- Email: [email protected]
Chronic regenerative changes in the spinal cord after cord compression injury in rats
Surg Neurol 1987;27:209-19
209
Chronic Regenerative Changes in the Spinal Cord after Cord Compression Injury in Rats M. Christopher Wallace, M.D., Charles H. Tator, M.D., F.R.C.S.(C), and Anthony J. Lewis, M.D., F.R.C.S.(C) Division of Neurosurgery and Playfair Neuroscience Unit, Toronto Western Hospital, and Department of Pathology, Sunnybrook Medical Centre, University of Toronto, Toronto, Ontario, Canada
Wallace MC, Tator CH, Lewis AJ. Chronic regenerative changes in the spinal cord after cord compression injury in rats. Surg Neurol 1987;27:209-19. Long-term regenerative changes and pathological effects after acute compression injury of the spinal cord were studied in rats. Twenty adult female Wistar rats underwent cord injury by the extradural clip compression technique at T6-7. Following injury, extradural electrodes attached to receiver-stimulators that were implanted subcutaneously were placed proximal and distal to the injury site. The animals were maintained in cages with electromagnetic fields created by encircling antennae. The control animals were in a field adjusted to a frequency below the sensitive frequency range of the receiver-stimulators so that they received no spinal cord stimulation. After 15-20 weeks of continuous spinal cord stimulation, histological sections of the cords were assessed and scored blindly for pathological changes including magnitude and extent of cord injury, and cystic cavitation, and for regenerative changes including proliferation of axons, Schwann cells and ependymal cells, and formation of myelin. In all 20 animals, there was a complete absence of normal cord tissue at the injury site, and cystic cavitation was frequently present at the injury site and beyond. Extensive regenerative changes were seen in all animals including regeneration of axons, Schwann cells and ependymal cells, and formation of myelin. Statistical analysis did not show a significant difference between treatment and control groups. KEYWORDS: Spinal cord injury; Pathology; Regeneration
The traditional view that regeneration does not occur in the central nervous system has been contested by many investigators [ 17,20,2 3,32]. Unfortunately, there have been few detailed studies of pathology and regenAddress reprint requests to: C.H. Tator, M.D., Division of Neurosurgery and Playfair Neuroscience Unit, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8.
© 1987 by ElsevierSciencePublishingCo., Inc.
erative capacity in the spinal cord in the chronic phase following experimental spinal cord injury [28,33]. In contrast, there has been extensive documentation of the pathology and regenerative changes in the acute phase after experimental spinal injury [1,3,4,8,12-14,20], including the accurate and still relevant descriptions o f the changes due to spinal cord transection by Ramon and Cajal [20]. The acute onset of hemorrhage followed by edema and central necrosis has been clearly demonstrated by Balentine [4], Ducker et al [8], Kao et al [12-14], and Wagner et al [28-30]. Since Ramon and Cajal's [20] major work on this subject, descriptions of the chronic alterations in morphology and regenerative capacity have been confined largely to postmortem examination in humans [10,11], with the exception o f a few experimental reports, such as the study of Wagner et al [30] in which the pathological progression from hemorrhage to cystic cavitation in the spinal cord was investigated up to 4 months after spinal cord injury induced by the weight-dropping technique. Although compression injury is one of the most c o m m o n types of cord injury in humans, the chronic pathology and regenerative changes following experimental compression injury have not previously been documented. The purpose of the present study was to document and quantify these changes in rats injured by extradural clip compression, and to correlate them with clinical recovery. The animals had undergone a 1 5 - 2 0 week course of extradural electrical stimulation--the techniques and results of which have been described elsewhere [ 3 1 ] - - a n d thus, the study also enabled us to assess the histological effect o f long-term electrical stimulation of the spinal cord.
Materials and Methods Experimental Protocol After undergoing a thoracic laminectomy under pentobarbital anesthesia (40 mg/kg intraperitoneally), 20 adult Wistar rats were subjected to a clip compression 0090-3019/87/$3.50
210
Surg Neurol 1987;27:209-19
Figure 1. Longitudinal section through the central portion of an injury site. A normal appearing root can be seen entrapped underneath an area of meningeal thickening at the top. The injury site does not contain any normal nervous tissue, and cystic cavitation is a predominant feature (6 ~m thick, Luxol fast blue stain, x 32).
injury of 125 gram force for 1 minute to the spinal cord at T6-7. The injury requires a two-level laminectomy and the application o f an aneurysm clip around the cord as previously described [21]. Electrodes were placed extradurally, with the cathode 7 mm proximal and the anode 7 mm distal, to the injury site, and were attached
Figure 2. Low-power view of the section shown in Figure 1. The posttraumatic cavity extends in a cephalad direction within this central coronal section well beyond the confines of the injury site (6/~m, Luxol fast blue. x 12).
Wallace et al
by wires to a receiver-stimulator (Medtronic 3521) implanted subcutaneously. Postoperatively, the animals were nursed on a bed of sawdust, food was provided ad libitum, but water was restricted during the night to prevent bladder distention. Bladders were emptied manually three times daily. The animal cages were made of plastic, and were specially equipped with encircling antennae to generate a pulsed electromagnetic field. The techniques and circuitry used for both treatment and control groups are described elsewhere [31]. Briefly, the treatment animals received extradural, alternating current stimulation in an electromagnetic field of 460 kHz, whereas the control animals received no extradural stimulation, as they were placed in a 400-kHz field, a frequency below the sensitivity of their tuned receiver-stimulators. Clinical assessment of neurological function was per-
Regenerative Changes in the Spinal Cord
formed weekly on the inclined plane. Animals were placed horizontally on an angled board to determine the maximum angle of inclination at which balance can be maintained. This technique has been used previously in this laboratory [22]. Electrophysiological assessment by measurement of the somatosensory evoked potential was performed with the rats under anesthesia on the day of sacrifice [31]. Ten animals (5 control, 5 treated) were killed at 15 weeks, and the remaining 10 animals (5 control, 5 treated) were killed at 20 weeks after the injury.
Histological Assessment Following electrophysiological assessment, animals were killed by an intracardiac injection of sodium pentobarbital. A complete laminectomy from C 1 to L4 was performed, and the entire spinal cord was removed and placed in 10% formalin for 72 hours. After fixation, a 3-cm-long segment o f the cord, with the injury site at its midpoint, was embedded in paraffin. The cord was sectioned longitudinally starting from the posterior surface and progressing in an anterior direction. Three adjacent sections 6 / z m thick were taken every 30/zm, and these were stained with Luxol fast blue (LFB)-hematoxylin and eosin, Masson, and Holmes' stains, respectively. Luxol fast blue was used for myelin staining, Masson for connective tissue, and Holmes' silver method for axons. The three stains allowed differentiation between myelin, connective tissue, and axons, and showed any associations between them. In most animals, three adjacent sections were obtained from each of six posteroanterior levels o f the cord. The 18 slides from each animal were assessed blindly by two individuals, without knowledge o f whether an animal belonged to the treatment or control group. Attention was paid to the severity of the pathological changes at the injury site levels, the persistence of any normal appearing cord tissue, and the amount o f scarring and cavitation, both at the injury site and beyond. Regenerative activity was assessed on the basis of the presence of Schwann cell activity, ependymal cell proliferation, new myelin formation, and the presence o f axons. For the first three features a simple, subjective rating scale was employed: I -- absent to minimal activity of the regenerative feature in question; II = moderate activity; and III = marked activity.
