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AChE-positive fiber growth after hippocampal fimbria transection and peripheral nerve homogenate implantation
03614230185$3.00 + .OO
Bruin Research Bufletin, Vol. 15, pp. 13-18, 1985.Q Ankho International Inc. Printed in the U.S.A.
AChE-Positive Fiber Growth After Hippocampal Fimbria Transection and Peripheral Nerve Homogenate Implantation1 JOHN S. WENDT Dallas Veterans Administration
Medical Center and University of Texas Health Science 4500 S. Lancaster Road, Dallas, TX 75216
Received 2 November
Center at Dallas
1984
WENDT, J. S. AChE-positive fiber growth after hip~ocampai fimbria transection and peripheral nerve homogenate BRAIN RES BULL 15(l) 13-18, 198X-Gelfoam treated with peripheral nerve homogenate was implanted into a site of hip~ampal fimbria transection in the adult rat to assess whether the homogenate might enhance growth of AChE-positive fibers into the lesion site and whether the fiber growth might be directed to the implants. Homogenate was prepared from intact peripheral nerve and in a few cases from degenerated nerve. Some implants were encased iu Silastic. Homogenate was also injected into the denervated hippocampus. The major finding was that AChE-positive fiber growth was associated with regions of high astroglial cell content in preference to the relatively hypocellular implants. No clear differences in AChE fiber sprouting into the lesion site, fiber growth into implants, or hippocampal reinnervation were noted between homogenate and saline-treated animals.
~rnp~a~fafion.
AChE fiber growth
Peripheral nerve homogenate implant
Astroglial cells
METHOD
ALTHOUGH axonal growth from central nervous system (CNS) neurons into peripheral nerve implants has now been demonstrated for a variety of CNS neurons in the adult rat Il. 11, 131, the mechanisms of ingrowth are not known. Possible mechanisms include (a) neurotropic substances [6-S] emanating from the implant that attract growing neurites toward the implant, (b) a suitable growth substratum within the implant to which axons adhere by a process of random search and selection, and (c) production of neuritepromoting factors [lo] that enhance regenerative axonal sprouting, resulting in a greater number of growing neurites encountering the peripheral nerve growth substratum. Also, combinations of these mechanisms may be operative for various neuronal populations. By investigating the relationships between regenerating CNS axons and components of peripheral nerve (e.g., extracellular matrix, Schwann cells, and peripheral nerve homogenate), it may be possible to assess which factors are most important in determining growth of axons into peripheral nerve implants in vivo. Results of such studies may have implications for focusing on particular strategies for promoting CNS regeneration. For example, peripheral nerve homogenate may contain neurotropic and neurite-promoting factors that might influence AChE-positive axonal growth in vivo in the absence of an implanted peripheral nerve tissue substratum. To test this possibility the present study examines the effect of peripheral nerve homogenate implanted into a site of fimbria transection on growth of AChE-positive axons.
Adult Sprague-Dawley rats received implants consisting of Gelfoam (Upjohn) soaked with either homogenate derived from autologous sciatic nerve or normal saline (controls). In most animals receiving homogenate the homogenate was prepared from intact sciatic nerve (homogenate of intact nerve or HIN); in a few animals homogenate was prepared from pre-degenerated nerve (HDN) which one week previously had been transected and allowed to undergo Wallerian degeneration. In an attempt to retain possible neurotropic/neurotrophic substances that might be lost through diffusion out of the Gelfoam matrix, some implants were encased in medical grade Silastic tubing (Storz Instrument Company) prior to implantation. The detailed experiment protocol is as follows: Unilateral hip~~pal funbria transections were performed in adult Sprague-Dawley rats as described previously 1131. Prior to all surgical manipulations, including sacrifice and perfusion, animals were anesthetized with pentobarbital (SO mg/kg). In experiments involving HDN the proximal right sciatic nerve was transected at the time of fimbria transection. One week later the animals were randomly assigned to experimental and control groups. In the experimental groups (but not controls) a 4 mm segment of right sciatic nerve (from the distal stump in those animals with prior sciatic nerve transections) was removed and immediately placed in approximately 50 ~1 of normal saline. Nerve fibers were teased apart throughly with micro dissecting tweezers. The
‘Presented in part at the thi~y-~fth fou~eenth
Hippocampal fimbtia transection
annual meeting of the American Academy of Neurology, annual meeting of the Society for Neuroscience, October, 1984.
