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An in vivo assay of neurotropic activity
Brain Research, 278 (1983) 229-231 Elsevier
229
An in vivo assay of neurotropic activity MICHAEL J. POLITIS and PETER S. SPENCER Institute of Neurotoxicology, Albert Einstein College of Medicine, Kennedy Center, 1410 Pelham Parkway S., Bronx, NY 10461 (U.S.A.) (Accepted July 5th, 1983) Key words: regeneration - - axonal guidance
Previous studies in this laboratory indicate that diffusible factors from distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerve fibers in vivo9. The present study was performed (a) to strengthen this hypothesis, and (b) to begin to determine the distribution and identity of putative neurotropic factors in peripheral nerves. Rat sciatic nerves were transected and inserted into the inlet end of a hollow 6 mm-long Y-shaped implant made of medical-grade Silastic tubing. To one of the outlet ends was attached an Elvax pellet impregnated with whole homogenate from a single sciatic nerve. The other outlet was attached to a pellet not containing homogenate. Homogenates assayed were obtained from distal stumps of transected nerves derived 14 days postoperatively or from unoperated sciatic nerves. After 3.5 weeks, nerve fiber bundles consisting of myelinated regenerating axons were only present in implant forks attached to pellets containing denervated nerve homogenate. This activity was heat- and trypsin-sensitive. These data (a) strengthen the hypothesis that diffusible factors from distal nerve stumps of transected nerves can support nerve fiber regeneration, (b) indicate that those factors are protein and/or protein dependent, and (c) suggest that unoperated peripheral nerve (at the levels assayed) either do not contain such factors or contain substances which inhibit such factors.
A x o n s from transected p e r i p h e r a l nerves can grow over gaps of up to 35 m m to reinnervate distal nerve stumps tt. In vitro studies have d e m o n s t r a t e d neuritic growth, often in circuitous routes, t o w a r d specific 'target tissue'3-5,7, possibly m e d i a t e d by diffusible ' n e u r o t r o p i c ' factors. In a previous study9, we re-examined the hypothesis, p r o p o s e d by Cajal 2 and later refuted by Weiss and Taylort2, that cells in distal stumps of transected p e r i p h e r a l nerves can exert a tropic effect on regenerating nerve fibers using surgical materials and assay p r o c e d u r e s not available to those investigators. In these studies, proximal stumps of transected rat or cat peripheral nerves were inserted into inlet ends of 6 mm-long, Y - s h a p e d implants m a d e of medicalgrade Silastic tubing. To one outlet was attached a ' n e u r o t r o p i c lure' (tissue from distal stump of transected nerve). T h e o t h e r outlet was left vacant, or attached to distal nerve stumps r e n d e r e d necrotic with dry ice and nucleic-acid-synthesis inhibitors 9 or tendon (unpublished observations). Several weeks later, regenerating axons (assessed morphologically and with the use of axonally t r a n s p o r t e d label) had 0006-8993/83/$03.00 (~ 1983 Elsevier Science Publishers B.V.
preferentially grown into implant forks attached to untreated distal nerve stump grafts. Insertion of a filter between the distally located grafts and outlet ends of the implant did not prevent this response. These data are consistent with the idea that diffusible factors from distal stumps of transected p e r i p h e r a l nerves can attract/support regenerating nerve fibers over distances of several m m in vivo. The present study was p e r f o r m e d (a) to strengthen this hypothesis, and (b) to begin to d e t e r m i n e the distribution and identity of putative neurotropic factors in peripheral nerve trunks. F o r this purpose, Elvax pellets ( D u p o n t Chemical Co., Willmington, D E ) were e m p l o y e d . Surgically i m p l a n t e d pellets containing vascularization-promoting factors have been used to induce and attract blood-vessel formation in retinat, 6. The same principle was e m p l o y e d in this study to induce axonal regeneration into an inorganic substrate (Silastic tubes). All surgical p r o c e d u r e s were p e r f o r m e d aseptically with animals under deep p e n t o b a r b i t a l anesthesia. Male S p r a g u e - D a w l e y rats (250 g b o d y weight) were used. The rat sciatic nerve was transected and the
230
Fig. 1. Cross-section through midportion of an implant fork attached to a pellet containing an untreated homogenate from dcnervatcd sciatic nerve. (A 1 #m section stained with toluidine blue, x 160).