Surg Neurol 1987;27:209-19
Table 1. Cystic Cavitation Extending more than 3 mm from
Injury Site Location from injury site
Group
No. of animals w i t h cavities
Cephalad
Caudad
Significance
Control Treatment
3/10 3/10
3~ 2
Ia 1
NS
q n one animal there was cavitation in both directions from the injury site.
myelination was determined by developing a linear model from functions of the categorical responses, and performing a least-squares analysis ( F U N C A T procedure, SAS, Carey, NC). As described below, the electrodes lost contact with the spinal cord in some animals, and therefore, the analyses were done with and without consideration for this factor. The level for statistical significance was set at p -- 0.05. Results
Clinical Assessment Ten animals were killed at 15 weeks, and 10 at 20 weeks. The results obtained from the inclined plane studies demonstrated no significant difference between treatment and control groups [31].
Pathological and Regenerative Changes General features. Gross examination revealed a pannus of extradural scar tissue up to 1 mm thick overlying the spinal cord at the injury site, which could be separated from the dura mater. There was considerable narrowing of the cord for 5-7 mm centered at the injury site and the dura mater was more opaque in this region. In some cases, areas of cystic cavitation within the spinal cord could be observed. Microscopically, the typical injury site was characterized by the absence of normal neural tissue including neurons, axons, and myelin, and the presence of necrosis and cystic cavities (Figure 1). T h e r e was a total loss of the gray-white interface and none of the normal tracts could be identified. The cavities were mostly located in the central areas of the cord, and occasionally a thin
T a b l e 2.
Schwann Cell Proliferation
Statistical Analyses The inclined plane and histological results were analyzed using the Student's t-test. Correlation between the histological results and the clinical results was assessed by analysis of covariance. Correlation between scores for Schwann cell activity, ependymal proliferation, and new
211
Degree of proliferation ~ Group
Minimal
Moderate
Marked
Significance
Control Treatment
2 2
7 5
1 3
NS
~See Methods section for definition of minimal, moderate, and marked Schwann cell proliferation.
212
Surg Neurol 1987;27:209-19
bridge of tissue coursed through them. Around the cavities, and sometimes within the cavities, there was cellular and myelin debris associated with numerous macrophages. The macrophages were often laden with lipid, and these were observed with LFB stain. N e w blood vessels with accompanying fibroblasts were often seen in the m o r e heavily scarred areas, where staining of collagen by the Masson stain was the greatest. However, it is noteworthy that dense scarring with collagen was rare, and in those cases, it did not involve the entire cross-sectional area of the cord at the injury site and was usually located peripherally. At the cephalad and caudad margins of the injury site, the cord tissue was markedly altered. There were no persisting neurons at the injury site, whereas at the margins, the anterior horn cells were abnormally swollen and pale, with relatively few neurons identified in the dorsal horns. Astrocytic proliferation was noted but no identifiable oligodendroglia could be observed for a considerable distance from the injury site. The axons seen coursing into the injury site were larger than normal, and the myelin surrounding them appeared to be thicker and more heavily stained by LFB than normal myelin in the spinal cord. In general, this altered central myelin had the same caliber and tinctorial properties as that in the adjacent nerve roots. The course of the myelinated and nonmyelinated axons at or near the injury location was random. The thicker and more heavily stained myelin was commonly associated with Schwann cells or ependymal cells, and indeed, there seemed to be more proliferation of myelinated fibers in areas populated by these two cell types. T h e r e were fewer axons and myelin in areas containing blood vessels, fibroblasts, and collagen. The nature and degree of these pathological and regenerative changes were very consistent among the 20 animals, indicating the consistency of the injury. Except for the persistence of a small number of myelinated axons at the peripheral margins of the injury site in a minority of the animals, the histological damage was complete in all 20 animals. N o obvious gross difference in histological pattern could be seen between treatment and control groups.
Cystic cavitation. Cystic cavitation at the site of compression injury was seen to some extent in every animal. The cavities, usually multiple, varied in size and were most often located in the central and dorsal regions of the injury site, occupying up to 9 0 % of the width of the spinal cord. The cavities were surrounded by a "pseudocapsule" of compressed nervous tissue. Although the fluid in these cysts was clear on gross examination at autopsy, subsequent microscopy revealed some cellular debris. For example, it was c o m m o n to find hemosiderin-laden mac-
Wallace et al
rophages circumferentially around the inner margin. In a majority of the animals, the cavities at the injury site were continuous with the central canal, in which case the ependymal cells lined the cavity for only a short distance, beyond which the wall of the cyst had a "pseudocapsule." Several animals had cavitation extending a considerable distance from the area of injury (Figure 2). W h e n these cavities extended m o r e than 3 m m from the injury site, they were arbitrarily designated as instances of posttraumatic syringomyelia. Such cavities were found in 6 out of 20 (30%) animals, and their frequency was not altered by treatment or by time of death (Table 1). Five of the six animals had cavities that extended cephalad and one extended in both directions from the injury site. The largest syrinx extended 6.5 m m from the injury site. (The width of the compression clip was only 1 mm.)
Schwann cellproliferation. Table 2 contains the scoring of Schwann cell proliferation. Schwann cells were found in abundance at or near the injury site at all posteroanterior levels of the spinal cord. They were easily identified as small, eosinophilic cells and were not confined to the dorsal root entry zones. They were most often present as sheets of cells (Figure 3), but occasionally demonstrated a tendency for whorl or knot formation resembling a schwannoma (Figure 4). Both nonmyelinated and myelinated axons were usually seen in association with the Schwann cells, and often their irregular courses brought them near clusters of Schwann cells. The origin of the Schwann cells could not be determined, although they were m o r e abundant at both the dorsal and ventral root entry zones. From these zones, islands of cells could be identified penetrating into the more central areas of the cord. Indeed, there was a surprisingly large amount of Schwann cell proliferation in the most anterior levels of the cord sections. The amount of Schwann cell proliferation had no effect on clinical function as measured by the inclined plane, and there was no statistical difference between experimental groups with respect to the degree of Schwann cell proliferation. In addition, there was no significant correlation between Schwann cell prolifera-
Figure 3 (opposite, top). A sheet of closely packed Schwann cells can be seen arising from the lower left-hand portion of the photograph, and extending into the central area of the figure. Numerous other Schwann cell nests are present elsewhere (6 gm, Luxol fast blue, x 200). Figure 4 (opposite, bottom). A collection of very densely packed Schwann cells resembling a peripheral schwannoma can be seen in the central portion of the figure. A few blood vessels are seen within the whorling pattern of Schwann cells (6 ~*m,Luxol fast blue, × 400).
u
~ rrqlr~ O
214
Surg Neurol 1987;27:209-19
tion and the presence of myelin or other cell types, including ependymal cells.
Myelinated fiber formation.
In many sections, the new myelin tubes in the cord were found to be continuous with myelinated axons from both the dorsal and ventral roots, although the origin of most of the myelinated axons could not be determined. As noted above, the myelin found at or near the injury site resembled myelin found in the roots and were both thicker and more intensely stained with LFB than normal central myelin within the normal cord (Figure 5). The axons within these myelin sheaths were thick in caliber (Figure 6) and their course was random, although they were more extensive peripherally. The degree of myelin formation at the injury site is shown in Table 3. The predominance of both moderate and marked scores is indicative of the exuberant myelination found in both experimental groups, with no significant difference between the groups. Analysis of the 20 animals, regardless of which group they were in, showed no significant correlation between the degree of Schwann cell activity and the presence of myelin sheaths, despite their anatomical association. In addition, the inclined plane performance was not significantly related to the myelin proliferation score. The presence o f myelin was scored separately in the following four segments of the cord with respect to the injury site; anterior, posterior, proximal, and distal. The scores for both treatment and control groups are shown in Table 4. There were no significant differences between groups for any of the four regions with respect to myelin proliferation, although there was a trend towards more moderate to marked myelin proliferation distally than proximally. In addition, there was no correlation between the inclined plane performance and myelin proliferation in any of the four regions.