13
San Diego, CA, April, 1983, and at the
14
teased nerve and fluid were than placed in a tissue grinder, and an aliquot (150 ~1) of normal saline was added. The nerve was then homogenized with a Teflon serrated-tipped pestle at 700 rpm for approximately 1 min. Microscopic slide smears of homogenate stained with cresyl violet or toluidine blue and examined by light microscopy disclosed amorphous material with few identifiable cells. In experimental animals, I ~1 of homogenate was injected under stereotaxic guidance into the ipsilateral dorsal hippocampus by means of a 30 gauge needle connected by tubing to a 5 ~1 Hamilton syringe attached to a Sage infusion pump. Control animals received corresponding intrahippocampal saline injections. The fimbria transection site was then exposed, and scar tissue was gently dissected with forceps and suction with a finely tapered Pasteur pipet, yielding the transection cavity. After hemostasis had been achieved, the implant was placed in the transection cavity. Implants not encased with Silastic tubing consisted of several small pieces of Gelfoam soaked with homogenate or saline (controls). Silastic implants consisted of Gelfoam, soaked with either peripheral nerve homogenate or saline, inserted into 1.5 mm long segments of 0.25x0.047” Silastic tubing, which were then inserted horizontally into the transection sites. In order to assess possible effects of the Silastic material itself, two sets of controls were used, again determined by random selection one week after fimbria transection. One control group received no further treatment after fimbria transection. The second control group received intrahippocampal injections and Silastic implants in which saline was substituted for homogenate. After post-implantation survival periods of 10 days to 14 weeks animals were anesthetized and were perfused with normal saline followed by 10% formalin in saline. The brains were then removed and embedded in egg yolk, fixed 24 hours in formaldehyde fumes followed by immersion in 10% formaldehyde -30% sucrose solution for an additional 24 hours. In the initial experiments in which Silastic was not used, brains were then sectioned horizontally on a freezing microtome. In subsequent experiments in which Silastic was used frozen brain slices were cut in a “sagitto-coronal” plane (45” between the sagittal and coronal planes) in such a way that the septum, implant within the timbria transection site, and hippocampus all could be viewed in a single tissue section. The position of the Silastic implants in the transection cavity was noted both at the time of frozen sectioning and by subsequent microscopic examination. All tissue sections (40 pm) were processed for AChE histochemistry using a modified Koelle [4] procedure with tetraisopropyl-pyrophosphoramide at a concentration of 4x 10e5 M to inhibit nonspecific cholinesterases. After AChE staining the sections were mounted on slides and counterstained with cresyl violet. Microscopic analysis was conducted without prior knowledge of whether Gelfoam implants had been soaked in homogenate or saline. AChE fiber growth within the lesion site was assessed qualitatively in terms of the relationship of the growing fibers to their surrounding environment and their orientation with respect to the implants. Axonal growth into the lesion site and implants was also assessed semiquantitatively as to whether it was substantial (approximating the amount of growth associated with implants of intact peripheral nerve), moderate (small clusters of fibers or several scattered fascicles of nerve fibers), or negligible (little or no AChE-positive axonal growth into the lesion site or implants). AChE stain in the denervated hippocampus was also assessed qualitatively. Several slides were examined by a
WFINUI
TABLE FIBER GROWTH No
silastic
Control HINf HDNrj
n*
1
INTO IMPLANTS
nf
Silastic
n
nf
5
1
9 3
2 I
Control-NIY Control-SI#
6 5
0
HIN HDN
5 3
0 2
*Number in group, inumber with fiber growth into implant. *homogenate of intact nerve, Phomogenate of degenerated nerve. fin0 implant, #Silastic implant.
board-certified neuropathologist (Dr. Charles order to classify cells within the lesion site.
