.L., Kupferman, A. and Catsimpoolas, N., Lipid angiogenic factor from omentum, J. Am. Med. Assoc., 252 (1984) 2034-2036. 6 Harman, K. and de la Torre, J.C., The influence of angiogenesis on reconnection prior to axonal elongation, Soc. Neurosci. Abstr., 14 (1988) 498. 7 Harman, K. and de la Torre, J.C., An improved model to study rat sciatic nerve regeneration, Soc. Neurosci. Abstr., 15 (1989) 884. 8 Harman, K., Katnick, J. and de la Torre, J.C., A quick and accurate line-sampling technique to quantify myelinated axons in peripheral nerve cross-sections, J. Neurosci. Methods, in press. 9 Kaplan, R., Zaheer, A., Jay, M. and Lim, R., Molecular cloning and expression of biologically active human glia maturation factor beta, J. Neurochem., in press. 10 Karaca, M., de la Torre, J.C., Goyal, R.N., Lach, B., Russell, N.A. and Benoit, B.G., Rat sciatic nerve regeneration using a collagen bioimplant, Turk. Neurosurg., 1 (1989) 4-11.
11 Kline, D.G., Hackett, E.R., Davis, G.D. and Myers, M.B., Effect of mobilization on the blood supply and regeneration of injured nerves, J. Surg. Res., 12 (1972) 254-266. 12 Lim, R. and Miller, J.F., Glia maturation factor influences recovery from injury in neonatal rat brains, Experientia, 41 (1985) 412-415. 13 Lim, R., Miller, J.E and Zaheer, A., Purification and characterization of glia maturation factor-beta: a growth regulator for neurons and glia, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3901-3905. 14 Lim, R. and Huang, L., Giia maturation factor-beta promotes the appearance of large neurofilament-rich neurons in injured rat brains, Brain Research, 504 (1989) 154-158. 15 Liuzzi, E, Proteolysis is a critical step in the physiological stop pathway: mechanisms involved in the blockade of axonal regeneration by mammalian astrocytes, Brain Research, 512 (1990) 277-283. 16 Longo, EM., Manthorpe, M., Skaper, S.D., Lundborg, G. and Varon, S., Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers, Brain Research, 261 (1983) 109-117. 17 Madison, R., da Silva, C.E, Dikkes, P., Sidman, R.L. and Chiu, T.-H. Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminin-containing gel, Exp. Neurol., 88 (1985) 767-772. 18 MUller, H., Williams, L.R. and Varon, S., Nerve regeneration chamber: evaluation of exogenous agents applied by multiple injections, Brain Research, 413 (1987) 320-326. 19 Politis, M.J., Ederle, K. and Spencer, P.S., Tropism in nerve regeneration in vivo, attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves, Brain Research, 253 (1982) 1-12. 20 Powell, H.C., Longo, F.M., LeBeau, J.M. and Myers, R.R.,
335 Abnormal nerve regeneration in galactose neuropathy, J. Neuropathol. Exp. Neurol., 45 (1986) 151-160. 21 Spungin, B. and Friedberg, I. The effect of leupeptin on the telescoping of Tetrahymena axomenes, Isr. J. Med. Sci., 23 (1987) 866. 22 Stefoski, D., Davis, E A . , Faut, M. and Schauf, C.L., 4-Aminopyridine improves clinical signs in multiple sclerosis, Ann. Neurol., 21 (1987) 71-77. 23 Targ, E.E, and Kocsis, J.D., 4-Aminopyridine leads to resto-
ration of conduction in demyelinated rat sciatic nerve, Brain Research, 328 (1985) 358-361. 24 Williams, L.R., Exogenous matrix precursors stimulate the temporal progress of nerve regeneration within a silicone chamber, Neurochem. Res., 12 (1987) 851-860. 25 Williams, L.R., Rat aorta isografts possess nerve regenerationpromoting properties in silicone Y chambers, Exp. Neurol., 97 (1987) 555-563.