Ependymal cell proliferation. One of the unexpected features was the marked degree of ependymal cell proliferation. The ependymal proliferation tended to be more pronounced centrally, although most animals showed at least some peripheral extension. In contrast to the normal pattern of ependymal cells lining the central canal, the proliferating ependymal cells formed cords of densely packed cells heaped on one another and tended to project towards the area of injury (Figure 7). In some instances, the cords of ependymal cells appeared to form a rosette of four to five cells, but the rosettes did not contain a distinct lumen. They were often seen beside bundles of myelinated axons. Ependymal cells and Schwann cells generally occupied the central and peripheral portions of the cord, respectively. When found in the same area, it was often difficult to distinguish
Wallace et al
where the two cell types (Figure 8) merged with one another. The ratings for ependymal cell proliferation are shown in Table 5 for control and treatment groups. Although there was a trend towards more marked proliferation in the treatment group, this was not statistically significant. In addition, there was no significant correlation between ependymal and Schwann cell proliferation and inclined plane performance.
Discussion The historical view that there is no regenerative activity in the spinal cord is easily refuted by the exuberant regenerative activity seen in all 20 experimental animals. Despite the virtually complete loss of normal architecture due to the severity of the injury, the regenerative capacity of a variety of cell types was demonstrated. The consistency of the injury was evident from the uniformly severe pathological changes. The studies performed on the inclined plane were consistent with severe injury of the spinal cord [21,22]. The theory that a connective tissue scar impedes the growth of axons has been postulated previously as an explanation for the failure of regeneration in the cord [10,23,33]. In the present study, heavy connective tissue scarring with collagen was not a prominent feature in most animals. Only two animals showed a dense scar composed of abundant fibroblast proliferation, collagen, and regenerating capillaries. Thus, the present study does not support the connective tissue barrier hypothesis for the lack of regeneration following injury. The paucity of persisting neurons or glial tissue at the injury site, with the exception ofastrocytes, has been documented by other investigators [4,11]. The most likely explanation is the greater sensitivity of neurons and oligodendroglia to the ischemia accompanying trauma. In some studies, neurons have been found to survive low forces of injury and appear normal with respect to shape and Nissl substance [20,28]. The compression injury produced by the 125-gram clip for 1 minute was severe enough to prevent neuronal survival at the injury site. The frequency and rapidity of onset of posttraumatic
Figure 5 (opposite, top). An area adjacent to the injury site demonstrating numerous newly myelinated fibers. The myelin tubes are much thicker and more deeply stained than central myelin. Note the random direction of the myelin tubes (6/xm, Luxol fast blue, x 400). Figure 6 (opposite, bottom). Holmes' silver stain was used to identify axons, Numerous axons can be seen coursing across the top portion of the figure, in association with numerous Schwann cells (6 txm, Holmes' stain, x 1250).
b
'1
t
216
Surg Neurol 1987;27:209-19
Wallace et al
Table 3. New Myeliu Formation Degree of formation~ Group
Minimal
Moderate
Marked
Significance
Control Treatment
0 1
3 4
7 5
NS
~See Methods section for definition of minimal, moderate, and marked myelin formation.
syringomyelia was striking. Its occurrence within 15-20 weeks in 30% of the animals is higher than estimates from human autopsy series [5,10,11,16,19]. The mechanisms underlying the formation of posttraumatic syringomyelia are unknown. It has been postulated that the cavities may develop from the hemorrhagic areas that occur in the acute phase of spinal cord injury [4,8,29]. The predominantly central location of the cavities in the present study parallels the distribution of hemorrhages in the acute phase at the injury site, and the hemosiderin-laden macrophages along the periphery would also be consistent with the theory that at least the cavities at the injury site were once hemorrhages. The extensive penetration of newly myelinated axons from proximal and distal segments towards the injury sites and along the sides of the cystic cavities was a prominent feature of the regenerative activity. Although most authors have ascribed the origin of these fibers to the dorsal roots [23,33], it is noteworthy that Ramon and Cajal [20] and Wohlfart [32] also implicated the ventral roots. Our studies showed that there was a similar degree of regeneration of myelinated axons in both the anterior and posterior portions of the cord. This would suggest origination from both the dorsal and ventral roots, although proof of an anatomical connection would require axonal tracing techniques such as horseradish peroxidase [7] or radioisotopically labeled amino acids [7]. Similarly, axons identified coursing through the injury site may have been regenerated central axons, but without a tracer technique this cannot be proven.
The presence of newly myelinated fibers adjacent to the ventral roots could be explained by a number of mechanisms including remyelination of axons not destroyed by the injury, collateral sprouting of efferent fibers from adjacent levels, or growth from interneurons or anterior horn cells in the cord adjacent to the injury site. The direction of growth could not be ascertained in this study. Even though there were numerous regenerated myelinated axons within or near the injury site, no correlation was found between the scores for myelin proliferation and the animals' clinical performance on the inclined plane. A likely explanation for this lack of correlation is that the regenerating fibers were not from the motor system. Other possible explanations include a clinical scale not sufficiently sensitive to detect small increments in performance, an insufficient posttraumatic assessment time, failure of the regenerating fibers to make functional connections with appropriate distal motor neurons, or a histological scoring scale that lacks precision. The inclined plane assessment technique has been shown to accurately reflect the degree of injury in a clear dose-response relationship [21]. The time required for regeneration to produce functional improvement on the basis of newly established connections is not known. The period of 15-20 weeks chosen in this study was more than adequate for the inclined plane technique to detect maximum functional recovery due to nonregenerative processes, but may not have been F i g u r e 7 (opposite, top). Ependymal proliferation is marked, extending from an area of the central canal seen at the left towards the margin of the injury site on the right. Note the adhesive, cordlike structure of the proliferating cells, and the more exuberant proliferation as they get closer to the injury site (6 ~m, Luxol fast blue, x200).
F i g u r e 8 (opposite, bottom). A t the margin of the injury site, proliferative activity of Schwann cells (lower left) and ependymal cells (right and central) can be seen. The ependymal cells have more intensely stained nuclei, and are more cordlike in their arrangement compared to the Schwann cells. In the center of the figure, differentiation of the two cell types becomesdifficult as they appear to merge (6 ~m, Luxol fast blue, x200).
Table 4. New Myelin Formation According to Location in the Cord Degree of formation
Site
Group
Minimal
Moderate
Marked
Significance
Proximal anterior
Control Treatment
4 4
4 3
2 3
NS
Proximal posterior
Control Treatment
7 7
1 3
2 0
NS
Distal anterior
Control Treatment
1 2
6 6
3 2
NS
Distal posterior
Control Treatment
1 4
4 3
5 3
NS
218
Surg Neurol 1987;27:209-19
Wallace et al
Table 5. Ependymal Cell Proliferation ~ Degree of proliferation Group
Minimal
Moderate
Marked
Significance
Control Treatment
7 3
2 5
1 2
NS
~See Methods section for definition of minimal, moderate, and marked ependymal cell proliferation.