proximal stump inserted into the single inlet end of a 6 m m q o n g Y-shaped Silastic tube with the aid of a 9.0 suture. To one of the paired outlet ends was attached (with 9.0 suture) an Elvax pellet (1 mm thick disc) containing homogenate from one sciatic nerve (obtained previously from separate animals and maintained for less than 7 days unhomogenized at - - 2 0 °C). The opposite outlet was attached to a pellet not containing homogenate. The interior of the implant was irrigated with saline for 1 min (then drained) to rehydrate the beads. Pellets were replaced as described above 7 days later. Animals were sacrificed 3.5 weeks after initial surgery and contents of the implant lumen (midportion of implant forks) fixed in 2.5% glutaraidehyde (in phosphate buffer, pH 7.4). Samples were then postfixed in buffered 2% osmium, dehydrated stepwise in increasing concentrations of alcohol, and embedded with epoxy resin. One micrometer sections were obtained and stained with toluidine blue. Elvax pellets were dissolved in methylene chloride (40% solution) at 40 °C. Homogenates of rat sciatic nerves (obtained with a Polytron homogenizer) were lyophilized and added to dissolved pellets, the mixture being dried under aseptic conditions and a partial vacuum. Pellets were stored at 5 °C for no more than 7 days. Homogenate from the equivalent of one sciatic nerve was mixed into each pellet. Animals were divided among 5 groups and treated as follows.
Group A. A pellet containing homogenate from unoperated sciatic nerve was placed opposite a pellet containing no homogenate (n = 6). Group B. Sciatic nerves were transected at the level of the sciatic notch and the distal stumps allowed to degenerate in the absence of regenerating axonst". Pellets were impregnated with homogenate from distal stumps obtained 14 days post-operatively (without the proximal-most 3 mm) and placed individually opposite a pellet containing no homogenate (n = ~). Group C. Homogenate obtained as in group B was exposed to trypsin (0.5%) for 60 min prior to lyophilization. This was followed by addition of trypsin inhibitor (n = 4) or albumin (to a final concentration o! 20%, n = 4) to inactivate and absorb the trypsin, respectively. Mixtures were then lyophilized and added to Elvax beads. Pellets infiltrated with trypsin plus inhibitor or albumin were placed in opposite outlets of Silastic implants. Group D. Homogenates from denervated nerve stumps were exposed to 80 °C for 10 min prior to lyophilization (n = 4). Mixtures were lyophylized, added to Elvax beads, and placed opposite beads containing no homogenate. Group E. Neither pellet contained homogenate (n = 2). In all cases in groups A, C, D and E, no axons had grown into implant lumens. Both implant forks were vacant or fluid-filled at time of sacrifice. However, in all animals in group B, a well-organized bundle ol
231 myelinated regenerating nerve fibers grew into the implant lumen, in 5 cases, exclusively t o w a r d the pellet containing nerve h o m o g e n a t e . One animal showed a small n u m b e r of fibers in the fork connected to the pellet without h o m o g e n a t e , but the n u m b e r of m y e l i n a t e d figures was several times less than in the opposite fork. The nerve fiber bundles consisted of an inner core of regenerating nerve fibers s u r r o u n d e d by circumferentially o r i e n t e d cells (Fig. 1). This a r r a n g e m e n t was similar to cases in which p e r i p h e r a l nerve grafts acted as neurotropic 'lures' at the outlet ends of the implants 9. This study indicates that (a) Elvax pellets mixed with h o m o g e n a t e from d e n e r v a t e d p e r i p h e r a l nerves can attract/support axonal r e g e n e r a t i o n , (b) this activity is heat- and trypsin-sensitive, and (c) h o m o g e nate from u n o p e r a t e d nerves does not exert this action. These d a t a lend support to the contention that distal stumps of transected p e r i p h e r a l nerve trunks contain diffusible factors (possibly substrate molecules) which can attract nerve fiber regeneration. In addition, these factors a p p e a r to be proteins or protein-dependent. U n o p e r a t e d p e r i p h e r a l nerve trunks (at least in the levels assayed in the present studies) a p p e a r either (a) not to contain these factors or (b) contain substances which inactivate these factors. It is not certain whether the mechanism by which pellets containing nerve h o m o g e n a t e attract regener-