Brain Research, 564 (1991) 332-335 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124927K
BRES 24927
Glia maturation factor 13 stimulates axon regeneration in transected rat sciatic nerve K a t h e r i n e H a r m a n 1, Julia Katnick 2, R a m o n Lim3, . Asgar Z a h e e r 3 and Jack C. de la Torre 2 1Physiotherapy Program, 2Division of Neurosurgery, University of Ottawa, Ottawa, Ont. (Canada) and 3Division of Neurochemistry and Neurobiology, Department of Neurology, University of lowa, Iowa City, IA (U.S.A.) (Accepted 13 August 1991) Key words: Sciatic nerve; Transection; Axon regeneration; Neurotrophic factor; Glia maturation factor fl
Rat sciatic nerves were bilaterally transected and repaired with an entubulation technique. The nerve interstump gap was filled with either collagen gel or collagen gel mixed with a putative neurotrophic factor (leupeptin, 4-aminopyridine, lipid angiogenicfactor or glia maturation factor fl (GMF-fl)). Six weeks after nerve transection, the myelinated distal stump axons were quantified for each nerve. Only the nerves treated with GMF-fl had significantly more axons than the control side. Peripheral nerves are known to regenerate after transection when adequate substrate and sufficient blood flow for the elongating axons are provided u'2°'25. Collagen gel has been used effectively as a substrate, supporting axonal regeneration 3'7'1° as well as blood vessel regrowth. Neurotrophic factors (NTF) have been found to modify peripheral nerve regeneration ~6"19"24, yet the key to understanding and improving functional outcome remains unclear. We hypothesized that by implanting a collagen/neurotrophic factor mixture (COL/NTF) into the interstump gap formed after transecting the rat sciatic nerve, the resultant axonal regeneration would be more abundant than with collagen only. Also, we believed that treatment with NTFs may provide clues with respect to events specifically occurring at the transection site. In this communication, we report that recombinant glia maturation factor fl (GMF-fl), a 17 kDa protein endogenous to the nervous system 9"13, appears to increase the number of regenerating axons when applied to the repair site after transection. Following anaesthesia with ketamine (60 mg/kg intramuscular) and pentobarbital (19 mg/kg intraperitoneal), both sciatic nerves of 16 female Sprague-Dawley rats (225-250 g) were exposed for transection. Each nerve was first anchored to polyethylene tubing (PE 280), then bathed in 15% polyvinyl alcohol:chlorpromazine solution 4. After flushing out the solution, the nerve was rapidly cooled with fluori-methane spray (15% dichlorodifluoromethane and 85% trichloromonofluoromethane
spray; Gebauer Chemical Co., Cleveland, OH) to render it firm 7, then quickly transected with an ultrafineedged razor blade. For each rat sciatic nerve, the 2 mm ( - 0.25 mm) interstump gap created was filled with either collagen (COL; collagen type I, 35 mg/ml; Collagen Corp., Palo Atlo, CA) or COL/NTF. Selection of NTF concentrations was based on previous reports showing the effectiveness of these compounds in protecting or stimulating axonal regeneration 1'2'5'13'~3-25. We then extrapolated these doses to make them appropriate for in situ application 6a6. The concentration of each NTF/ml collagen was: 4-aminopyridine 0.2 mg; lipid angiogenic factor 2.0 mg; leupeptin 0.2 mg; GMF-fl 20 ng/ml. Each preparation required 0.025 ml of collagen to fill the gap. Six weeks later, the rats were anaesthetized and perfused through the left cardiac ventricle with 500 ml heparinized saline and then 500 ml 10% buffered formalin. Both sciatic nerves were then removed and processed for semi-thin (0.5 #m) cross-sections. During surgery, a reference marker was placed at the distal stump:collagen interface and was later sighted when microtome sectioning. This marker served as a starting point from which we measured the distance each section was taken from the transection site. The collagen gel is mostly absorbed by the peripheral nerve tissue after 6 weeks but a few particles migrated for short distances into the distal nerve stump. Therefore, the first section that was completely free of collagen particles was chosen for analysis. A section from the treated sciatic nerve in the opposite leg
Correspondence: K. Harman, Physiotherapy Program, University of Ottawa, 451 Smyth Road, Room 2047, Ottawa, Ont. K1H 8M5, Canada. Fax: (1) (613) 738-3191.