sufficiently long to detect functional recovery on the basis of regenerative processes. The close spatial relationship between Schwann cells and myelinated axons was evident in most sections, but there was no statistical correlation between the degrees of regenerative activity of Schwann cells, axons, and new myelin tubes. Whether or not the axons preceded the Schwann cells or vice versa could not be determined. Axons, both myelinated and nonmyelinated, were more numerous in association with sheets of Schwann cells than with knots or clumps of these supportive cells. Although the presence of proliferating Schwann cells is probably a favorable sign of regeneration, the frequent clumping and whorling of the cells as seen in a schwannoma may have been detrimental to the fate of a growing axon in terms of establishing a functional connection. Thus, if indeed the Schwann cell tube provides direction for the growing axon, as in regeneration in the peripheral nervous system, and its function is interrupted by Schwann cell clumping, then the cells' subsequent function of myelination would be completed upon misdirected axons. The cell that demonstrated a surprising degree of proliferation was the ependymal cell. In the rat, the central canal is normally patent and lined by simple, cuboidal, ciliated ependymal cells. There was no difficulty in recognizing proliferative activity of the cells in the central sections of the cord. As noted above, the cords of proliferating ependymal cells extended toward the injury site from both the proximal and distal segments of the cord and were often associated with axons. It is interesting to speculate that these cells may have some neurotrophic function. Ramon and Cajal [20] discussed the importance of a "mesodermal or ectodermal influence" for the regenerative attempts of central axons, but they postulated a role for the Schwann cells in this regard. The possible role of ependymal cells in regeneration in the mammalian central nervous system has not been addressed, although in lower animals capable of spinal cord regeneration, ependymal cells play a major role [9,18]. For example, after amputating the tail of the newt, ependymal cells were found to proliferate into the regenerating stump before the growing axons, and the clefts between these proliferating ependymal cells afford a directional scaffolding for the regenerating
axons [9,18]. In higher vertebrates, however, evidence for ependymal cells having a role in regeneration is minimal. Sugar and Gerrard [23] noted "rosette-forming cells" associated with regenerating fibers in the transected rat spinal cord, but they did not refer to them as ependymal cells. Recently, Vaguero et al [27] documented significant ependymal cell proliferation following spinal cord transection in the rat, but they did not comment on any association with regenerative activity of axons. To date, only Matthews et al [15] have demonstrated a relationship between the regenerating axons in the transected rat spinal cord and the proliferating sheets of ependymal cells. With electron microscopy they found an invagination of the ependymal cells by growing axons, similar to that seen in the newt [9,19]. Our results did not show such an intimate anatomical relationship between axons and regenerating ependyma, but the pattern and extent of the proliferating sheets of ependymal cells is consistent with that found by other investigators [ 15,27]. It remains an attractive hypothesis that because of their neuroblast origin and ability to proliferate, the regenerating ependymal cells are capable of exerting a growth stimulating influence on damaged axons in the central nervous system. Despite the observed ependymal cell proliferation, we found no significant association between clinical performance and other regenerative activity. It is possible that increased proliferation or improved migration of the ependymal cells to the axons located more peripherally within the cord might result in improved function. Unfortunately, methods of improving ependymal cell proliferation or migration are completely unknown. In conclusion, this study has shown exuberant regenerative activity of Schwann cells, ependymal cells, and myelinated fibers in the spinal cord following experimental spinal cord injury. Of particular interest is the proliferation of ependymal cells and their migration from the central canal.
The technical assistance of Mrs. L. Marmash, Mrs. B. Young, Mrs. E. Forsythe, and Miss M. Vespa is gratefully acknowledged. Dr. M.C. Wallace was a Fellow of the Medical Research Council of Canada. This research was supported by the Medical Research Council of Canada (grant MT 4046).
References 1. Allen AR. Remarks on the histological changes in the spinal cord due to impact: an experimental study. J Nerv Ment Dis 1914;41:141-7. 2. Allen AR. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column: preliminary report. JAMA 1911;57:878-80. 3. Assenmacher DR, Ducker TB. Experimental traumatic paraple-
R e g e n e r a t i v e C h a n g e s in the Spinal C o r d
gia: the vascular and pathological changes seen in reversible and irreversible spinal cord lesions. J Bone Joint Surg 1971;53A:671-80. 4. Balentine JD. Pathology of experimental spinal cord trauma. I. The necrotic lesion as a function of vascular injury. Lab Invest 1978;39;236-53. 5. Barnett HJM, Botterell EH, Jousse AT, Wynn-Jones M. Progressive myelopathy as a sequel to traumatic paraplegia. Brain 1966;89:159-73. 6. Bernstein JJ, Wells MR, Bernstein ME. The effect of puromycin treatment on the regeneration of hemisected and transected rat spinal cord. J Neurocytol 1978;7:215-28. 7. Cowan WM, Cuenod M. The use of axonal transport for studies of neuronal connectivity. Amsterdam, Oxford, New York: Elsevier Science Publishing, 1975. 8. Ducker TB, Kindt GW, Kempe LG. Pathological finding in acute experimental spinal cord trauma. J Neurosurg 1971;35:700-8. 9. Egar M, Singer M. The role of ependyma in spinal cord regeneration in the urodele. Triturus. Exp Neurol 1972;37:422-30. I0. Holmes G. Spinal injuries of warfare. Br Med J 1915;2:769-74. 11. Hughes JT. Pathology of the spinal cord. 2nd ed. Philadelphia, London, Toronto: WB Saunders, 1978. 12. Kao CC, Chang LW. The mechanism of spinal cord cavitation following spinal cord transection. Part I: a correlated histochemical study. J Neurosurg 1977;46:197-209. 13. Kao CC, Chang LW, Bloodworth JMB Jr. The mechanism of spinal cord cavitation following spinal cord transection. Part 2: electron microscopic observations. J Neurosurg 1977;46:745-56. 14. Kao CC, Chang LW, Bloodworth JMB Jr. The mechanism of spinal cavitation following spinal cord transection. Part 3: delayed grafting with and without spinal cord retransection. J Neurosurg 1977;46:757-66. 15. Matthews MA, St. Onge MF, Faciane CL. An electron microscopic analysis of abnormal ependymal cell proliferation and envelopment of sprouting axons following spinal cord transection in the rat. Acta Neuropathol 1979,45:27-36. 16. McLean DR, Miller JDR, Allen PBR, Ezzeddin SA. Posttraumatic syringomyelia. J Neurosurg 1973;39:485-92. 17. Moore RY. Regeneration in the mammalian nervous system. Ann NY Acad Sci 1980;339:102-14. 18. Norlander RH, Singer M. The role of ependyma in regeneration
Surg N e u r o l 1987;27:209-19
219
of the spinal cord in the urodele amphibian tail. J Comp Neurol 1978;180:349-74. 19. Nurick S, Russell JA, Deck MDF. Cystic degeneration of the spinal cord following spinal cord injury. Brain 1970;93:211-22. 20. Ramon Y, Cajal S. Degeneration and regeneration of the nervous system. Volumes 1 and 2. RM May translator. London: Oxford University Press, 1928. 21. Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978;9:39-43. 22. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg 1977;47:577-81. 23. Sugar O, Gerrard RW. Spinal cord regeneration in the rat. J Neurophysiol 1940;3:1-19. 24. Tarlov IM, Klinger I. Spinal cord compression studies. II. Time limits for recovery after acute compression in dogs. Arch Neurol 1954;71:271-90. 25. Tarlov M. Spinal cord compression studies. III. Time limits for recovery after gradual compression in dogs. Arch Neurol 1954;71:588-97. 26. Tator CH, Rivlin AS, Lewis AJ, Schmoll B. Effect of acute spinal cord injury on axonal counts in the pyramidal tract of rats. J Neurosurg 1984;61 : 118-23. 27. Vaguero J, Ramiro MJ, Oya S, CatezudoJM. Ependymal reaction after experimental spinal cord injury. Acta Neurochir (Wien) 1981;55:295-302. 28. Wagner FCJr, Dohrmann GJ. Alterations in nerve cells and myelinated fibres in spinal cord injury. Surg Neurol 1975;3:125-31. 29. Wagner FC Jr, Dohrmann GJ, Bucy PC. Histopathology of transitory traumatic paraplegia in the monkey. J Neurosurg 1971;35:272-6. 30. Wagner FC Jr, Van Gilder JC, Dohrmann GJ. Pathological changes from acute to chronic in experimental spinal cord trauma. J Neurosurg 1978;48:92-8. 31. Wallace MC, Tator CH, Gentles W. Effect of electrical stimulation of the spinal cord on recovery from acute spinal cord injury in rats. (submitted for publication). 32. Wohlfart G. Degeneration and regeneration in the nervous system. World Neurol 1961 ;2:187-98. 33. Wolman L. The neuropathology of traumatic paraplegia. A critical historical review. Paraplegia 1964;1:233-51.