1 Ben Ezra, D., Neovasculogenic ability of prostaglandin, growth factor and synthetic chemoattractants, Amer. J. Ophthal., 86 (1978) 455-460. 2 Cajal, S. Ram6n y, Experiments dealing with the transplantation of nerves or their products designed to prove especially an attractive or neurotropic action on nerve sprouts. In Degeneration and Regeneration of the Nervous System, Hafner Press, New York, 1968, pp. 329-361. 3 Coughlin, M. D., Target organ stimulation of parasympathetic nerve growth in the developing mouse submandibular gland, Develop. Biol., 43 (1975) 140-158. 4 Coughlin, M. and Rathbone, M. P., Factors involved in the stimulation of parasympathetic nerve outgrowth, Develop. Biol., 61 (1977) 131-139. 5 Crain, S. and Peterson, E. R., Selective innervation of target regions within fetal mouse spinal cord and medulla explants by isolated dorsal root ganglia in organotypic cocultures, Develop. Brain Res., 2 (1982) 341-362. 6 D'Amore, P. A. and Brunsun, S. K., Endothelial cell growth factor/angiogenesis factor from bovine retina, J. Cell Biol., 91 (1981) 196a. 7 Ebendal, T. and Jacobson, C. O., Tissue explants affecting
ating nerve fibers is the same as for distally-located peripheral nerve grafts. F o r instance, it remains to be d e t e r m i n e d if the p r i m a r y ' a t t r a c t a n t ' effect is exerted on the tips of injured axons or cells in their vicinity. If the latter is so, extension of axonal growth cones into implant lumens could 'passively' follow these cells or an acellular matrix p r o d u c e d by them. A detailed ultrastructural study of lumenal contents throughout the first two post-operative weeks in preparations in which pellets vs nerve grafts are utilized as 'lures' will be required to address this issue. A n u m b e r of in vitro assay p r o c e d u r e s for neurotropic activity are available (e.g. refs. 4 and 8). The p r e p a r a t i o n p r e s e n t e d here is a potential tool for assay of neurotropic activity in vivo. F u r t h e r , this assay can be used to assess changes in behavior of injured axons and associated cells in the absence of primary alterations in neuronal cell bodies (i.e., n e u r o t r o p h i c activity). The p r e p a r a t i o n could be utilized to assay the ability of (a) whole tissue h o m o g e n a t e s (e.g. from various d e v e l o p m e n t a l or post-traumatic stages), (b) specific growth-promoting factors (e.g. nerve growth factor) or (c) substrate c o m p o n e n t s (e.g. laminin) for their ability to direct/support nerve fiber regeneration. Such studies could be helpful in elucidating the mechanisms which direct the growth of regenerating axons in vivo.
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extension and orientation of axons in cultures of chick ganglia, Exp. CellRes., 105 (1977) 379-387. Gundersen, R. W. and Barret, J. N., Characterization of the turning response of dorsal root neurites towards nerve growth factor, J. Cell Biol., 87 (1980) 546-550. 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. Politis, M. J. and Spencer, P. S., A method to separate spatially the temporal sequence of axon-Schwann cell interaction during nerve regeneration, J. Neurocytol., l0 (1980) 221-232. Sunderland (1963), Quoted by G. Lunborg. In A. Gorio, H. Milesi and S. Mingrino (Eds.), Symposium on PostTraumatic Peripheral Nerve Regeneration, Raven Press, New York, p. 241. Weiss, P. and Taylor, A. C., Further evidence against 'neurotropism' in nerve regeneration, J. exp. Zool., 95 (1944) 233-257.