333 was then taken at a matching distance. W h e n matching of the distances in the two nerves could not be achieved, the rat preparation was rejected from the study. All sciatic nerve cross-sections were stained with Toluidine blue and their images were projected by camera lucida directly onto a digitizing tablet where the axons were quantified using our line-sampling technique 8. All nerve cross-sections were analyzed by comparing the axon counts at matching distances in nerves treated with C O L vs C O L / N T F in terms of percentage change (Table I). Also, Student's t-test and A N O V A were applied to the data. Each animal served as its own control. Table I lists the distance from the transection site and the number of axons in each nerve section. The nerves treated with GMF-fl were remarkably similar with a 4 8 52% increase in the number of axons as compared to their controls. This increase was statistically significant using a paired t-test at < 0.05 confidence level. In the other 3 groups we found significant variability, with increases and decreases in the number of distal stump axons as compared to their controls, and there was no statistical significance when comparing these groups. Our premise for this study was that after nerve transection, repair involving the application of a C O L / N T F mixture at the transection site would improve axonal regeneration when compared to nerves treated with C O L
only. Others have reported growth stimulation after application of NTFs at the transection site 17'18'24. Lipid angiogenic factor 5, extracted from the omentum, has been shown to strongly stimulate angiogenesis, an essential element in neuronal regeneration TM. GMF-fl is a brain protein that has been implicated in neuronal recovery following injury TM. These two substances have not previously been tested in the peripheral nerve following transection. 4-Aminopyridine is a potassium channel blocker that has been reported to provide temporary conduction improvement to heat-injured and demyelinated nerves, but has only been administered intravenously or in vitro 22'23. Leupeptin is a calcium-activated neutral protease inhibitor that has been reported to enhance primate peripheral nerve regrowth 1 and appears to protect axonal structure 21. Each N T F held the prospect of exerting a stimulatory or protective effect on regenerating axons when applied at the transection and repair site. We previously reported axonal elongation and angiogenesis when transected rat sciatic nerve was repaired with our entubulation technique containing collagen gel 3'7. Our linesampling technique has been shown to be highly accurate for quantifying myelinated axons in sciatic nerve cross-sections 8. Two factors prevented us from studying all nerve sec-
TABLE I Percent difference in axonal counts between collagen and COLINTF treated transected sciatic nerves
Four neurotrophic factors mixed with collagen (COL/NTF) were studied. The table indicates the axonal count at a specific distance distal to the transection site for each nerve section. The last column lists the percent difference between the axonal counts of nerves treated with COL/NTF mixture and treated with collagen only (COL). The percent difference was calculated as follows: % difference =
no. of axons (COL/N'IT) - no. of axons (COL) x I00 no. of axons (COL)
Collagen
Collagen/neurotrophic factor
Difference (%)
Axonal counts
Distance (l~m)
Axonal counts
Distance (~m)
4-Aminopyridine (n = 3)
7909 16,243 13,120
700 675 1200
19,309 9678 15,528
700 675 1200
144 --40 18
Lipid angiogenic factor (n = 3)
11,861 13,872 19,225
1200 975 1200
11,932 15,875 14,366
1200 975 1200
0.59 14 -25
Leupeptin (n = 3)
10,682 11,445 11,094
1800 1200 900
10,993 7995 11,166
1800 1200 900
2.9 -30 0.6
Glia maturation factor fl (n = 3)
18,489 10,895 15,186
900 1000 900
27,847 16,099 23,020
900 1000 900
* GMF-fl (COL vs COL/NTF) statistically different using a paired t-test, P < 0.05.
50* 48* 51"
334 tions at exactly the same site in the distal stump. First, small particles of collagen gel were displaced into the degenerated distal stump, presumably due to proximodistal pressure from migrating tissue elements (no such displacement was noted in the proximal stump). In order to have a clear section, we cut distally until no collagen was observed. The second factor was that 25% of the nerves trifurcated more proximally than others, limiting the extent distally we could cut on a single nerve trunk. These two factors resulted in matched pairs of nerve sections at different distances from the reference point. Despite this, all sections were taken within a range of 700 #m. Results were analyzed in pairs by comparing axonal counts in C O L vs C O L / N T F (left to right legs of the same animal) treated nerves. These comparisons were expressed in terms of percentage change of axonal counts. We have used axonal counts in the distal stump to measure the effect of each N T F because once they have crossed the interstump gap, the axons have overcome a primary obstacle in recovery after transection. Although we assume increased axon regeneration favours improved function, there is no evidence that this premise is valid. Studies involving the evaluation of the density of target
Treatment at the transection site with 20 ng/ml GMF-fl in collagen results in significantly more axons in the regenerate. O u r finding supports an earlier report of decreased post-traumatic brain atrophy with GMF-fl treatm e n t 12. Bosch et al.2 demonstrated that nerve injury induces
the
production
of
endogenous
GMF-fl
in
Schwann cells. O u r results suggest a role for GMF-fl in peripheral axon regeneration. A dose-response study should establish the optimal GMF-fl concentration. The absence of a positive relationship between axonal counts and treatment with the other NTFs may be due to suboptimal concentrations in the collagen gel, the inability of those NTFs to exert a focal effect on regenerating axons in this preparation or by some other unk n o w n cause. The implantation of a C O L / N T F mixture in our entubulation preparation provides a useful and quantifiable technique for screening potential enhancers of peripheral axon growth.
tissue reinnervation and functional improvement are necessary to establish this correlation.
This project was supported by the Easter Seal Research Institute (Ontario) to J.C.T. and by the following grants to R.L.: Department of Veteran's Affairs Merit Review Award, Grant BNS8917665 from the National Science Foundation, Grant DK-25295 from the Diabetes-Endocrinology Center, and Grant LA-9001-1 from the American Paralysis Association.