209
Chronic Regenerative Changes in the Spinal Cord after Cord Compression Injury in Rats M. Christopher Wallace, M.D., Charles H. Tator, M.D., F.R.C.S.(C), and Anthony J. Lewis, M.D., F.R.C.S.(C) Division of Neurosurgery and Playfair Neuroscience Unit, Toronto Western Hospital, and Department of Pathology, Sunnybrook Medical Centre, University of Toronto, Toronto, Ontario, Canada
Wallace MC, Tator CH, Lewis AJ. Chronic regenerative changes in the spinal cord after cord compression injury in rats. Surg Neurol 1987;27:209-19. Long-term regenerative changes and pathological effects after acute compression injury of the spinal cord were studied in rats. Twenty adult female Wistar rats underwent cord injury by the extradural clip compression technique at T6-7. Following injury, extradural electrodes attached to receiver-stimulators that were implanted subcutaneously were placed proximal and distal to the injury site. The animals were maintained in cages with electromagnetic fields created by encircling antennae. The control animals were in a field adjusted to a frequency below the sensitive frequency range of the receiver-stimulators so that they received no spinal cord stimulation. After 15-20 weeks of continuous spinal cord stimulation, histological sections of the cords were assessed and scored blindly for pathological changes including magnitude and extent of cord injury, and cystic cavitation, and for regenerative changes including proliferation of axons, Schwann cells and ependymal cells, and formation of myelin. In all 20 animals, there was a complete absence of normal cord tissue at the injury site, and cystic cavitation was frequently present at the injury site and beyond. Extensive regenerative changes were seen in all animals including regeneration of axons, Schwann cells and ependymal cells, and formation of myelin. Statistical analysis did not show a significant difference between treatment and control groups. KEYWORDS: Spinal cord injury; Pathology; Regeneration
The traditional view that regeneration does not occur in the central nervous system has been contested by many investigators [ 17,20,2 3,32]. Unfortunately, there have been few detailed studies of pathology and regenAddress reprint requests to: C.H. Tator, M.D., Division of Neurosurgery and Playfair Neuroscience Unit, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8.
© 1987 by ElsevierSciencePublishingCo., Inc.
erative capacity in the spinal cord in the chronic phase following experimental spinal cord injury [28,33]. In contrast, there has been extensive documentation of the pathology and regenerative changes in the acute phase after experimental spinal injury [1,3,4,8,12-14,20], including the accurate and still relevant descriptions o f the changes due to spinal cord transection by Ramon and Cajal [20]. The acute onset of hemorrhage followed by edema and central necrosis has been clearly demonstrated by Balentine [4], Ducker et al [8], Kao et al [12-14], and Wagner et al [28-30]. Since Ramon and Cajal's [20] major work on this subject, descriptions of the chronic alterations in morphology and regenerative capacity have been confined largely to postmortem examination in humans [10,11], with the exception o f a few experimental reports, such as the study of Wagner et al [30] in which the pathological progression from hemorrhage to cystic cavitation in the spinal cord was investigated up to 4 months after spinal cord injury induced by the weight-dropping technique. Although compression injury is one of the most c o m m o n types of cord injury in humans, the chronic pathology and regenerative changes following experimental compression injury have not previously been documented. The purpose of the present study was to document and quantify these changes in rats injured by extradural clip compression, and to correlate them with clinical recovery. The animals had undergone a 1 5 - 2 0 week course of extradural electrical stimulation--the techniques and results of which have been described elsewhere [ 3 1 ] - - a n d thus, the study also enabled us to assess the histological effect o f long-term electrical stimulation of the spinal cord.
Materials and Methods Experimental Protocol After undergoing a thoracic laminectomy under pentobarbital anesthesia (40 mg/kg intraperitoneally), 20 adult Wistar rats were subjected to a clip compression 0090-3019/87/$3.50
210
Surg Neurol 1987;27:209-19
Figure 1. Longitudinal section through the central portion of an injury site. A normal appearing root can be seen entrapped underneath an area of meningeal thickening at the top. The injury site does not contain any normal nervous tissue, and cystic cavitation is a predominant feature (6 ~m thick, Luxol fast blue stain, x 32).
injury of 125 gram force for 1 minute to the spinal cord at T6-7. The injury requires a two-level laminectomy and the application o f an aneurysm clip around the cord as previously described [21]. Electrodes were placed extradurally, with the cathode 7 mm proximal and the anode 7 mm distal, to the injury site, and were attached
Figure 2. Low-power view of the section shown in Figure 1. The posttraumatic cavity extends in a cephalad direction within this central coronal section well beyond the confines of the injury site (6/~m, Luxol fast blue. x 12).
Wallace et al
by wires to a receiver-stimulator (Medtronic 3521) implanted subcutaneously. Postoperatively, the animals were nursed on a bed of sawdust, food was provided ad libitum, but water was restricted during the night to prevent bladder distention. Bladders were emptied manually three times daily. The animal cages were made of plastic, and were specially equipped with encircling antennae to generate a pulsed electromagnetic field. The techniques and circuitry used for both treatment and control groups are described elsewhere [31]. Briefly, the treatment animals received extradural, alternating current stimulation in an electromagnetic field of 460 kHz, whereas the control animals received no extradural stimulation, as they were placed in a 400-kHz field, a frequency below the sensitivity of their tuned receiver-stimulators. Clinical assessment of neurological function was per-
Regenerative Changes in the Spinal Cord
formed weekly on the inclined plane. Animals were placed horizontally on an angled board to determine the maximum angle of inclination at which balance can be maintained. This technique has been used previously in this laboratory [22]. Electrophysiological assessment by measurement of the somatosensory evoked potential was performed with the rats under anesthesia on the day of sacrifice [31]. Ten animals (5 control, 5 treated) were killed at 15 weeks, and the remaining 10 animals (5 control, 5 treated) were killed at 20 weeks after the injury.
Histological Assessment Following electrophysiological assessment, animals were killed by an intracardiac injection of sodium pentobarbital. A complete laminectomy from C 1 to L4 was performed, and the entire spinal cord was removed and placed in 10% formalin for 72 hours. After fixation, a 3-cm-long segment o f the cord, with the injury site at its midpoint, was embedded in paraffin. The cord was sectioned longitudinally starting from the posterior surface and progressing in an anterior direction. Three adjacent sections 6 / z m thick were taken every 30/zm, and these were stained with Luxol fast blue (LFB)-hematoxylin and eosin, Masson, and Holmes' stains, respectively. Luxol fast blue was used for myelin staining, Masson for connective tissue, and Holmes' silver method for axons. The three stains allowed differentiation between myelin, connective tissue, and axons, and showed any associations between them. In most animals, three adjacent sections were obtained from each of six posteroanterior levels o f the cord. The 18 slides from each animal were assessed blindly by two individuals, without knowledge o f whether an animal belonged to the treatment or control group. Attention was paid to the severity of the pathological changes at the injury site levels, the persistence of any normal appearing cord tissue, and the amount o f scarring and cavitation, both at the injury site and beyond. Regenerative activity was assessed on the basis of the presence of Schwann cell activity, ependymal cell proliferation, new myelin formation, and the presence o f axons. For the first three features a simple, subjective rating scale was employed: I -- absent to minimal activity of the regenerative feature in question; II = moderate activity; and III = marked activity.
Surg Neurol 1987;27:209-19
Table 1. Cystic Cavitation Extending more than 3 mm from
Injury Site Location from injury site
Group
No. of animals w i t h cavities
Cephalad
Caudad
Significance
Control Treatment
3/10 3/10
3~ 2
Ia 1
NS
q n one animal there was cavitation in both directions from the injury site.
myelination was determined by developing a linear model from functions of the categorical responses, and performing a least-squares analysis ( F U N C A T procedure, SAS, Carey, NC). As described below, the electrodes lost contact with the spinal cord in some animals, and therefore, the analyses were done with and without consideration for this factor. The level for statistical significance was set at p -- 0.05. Results
Clinical Assessment Ten animals were killed at 15 weeks, and 10 at 20 weeks. The results obtained from the inclined plane studies demonstrated no significant difference between treatment and control groups [31].