229
An in vivo assay of neurotropic activity MICHAEL J. POLITIS and PETER S. SPENCER Institute of Neurotoxicology, Albert Einstein College of Medicine, Kennedy Center, 1410 Pelham Parkway S., Bronx, NY 10461 (U.S.A.) (Accepted July 5th, 1983) Key words: regeneration - - axonal guidance
Previous studies in this laboratory indicate that diffusible factors from distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerve fibers in vivo9. The present study was performed (a) to strengthen this hypothesis, and (b) to begin to determine the distribution and identity of putative neurotropic factors in peripheral nerves. Rat sciatic nerves were transected and inserted into the inlet end of a hollow 6 mm-long Y-shaped implant made of medical-grade Silastic tubing. To one of the outlet ends was attached an Elvax pellet impregnated with whole homogenate from a single sciatic nerve. The other outlet was attached to a pellet not containing homogenate. Homogenates assayed were obtained from distal stumps of transected nerves derived 14 days postoperatively or from unoperated sciatic nerves. After 3.5 weeks, nerve fiber bundles consisting of myelinated regenerating axons were only present in implant forks attached to pellets containing denervated nerve homogenate. This activity was heat- and trypsin-sensitive. These data (a) strengthen the hypothesis that diffusible factors from distal nerve stumps of transected nerves can support nerve fiber regeneration, (b) indicate that those factors are protein and/or protein dependent, and (c) suggest that unoperated peripheral nerve (at the levels assayed) either do not contain such factors or contain substances which inhibit such factors.
A x o n s from transected p e r i p h e r a l nerves can grow over gaps of up to 35 m m to reinnervate distal nerve stumps tt. In vitro studies have d e m o n s t r a t e d neuritic growth, often in circuitous routes, t o w a r d specific 'target tissue'3-5,7, possibly m e d i a t e d by diffusible ' n e u r o t r o p i c ' factors. In a previous study9, we re-examined the hypothesis, p r o p o s e d by Cajal 2 and later refuted by Weiss and Taylort2, that cells in distal stumps of transected p e r i p h e r a l nerves can exert a tropic effect on regenerating nerve fibers using surgical materials and assay p r o c e d u r e s not available to those investigators. In these studies, proximal stumps of transected rat or cat peripheral nerves were inserted into inlet ends of 6 mm-long, Y - s h a p e d implants m a d e of medicalgrade Silastic tubing. To one outlet was attached a ' n e u r o t r o p i c lure' (tissue from distal stump of transected nerve). T h e o t h e r outlet was left vacant, or attached to distal nerve stumps r e n d e r e d necrotic with dry ice and nucleic-acid-synthesis inhibitors 9 or tendon (unpublished observations). Several weeks later, regenerating axons (assessed morphologically and with the use of axonally t r a n s p o r t e d label) had 0006-8993/83/$03.00 (~ 1983 Elsevier Science Publishers B.V.
preferentially grown into implant forks attached to untreated distal nerve stump grafts. Insertion of a filter between the distally located grafts and outlet ends of the implant did not prevent this response. These data are consistent with the idea that diffusible factors from distal stumps of transected p e r i p h e r a l nerves can attract/support regenerating nerve fibers over distances of several m m in vivo. The present study was p e r f o r m e d (a) to strengthen this hypothesis, and (b) to begin to d e t e r m i n e the distribution and identity of putative neurotropic factors in peripheral nerve trunks. F o r this purpose, Elvax pellets ( D u p o n t Chemical Co., Willmington, D E ) were e m p l o y e d . Surgically i m p l a n t e d pellets containing vascularization-promoting factors have been used to induce and attract blood-vessel formation in retinat, 6. The same principle was e m p l o y e d in this study to induce axonal regeneration into an inorganic substrate (Silastic tubes). All surgical p r o c e d u r e s were p e r f o r m e d aseptically with animals under deep p e n t o b a r b i t a l anesthesia. Male S p r a g u e - D a w l e y rats (250 g b o d y weight) were used. The rat sciatic nerve was transected and the
230
Fig. 1. Cross-section through midportion of an implant fork attached to a pellet containing an untreated homogenate from dcnervatcd sciatic nerve. (A 1 #m section stained with toluidine blue, x 160).