1 Badalamente, M.A., Hurst, L.C. and Stracher, A., Neuromuscular recovery using calcium protease inhibition after median nerve repair in primates, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5983-5987. 2 Bosch, E.P., Zhong, W. and Lim, R., Axonai signals regulate expression of glia maturation factor-beta in Schwann cells: immunohistochemical study of injured sciatic nerve and cultured Sehwann cells, J. Neurosci., 9 (1989) 3690-3698. 3 de la Torre, J.C., Karaca, M., Merali, Z., Fortin, T. and Richard, M., Laser of razor? A novel experimental peripheral nerve repair technique, Neurosurgery, 22 (1988) 531-538. 4 de Medinaceli, L. and Church, A.C., Peripheral nerve reconnection: inhibition of early degenerative processes through the use of a novel fluid medium, Exp. Neurol., 84 (1984) 396-408. 5 Goldsmith, H.S., Griffith, A.L., Kupferman, A. and Catsimpoolas, N., Lipid angiogenic factor from omentum, J. Am. Med. Assoc., 252 (1984) 2034-2036. 6 Harman, K. and de la Torre, J.C., The influence of angiogenesis on reconnection prior to axonal elongation, Soc. Neurosci. Abstr., 14 (1988) 498. 7 Harman, K. and de la Torre, J.C., An improved model to study rat sciatic nerve regeneration, Soc. Neurosci. Abstr., 15 (1989) 884. 8 Harman, K., Katnick, J. and de la Torre, J.C., A quick and accurate line-sampling technique to quantify myelinated axons in peripheral nerve cross-sections, J. Neurosci. Methods, in press. 9 Kaplan, R., Zaheer, A., Jay, M. and Lim, R., Molecular cloning and expression of biologically active human glia maturation factor beta, J. Neurochem., in press. 10 Karaca, M., de la Torre, J.C., Goyal, R.N., Lach, B., Russell, N.A. and Benoit, B.G., Rat sciatic nerve regeneration using a collagen bioimplant, Turk. Neurosurg., 1 (1989) 4-11.
11 Kline, D.G., Hackett, E.R., Davis, G.D. and Myers, M.B., Effect of mobilization on the blood supply and regeneration of injured nerves, J. Surg. Res., 12 (1972) 254-266. 12 Lim, R. and Miller, J.F., Glia maturation factor influences recovery from injury in neonatal rat brains, Experientia, 41 (1985) 412-415. 13 Lim, R., Miller, J.E and Zaheer, A., Purification and characterization of glia maturation factor-beta: a growth regulator for neurons and glia, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3901-3905. 14 Lim, R. and Huang, L., Giia maturation factor-beta promotes the appearance of large neurofilament-rich neurons in injured rat brains, Brain Research, 504 (1989) 154-158. 15 Liuzzi, E, Proteolysis is a critical step in the physiological stop pathway: mechanisms involved in the blockade of axonal regeneration by mammalian astrocytes, Brain Research, 512 (1990) 277-283. 16 Longo, EM., Manthorpe, M., Skaper, S.D., Lundborg, G. and Varon, S., Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers, Brain Research, 261 (1983) 109-117. 17 Madison, R., da Silva, C.E, Dikkes, P., Sidman, R.L. and Chiu, T.-H. Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminin-containing gel, Exp. Neurol., 88 (1985) 767-772. 18 MUller, H., Williams, L.R. and Varon, S., Nerve regeneration chamber: evaluation of exogenous agents applied by multiple injections, Brain Research, 413 (1987) 320-326. 19 Politis, M.J., Ederle, K. and Spencer, P.S., Tropism in nerve regeneration in vivo, attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves, Brain Research, 253 (1982) 1-12. 20 Powell, H.C., Longo, F.M., LeBeau, J.M. and Myers, R.R.,
335 Abnormal nerve regeneration in galactose neuropathy, J. Neuropathol. Exp. Neurol., 45 (1986) 151-160. 21 Spungin, B. and Friedberg, I. The effect of leupeptin on the telescoping of Tetrahymena axomenes, Isr. J. Med. Sci., 23 (1987) 866. 22 Stefoski, D., Davis, E A . , Faut, M. and Schauf, C.L., 4-Aminopyridine improves clinical signs in multiple sclerosis, Ann. Neurol., 21 (1987) 71-77. 23 Targ, E.E, and Kocsis, J.D., 4-Aminopyridine leads to resto-
ration of conduction in demyelinated rat sciatic nerve, Brain Research, 328 (1985) 358-361. 24 Williams, L.R., Exogenous matrix precursors stimulate the temporal progress of nerve regeneration within a silicone chamber, Neurochem. Res., 12 (1987) 851-860. 25 Williams, L.R., Rat aorta isografts possess nerve regenerationpromoting properties in silicone Y chambers, Exp. Neurol., 97 (1987) 555-563.