White)
in
RESULTS
The results of this study are based on observations from thirty-six animals (Table 1). Sixteen were controls (5 with saline-soaked Gelfoam, 6 with no treatment after the initial surgery, and 5 with Silastic implants). Fourteen had received HIN implants, of which 5 consisted of Silastic implants. Six animals had received HDN implants, 3 with Silastic implants. Among the 17 animals with non-Silastic implants, the implant was appropriately positioned in the transection site of all but one animal. In 9 of 13 animals with Silastic implants appropriate horizontal placement within the fimbria transection site was noted. Three Silastic implants (one eachcontrol, HIN, HDN) were more superficially located in the cavity, while one (HDN) had assumed a vertical position adjacent to the septum. Some growth of AChE-positive fibers into the lesion site was seen in most animals, and there was considerable variability in the amount of fiber growth among different animals within each group. Although moderate AChE-positive fiber growth was seen in the majority of animals (both controls and experimentals), growth of AChE-positive fascicles into the Gelfoam implants (Table 1) was noted in only six animals (1 control, 2 HIN, 1 HDN, and 2 HDN with Silastic). Extensive growth of AChE-positive fibers consistent with that expected after implantation of intact peripheral nerve was seen in only one animal, which had received a Silastic-HDN implant (described in detail below). AChE-positive fiber density in the denervated hippocampus was sparse regardless of treatment, although somewhat more prominent in the rostromedial region (not the site of homogenate injection). The most notable finding of this study was that AChEpositive fiber growth was largely limited to regions of high cellular content. The cellular morphology-consisting of predominantly cuboidal cells with eccentrically placed, light-stained nuclei in a stromal framework associated with abundant capillary vascularity-was characteristic of astroglial cells (Figs. l-3). Most implants consisted of a loose Gelfoam matrix with few cells and contained few, if any, AChEpositive fascicles. In most cases AChE-positive fascicles in cellular regions adjacent to Gelfoam implants appeared neither to enter the implants nor to be directed toward them. Figure 1 shows the rostra] aspect of a saline-soaked Gelfoam implant which had been encased in Silastic (the Silastic material itself was not preserved in tissue sections). Fascicles of AChF-
AChE-POSITIVE
FIBER GROWTH
FIG. 1. Gelfoam implant (i) treated with saline and encased in Silastic adjacent to cellular region fc) of the rostal portion of the lesion site near the septum. Cuboidal cells with light-staining nuclei are astroglial cells (hollow arrows). Occasional fibroblasts (short solid arrow) are identified by slender dark-staining nuclei. Note that the AChE-stained fascicles (long arrow) present in the cellular region do not appear to extend into the implant. Thirteen weeks post-implantation. fx200.)
positive fibers grew through a region of predominantly astroglial cells near the rostra1 fimbria stump but did not appear to extend into the relatively acellular implant matrix. Similarly, in Fig. 2 a strand of AChE-positive fibers that appeared to originate from the caudate nucleus extended along an astroglial tissue bridge adjacent to a Silastic implant containing Gelfoam treated with HDN. The fibers grew along the cellular substratum without invading the relatively hy~cellular implant. The few implants containing AChE-positive fascicles also contained more cells in the vicinity of the axons. A single implant exhibited ingrowth of AChE-positive fibers to an extent normally observed after implantation of whole peripheral nerve (Fig. 3). This implant consisted of Silastic tubing containing Gelfoam treated with HDN. The implant had assumed a vertical position immediately adjacent to the septum. Gelfoam was not identified within or around the implant. In its place was a dense array of predominantly astroglial cells extending throughout the implant and around its outer margins. Dense fascicles of AChEpositive fibers appearing to originate largely from the septum coursed through the rostal aspect of the interior of the implant. Other fascicles grew along the exterior caudal margin of the implant. The cells within the implant appeared to be arranged in two types of configurations. Within the caudal portion of the im-
plant furthest from the septum, cells appeared to assume a random cotdiguration. This portion of the implant was largely devoid of AChE-positive fiber growth. By contrast, in the rostra1 portion of the implant cells appeared to be oriented in a compact linear array in parallel to the path taken by AChEpositive axons (Fig. 3b). DISCUSSION
The results indicate that after hippocampal fimbria tmnsection and implantation of Gelfoam containing either peripheral nerve homogenate or saline, AChE-positive axonal growth occurs primarily in regions of high astroglial cell content. Most of the Gelfoam implants were relatively hypocellular and contained no AChE fascicles. This outcome was observed whether or not the Gelfoam implants had been treated with peripheral nerve homogenate. The few implants that contained AChE-positive fascicles also contained a higher concentration of cells. In the implant containing AChE-positive fascicle ingrowth comparable to that normally seen with implants of intact peripheral nerve, the loose Gelfoam matrix had been replaced by a compact array of predominantly astroglial cells. Along the path of axonal growth within this implant the cells appeared to assume a linear orientation.
16
WEN
0’1
FIG. 2. Gelfoam implant (i) treated with HDN and encased in Silastic adjacent to a band of tissue composed predomina~ti~ of astroglial ceils (c) extending from the caudate nucleus (below figure). AChE-positive fibers (solid arrows) course through the band of ceiiular tissue without turning toward or entering the implant. Cells containing pigmented material within the implant are macrophages (hollow arrow). Six weeks cost-implantation. I x 200.)