proximal stump inserted into the single inlet end of a 6 m m q o n g Y-shaped Silastic tube with the aid of a 9.0 suture. To one of the paired outlet ends was attached (with 9.0 suture) an Elvax pellet (1 mm thick disc) containing homogenate from one sciatic nerve (obtained previously from separate animals and maintained for less than 7 days unhomogenized at - - 2 0 °C). The opposite outlet was attached to a pellet not containing homogenate. The interior of the implant was irrigated with saline for 1 min (then drained) to rehydrate the beads. Pellets were replaced as described above 7 days later. Animals were sacrificed 3.5 weeks after initial surgery and contents of the implant lumen (midportion of implant forks) fixed in 2.5% glutaraidehyde (in phosphate buffer, pH 7.4). Samples were then postfixed in buffered 2% osmium, dehydrated stepwise in increasing concentrations of alcohol, and embedded with epoxy resin. One micrometer sections were obtained and stained with toluidine blue. Elvax pellets were dissolved in methylene chloride (40% solution) at 40 °C. Homogenates of rat sciatic nerves (obtained with a Polytron homogenizer) were lyophilized and added to dissolved pellets, the mixture being dried under aseptic conditions and a partial vacuum. Pellets were stored at 5 °C for no more than 7 days. Homogenate from the equivalent of one sciatic nerve was mixed into each pellet. Animals were divided among 5 groups and treated as follows.
Group A. A pellet containing homogenate from unoperated sciatic nerve was placed opposite a pellet containing no homogenate (n = 6). Group B. Sciatic nerves were transected at the level of the sciatic notch and the distal stumps allowed to degenerate in the absence of regenerating axonst". Pellets were impregnated with homogenate from distal stumps obtained 14 days post-operatively (without the proximal-most 3 mm) and placed individually opposite a pellet containing no homogenate (n = ~). Group C. Homogenate obtained as in group B was exposed to trypsin (0.5%) for 60 min prior to lyophilization. This was followed by addition of trypsin inhibitor (n = 4) or albumin (to a final concentration o! 20%, n = 4) to inactivate and absorb the trypsin, respectively. Mixtures were then lyophilized and added to Elvax beads. Pellets infiltrated with trypsin plus inhibitor or albumin were placed in opposite outlets of Silastic implants. Group D. Homogenates from denervated nerve stumps were exposed to 80 °C for 10 min prior to lyophilization (n = 4). Mixtures were lyophylized, added to Elvax beads, and placed opposite beads containing no homogenate. Group E. Neither pellet contained homogenate (n = 2). In all cases in groups A, C, D and E, no axons had grown into implant lumens. Both implant forks were vacant or fluid-filled at time of sacrifice. However, in all animals in group B, a well-organized bundle ol
231 myelinated regenerating nerve fibers grew into the implant lumen, in 5 cases, exclusively t o w a r d the pellet containing nerve h o m o g e n a t e . One animal showed a small n u m b e r of fibers in the fork connected to the pellet without h o m o g e n a t e , but the n u m b e r of m y e l i n a t e d figures was several times less than in the opposite fork. The nerve fiber bundles consisted of an inner core of regenerating nerve fibers s u r r o u n d e d by circumferentially o r i e n t e d cells (Fig. 1). This a r r a n g e m e n t was similar to cases in which p e r i p h e r a l nerve grafts acted as neurotropic 'lures' at the outlet ends of the implants 9. This study indicates that (a) Elvax pellets mixed with h o m o g e n a t e from d e n e r v a t e d p e r i p h e r a l nerves can attract/support axonal r e g e n e r a t i o n , (b) this activity is heat- and trypsin-sensitive, and (c) h o m o g e nate from u n o p e r a t e d nerves does not exert this action. These d a t a lend support to the contention that distal stumps of transected p e r i p h e r a l nerve trunks contain diffusible factors (possibly substrate molecules) which can attract nerve fiber regeneration. In addition, these factors a p p e a r to be proteins or protein-dependent. U n o p e r a t e d p e r i p h e r a l nerve trunks (at least in the levels assayed in the present studies) a p p e a r either (a) not to contain these factors or (b) contain substances which inactivate these factors. It is not certain whether the mechanism by which pellets containing nerve h o m o g e n a t e attract regener-