Pathological and Regenerative Changes General features. Gross examination revealed a pannus of extradural scar tissue up to 1 mm thick overlying the spinal cord at the injury site, which could be separated from the dura mater. There was considerable narrowing of the cord for 5-7 mm centered at the injury site and the dura mater was more opaque in this region. In some cases, areas of cystic cavitation within the spinal cord could be observed. Microscopically, the typical injury site was characterized by the absence of normal neural tissue including neurons, axons, and myelin, and the presence of necrosis and cystic cavities (Figure 1). T h e r e was a total loss of the gray-white interface and none of the normal tracts could be identified. The cavities were mostly located in the central areas of the cord, and occasionally a thin
T a b l e 2.
Schwann Cell Proliferation
Statistical Analyses The inclined plane and histological results were analyzed using the Student's t-test. Correlation between the histological results and the clinical results was assessed by analysis of covariance. Correlation between scores for Schwann cell activity, ependymal proliferation, and new
211
Degree of proliferation ~ Group
Minimal
Moderate
Marked
Significance
Control Treatment
2 2
7 5
1 3
NS
~See Methods section for definition of minimal, moderate, and marked Schwann cell proliferation.
212
Surg Neurol 1987;27:209-19
bridge of tissue coursed through them. Around the cavities, and sometimes within the cavities, there was cellular and myelin debris associated with numerous macrophages. The macrophages were often laden with lipid, and these were observed with LFB stain. N e w blood vessels with accompanying fibroblasts were often seen in the m o r e heavily scarred areas, where staining of collagen by the Masson stain was the greatest. However, it is noteworthy that dense scarring with collagen was rare, and in those cases, it did not involve the entire cross-sectional area of the cord at the injury site and was usually located peripherally. At the cephalad and caudad margins of the injury site, the cord tissue was markedly altered. There were no persisting neurons at the injury site, whereas at the margins, the anterior horn cells were abnormally swollen and pale, with relatively few neurons identified in the dorsal horns. Astrocytic proliferation was noted but no identifiable oligodendroglia could be observed for a considerable distance from the injury site. The axons seen coursing into the injury site were larger than normal, and the myelin surrounding them appeared to be thicker and more heavily stained by LFB than normal myelin in the spinal cord. In general, this altered central myelin had the same caliber and tinctorial properties as that in the adjacent nerve roots. The course of the myelinated and nonmyelinated axons at or near the injury location was random. The thicker and more heavily stained myelin was commonly associated with Schwann cells or ependymal cells, and indeed, there seemed to be more proliferation of myelinated fibers in areas populated by these two cell types. T h e r e were fewer axons and myelin in areas containing blood vessels, fibroblasts, and collagen. The nature and degree of these pathological and regenerative changes were very consistent among the 20 animals, indicating the consistency of the injury. Except for the persistence of a small number of myelinated axons at the peripheral margins of the injury site in a minority of the animals, the histological damage was complete in all 20 animals. N o obvious gross difference in histological pattern could be seen between treatment and control groups.
Cystic cavitation. Cystic cavitation at the site of compression injury was seen to some extent in every animal. The cavities, usually multiple, varied in size and were most often located in the central and dorsal regions of the injury site, occupying up to 9 0 % of the width of the spinal cord. The cavities were surrounded by a "pseudocapsule" of compressed nervous tissue. Although the fluid in these cysts was clear on gross examination at autopsy, subsequent microscopy revealed some cellular debris. For example, it was c o m m o n to find hemosiderin-laden mac-
Wallace et al
rophages circumferentially around the inner margin. In a majority of the animals, the cavities at the injury site were continuous with the central canal, in which case the ependymal cells lined the cavity for only a short distance, beyond which the wall of the cyst had a "pseudocapsule." Several animals had cavitation extending a considerable distance from the area of injury (Figure 2). W h e n these cavities extended m o r e than 3 m m from the injury site, they were arbitrarily designated as instances of posttraumatic syringomyelia. Such cavities were found in 6 out of 20 (30%) animals, and their frequency was not altered by treatment or by time of death (Table 1). Five of the six animals had cavities that extended cephalad and one extended in both directions from the injury site. The largest syrinx extended 6.5 m m from the injury site. (The width of the compression clip was only 1 mm.)
Schwann cellproliferation. Table 2 contains the scoring of Schwann cell proliferation. Schwann cells were found in abundance at or near the injury site at all posteroanterior levels of the spinal cord. They were easily identified as small, eosinophilic cells and were not confined to the dorsal root entry zones. They were most often present as sheets of cells (Figure 3), but occasionally demonstrated a tendency for whorl or knot formation resembling a schwannoma (Figure 4). Both nonmyelinated and myelinated axons were usually seen in association with the Schwann cells, and often their irregular courses brought them near clusters of Schwann cells. The origin of the Schwann cells could not be determined, although they were m o r e abundant at both the dorsal and ventral root entry zones. From these zones, islands of cells could be identified penetrating into the more central areas of the cord. Indeed, there was a surprisingly large amount of Schwann cell proliferation in the most anterior levels of the cord sections. The amount of Schwann cell proliferation had no effect on clinical function as measured by the inclined plane, and there was no statistical difference between experimental groups with respect to the degree of Schwann cell proliferation. In addition, there was no significant correlation between Schwann cell prolifera-
Figure 3 (opposite, top). A sheet of closely packed Schwann cells can be seen arising from the lower left-hand portion of the photograph, and extending into the central area of the figure. Numerous other Schwann cell nests are present elsewhere (6 gm, Luxol fast blue, x 200). Figure 4 (opposite, bottom). A collection of very densely packed Schwann cells resembling a peripheral schwannoma can be seen in the central portion of the figure. A few blood vessels are seen within the whorling pattern of Schwann cells (6 ~*m,Luxol fast blue, × 400).
u
~ rrqlr~ O
214
Surg Neurol 1987;27:209-19
tion and the presence of myelin or other cell types, including ependymal cells.
Myelinated fiber formation.
In many sections, the new myelin tubes in the cord were found to be continuous with myelinated axons from both the dorsal and ventral roots, although the origin of most of the myelinated axons could not be determined. As noted above, the myelin found at or near the injury site resembled myelin found in the roots and were both thicker and more intensely stained with LFB than normal central myelin within the normal cord (Figure 5). The axons within these myelin sheaths were thick in caliber (Figure 6) and their course was random, although they were more extensive peripherally. The degree of myelin formation at the injury site is shown in Table 3. The predominance of both moderate and marked scores is indicative of the exuberant myelination found in both experimental groups, with no significant difference between the groups. Analysis of the 20 animals, regardless of which group they were in, showed no significant correlation between the degree of Schwann cell activity and the presence of myelin sheaths, despite their anatomical association. In addition, the inclined plane performance was not significantly related to the myelin proliferation score. The presence o f myelin was scored separately in the following four segments of the cord with respect to the injury site; anterior, posterior, proximal, and distal. The scores for both treatment and control groups are shown in Table 4. There were no significant differences between groups for any of the four regions with respect to myelin proliferation, although there was a trend towards more moderate to marked myelin proliferation distally than proximally. In addition, there was no correlation between the inclined plane performance and myelin proliferation in any of the four regions.