The ass~iation between Ache-~sitive axonal growth and regions of high astroglial cell content suggests that the cells provide a positive environment for axonal growth whereas the relatively hypocellular Gelfoam implants do not. AChE-positive axonal growth in the present study overall, however, was far less than that previously demonstrated with implants of intact peripheral nerve [ 131, indicating that the latter provide a much more favorable growth environment. Whether a favorable growth environment is determined p~rna~ly by the nature of the substratum remains unclear; however, pronounced axonal growth associated with what appeared to be a linear orientation of cells in one implant suggests that cellular conjuration may have had some relationship to axonal path finding. Reactive gliosis is generally believed to inhibit CNS regeneration in mammals [9]. Ahhough the present study does not prove that the astroglial cells associated with axonal growth are reactive astrocytes, the findings suggest that such inhibition is not absolute or universal. Recent in vitro studies also have demonstrated mammalian CNS neurite outgrowth over astroglial cells [2,5]. In addition, laminin, a glycoprotein that may be an important component of axonal growth substrata, has been identified in reactive astrocytes in the adult rat [33. FalIon [2] points out that axonal growth inhibition in vivo may be due to the unfavorable geometry of the astrocy-
tic scar rather than to an inhibitory property of the astrocyte surface. The lack of obvious effect of Gelfoam implants treated with peripheral nerve homogenate on AChE-positive axonal growth in this study does not exclude the possibiIity that peripheral nerve may contain neurotropic and neuritepromoting factors that influence AChE-positive axonal growth. First, such factors may have diffused out of the lesion site before exerting an effect on axonal growth. The purpose of encasing Gelfoam implants in Silastic was to inhibit diffusion, but it is not known how effective that strategy was. Second, continuous synthesis of tropic/trophic factors (such as by Schwann cells) might be required to demonstrate an effect, rather than one-time administration. Third, possible tropic/trophic factors might be present in higher concentration in degenerating peripheral nerve than in intact peripheral nerve; the WDN ovulation examined in the present study was too small to exclude possible differences between the HIN and HDN ~puIations. Fourth, possible tropicltrophic effects may have been masked by other factors in the growth environment. Recent studies of peripheral nerve regeneration have confirmed the existence of diffusible neurotropic substances from nerve tissue [(i-S]-in eontrast to earlier studies 1121. The use of different growth substrata is a possible explanation for the differences in results:
AChE-POSITIVE
FIBER GROWTH
FIG. 3a. Implant (i) of Gelfoam treated with HDN and encased in Silastic in vertical position adjacent to the septum (s) ventrally and corpus callosum dorsally. The implant has been heavily infiltrated with predominantly astroglial cells, and no Gelfoam is seen. AChE-positive fibers, some of which appear to originate from the septum, invade the implant (arrow). Eleven weeks post-implantation. (~74.) 3b. Higher magnification of the implant from Fig. 3a shows AChE-positive fibers (arrow) contained in a region of astroglial cells with an apparent linear orientation (a). Astroglial cells elsewhere appear to have a random orientation (b).
the vascular tissue used in the earlier experiments may have obscured neurotropic influences that have been revealed after replacing vascular tissue with inorganic tubing. Similarly, in the present study the cel~uIar growth environment in some manner may have obscured possible tropickrophic activity of the homogenate.
ACKNOWLEDGEMENTS
Dr. Charles White’s analysis of cellular detail, Dr. Isaac Crawford’s helpful su~estions in writing the manuscript, and Mrs. Karen Ayyad’s diligent technical assistance are gratefully appreciated. Supported by a V. A. Merit Review.
REFERENCES Benfey, M. and A. J. Aguayo. Extensive elongation of axons from rat brain into peripheral nerve grafts. Nnturr 1%: 15@152, 1982. Fallon, J. R. Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro. J Ce// Eiol 100: 198-207, 1985. Liesi, P., S. Kaakkole, D. Dahl and A. Vaheri. Laminin is induced in astrocvtes of adult brain bv iniurv. EMBO J 3: 683-686, Matthews, D. A., J. V. Nadler, G. S. Lynch and C. W. Cotman. Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity. DebI Biol 36: 130-141, 1974.
5. Noble, M., J. Fok-Seang and J. Cohen. Glia are a unique substrate for the vitro growth of central nervous system neurons. J Neurosci 4: 1892-1903, 1984. 6. Ochi, M. Experimental study on orientation of regenerating fibers in the severed verivheral nerve. Niroshimo J Med Sci 32: 389-406, 1983. * 7. Politis, M. J., K. Ederle and P. S. Spencer. Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves. Bruin Res 25% f-2, 1982. 8. Politis, M. H. and P. S. Spencer. An in vivo assay of neurotropic activity. Brain Res 278: 229-231, 1983.
18
9. Reier, P. J., L. J. Stensaas and L. Guth. The astrocytic scar as an impediment to regeneration in the central nervous system. In: Spinal Cord Reconstruction, edited by C. C. Kao, R. P. Bunge and P. J. Reier. New York: Raven Press, 1983, pp. 163195. 10. Richardson, P. M. and T. Ependal. Nerve growth activities in rat peripheral nerve. Brain Res 246: 57-64, 1982.