1 Ben Ezra, D., Neovasculogenic ability of prostaglandin, growth factor and synthetic chemoattractants, Amer. J. Ophthal., 86 (1978) 455-460. 2 Cajal, S. Ram6n y, Experiments dealing with the transplantation of nerves or their products designed to prove especially an attractive or neurotropic action on nerve sprouts. In Degeneration and Regeneration of the Nervous System, Hafner Press, New York, 1968, pp. 329-361. 3 Coughlin, M. D., Target organ stimulation of parasympathetic nerve growth in the developing mouse submandibular gland, Develop. Biol., 43 (1975) 140-158. 4 Coughlin, M. and Rathbone, M. P., Factors involved in the stimulation of parasympathetic nerve outgrowth, Develop. Biol., 61 (1977) 131-139. 5 Crain, S. and Peterson, E. R., Selective innervation of target regions within fetal mouse spinal cord and medulla explants by isolated dorsal root ganglia in organotypic cocultures, Develop. Brain Res., 2 (1982) 341-362. 6 D'Amore, P. A. and Brunsun, S. K., Endothelial cell growth factor/angiogenesis factor from bovine retina, J. Cell Biol., 91 (1981) 196a. 7 Ebendal, T. and Jacobson, C. O., Tissue explants affecting
ating nerve fibers is the same as for distally-located peripheral nerve grafts. F o r instance, it remains to be d e t e r m i n e d if the p r i m a r y ' a t t r a c t a n t ' effect is exerted on the tips of injured axons or cells in their vicinity. If the latter is so, extension of axonal growth cones into implant lumens could 'passively' follow these cells or an acellular matrix p r o d u c e d by them. A detailed ultrastructural study of lumenal contents throughout the first two post-operative weeks in preparations in which pellets vs nerve grafts are utilized as 'lures' will be required to address this issue. A n u m b e r of in vitro assay p r o c e d u r e s for neurotropic activity are available (e.g. refs. 4 and 8). The p r e p a r a t i o n p r e s e n t e d here is a potential tool for assay of neurotropic activity in vivo. F u r t h e r , this assay can be used to assess changes in behavior of injured axons and associated cells in the absence of primary alterations in neuronal cell bodies (i.e., n e u r o t r o p h i c activity). The p r e p a r a t i o n could be utilized to assay the ability of (a) whole tissue h o m o g e n a t e s (e.g. from various d e v e l o p m e n t a l or post-traumatic stages), (b) specific growth-promoting factors (e.g. nerve growth factor) or (c) substrate c o m p o n e n t s (e.g. laminin) for their ability to direct/support nerve fiber regeneration. Such studies could be helpful in elucidating the mechanisms which direct the growth of regenerating axons in vivo.
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extension and orientation of axons in cultures of chick ganglia, Exp. CellRes., 105 (1977) 379-387. Gundersen, R. W. and Barret, J. N., Characterization of the turning response of dorsal root neurites towards nerve growth factor, J. Cell Biol., 87 (1980) 546-550. 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. Politis, M. J. and Spencer, P. S., A method to separate spatially the temporal sequence of axon-Schwann cell interaction during nerve regeneration, J. Neurocytol., l0 (1980) 221-232. Sunderland (1963), Quoted by G. Lunborg. In A. Gorio, H. Milesi and S. Mingrino (Eds.), Symposium on PostTraumatic Peripheral Nerve Regeneration, Raven Press, New York, p. 241. Weiss, P. and Taylor, A. C., Further evidence against 'neurotropism' in nerve regeneration, J. exp. Zool., 95 (1944) 233-257.