Ependymal cell proliferation. One of the unexpected features was the marked degree of ependymal cell proliferation. The ependymal proliferation tended to be more pronounced centrally, although most animals showed at least some peripheral extension. In contrast to the normal pattern of ependymal cells lining the central canal, the proliferating ependymal cells formed cords of densely packed cells heaped on one another and tended to project towards the area of injury (Figure 7). In some instances, the cords of ependymal cells appeared to form a rosette of four to five cells, but the rosettes did not contain a distinct lumen. They were often seen beside bundles of myelinated axons. Ependymal cells and Schwann cells generally occupied the central and peripheral portions of the cord, respectively. When found in the same area, it was often difficult to distinguish
Wallace et al
where the two cell types (Figure 8) merged with one another. The ratings for ependymal cell proliferation are shown in Table 5 for control and treatment groups. Although there was a trend towards more marked proliferation in the treatment group, this was not statistically significant. In addition, there was no significant correlation between ependymal and Schwann cell proliferation and inclined plane performance.
Discussion The historical view that there is no regenerative activity in the spinal cord is easily refuted by the exuberant regenerative activity seen in all 20 experimental animals. Despite the virtually complete loss of normal architecture due to the severity of the injury, the regenerative capacity of a variety of cell types was demonstrated. The consistency of the injury was evident from the uniformly severe pathological changes. The studies performed on the inclined plane were consistent with severe injury of the spinal cord [21,22]. The theory that a connective tissue scar impedes the growth of axons has been postulated previously as an explanation for the failure of regeneration in the cord [10,23,33]. In the present study, heavy connective tissue scarring with collagen was not a prominent feature in most animals. Only two animals showed a dense scar composed of abundant fibroblast proliferation, collagen, and regenerating capillaries. Thus, the present study does not support the connective tissue barrier hypothesis for the lack of regeneration following injury. The paucity of persisting neurons or glial tissue at the injury site, with the exception ofastrocytes, has been documented by other investigators [4,11]. The most likely explanation is the greater sensitivity of neurons and oligodendroglia to the ischemia accompanying trauma. In some studies, neurons have been found to survive low forces of injury and appear normal with respect to shape and Nissl substance [20,28]. The compression injury produced by the 125-gram clip for 1 minute was severe enough to prevent neuronal survival at the injury site. The frequency and rapidity of onset of posttraumatic
Figure 5 (opposite, top). An area adjacent to the injury site demonstrating numerous newly myelinated fibers. The myelin tubes are much thicker and more deeply stained than central myelin. Note the random direction of the myelin tubes (6/xm, Luxol fast blue, x 400). Figure 6 (opposite, bottom). Holmes' silver stain was used to identify axons, Numerous axons can be seen coursing across the top portion of the figure, in association with numerous Schwann cells (6 txm, Holmes' stain, x 1250).
b
'1
t
216
Surg Neurol 1987;27:209-19
Wallace et al
Table 3. New Myeliu Formation Degree of formation~ Group
Minimal
Moderate
Marked
Significance
Control Treatment
0 1
3 4
7 5
NS
~See Methods section for definition of minimal, moderate, and marked myelin formation.
syringomyelia was striking. Its occurrence within 15-20 weeks in 30% of the animals is higher than estimates from human autopsy series [5,10,11,16,19]. The mechanisms underlying the formation of posttraumatic syringomyelia are unknown. It has been postulated that the cavities may develop from the hemorrhagic areas that occur in the acute phase of spinal cord injury [4,8,29]. The predominantly central location of the cavities in the present study parallels the distribution of hemorrhages in the acute phase at the injury site, and the hemosiderin-laden macrophages along the periphery would also be consistent with the theory that at least the cavities at the injury site were once hemorrhages. The extensive penetration of newly myelinated axons from proximal and distal segments towards the injury sites and along the sides of the cystic cavities was a prominent feature of the regenerative activity. Although most authors have ascribed the origin of these fibers to the dorsal roots [23,33], it is noteworthy that Ramon and Cajal [20] and Wohlfart [32] also implicated the ventral roots. Our studies showed that there was a similar degree of regeneration of myelinated axons in both the anterior and posterior portions of the cord. This would suggest origination from both the dorsal and ventral roots, although proof of an anatomical connection would require axonal tracing techniques such as horseradish peroxidase [7] or radioisotopically labeled amino acids [7]. Similarly, axons identified coursing through the injury site may have been regenerated central axons, but without a tracer technique this cannot be proven.
The presence of newly myelinated fibers adjacent to the ventral roots could be explained by a number of mechanisms including remyelination of axons not destroyed by the injury, collateral sprouting of efferent fibers from adjacent levels, or growth from interneurons or anterior horn cells in the cord adjacent to the injury site. The direction of growth could not be ascertained in this study. Even though there were numerous regenerated myelinated axons within or near the injury site, no correlation was found between the scores for myelin proliferation and the animals' clinical performance on the inclined plane. A likely explanation for this lack of correlation is that the regenerating fibers were not from the motor system. Other possible explanations include a clinical scale not sufficiently sensitive to detect small increments in performance, an insufficient posttraumatic assessment time, failure of the regenerating fibers to make functional connections with appropriate distal motor neurons, or a histological scoring scale that lacks precision. The inclined plane assessment technique has been shown to accurately reflect the degree of injury in a clear dose-response relationship [21]. The time required for regeneration to produce functional improvement on the basis of newly established connections is not known. The period of 15-20 weeks chosen in this study was more than adequate for the inclined plane technique to detect maximum functional recovery due to nonregenerative processes, but may not have been F i g u r e 7 (opposite, top). Ependymal proliferation is marked, extending from an area of the central canal seen at the left towards the margin of the injury site on the right. Note the adhesive, cordlike structure of the proliferating cells, and the more exuberant proliferation as they get closer to the injury site (6 ~m, Luxol fast blue, x200).
F i g u r e 8 (opposite, bottom). A t the margin of the injury site, proliferative activity of Schwann cells (lower left) and ependymal cells (right and central) can be seen. The ependymal cells have more intensely stained nuclei, and are more cordlike in their arrangement compared to the Schwann cells. In the center of the figure, differentiation of the two cell types becomesdifficult as they appear to merge (6 ~m, Luxol fast blue, x200).
Table 4. New Myelin Formation According to Location in the Cord Degree of formation
Site
Group
Minimal
Moderate
Marked
Significance
Proximal anterior
Control Treatment
4 4
4 3
2 3
NS
Proximal posterior
Control Treatment
7 7
1 3
2 0
NS
Distal anterior
Control Treatment
1 2
6 6
3 2
NS
Distal posterior
Control Treatment
1 4
4 3
5 3
NS
218
Surg Neurol 1987;27:209-19
Wallace et al
Table 5. Ependymal Cell Proliferation ~ Degree of proliferation Group
Minimal
Moderate
Marked
Significance
Control Treatment
7 3
2 5
1 2
NS
~See Methods section for definition of minimal, moderate, and marked ependymal cell proliferation.