WEND’1
II. Richardson, P. M., V. M. K. Issa and A. J. Aguayo. Regeneration of long spinal axons in the rat. J Neuroc,yro/ 13: 16.5-182, 1984. 12. Weiss, P. and A. C. Taylor. Further evidence against neurotropism in nerve regeneration. J Exp Zoo1 95: 233-257. 1944. 13. Wendt, J. S., G. E. Fagg and C. W. Cotman. Regeneration of rat hippocampal timbria fibers after fimbria transection and peripheral nerve or fetal hippocampal implantation. E.rp Naurol 79: 452-461. 1983.
Bruin Research Bufletin, Vol. 15, pp. 13-18, 1985.Q Ankho International Inc. Printed in the U.S.A.
AChE-Positive Fiber Growth After Hippocampal Fimbria Transection and Peripheral Nerve Homogenate Implantation1 JOHN S. WENDT Dallas Veterans Administration
Medical Center and University of Texas Health Science 4500 S. Lancaster Road, Dallas, TX 75216
Received 2 November
Center at Dallas
1984
WENDT, J. S. AChE-positive fiber growth after hip~ocampai fimbria transection and peripheral nerve homogenate BRAIN RES BULL 15(l) 13-18, 198X-Gelfoam treated with peripheral nerve homogenate was implanted into a site of hip~ampal fimbria transection in the adult rat to assess whether the homogenate might enhance growth of AChE-positive fibers into the lesion site and whether the fiber growth might be directed to the implants. Homogenate was prepared from intact peripheral nerve and in a few cases from degenerated nerve. Some implants were encased iu Silastic. Homogenate was also injected into the denervated hippocampus. The major finding was that AChE-positive fiber growth was associated with regions of high astroglial cell content in preference to the relatively hypocellular implants. No clear differences in AChE fiber sprouting into the lesion site, fiber growth into implants, or hippocampal reinnervation were noted between homogenate and saline-treated animals.
~rnp~a~fafion.
AChE fiber growth
Peripheral nerve homogenate implant
Astroglial cells
METHOD
ALTHOUGH axonal growth from central nervous system (CNS) neurons into peripheral nerve implants has now been demonstrated for a variety of CNS neurons in the adult rat Il. 11, 131, the mechanisms of ingrowth are not known. Possible mechanisms include (a) neurotropic substances [6-S] emanating from the implant that attract growing neurites toward the implant, (b) a suitable growth substratum within the implant to which axons adhere by a process of random search and selection, and (c) production of neuritepromoting factors [lo] that enhance regenerative axonal sprouting, resulting in a greater number of growing neurites encountering the peripheral nerve growth substratum. Also, combinations of these mechanisms may be operative for various neuronal populations. By investigating the relationships between regenerating CNS axons and components of peripheral nerve (e.g., extracellular matrix, Schwann cells, and peripheral nerve homogenate), it may be possible to assess which factors are most important in determining growth of axons into peripheral nerve implants in vivo. Results of such studies may have implications for focusing on particular strategies for promoting CNS regeneration. For example, peripheral nerve homogenate may contain neurotropic and neurite-promoting factors that might influence AChE-positive axonal growth in vivo in the absence of an implanted peripheral nerve tissue substratum. To test this possibility the present study examines the effect of peripheral nerve homogenate implanted into a site of fimbria transection on growth of AChE-positive axons.
Adult Sprague-Dawley rats received implants consisting of Gelfoam (Upjohn) soaked with either homogenate derived from autologous sciatic nerve or normal saline (controls). In most animals receiving homogenate the homogenate was prepared from intact sciatic nerve (homogenate of intact nerve or HIN); in a few animals homogenate was prepared from pre-degenerated nerve (HDN) which one week previously had been transected and allowed to undergo Wallerian degeneration. In an attempt to retain possible neurotropic/neurotrophic substances that might be lost through diffusion out of the Gelfoam matrix, some implants were encased in medical grade Silastic tubing (Storz Instrument Company) prior to implantation. The detailed experiment protocol is as follows: Unilateral hip~~pal funbria transections were performed in adult Sprague-Dawley rats as described previously 1131. Prior to all surgical manipulations, including sacrifice and perfusion, animals were anesthetized with pentobarbital (SO mg/kg). In experiments involving HDN the proximal right sciatic nerve was transected at the time of fimbria transection. One week later the animals were randomly assigned to experimental and control groups. In the experimental groups (but not controls) a 4 mm segment of right sciatic nerve (from the distal stump in those animals with prior sciatic nerve transections) was removed and immediately placed in approximately 50 ~1 of normal saline. Nerve fibers were teased apart throughly with micro dissecting tweezers. The
‘Presented in part at the thi~y-~fth fou~eenth
Hippocampal fimbtia transection
annual meeting of the American Academy of Neurology, annual meeting of the Society for Neuroscience, October, 1984.
13
San Diego, CA, April, 1983, and at the
14
teased nerve and fluid were than placed in a tissue grinder, and an aliquot (150 ~1) of normal saline was added. The nerve was then homogenized with a Teflon serrated-tipped pestle at 700 rpm for approximately 1 min. Microscopic slide smears of homogenate stained with cresyl violet or toluidine blue and examined by light microscopy disclosed amorphous material with few identifiable cells. In experimental animals, I ~1 of homogenate was injected under stereotaxic guidance into the ipsilateral dorsal hippocampus by means of a 30 gauge needle connected by tubing to a 5 ~1 Hamilton syringe attached to a Sage infusion pump. Control animals received corresponding intrahippocampal saline injections. The fimbria transection site was then exposed, and scar tissue was gently dissected with forceps and suction with a finely tapered Pasteur pipet, yielding the transection cavity. After hemostasis had been achieved, the implant was placed in the transection cavity. Implants not encased with Silastic tubing consisted of several small pieces of Gelfoam soaked with homogenate or saline (controls). Silastic implants consisted of Gelfoam, soaked with either peripheral nerve homogenate or saline, inserted into 1.5 mm long segments of 0.25x0.047” Silastic tubing, which were then inserted horizontally into the transection sites. In order to assess possible effects of the Silastic material itself, two sets of controls were used, again determined by random selection one week after fimbria transection. One control group received no further treatment after fimbria transection. The second control group received intrahippocampal injections and Silastic implants in which saline was substituted for homogenate. After post-implantation survival periods of 10 days to 14 weeks animals were anesthetized and were perfused with normal saline followed by 10% formalin in saline. The brains were then removed and embedded in egg yolk, fixed 24 hours in formaldehyde fumes followed by immersion in 10% formaldehyde -30% sucrose solution for an additional 24 hours. In the initial experiments in which Silastic was not used, brains were then sectioned horizontally on a freezing microtome. In subsequent experiments in which Silastic was used frozen brain slices were cut in a “sagitto-coronal” plane (45” between the sagittal and coronal planes) in such a way that the septum, implant within the timbria transection site, and hippocampus all could be viewed in a single tissue section. The position of the Silastic implants in the transection cavity was noted both at the time of frozen sectioning and by subsequent microscopic examination. All tissue sections (40 pm) were processed for AChE histochemistry using a modified Koelle [4] procedure with tetraisopropyl-pyrophosphoramide at a concentration of 4x 10e5 M to inhibit nonspecific cholinesterases. After AChE staining the sections were mounted on slides and counterstained with cresyl violet. Microscopic analysis was conducted without prior knowledge of whether Gelfoam implants had been soaked in homogenate or saline. AChE fiber growth within the lesion site was assessed qualitatively in terms of the relationship of the growing fibers to their surrounding environment and their orientation with respect to the implants. Axonal growth into the lesion site and implants was also assessed semiquantitatively as to whether it was substantial (approximating the amount of growth associated with implants of intact peripheral nerve), moderate (small clusters of fibers or several scattered fascicles of nerve fibers), or negligible (little or no AChE-positive axonal growth into the lesion site or implants). AChE stain in the denervated hippocampus was also assessed qualitatively. Several slides were examined by a
WFINUI
TABLE FIBER GROWTH No
silastic
Control HINf HDNrj
n*
1
INTO IMPLANTS
nf
Silastic
n
nf
5
1
9 3
2 I
Control-NIY Control-SI#
6 5
0
HIN HDN
5 3
0 2
*Number in group, inumber with fiber growth into implant. *homogenate of intact nerve, Phomogenate of degenerated nerve. fin0 implant, #Silastic implant.
board-certified neuropathologist (Dr. Charles order to classify cells within the lesion site.