sufficiently long to detect functional recovery on the basis of regenerative processes. The close spatial relationship between Schwann cells and myelinated axons was evident in most sections, but there was no statistical correlation between the degrees of regenerative activity of Schwann cells, axons, and new myelin tubes. Whether or not the axons preceded the Schwann cells or vice versa could not be determined. Axons, both myelinated and nonmyelinated, were more numerous in association with sheets of Schwann cells than with knots or clumps of these supportive cells. Although the presence of proliferating Schwann cells is probably a favorable sign of regeneration, the frequent clumping and whorling of the cells as seen in a schwannoma may have been detrimental to the fate of a growing axon in terms of establishing a functional connection. Thus, if indeed the Schwann cell tube provides direction for the growing axon, as in regeneration in the peripheral nervous system, and its function is interrupted by Schwann cell clumping, then the cells' subsequent function of myelination would be completed upon misdirected axons. The cell that demonstrated a surprising degree of proliferation was the ependymal cell. In the rat, the central canal is normally patent and lined by simple, cuboidal, ciliated ependymal cells. There was no difficulty in recognizing proliferative activity of the cells in the central sections of the cord. As noted above, the cords of proliferating ependymal cells extended toward the injury site from both the proximal and distal segments of the cord and were often associated with axons. It is interesting to speculate that these cells may have some neurotrophic function. Ramon and Cajal [20] discussed the importance of a "mesodermal or ectodermal influence" for the regenerative attempts of central axons, but they postulated a role for the Schwann cells in this regard. The possible role of ependymal cells in regeneration in the mammalian central nervous system has not been addressed, although in lower animals capable of spinal cord regeneration, ependymal cells play a major role [9,18]. For example, after amputating the tail of the newt, ependymal cells were found to proliferate into the regenerating stump before the growing axons, and the clefts between these proliferating ependymal cells afford a directional scaffolding for the regenerating
axons [9,18]. In higher vertebrates, however, evidence for ependymal cells having a role in regeneration is minimal. Sugar and Gerrard [23] noted "rosette-forming cells" associated with regenerating fibers in the transected rat spinal cord, but they did not refer to them as ependymal cells. Recently, Vaguero et al [27] documented significant ependymal cell proliferation following spinal cord transection in the rat, but they did not comment on any association with regenerative activity of axons. To date, only Matthews et al [15] have demonstrated a relationship between the regenerating axons in the transected rat spinal cord and the proliferating sheets of ependymal cells. With electron microscopy they found an invagination of the ependymal cells by growing axons, similar to that seen in the newt [9,19]. Our results did not show such an intimate anatomical relationship between axons and regenerating ependyma, but the pattern and extent of the proliferating sheets of ependymal cells is consistent with that found by other investigators [ 15,27]. It remains an attractive hypothesis that because of their neuroblast origin and ability to proliferate, the regenerating ependymal cells are capable of exerting a growth stimulating influence on damaged axons in the central nervous system. Despite the observed ependymal cell proliferation, we found no significant association between clinical performance and other regenerative activity. It is possible that increased proliferation or improved migration of the ependymal cells to the axons located more peripherally within the cord might result in improved function. Unfortunately, methods of improving ependymal cell proliferation or migration are completely unknown. In conclusion, this study has shown exuberant regenerative activity of Schwann cells, ependymal cells, and myelinated fibers in the spinal cord following experimental spinal cord injury. Of particular interest is the proliferation of ependymal cells and their migration from the central canal.
The technical assistance of Mrs. L. Marmash, Mrs. B. Young, Mrs. E. Forsythe, and Miss M. Vespa is gratefully acknowledged. Dr. M.C. Wallace was a Fellow of the Medical Research Council of Canada. This research was supported by the Medical Research Council of Canada (grant MT 4046).
References 1. Allen AR. Remarks on the histological changes in the spinal cord due to impact: an experimental study. J Nerv Ment Dis 1914;41:141-7. 2. Allen AR. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column: preliminary report. JAMA 1911;57:878-80. 3. Assenmacher DR, Ducker TB. Experimental traumatic paraple-
R e g e n e r a t i v e C h a n g e s in the Spinal C o r d
gia: the vascular and pathological changes seen in reversible and irreversible spinal cord lesions. J Bone Joint Surg 1971;53A:671-80. 4. Balentine JD. Pathology of experimental spinal cord trauma. I. The necrotic lesion as a function of vascular injury. Lab Invest 1978;39;236-53. 5. Barnett HJM, Botterell EH, Jousse AT, Wynn-Jones M. Progressive myelopathy as a sequel to traumatic paraplegia. Brain 1966;89:159-73. 6. Bernstein JJ, Wells MR, Bernstein ME. The effect of puromycin treatment on the regeneration of hemisected and transected rat spinal cord. J Neurocytol 1978;7:215-28. 7. Cowan WM, Cuenod M. The use of axonal transport for studies of neuronal connectivity. Amsterdam, Oxford, New York: Elsevier Science Publishing, 1975. 8. Ducker TB, Kindt GW, Kempe LG. Pathological finding in acute experimental spinal cord trauma. J Neurosurg 1971;35:700-8. 9. Egar M, Singer M. The role of ependyma in spinal cord regeneration in the urodele. Triturus. Exp Neurol 1972;37:422-30. I0. Holmes G. Spinal injuries of warfare. Br Med J 1915;2:769-74. 11. Hughes JT. Pathology of the spinal cord. 2nd ed. Philadelphia, London, Toronto: WB Saunders, 1978. 12. Kao CC, Chang LW. The mechanism of spinal cord cavitation following spinal cord transection. Part I: a correlated histochemical study. J Neurosurg 1977;46:197-209. 13. Kao CC, Chang LW, Bloodworth JMB Jr. The mechanism of spinal cord cavitation following spinal cord transection. Part 2: electron microscopic observations. J Neurosurg 1977;46:745-56. 14. Kao CC, Chang LW, Bloodworth JMB Jr. The mechanism of spinal cavitation following spinal cord transection. Part 3: delayed grafting with and without spinal cord retransection. J Neurosurg 1977;46:757-66. 15. Matthews MA, St. Onge MF, Faciane CL. An electron microscopic analysis of abnormal ependymal cell proliferation and envelopment of sprouting axons following spinal cord transection in the rat. Acta Neuropathol 1979,45:27-36. 16. McLean DR, Miller JDR, Allen PBR, Ezzeddin SA. Posttraumatic syringomyelia. J Neurosurg 1973;39:485-92. 17. Moore RY. Regeneration in the mammalian nervous system. Ann NY Acad Sci 1980;339:102-14. 18. Norlander RH, Singer M. The role of ependyma in regeneration
Surg N e u r o l 1987;27:209-19
219
of the spinal cord in the urodele amphibian tail. J Comp Neurol 1978;180:349-74. 19. Nurick S, Russell JA, Deck MDF. Cystic degeneration of the spinal cord following spinal cord injury. Brain 1970;93:211-22. 20. Ramon Y, Cajal S. Degeneration and regeneration of the nervous system. Volumes 1 and 2. RM May translator. London: Oxford University Press, 1928. 21. Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978;9:39-43. 22. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg 1977;47:577-81. 23. Sugar O, Gerrard RW. Spinal cord regeneration in the rat. J Neurophysiol 1940;3:1-19. 24. Tarlov IM, Klinger I. Spinal cord compression studies. II. Time limits for recovery after acute compression in dogs. Arch Neurol 1954;71:271-90. 25. Tarlov M. Spinal cord compression studies. III. Time limits for recovery after gradual compression in dogs. Arch Neurol 1954;71:588-97. 26. Tator CH, Rivlin AS, Lewis AJ, Schmoll B. Effect of acute spinal cord injury on axonal counts in the pyramidal tract of rats. J Neurosurg 1984;61 : 118-23. 27. Vaguero J, Ramiro MJ, Oya S, CatezudoJM. Ependymal reaction after experimental spinal cord injury. Acta Neurochir (Wien) 1981;55:295-302. 28. Wagner FCJr, Dohrmann GJ. Alterations in nerve cells and myelinated fibres in spinal cord injury. Surg Neurol 1975;3:125-31. 29. Wagner FC Jr, Dohrmann GJ, Bucy PC. Histopathology of transitory traumatic paraplegia in the monkey. J Neurosurg 1971;35:272-6. 30. Wagner FC Jr, Van Gilder JC, Dohrmann GJ. Pathological changes from acute to chronic in experimental spinal cord trauma. J Neurosurg 1978;48:92-8. 31. Wallace MC, Tator CH, Gentles W. Effect of electrical stimulation of the spinal cord on recovery from acute spinal cord injury in rats. (submitted for publication). 32. Wohlfart G. Degeneration and regeneration in the nervous system. World Neurol 1961 ;2:187-98. 33. Wolman L. The neuropathology of traumatic paraplegia. A critical historical review. Paraplegia 1964;1:233-51.