White)
in
RESULTS
The results of this study are based on observations from thirty-six animals (Table 1). Sixteen were controls (5 with saline-soaked Gelfoam, 6 with no treatment after the initial surgery, and 5 with Silastic implants). Fourteen had received HIN implants, of which 5 consisted of Silastic implants. Six animals had received HDN implants, 3 with Silastic implants. Among the 17 animals with non-Silastic implants, the implant was appropriately positioned in the transection site of all but one animal. In 9 of 13 animals with Silastic implants appropriate horizontal placement within the fimbria transection site was noted. Three Silastic implants (one eachcontrol, HIN, HDN) were more superficially located in the cavity, while one (HDN) had assumed a vertical position adjacent to the septum. Some growth of AChE-positive fibers into the lesion site was seen in most animals, and there was considerable variability in the amount of fiber growth among different animals within each group. Although moderate AChE-positive fiber growth was seen in the majority of animals (both controls and experimentals), growth of AChE-positive fascicles into the Gelfoam implants (Table 1) was noted in only six animals (1 control, 2 HIN, 1 HDN, and 2 HDN with Silastic). Extensive growth of AChE-positive fibers consistent with that expected after implantation of intact peripheral nerve was seen in only one animal, which had received a Silastic-HDN implant (described in detail below). AChE-positive fiber density in the denervated hippocampus was sparse regardless of treatment, although somewhat more prominent in the rostromedial region (not the site of homogenate injection). The most notable finding of this study was that AChEpositive fiber growth was largely limited to regions of high cellular content. The cellular morphology-consisting of predominantly cuboidal cells with eccentrically placed, light-stained nuclei in a stromal framework associated with abundant capillary vascularity-was characteristic of astroglial cells (Figs. l-3). Most implants consisted of a loose Gelfoam matrix with few cells and contained few, if any, AChEpositive fascicles. In most cases AChE-positive fascicles in cellular regions adjacent to Gelfoam implants appeared neither to enter the implants nor to be directed toward them. Figure 1 shows the rostra] aspect of a saline-soaked Gelfoam implant which had been encased in Silastic (the Silastic material itself was not preserved in tissue sections). Fascicles of AChF-
AChE-POSITIVE
FIBER GROWTH
FIG. 1. Gelfoam implant (i) treated with saline and encased in Silastic adjacent to cellular region fc) of the rostal portion of the lesion site near the septum. Cuboidal cells with light-staining nuclei are astroglial cells (hollow arrows). Occasional fibroblasts (short solid arrow) are identified by slender dark-staining nuclei. Note that the AChE-stained fascicles (long arrow) present in the cellular region do not appear to extend into the implant. Thirteen weeks post-implantation. fx200.)
positive fibers grew through a region of predominantly astroglial cells near the rostra1 fimbria stump but did not appear to extend into the relatively acellular implant matrix. Similarly, in Fig. 2 a strand of AChE-positive fibers that appeared to originate from the caudate nucleus extended along an astroglial tissue bridge adjacent to a Silastic implant containing Gelfoam treated with HDN. The fibers grew along the cellular substratum without invading the relatively hy~cellular implant. The few implants containing AChE-positive fascicles also contained more cells in the vicinity of the axons. A single implant exhibited ingrowth of AChE-positive fibers to an extent normally observed after implantation of whole peripheral nerve (Fig. 3). This implant consisted of Silastic tubing containing Gelfoam treated with HDN. The implant had assumed a vertical position immediately adjacent to the septum. Gelfoam was not identified within or around the implant. In its place was a dense array of predominantly astroglial cells extending throughout the implant and around its outer margins. Dense fascicles of AChEpositive fibers appearing to originate largely from the septum coursed through the rostal aspect of the interior of the implant. Other fascicles grew along the exterior caudal margin of the implant. The cells within the implant appeared to be arranged in two types of configurations. Within the caudal portion of the im-
plant furthest from the septum, cells appeared to assume a random cotdiguration. This portion of the implant was largely devoid of AChE-positive fiber growth. By contrast, in the rostra1 portion of the implant cells appeared to be oriented in a compact linear array in parallel to the path taken by AChEpositive axons (Fig. 3b). DISCUSSION
The results indicate that after hippocampal fimbria tmnsection and implantation of Gelfoam containing either peripheral nerve homogenate or saline, AChE-positive axonal growth occurs primarily in regions of high astroglial cell content. Most of the Gelfoam implants were relatively hypocellular and contained no AChE fascicles. This outcome was observed whether or not the Gelfoam implants had been treated with peripheral nerve homogenate. The few implants that contained AChE-positive fascicles also contained a higher concentration of cells. In the implant containing AChE-positive fascicle ingrowth comparable to that normally seen with implants of intact peripheral nerve, the loose Gelfoam matrix had been replaced by a compact array of predominantly astroglial cells. Along the path of axonal growth within this implant the cells appeared to assume a linear orientation.
16
WEN
0’1
FIG. 2. Gelfoam implant (i) treated with HDN and encased in Silastic adjacent to a band of tissue composed predomina~ti~ of astroglial ceils (c) extending from the caudate nucleus (below figure). AChE-positive fibers (solid arrows) course through the band of ceiiular tissue without turning toward or entering the implant. Cells containing pigmented material within the implant are macrophages (hollow arrow). Six weeks cost-implantation. I x 200.)
The ass~iation between Ache-~sitive axonal growth and regions of high astroglial cell content suggests that the cells provide a positive environment for axonal growth whereas the relatively hypocellular Gelfoam implants do not. AChE-positive axonal growth in the present study overall, however, was far less than that previously demonstrated with implants of intact peripheral nerve [ 131, indicating that the latter provide a much more favorable growth environment. Whether a favorable growth environment is determined p~rna~ly by the nature of the substratum remains unclear; however, pronounced axonal growth associated with what appeared to be a linear orientation of cells in one implant suggests that cellular conjuration may have had some relationship to axonal path finding. Reactive gliosis is generally believed to inhibit CNS regeneration in mammals [9]. Ahhough the present study does not prove that the astroglial cells associated with axonal growth are reactive astrocytes, the findings suggest that such inhibition is not absolute or universal. Recent in vitro studies also have demonstrated mammalian CNS neurite outgrowth over astroglial cells [2,5]. In addition, laminin, a glycoprotein that may be an important component of axonal growth substrata, has been identified in reactive astrocytes in the adult rat [33. FalIon [2] points out that axonal growth inhibition in vivo may be due to the unfavorable geometry of the astrocy-
tic scar rather than to an inhibitory property of the astrocyte surface. The lack of obvious effect of Gelfoam implants treated with peripheral nerve homogenate on AChE-positive axonal growth in this study does not exclude the possibiIity that peripheral nerve may contain neurotropic and neuritepromoting factors that influence AChE-positive axonal growth. First, such factors may have diffused out of the lesion site before exerting an effect on axonal growth. The purpose of encasing Gelfoam implants in Silastic was to inhibit diffusion, but it is not known how effective that strategy was. Second, continuous synthesis of tropic/trophic factors (such as by Schwann cells) might be required to demonstrate an effect, rather than one-time administration. Third, possible tropic/trophic factors might be present in higher concentration in degenerating peripheral nerve than in intact peripheral nerve; the WDN ovulation examined in the present study was too small to exclude possible differences between the HIN and HDN ~puIations. Fourth, possible tropicltrophic effects may have been masked by other factors in the growth environment. Recent studies of peripheral nerve regeneration have confirmed the existence of diffusible neurotropic substances from nerve tissue [(i-S]-in eontrast to earlier studies 1121. The use of different growth substrata is a possible explanation for the differences in results:
AChE-POSITIVE
FIBER GROWTH
FIG. 3a. Implant (i) of Gelfoam treated with HDN and encased in Silastic in vertical position adjacent to the septum (s) ventrally and corpus callosum dorsally. The implant has been heavily infiltrated with predominantly astroglial cells, and no Gelfoam is seen. AChE-positive fibers, some of which appear to originate from the septum, invade the implant (arrow). Eleven weeks post-implantation. (~74.) 3b. Higher magnification of the implant from Fig. 3a shows AChE-positive fibers (arrow) contained in a region of astroglial cells with an apparent linear orientation (a). Astroglial cells elsewhere appear to have a random orientation (b).
the vascular tissue used in the earlier experiments may have obscured neurotropic influences that have been revealed after replacing vascular tissue with inorganic tubing. Similarly, in the present study the cel~uIar growth environment in some manner may have obscured possible tropickrophic activity of the homogenate.
ACKNOWLEDGEMENTS
Dr. Charles White’s analysis of cellular detail, Dr. Isaac Crawford’s helpful su~estions in writing the manuscript, and Mrs. Karen Ayyad’s diligent technical assistance are gratefully appreciated. Supported by a V. A. Merit Review.
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5. Noble, M., J. Fok-Seang and J. Cohen. Glia are a unique substrate for the vitro growth of central nervous system neurons. J Neurosci 4: 1892-1903, 1984. 6. Ochi, M. Experimental study on orientation of regenerating fibers in the severed verivheral nerve. Niroshimo J Med Sci 32: 389-406, 1983. * 7. Politis, M. J., K. Ederle and P. S. Spencer. Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves. Bruin Res 25% f-2, 1982. 8. Politis, M. H. and P. S. Spencer. An in vivo assay of neurotropic activity. Brain Res 278: 229-231, 1983.
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9. Reier, P. J., L. J. Stensaas and L. Guth. The astrocytic scar as an impediment to regeneration in the central nervous system. In: Spinal Cord Reconstruction, edited by C. C. Kao, R. P. Bunge and P. J. Reier. New York: Raven Press, 1983, pp. 163195. 10. Richardson, P. M. and T. Ependal. Nerve growth activities in rat peripheral nerve. Brain Res 246: 57-64, 1982.
WEND’1
II. Richardson, P. M., V. M. K. Issa and A. J. Aguayo. Regeneration of long spinal axons in the rat. J Neuroc,yro/ 13: 16.5-182, 1984. 12. Weiss, P. and A. C. Taylor. Further evidence against neurotropism in nerve regeneration. J Exp Zoo1 95: 233-257. 1944. 13. Wendt, J. S., G. E. Fagg and C. W. Cotman. Regeneration of rat hippocampal timbria fibers after fimbria transection and peripheral nerve or fetal hippocampal implantation. E.rp Naurol 79: 452-461. 1983.